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https://openalex.org/W2974431181	https://europepmc.org/articles/pmc6801817?pdf=render	English	null	Production Losses Associated with Alcohol-Attributable Mortality in the European Union	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	9,052	Received: 20 August 2019; Accepted: 20 September 2019; Published: 21 September 2019 Abstract: The economic aspects of alcohol misuse are attracting increasing attention from policy makers and researchers but the evidence on the economic burden of this substance is hardly comparable internationally. This study aims to overcome this problem by estimating production losses (indirect costs) associated with alcohol-attributable mortality in 28 European Union (EU) countries in the year 2016. This study applies the prevalence-based top–down approach, societal perspective and human capital method to sex- and age-speciﬁc data on alcohol-related mortality at working age. The alcohol-attributable mortality data was taken from estimates based on the Global Burden of Disease Study 2016. Uniform data on labor and economic measures from the Eurostat database was used. The total production losses associated with alcohol-related deaths in the EU in 2016 were €32.1 billion. The per capita costs (share of costs in gross domestic product (GDP)) were €62.88 (0.215%) for the whole EU and ranged from €17.29 (0.062%) in Malta to €192.93 (0.875%) in Lithuania. On average, 81% of the losses were associated with male deaths and mortality among those aged 50–54 years generated the highest burden. Because alcohol is a major avoidable factor for mortality, public health community actions aimed at limiting this substance misuse might not only decrease the health burden but also contribute to the economic welfare of European societies. Keywords: production losses; indirect costs; alcohol mortality; human capital method; European Union International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2019, 16, 3536; doi:10.3390/ijerph16193536 Article Production Losses Associated with Alcohol-Attributable Mortality in the European Union Bła˙zej Łyszczarz D f P bli H l h F l f H l h S i Ni l C i U i i i T ´ Bła˙zej Łyszczarz a ej ys c a Department of Public Health, Faculty of Health Sciences, Nicolaus Copernicus University in Toru´n, 85-830 Bydgoszcz, Poland; blazej@cm.umk.pl; Tel.: +48-52-585-5409 1. Introduction Alcohol is a major avoidable risk factor for mortality and morbidity worldwide [1–3]. As such, the misuse of alcohol is a public health concern; this is particularly relevant in the European context, because the consumption of the substance is higher there than in any other region [4,5]. Alcohol is responsible for a number of adverse health eﬀects and is the third leading risk for the burden of disease in Europe [6]; in the European Union (EU), 1 in 7 males and 1 in 13 females aged 15–64 years die of alcohol-attributable causes, corresponding to 11.8% of all deaths in this age category [7]. This considerable health impact of alcohol translates to economic consequences which result in a decrease in the welfare of societies. The resources spent on tackling the detrimental health eﬀects of alcohol (‘direct costs’ of health care, crime, property loss, welfare assistance, i.a.) cannot be employed for other, probably more eﬃcient, purposes. Moreover, a certain amount of the potential economic output is not produced because of alcohol eﬀects and this category of losses (referred to as ‘indirect costs’) encompasses premature mortality, inability to work, decreased productivity and incarceration [8–11]. Some studies also attempt to attribute economic value to pain, suﬀering and the decline in the quality of life due to alcohol (‘intangible costs’) [12,13]; however, this cost category does not represent either the real or potential loss of material resources. The literature on the economic consequences of alcohol is vast and growing rapidly. Only in the last 15 years, several systematic reviews on the economic impact of alcohol consumption [4,8,10,14], www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph 2 of 14 Int. J. Environ. Res. Public Health 2019, 16, 3536 heavy drinking and alcohol dependence [9,15] have been published. Studies from individual states include both estimates from high-income countries (e.g., Belgium [12,16], the United States [11,17], Germany [18–20], the United Kingdom [13,21], Estonia [22] and Portugal [23]) and middle-income states (e.g., Russia [24], Sri Lanka [25] and Thailand [26]). Despite this abundance of evidence, the comparability of ﬁndings from particular countries is extremely limited because of methodological heterogeneity (diﬀerent deﬁnitions, data sources, cost categories and calculation methods), resulting in a broad range of estimates [8,9]. A recent systematic review of alcohol cost estimates in the EU reports an economic burden ranging from 0.12% of gross domestic product (GDP) in Portugal to 3.47% of GDP in Sweden [10]. 1. Introduction Furthermore, two studies from the same country (Scotland) and similar period estimate the burden of alcohol at 2.03% of GDP in 2007 [27] and 1.13% of GDP in 2009–2010 [13]. Such a variation in the results makes the conclusions drawn ambiguous and precludes assessing the cost-eﬀectiveness of alcohol-targeted policies across countries, which is one of the purposes of social cost studies [10]. This study aims to contribute to the literature by providing highly comparable estimates on one of the indirect cost categories, namely the production losses associated with alcohol-attributable mortality. Clearly, the cost of mortality constitutes only a single component of alcohol economic burden; however, several cost-of-illness studies show that its magnitude is considerable. The cost of mortality constituted the following shares of total alcohol-attributable costs (or production losses): 27.0% (or 35.2%) in Germany [19]; 30.2% (42.0%) in the US [11]; 43.8% (61.8%) in Belgium [12]; 45.0% (68.8%) in another study from Germany [18] and as much as 66.7% (69.6%) in Thailand [26]. These ﬁgures show that mortality is a major burden category in the cost of alcohol misuse. Thus, estimating the losses due to alcohol-attributable mortality in a wide range of European countries is the ﬁrst contribution which provides highly comparable estimates across numerous economies. This comparability can be achieved with the beneﬁts of data collection consistency in the EU; data regarding mortality as well as labor and economic measures is collected and reported with high uniformity. Additionally, although this study is concerned with a single component of economic burden, the indirect costs of mortality are subject to applied research in numerous health problems (particularly in cancer [28–30], but also in alcoholism [31]) and such estimates provide useful insights into the economic burden of illness. Hence, the purpose of this study was to provide the cost estimates of alcohol-attributable mortality in 28 EU countries (for the year 2016) and, as such, to overcome one of the main shortcomings of the previous studies which is the incomparability of ﬁndings from various geographical settings. 2. Materials and Methods Using these country-speciﬁc labor market statistics I identiﬁed the average time of work lost due to premature deaths associated with alcohol, separately for men and women at every 5-year interval of age for each of the 28 states. In this study, the mean productivity of a person was proxied by per worker GDP adjusted for the decreasing marginal productivity of labor. This adjustment requires multiplying the value of production by the correction coeﬃcient (here: 0.65) and this reﬂects a fact that each incremental worker produces output that is lower than on average. The use of the coeﬃcient results from the law of diminishing marginal productivity, which—applied to the present context—means that the productivity gained thorough avoidance of early deaths would be lower than the output of an average worker in the economy (for more details see [42,43]). The GDP was also adjusted for the purchasing power parity (PPP) [44] and this allowed accounting for price diﬀerences among the 28 EU countries. The sex-speciﬁc number of deaths at each 5-year interval from 15–19 years to 65–69 years was taken from an analysis of alcohol use and burden based on the Global Burden of Disease (GBD) Study 2016 [2]. This study estimated the number of sex- and age-speciﬁc deaths attributable to alcohol and associated with the following outcomes: atrial ﬁbrillation and ﬂutter; breast cancer; cirrhosis and other chronic liver diseases; colon and rectum cancer; diabetes mellitus; epilepsy; esophageal cancer; hemorrhagic stroke; hypertensive stroke disease; interpersonal violence; ischemic heart disease; ischemic stroke; larynx cancer; lip and oral cavity cancer; liver cancer; lower respiratory infections; pharynx and nasopharynx cancer; pancreatitis; self-harm; transport injuries; tuberculosis; unintentional injuries [2]. These estimates are based on the notion of population attributable fraction which represents the share of deaths that would have been avoided if the exposure to alcohol in the past had been reduced to the counterfactual level of the theoretical minimum alcohol exposure [2]. Because the number of deaths in the GBD’s estimates is reported in 5-year intervals, I assumed that each death occurred at the middle age of each interval, e.g., at the age of 47 for the 45–49 years interval. A half-cycle adjustment was applied to account for the fact that deaths occur throughout the whole year; therefore, with this assumption, the present deaths are considered to happen in the middle of the year [12,18,45]. 2. Materials and Methods Similar to a majority of previous research, this study uses the prevalence-based [18,24,26], top–down approach [27,32,33], societal perspective [34–36] and the human capital method (HCM) to estimate the production losses (indirect costs) associated with deaths attributable to alcohol in 28 EU countries in 2016. The top–down approach was used because for a study based on mortality data, central registries provide more accurate ﬁgures than data collected for small samples such as in the bottom–up alternative. Moreover, more previous studies on the cost of alcohol relied on the top–down approach as shown in a systematic review by Laramée et al. [9]. The research only accounts for losses in formal economy and, as such, I have not included the costs resulting from informal economic activities (e.g., housekeeping or informal caregiving) undone because of alcohol-related deaths. This approach results from the fact that no comparable data on informal production levels for the range of countries investigated here exists. Therefore, only those death cases which occurred at the working age were included in the estimates. The choice of the HCM in cost valuation means that a premature mortality case translates to a discounted value of output that would be produced if the one who died was still alive and working until the average age of retirement [37–39]. A set of sex-speciﬁc, country-level labor market measures was used to identify the average time a person at a particular age would work if they had not died; these indicators were: 3 of 14 Int. J. Environ. Res. Public Health 2019, 16, 3536 • The average age of starting ﬁrst regular job (data for 2015; obtained from Eurostat on request. Data for Denmark and Sweden were not available and so, for these two countries, I used a sex-speciﬁc average value for the remaining 26 EU countries); • The average age of starting ﬁrst regular job (data for 2015; obtained from Eurostat on request. Data for Denmark and Sweden were not available and so, for these two countries, I used a sex-speciﬁc average value for the remaining 26 EU countries); • The average eﬀective age of exit from labor market [40]; and • The employment rate for population aged 15–64 [41]. The future values of the above measures were assumed to be constant because no reliable and comparable predictions in this respect were available for the range of countries investigated. 3.1. Alcohol-Attributable Deaths at Working Age The estimated number of alcohol-attributable deaths at working age in the year 2016 was 137,122 in the whole European Union and 79% of these cases (108,360) were deaths among men. Germany experienced the highest absolute burden of 24,137 deaths, followed by Poland (18,044) and France (16,136). On the other hand, there were less than 200 deaths at working age due to alcohol in Malta (28), Cyprus (115) and Luxembourg (120). A vast majority of these deaths concerned men and the share of their deaths in total ranged from 63.4% in the United Kingdom to 89.8% in Slovakia. The gender structure of the deaths exhibits a clear geographical and socio-economic pattern. In all the countries of Central and Eastern Europe (CEE), the share of male deaths was >84% of the total number (86.5% on average), while the corresponding proportion was 15.5; 10.5; and 8.1 percentage points lower in Northern, Southern and Western Europe, respectively. Moreover, the other (non-CEE) countries that joined the EU in the 21st century (Lithuania, Latvia, Estonia, Croatia, Slovenia and Cyprus) had an enormously high share of men’s deaths too (Table 1). Table 1. Estimated number and incidence of alcohol-attributable deaths at working age and consumption of alcohol in 28 European Union countries, 2016. 2. Materials and Methods The future costs were discounted using a 5% rate, while the potential per worker GDP growth rates for each future decade for the particular countries [40] were used to reﬂect the paths of their economies’ growth. No sex- or age-speciﬁc data on per worker GDP were available; thus, the estimates average the losses using data for whole populations and do not reﬂect the speciﬁc productivity of those dying due to alcohol misuse. This simpliﬁcation might bias the results upward; this would be the case if alcohol-attributable deaths were more common among the less productive. A one-way deterministic sensitivity analysis was performed to test how the parameter changes aﬀect the results. A 3.5% discount rate and no discounting were used instead of the 5% in the base scenario. Moreover, I applied a ±0.05 variation in the coeﬃcient correcting for marginal labor productivity and replaced GDP by gross value added as a measure of productivity. Additionally, the lower and upper bounds of 95% conﬁdence intervals for the number of alcohol-attributable deaths as reported in GBD study [2] were used. The study used only data that is publicly available or obtainable on request. It did not involve any human participants; therefore, no approval from the ethics committee was sought. 4 of 14 Int. J. Environ. Res. Public Health 2019, 16, 3536 3. Results 3.1. Alcohol-Attributable Deaths at Working Age Public Health 2019, 16, 3536 Table 1 also shows the incidence of (estimated) alcohol-attributable deaths per 10,000 population by gender; the countries have been sorted in descending order of male deaths. There were 4.34 (1.10) deaths associated with alcohol per 10,000 men (women) in the whole European Union in 2016. These deaths were relatively most common in the three Baltic states, Romania and Hungary. On the other hand, the rates for Malta were several times lower than in the countries with the greatest burden. There was not much diﬀerence in the incidence of women’s deaths between three European sub-regions (CEE, Northern and Western: 1.27; 1.24; and 1.20 per 10,000, respectively), while the rate for Southern Europe was noticeably lower (0.75 per 10,000). On the contrary, men’s mortality incidence was much more diversiﬁed between the sub-regions; the rate for the CEE countries was more than twice as large (three times) as that for the Northern and Western Europe (Southern Europe). There is also a variation in the consumption of alcohol which ranges from 7.5 liters of pure alcohol per person aged 15+ per year in Italy to 15.0 liters in Lithuania (data refers to recorded and unrecorded consumption) (Table 1). 3.1. Alcohol-Attributable Deaths at Working Age Number Per 10,000 Population Alcohol Consumption (Litres) Women Men Total % of Men’s Deaths Women Men Central and Eastern Europe 5923 38,072 43,996 86.5% 1.27 8.65 12.2 Romania 1875 10,340 12,215 84.6% 1.86 10.74 12.6 Hungary 806 4734 5540 85.4% 1.57 10.11 11.4 Poland 2238 15,806 18,044 87.6% 1.14 8.60 11.6 Bulgaria 316 2727 3042 89.6% 0.86 7.87 12.7 Slovakia 214 1885 2099 89.8% 0.77 7.12 11.5 Czechia 474 2581 3055 84.5% 0.88 4.97 14.4 Northern Europe 6162 15,086 21,248 71.0% 1.24 3.14 11.3 Lithuania 337 2108 2445 86.2% 2.18 15.96 15.0 Latvia 199 1122 1321 84.9% 1.88 12.46 12.9 Estonia 144 746 890 83.8% 2.06 12.09 11.6 Finland 308 1347 1656 81.4% 1.11 4.98 10.7 Denmark 446 1265 1710 73.9% 1.55 4.44 10.4 Ireland 272 716 988 72.4% 1.13 3.04 13.0 United Kingdom 3932 6809 10,741 63.4% 1.18 2.11 11.4 Sweden 524 973 1497 65.0% 1.06 1.96 9.2 Southern Europe 5243 19,031 24,275 78.4% 0.75 2.88 9.1 Croatia 189 1323 1512 87.5% 0.88 6.57 8.9 Portugal 556 3028 3584 84.5% 1.02 6.19 12.3 Slovenia 74 413 487 84.7% 0.72 4.03 12.6 Spain 2131 7185 9316 77.1% 0.90 3.15 10.0 Greece 284 1256 1541 81.5% 0.51 2.41 10.4 Cyprus 16 99 115 85.7% 0.38 2.38 10.8 Italy 1986 5706 7692 74.2% 0.64 1.94 7.5 Malta 6 21 28 78.0% 0.27 0.94 8.1 Western Europe 11,433 36,171 47,604 76.0% 1.20 3.95 11.6 Germany 5957 18,180 24,137 75.3% 1.43 4.48 13.4 France 3578 12,558 16,136 77.8% 1.04 3.88 10.0 Austria 436 1664 2100 79.2% 0.98 3.88 11.6 Belgium 670 1835 2505 73.2% 1.17 3.29 12.1 Luxembourg 32 88 120 73.1% 1.11 3.00 13.0 Netherlands 760 1846 2606 70.8% 0.88 2.19 8.7 European Union 28,762 108,360 137,122 79.0% 1.10 4.34 11.0 Notes: Due to rounding, the ‘Total’ number of deaths might not equal the sum of values for men and women. The average numbers of deaths per 10,000 population for sub-regions and the whole EU and alcohol consumption ﬁgures (bold font used to mark that the values refer to group of countries and not single states) are population-weighted averages. ‘Alcohol consumption’ illustrates the total (recorded and unrecorded) alcohol consumption of pure alcohol consumed per person aged 15+ per year [46]. Table 1. Estimated number and incidence of alcohol-attributable deaths at working age and consumption of alcohol in 28 European Union countries, 2016. 5 of 14 Int. J. Environ. Res. 3.2. Production Losses Associated with Alcohol-Attributable Deaths J. Environ. Res. Public Health 2019, 16, 3536 6 of 14 Table 2. Cont. Table 2. Cont. Per Capita Cost (€ PPP) Total Cost (€ PPP) Women Men Total % of Men’s Costs Western Europe 68.96 2,779,978,519 10,108,405,118 12,888,383,637 78.4% Luxembourg 128.44 17,174,562 57,580,230 74,754,791 77.0% Germany 79.35 1,486,429,987 5,048,038,348 6,534,468,335 77.3% Austria 71.30 110,935,938 511,981,436 622,917,374 82.2% France 62.69 807,345,501 3,383,954,136 4,191,299,638 80.7% Belgium 60.76 157,787,776 530,748,450 688,536,227 77.1% Netherlands 45.59 200,304,755 576,102,518 776,407,273 74.2% European Union 62.88 6,115,124,781 26,007,783,271 32,122,908,052 81.0% Notes: € PPP—euro adjusted for purchasing power parity. The average per capita costs and the share of costs attributable to men’s deaths for sub-regions and the whole EU (bold font used to mark that the values refer to group of countries and not single states) are population-weighted averages. Figure 1 shows the production losses associated with alcohol mortality in relation to GDP and this allows assessing the burden of deaths in relation to the size of the European economies. Overall, the share of GDP lost due to alcohol deaths was 0.215% for the whole EU. Four countries experienced a burden exceeding 0.5% of GDP and these were Lithuania (0.875% of GDP), Latvia (0.751%), Estonia (0.640%) and Romania (0.626%). Malta was the country with the lowest burden of 0.062% of GDP and the other countries with a cost of <0.15% of GDP were Italy (0.104%), Greece and the Netherlands (0.122% both), Sweden (0.131%) and Cyprus (0.134%) (Figure 1). Int. J. Environ. Res. Public Health 2019, 16, x 7 of 16 Figure 1. Production losses associated with alcohol-attributable mortality as a share of gross domestic product in 28 European Union countries, 2016. Notes: The bar colors mark a geographic group of countries as follows (the values in brackets show sub-regional averages): green—Central and Eastern Europe (0.444% of GDP); blue—Northern Europe (0.192% of GDP); orange—Southern Europe (0.146% of GDP); grey—Western Europe (0.200% of GDP); black—the average European Union value. The age distribution of losses shows that the deaths of those between 50 and 54 generated the highest burden (22.8% of the total cost among women and 20.6% among men). 3.2. Production Losses Associated with Alcohol-Attributable Deaths The total production losses associated with alcohol-attributable mortality in 28 EU countries in 2016 were €32.1 billion adjusted for PPP (hereafter, all the Euro values are adjusted for PPP). This cost was highest in Germany (€6.5 billion), France (€4.2 billion), Poland (€3.5 billion), Romania and Spain (€2.1 billion both). On the other hand, the economic burden was lower than €100 million in Malta (€7.9 million), Cyprus (€28.0 million), Luxembourg (€74.8 million) and Slovenia (€88.9 million). The relative magnitude of the economic burden of alcohol-attributable deaths was measured by the per capita indirect cost; for the all 28 EU countries, this cost was €62.88. Lithuania was a country with the highest per capita cost of €192.93, followed by the two other Baltic states—Estonia (€143.95) and Latvia (€141.10)—as well as Luxembourg (€128.44). The per head losses were lowest in four Mediterranean countries: Malta (€17.29), Greece (€24.14), Italy (€29.45) and Cyprus (€32.84). The gender structure of the production losses clearly exhibits the gender pattern of mortality. Therefore, the costs attributable to male deaths was 81.0% of the total cost for the 28 EU countries and ranged from 67.1% (Sweden) to 91.0% (Slovakia) (Table 2). Table 2. Total and per capita production losses associated with alcohol-attributable mortality in 28 European Union countries, 2016. Per Capita Cost (€ PPP) Total Cost (€ PPP) Women Men Total % of Men’s Costs Central and Eastern Europe 87.91 793,907,451 7,172,161,352 7,966,068,804 90.0% Romania 108.72 216,877,078 1,925,090,280 2,141,967,358 89.9% Poland 93.34 325,329,237 3,218,780,378 3,544,109,615 90.8% Hungary 85.29 96,058,299 740,940,071 836,998,370 88.5% Slovakia 75.17 36,584,184 371,650,049 408,234,233 91.0% Czechia 63.77 83,807,460 590,058,364 673,865,824 87.6% Bulgaria 50.63 35,251,192 325,642,211 360,893,403 90.2% Northern Europe 62.21 1,570,442,055 4,503,711,353 6,074,153,409 74.1% Lithuania 192.93 62,226,095 491,146,991 553,373,086 88.8% Estonia 143.95 26,242,498 163,170,869 189,413,367 86.1% Latvia 141.10 39,743,853 236,745,286 276,489,139 85.6% Ireland 111.08 119,634,913 408,585,212 528,220,124 77.4% Denmark 87.59 116,146,423 385,586,701 501,733,125 76.9% Finland 83.25 79,100,833 378,379,100 457,479,933 82.7% United Kingdom 47.32 975,017,362 2,128,982,695 3,104,000,057 68.6% Sweden 46.70 152,330,079 311,114,500 463,444,579 67.1% Southern Europe 38.26 970,796,755 4,223,505,448 5,194,302,203 81.3% Portugal 68.73 96,220,541 613,473,616 709,694,157 86.4% Croatia 47.91 20,113,693 179,789,308 199,903,001 89.9% Spain 45.48 451,909,598 1,662,360,852 2,114,270,450 78.6% Slovenia 43.06 12,082,926 76,844,881 88,927,807 86.4% Cyprus 32.84 3,415,910 24,546,596 27,962,507 87.8% Italy 29.45 351,154,392 1,434,396,088 1,785,550,480 80.3% Greece 24.14 34,767,622 225,353,350 260,120,973 86.6% Malta 17.29 1,132,072 6,740,756 7,872,828 85.6% Table 2. Total and per capita production losses associated with alcohol-attributable mortality in 28 European Union countries, 2016. Int. 3.2. Production Losses Associated with Alcohol-Attributable Deaths However, the age 0.062 0.104 0.122 0.122 0.131 0.134 0.151 0.168 0.170 0.177 0.178 0.190 0.205 0.215 0.215 0.218 0.238 0.249 0.262 0.268 0.304 0.334 0.357 0.437 0.463 0.626 0.640 0.751 0.875 0.0 0.2 0.4 0.6 0.8 1.0 Malta Italy Greece Netherlands Sweden Cyprus United Kingdom Luxembourg Spain Belgium Slovenia Austria France European Union Ireland Germany Denmark Czechia Finland Croatia Portugal Slovakia Bulgaria Hungary Poland Romania Estonia Latvia Lithuania Cost of alcohol attributable mortality as a share of GDP (%) Figure 1. Production losses associated with alcohol-attributable mortality as a share of gross domestic product in 28 European Union countries, 2016. Notes: The bar colors mark a geographic group of countries as follows (the values in brackets show sub-regional averages): green—Central and Eastern Europe (0.444% of GDP); blue—Northern Europe (0.192% of GDP); orange—Southern Europe (0.146% of GDP); grey—Western Europe (0.200% of GDP); black—the average European Union value. 0.062 0.104 0.122 0.122 0.131 0.134 0.151 0.168 0.170 0.177 0.178 0.190 0.205 0.215 0.215 0.218 0.238 0.249 0.262 0.268 0.304 0.334 0.357 0.437 0.463 0.626 0.640 0.751 0.875 0.0 0.2 0.4 0.6 0.8 1.0 Malta Italy Greece Netherlands Sweden Cyprus United Kingdom Luxembourg Spain Belgium Slovenia Austria France European Union Ireland Germany Denmark Czechia Finland Croatia Portugal Slovakia Bulgaria Hungary Poland Romania Estonia Latvia Lithuania Cost of alcohol attributable mortality as a share of GDP (%) Cost of alcohol attributable mortality as a share of GDP (%) Figure 1. Production losses associated with alcohol-attributable mortality as a share of gross domestic product in 28 European Union countries, 2016. Notes: The bar colors mark a geographic group of countries as follows (the values in brackets show sub-regional averages): green—Central and Eastern Europe (0.444% of GDP); blue—Northern Europe (0.192% of GDP); orange—Southern Europe (0.146% of GDP); grey—Western Europe (0.200% of GDP); black—the average European Union value. The age distribution of losses shows that the deaths of those between 50 and 54 generated the Figure 1. Production losses associated with alcohol-attributable mortality as a share of gross domestic product in 28 European Union countries, 2016. Notes: The bar colors mark a geographic group of countries as follows (the values in brackets show sub-regional averages): green—Central and Eastern Europe (0.444% of GDP); blue—Northern Europe (0.192% of GDP); orange—Southern Europe (0.146% of GDP); grey—Western Europe (0.200% of GDP); black—the average European Union value. 7 of 14 Int. J. Environ. Res. 3.2. Production Losses Associated with Alcohol-Attributable Deaths Public Health 2019, 16, 3536 Considering the sub-regional analysis, the share of GDP lost was highest in the CEE countries (0.444%), followed by the western (0.200%), northern (0.192%) and southern states (0.146%). Noticeably, there is a high variation in this measure among the northern countries; the three Baltic states are the ones with the greatest share of GDP lost among all EU countries, while this value is much lower for other countries of the region (Figure 1). The age distribution of losses shows that the deaths of those between 50 and 54 generated the highest burden (22.8% of the total cost among women and 20.6% among men). However, the age distribution of cost diﬀered across the genders; for the young population (aged 20 to 34 years), the share of cost in the total losses for particular age intervals was higher for men (e.g., 5.2% for men and 3.1% for women among those aged 20–24 years). On the other hand, the corresponding shares were higher for women aged 35–54 years (Figure 2). Int. J. Environ. Res. Public Health 2019, 16, x 8 of 16 Figure 2. Age distribution of (a) female and (b) male production losses associated with alcohol- attributable mortality in 28 European Union countries, 2016. Figure 2. Age distribution of (a) female and (b) male production losses associated with alcohol-attributable mortality in 28 European Union countries, 2016. Figure 2. Age distribution of (a) female and (b) male production losses associated with alcohol- attributable mortality in 28 European Union countries, 2016. Figure 2. Age distribution of (a) female and (b) male production losses associated with alcohol-attributable mortality in 28 European Union countries, 2016. Figure 3 shows an association between the production losses estimated in this study and three socio-economic measures that potentially affect the magnitude of economic burden related to alcohol mortality, namely (a) per capita consumption of pure alcohol, (b) per capita GDP (€ PPP) and (c) share of GDP spent on health care. There is a substantial relationship (R2 = 0.393) between alcohol Figure 3 shows an association between the production losses estimated in this study and three socio-economic measures that potentially aﬀect the magnitude of economic burden related to alcohol mortality, namely (a) per capita consumption of pure alcohol, (b) per capita GDP (€ PPP) and (c) share of Int. J. Environ. Res. Public Health 2019, 16, 3536 8 of 14 GDP spent on health care. 3.3. Sensitivity Analysis The results of a one-way sensitivity analysis show a magnitude of changes in the present estimates resulting from the parameter variation. The use of the 3.5% discount rate resulted in an average change of 12.6% in the total production losses, with a minimum (maximum) change of 10.6% (15.5%). With no discounting, the cost estimates varied from 47.5% (Germany) to 77.1% (Cyprus), depending on the country analysis. A ±0.05 variation in the coeﬃcient adjusting for decreasing marginal productivity changed the estimates by ±7.7%. When gross value added was used instead of GDP, the results decreased by 12.1% on average (range: −5.7% to −17.9%). The use of the lower (upper) bound of conﬁdence interval for the estimated number of alcohol-attributable deaths reported in the GBD study [2] resulted in a change from the base scenario, ranging from −20.8% to −51.6% (20.7% to 67.9%) (Table 3). Table 3. Sensitivity analysis for estimates of production losses associated with alcohol-attributable mortality in 28 European Union countries, 2016. Table 3. Sensitivity analysis for estimates of production losses associated with alcohol-attributable mortality in 28 European Union countries, 2016. Average Change from BS 1 Minimum Change from Base Scenario Maximum Change from Base Scenario Discount rate (BS: 5%) 3.5% 12.6% 10.6% (Germany) 15.5% (Cyprus) 0% 58.3% 47.5% (Germany) 77.1% (Cyprus) Coeﬃcient to adjust for decreasing marginal labor productivity (BS: 0.65) 0.6 −7.7% −7.7% (all countries) 0.7 7.7% 7.7% (all countries) Productivity measure (BS: gross domestic product) Gross value added −12.1% −5.7% (Ireland) −17.9% (Croatia) 95% conﬁdence interval for the number of alcohol-attributable deaths (BS: point estimate) Lower bound −31.7% −20.8% (Portugal) −51.6% (Malta) Upper bound 34.9% 20.7% (Lithuania) 67.9% (Malta) 1 Notes: BS—base scenario; results for BS shown in Table 2. Notes: BS—base scenario; results for BS shown in Table 2. 3.2. Production Losses Associated with Alcohol-Attributable Deaths There is a substantial relationship (R2 = 0.393) between alcohol consumption and production losses expressed as a share of GDP. For the countries where people drink more alcohol these losses are higher; however, with growing consumption levels, the variation in costs increases. There also seems to be a relationship between the economic power of a country and the cost of alcohol deaths; the countries with higher per person GDP generally lose less in terms of the indirect costs of mortality, but this relationship is weaker (R2 = 0.230). Additionally, there is a negative and considerable association (R2 = 0.359) between expenditure on health and the production losses estimated. The countries spending a higher share of their GDP on health generally experience a lower economic burden of alcohol-related deaths and there is a higher variation in indirect cost among the countries spending lower shares of their GDP on health. Int. J. Environ. Res. Public Health 2019, 16, x 10 of 16 Figure 3. Association between production losses due to alcohol-attributable mortality and (a) consumption of pure alcohol per person aged 15+ per year (litres); (b) GDP per capita (€ PPP); and (c) health care expenditure as a share of GDP. Notes: GDP—gross domestic product; PPP—purchasing power parity. Source: own calculations. Figure 3. Association between production losses due to alcohol-attributable mortality and (a) consumption of pure alcohol per person aged 15+ per year (litres); (b) GDP per capita (€ PPP); and (c) health care expenditure as a share of GDP. Notes: GDP—gross domestic product; PPP—purchasing power parity. Source: own calculations. Figure 3. Association between production losses due to alcohol-attributable mortality and (a) consumption of pure alcohol per person aged 15+ per year (litres); (b) GDP per capita (€ PPP); and (c) health care expenditure as a share of GDP. Notes: GDP—gross domestic product; PPP—purchasing power parity. Source: own calculations. Figure 3. Association between production losses due to alcohol-attributable mortality and (a) consumption of pure alcohol per person aged 15+ per year (litres); (b) GDP per capita (€ PPP); and (c) health care expenditure as a share of GDP. Notes: GDP—gross domestic product; PPP—purchasing power parity. Source: own calculations. Int. J. Environ. Res. Public Health 2019, 16, 3536 9 of 14 4. Discussion Using the cost-of-illness methodology, this study estimated the production losses (indirect costs) associated with alcohol-attributable mortality in 28 European Union countries in the year 2016. The results show that the health and economic burden of alcohol varies notably throughout the countries and European sub-regions. The estimated number of deaths attributable to alcohol consumption in the EU was 137,122 in 2016 and 79% of these death cases occurred among men. The number of deaths at working age per 10,000 males was more than ten times higher in ﬁve countries—with the highest incidence (three Baltic states, Romania and Hungary) compared to Malta, where the burden was the lowest. The diﬀerence between the countries in the rate of women mortality was also high, with an eight-fold diﬀerence between the best- and worst-performing states. These alcohol-related mortality diﬀerences between the countries are clearly much deeper than for the overall mortality rates; the lowest value of 829 deaths per 100,000 inhabitants was in Spain and the respective value for the country with highest rate was <2 times greater (1602 in Bulgaria) [47]. The burden of mortality at working age was the most severe in Central and Eastern Europe and this was particularly notable for men; the lowest incidence was generally observed in Southern Europe. Moreover, a clear socio-economic pattern emerges from the present estimates; the post-communist countries that joined the EU in the 21st century experienced a much greater mortality burden and all of them had an enormously high share of male deaths. This pattern was observed in all the CEE states, the three Baltic republics as well as in Croatia and Slovenia and was previously described in epidemiological, demographic and social literature [48–50]. 10 of 14 Int. J. Environ. Res. Public Health 2019, 16, 3536 The production losses associated with alcohol-attributable deaths clearly exhibit patterns of mortality. The three countries with the greatest production losses were the Baltic republics, where the indirect costs constituted 0.640% to 0.875% of their GDP and this translated to per capita losses of €141.10–€192.93. Again, these values were several times higher than in the southern countries with the lowest costs (Malta, Greece and Italy; 0.062% to 0.122% of GDP and €17.29–€29.45 per capita). Overall, the diversity of the economic burden associated with alcohol-attributable mortality was large and this reﬂects not only the mortality diﬀerences but also market labor and economic conditions in particular economies. 4. Discussion The massive mortality in the Baltic states was reinforced by relatively long labor market activity, high employment rates among women, high prospects of economic growth and more deaths at younger ages. On the other hand, the lowest costs observed in the Mediterranean countries were not only due to relatively low death rates but also because of low employment and slow dynamics of projected economic development in the future. Considering the age distribution of mortality costs, the highest economic burden was identiﬁed for those at the age of 50–54 and 45–49 years. The age distribution of costs was similar for both genders with a slightly higher magnitude of cost shares among middle-aged women (40–54 years) and young men (see Figure 2). This gender diﬀerence clearly reﬂects the higher mortality of men at younger ages, which is a well-recognized phenomenon in epidemiological literature [51,52]. More generally, the higher costs associated with male mortality result from several reasons. First of all, male mortality rates are much higher than the female ones and this results from men’s higher consumption of alcohol, among others. Moreover, male employment rates are higher, and this fact strengthens the mortality eﬀect in terms of higher economic losses among men. Possibly, also other labor market characteristics of men and women may play a role in the cost distribution by gender. Unfortunately, with the data available, I was not able to assess such issues originating from the gender diﬀerences in labor market as (potential) productivity variation between men and women. Particularly, no data on per worker GDP broken down by gender was available and the only measure that was possible to use was gender pay gap. However, I decided not to use it because it is argued that payment gap reﬂects rather market discrimination and not real productivity diﬀerences [53]. This choice was further justiﬁed by the fact that, in my study, productivity was proxied by GDP and not earnings. This study also investigated the associations between indirect costs of alcohol-attributable mortality and selected socio-macroeconomic measures. According to this analysis, the countries with higher alcohol consumption suﬀer from greater mortality costs and this relationship is quite strong (R2 = 0.393). Obviously, this ﬁnding is not surprising. Moreover, the negative and notable relationship (R2 = 0.359) shows that the countries spending higher shares of their GDP on health experience a lower economic burden of alcohol-related mortality. 4. Discussion This last association clearly does not prove a causal relationship; however, it suggests that investing in health care might be a way to limit economic losses of alcohol consumption. This last statement requires more insightful analysis and could be useful in formulating a hypothesis which could be tested in future research. The stability of estimates reported here was tested using a one-way deterministic sensitivity analysis. The results were most prone to changes in the discount rate and with no discounting, the losses were 58.3% higher on average. This relatively high variation should not be surprising because a vast majority of the losses attributable to a mortality case occur in the future. Particularly, for those dying at young ages, the losses which span to decades towards the future are much lower when discounted. The other sensitivity scenario which resulted in a notable variation in results (ca. ±1/3 of costs on average) was the use of 95% conﬁdence intervals for a number of deaths attributable to alcohol. For all other parameter changes used, the variation in estimates was not that meaningful. Table 4 compares the present estimates with previous ﬁndings on the costs of alcohol misuse. For this comparison, I selected those studies that referred to EU countries and reported a separate category of mortality costs. Moreover, to allow for comparability, I expressed the losses associated with alcohol-attributable mortality as a share of GDP. In some cases my estimates are higher than in the previous research (e.g., from Belgium [12,54], Ireland [55], and Portugal [56]) while for other countries, 11 of 14 Int. J. Environ. Res. Public Health 2019, 16, 3536 the opposite is true (e.g., Estonian [22], German [18] and French [57] estimates). It is beyond the scope of this discussion to elaborate on the sources of these diﬀerences that result from methodological variation between the studies (see, for example, the diﬀerence in discount rates in Table 4) and the year of particular studies. Nonetheless, the comparison clearly exhibits the problematic nature of drawing comparisons between the costs of alcohol in various countries. The present study aims to overcome this problem by providing estimates on the production losses associated with alcohol-related mortality for a range of countries and obtained with uniform data and methods. Table 4. Comparison of production losses associated with mortality in selected studies from the European Union (2000 and onwards). 5. Limitations of the Study This study provides the ﬁrst internationally comparable estimates on the cost of alcohol-attributable mortality for a range of countries. Despite this important contribution, the present analysis has to be interpreted with caution because some important caveats apply to both the design and the scope of the research. Firstly, the costs estimated here only account for those alcohol-related problems that result in death. Therefore, the results do not provide ﬁgures on the overall economic burden of alcohol. Particularly, direct health care costs, crime and traﬃc costs as well as intangible costs, which are all a potentially important economic burden, were not considered here. Nevertheless, the assessment of mortality costs is important because they constitute a large share of total costs as explained in the introduction. Moreover, research on this single cost category is common in cost-of-illness literature [28–31]. Secondly, the number of deaths due to alcohol consumption which was taken from the GBD study was based on estimations that also have some limits of their own as pointed in the source paper [2]. These limitations were: potential failure to fully capture illicit or unrecorded production; use of American data to estimate motor vehicle harm; lack of data on harm caused to others from alcohol-attributable interpersonal violence; the exclusion of the population younger than 15 years; and inability to include some outcomes for which alcohol might be a risk factor such as dementia and psoriasis [2]. Overall, these limitations resulting from data constraints are likely to underestimate the burden of alcohol as stated in the GBD study [2]. Thirdly, there are further concerns about the appropriateness of the input data used. Particularly, the study uses average values of labor and economic measures which might bias the results. For example, the employment rate among those dying prematurely because of alcohol misuse might be diﬀerent than in the total population. One might expect this rate to be lower among alcohol misusers and if this is the case, the production losses estimated here are biased upward. The same reasoning applies for the use of average per worker GDP as a productivity measure; potentially some of those dying because of alcohol might be less productive than on average. This shortcoming of my analysis has to be kept in mind when interpreting the results; however, it needs to be stressed that my approach is not diﬀerent from other studies which use aggregated data. 4. Discussion Study Country (Year) Losses in the Compared Study 1 Losses in the Present Study DS % of GDP Lievens et al. [54] Belgium (2012) 0.134% 0.177% n.a. Verhaeghe et al. [12] Belgium (2012) 0.103% 0.177% 0% Byrne [55] Ireland (2007) 0.056% 0.215% n.a. Saar [22] Estonia (2006) 1.825% (1.052%) 2 0.640% 4% (10%) Konnopka, König [18] Germany (2002) 0.497% 0.218% 5% Lima, Esquerdo [56] Portugal (1995) 0.083% 0.304% 5% Fenoglio et al. [57] France (1997) 0.704% 0.205% 6% Jarl et al. [58] Sweden (2002) 0.119% (0.331%) 3 0.131% 3% Cabinet Oﬃce [59] UK (2001) 0.200% (0,220%) 4 0.151% n.a. Notes: 1 Own calculations based on Eurostat’s data on GDP and costs taken from respective studies; DS—discount rate; 2 4% (10%) discount rates; 3 net (gross) losses; 4 low (high) estimate; n.a.—details not available/reported. Table 4. Comparison of production losses associated with mortality in selected studies from the European Union (2000 and onwards). Notes: 1 Own calculations based on Eurostat’s data on GDP and costs taken from respective studies; DS—discount rate; 2 4% (10%) discount rates; 3 net (gross) losses; 4 low (high) estimate; n.a.—details not available/reported. 5. Limitations of the Study Only those studies relying on individual Int. J. Environ. Res. Public Health 2019, 16, 3536 12 of 14 data can overcome this problem, but they are prone to other issues, e.g., the potentially problematic generalization of results obtained with sample data to population level. data can overcome this problem, but they are prone to other issues, e.g., the potentially problematic generalization of results obtained with sample data to population level. References 1. Global Status Report on Alcohol and Health 2018; World Health Organization: Geneva, Switzerland, 2018. 1. Global Status Report on Alcohol and Health 2018; World Health Organization: Geneva, Switzerland, 2018. . Global Status Report on Alcohol and Health 2018; World Health Organization: Geneva, Switzerland, 2018. 2. GBD 2016 Alcohol Collaborators. Alcohol use and burden for 195 countries and territories, 1990–2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet 2018, 392, 1015–1035. [CrossRef] y y y , , [ ] 3. 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https://openalex.org/W4386559412	https://recyt.fecyt.es/index.php/retos/article/download/97357/73874	es		Modelos de enseñanza del deporte: Un estudio bibliométrico (Teaching models of sport: A bibliometric study)	Retos digital/Retos	2,023	cc-by	5,055	2023, Retos, 50, 936-942 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (https://recyt.fecyt.es/index.php/retos/index) Modelos de enseñanza del deporte: Un estudio bibliométrico Teaching models of sport: A bibliometric study Sandy Dorian Isla Alcoser, Ana Flávia Backes, Allana Alencar, Juarez Vieira do Nascimento Universidad Nacional Mayor de San Marcos (Peru), Universidade Federal de Santa Catarina (Brasil), **Universidade do Estado de Santa Catarina (Brasil) Resumen. El deporte fomenta un entorno ideal para potenciar el desarrollo integral de las personas por medio de diferentes modelos de enseñanza, estos no se podrían llevar a cabo sin una adecuada metodología y aplicación en niños, adolescentes o adultos. El principal objetivo fue realizar un análisis bibliométrico sobre de los aspectos importantes en la literatura científica acerca del deporte visto en materia de “modelos de enseñanza o instruccionales”, para lo cual 300 artículos fueron seleccionados de la base de datos de Scopus y Web of Science. Esta investigación utilizó el software R, el paquete de datos bibliometrix, para llevar a cabo los análisis descriptivos de autores, y análisis de colaboración. El periodo de análisis fue de 2002:2021. De forma adicional, se realizó un análisis del contenido de los artículos más relevantes en base al impacto según la búsqueda bibliométrica. Los resultados indican que una producción científica posee una gran ascendencia de estudios en el último periodo (2012 - 2021). Gil Arias, Kirk y Davis son los autores más productivos, China posee a la mayor cantidad de autores corresponsales; La universidad de Granada es la que está investigando en mayor proporción. Las keywords de acuerdo a su fuerza de conexión y frecuencia fueron: teaching, sports, physical education, humans, physical education and training. Del análisis de contenido, se deduce de los autores que es necesario un enfoque más formativo respecto a la naturaleza del deporte como parte fundamental del proceso de asimilación. Se concluye que se requiere mayor información referente al tema de estudio y cooperación a nivel internacional. Palabras claves: bibliometría, modelos de enseñanza, enseñanza, deportes, educación física. Abstract. Sport promotes an ideal environment to enhance the integral development of people through different teaching models, which could not be carried out without an adequate methodology and application in children, adolescents, or adults. The main objective was to carry out a bibliometric analysis of the important aspects of the scientific literature on sports seen in terms of “teaching models, instructional models”, for which 300 articles were selected from the Scopus and Web of Science databases. This research used the R software, the bibliometrix data package, to perform the descriptive analysis of the authors and collaborative analysis. The analysis period was 2002:2021. In addition, a content analysis of the most relevant articles was performed based on the impact according to the bibliometric search. The results indicate that scientific production has a great ancestry of studies in the last period (2012 - 2021). Gil Arias, Kirk, and Davis are the most productive authors, China has the highest number of corresponding authors; The University of Granada is the one that researches the most. The keywords according to binding strength and frequency were: teaching, sports, physical education, humans, physical education, and training. From the content analysis, it is deduced from the authors that a more formative approach is needed regarding the nature of sports as a fundamental part of the assimilation process. It is concluded that more information is needed on the topic of study and cooperation at an international level. Keywords: bibliometrics, teaching models, teaching, sports, physical education. Fecha recepción: 25-11-22. Fecha de aceptación: 11-08-23 Sandy Dorian Isla Alcoser sislaa@unmsm.edu.pe Introducción Las clases han sido señaladas como un entorno ideal para potenciar la práctica de actividad física, así como la adquisición de estilos de vida entre los adolescentes y adultos (Langford, et al., 2015). Por lo que su enseñanza y difusión en clases es fundamental para lograr buenos resultados en la salud física y mental de los estudiantes. Así el deporte ha sido un contenido central del currículo escolar de Educación Física en muchos países del mundo (Bradley, Board, Archer & Morgans, 2022). Actualmente los modelos de enseñanza del deporte expresan perspectivas y diversas comprensiones de su papel en la formación de las capacidades humanas, especialmente en lo que concierne se refiere al trato con niños y adolescentes (Kirk, 2013). Por ello, existe la necesidad de orientaciones adecuadas y actualizadas para seguir los caminos de la enseñanza deportiva y en los diversos ámbitos y campos de acción e intervención de la Educación Física. Por otro lado, el deporte es una parte importante de la educación física, pero, no se enseña bien, por lo que es, tergiversado y distorsionado en el enfoque tradicional de múltiples actividades, y se pierde el principal valor educativo del deporte (Casey & Kirk, 2021). La enseñanza del deporte es una práctica establecida en - 936 - la educación que puede adoptar un modelo de aprendizaje que esté en sintonía con la participación activa del estudiante en sus procesos de aprendizaje y su participación activa en lugar de su adquisición pasiva (Bradley, et al., 2022). En la literatura internacional, los modelos de enseñanza centrados en el estudiante son: modelo de enseñanza por medio de la comprensión del juego (MEC), modelo deportivo educacional (MDE), modelo de aprendizaje cooperativo (MAC), modelo de enseñanza por descubrimiento (MEPD) y modelo de enseñanza de la responsabilidad personal y social (MERPS) (Casey & Kirk, 2020). Algunos ensayos teóricos y estudios de revisión han abordado la diversidad de modelos de enseñanza o instruccionales, destacando los beneficios en la motivación, implicación y mejora del aprendizaje de los estudiantes (Hernando-Garijo, Hortigüela-Alcalá, Sánchez-Miguel & González-Víllora, 2021), las propuestas metodológicas para implementar el aprendizaje cooperativo (Martín & Jiménez, 2021; Silva, Farias, Moreno Arroyo & Mesquita, 2021), los principios pedagógicos de las prácticas orientadas a la enseñanza para la comprensión (Díaz, Palomino & Luna, 2020; Gómez, et al., 2023) y constructivismo (Backes, et al., Retos, número 50, 2023 (4º trimestre) 2023, Retos, 50, 936-942 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (https://recyt.fecyt.es/index.php/retos/index) 2023), enfoques diferentes y complementarios de la enseñanza del deporte (Graça & Mesquita, 2007; Mesquita, Pereira & Graça, 2009) y modulación de conductas técnicotácticas (Borges, Praça, Figueiredo, Vieira & Costa, 2022; Cantos & Hernández, 2019). Investigadores de diferentes países han concentrado esfuerzos para comprender mejor, explorar y aplicar nuevos modelos de enseñanza o instruccionales que se basen en la práctica, pero no fue posible encontrar en la literatura consultada un estudio sobre los autores, afiliaciones, colaboraciones científicas y revistas que concentran publicaciones sobre este tema investigativo. Considerando que la bibliometría ayuda a identificar tendencias de investigación, brinda información sobre aspectos específicos y generales a lo largo del tiempo, analiza la evolución de autores y redes de colaboración (Virú-Vásquez, et al., 2022), la presente investigación tiene como objetivo realizar un análisis bibliométrico de aspectos importantes en la literatura científica sobre el deporte en cuanto a modelos de enseñanza o instruccionales, temas pedagógicos para los cuales se realizó análisis descriptivo de autores y análisis de colaboración mediante bibliometrix, un paquete de datos del software R. Métodos Data y criterio de búsqueda El análisis bibliométrico se llevó a cabo siguiendo el procedimiento descrito por Zupic & Čater (2014), se aprecia cinco etapas, las cuales corresponden a estudio del diseño, recolección de la data, análisis de la data, visualización de la data y finalmente, la interpretación (figura 1). con “Deportes.”, haciendo uso del operador booleano “AND”. El periodo de búsqueda se realizó en mayo del 2022 en la base de datos de Scopus y Web of Science, la que incluye una gran cantidad de base de datos, abstracts, autores, entre otros. para lo cual el criterio de búsqueda fue el siguiente (KEY (teaching AND models) OR KEY (instructional AND models) OR KEY (models AND based AND practice) OR KEY (pedagogical AND tasks) AND KEY (sports)) AND PUBYEAR < 2022, donde se excluyó el año 2022. Se consideraron como criterios de inclusión todos los estudios empíricos/artículos originales, disponibles en su totalidad, y que abordaran, de alguna manera, los modelos de enseñanza basados en la práctica del deporte. Se excluyeron del análisis los artículos de opinión y las cartas abiertas. Los procedimientos de búsqueda y selección de los artículos fueron realizados de forma independiente por investigadores y, en un segundo momento, se cruzó la información para asegurar los criterios de elegibilidad de los datos. Para obtener el consenso necesario en los análisis resultantes de este proceso, se realizó una reunión entre los investigadores para analizar las discrepancias (Gomes & Caminha, 2014). La selección final sumó 300 artículos. Como recurso computacional para la manipulación y análisis de datos se utilizó el software R 3.5.2 (R Core Team, 2018), junto con el IDE RStudio 1.1.463. Todos los análisis fueron soportados por el paquete “Bibliometrix” (Aria & Cuccurullo, 2017), utilizando biblioshiny a través de la interfaz shiny. Los datos exportados incluyeron la siguiente información: número de documentos publicados en cada año, tipos de documentos publicados, áreas temáticas, nombres de revistas, nombres de autores, país y afiliación institucional de los autores que participaron en la publicación de la literatura recuperada. Resultados y discusión Figura 1. Fases de la investigación. Fuente: Adaptado de Secinaro et al. (2020) Para llevar a cabo el diseño de estudio, las preguntas son definidas con el método bibliométrico adecuado. El propósito del estudio utiliza los términos “Modelos de enseñanza”, “Modelos instruccionales”, “pedagogical tasks” y en unión Análisis descriptivo La tabla 1, se ilustra la información principal del presente análisis bibliométrico que fue realizado en la investigación, con un total de 300 artículos que fueron publicados en el periodo de 2002:2021, se resalta que un total de 813 autores publicaron en dicho periodo, con un total de 764 palabras clave, generando un promedio de 9.904 citas por documento. Los autores que publicaron de manera individual fueron 68. Por otro lado, la cantidad de citaciones promedio por documento es de 6.216. Tabla 1 Información principal Información prinicipal Documentos Sources Palabras clave de autores Periodo Autores Autores de documentos de un solo autor Autores por documento Coautores por documentos Citas promedio por documentos - 937 - Explicación Número total de documentos La distribución de la frecuencia de sources en revistas Número total de palabras clave Años de publicación Número total de autores El número de autores individuales por artículos Promedio de autores en cada documento Promedio de autores en cada documento Promedio de citas en cada documento Número 300 177 764 2002:2021 813 68 2.59 2.97 6.216 Retos, número 50, 2023 (4º trimestre) 2023, Retos, 50, 936-942 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (https://recyt.fecyt.es/index.php/retos/index) La producción científica anual referente a los modelos de enseñanza del deporte a lo largo de los años (figura 2), muestran una curva ascendente en los tres periodos. El periodo I (2002-2014) con un total de 13 artículos, el periodo II (2015-2018) con un total de 61 artículos, el periodo III (2019-2021) con un total de 139, lo cual muestra un incremento de artículos en los últimos años. Esto puede deberse a la posible consolidación de una línea de investigación, aproximación del binomio teoría y práctica y articulación de este paradigma en la práctica (Fernandez-Rio & Iglesias, 2022). tribuyó con al menos 1 artículo, el 7% con al menos 2 artículos, y el 0.3% de los artículos con al menos 6 artículos, según como se muestra en la figura 4. La implicación de investigadores de diferentes países y continentes sugiere la expansión de esta línea de investigación en el campo de la Pedagogía del Deporte a nivel mundial. Figura 4. Productividad de los autores por medio de la ley de Lotka Figura 2. Producción científica anual Autores Los autores más relevantes con la mayor cantidad de documentos publicados de acuerdo al campo de estudio (top 20) se ilustran en la figura 3. Se muestran cuatro grupos claramente diferenciados de acuerdo a la cantidad de documentos. El primer grupo lo conforma Gil Arias y Kirk con un total de 6 documentos; el segundo grupo conformado por el autor Davids, posee 5 documentos, el tercer grupo está conformado por 5 autores: Borysova, Casey, Harvey, Luguetti y Oliver con 4 documentos, el último grupo con un total de 3 documentos esta representado por los autores. La ley de Lotka es uno de los métodos para determinar la productividad científica de un autor en un tema determinado durante un período de tiempo determinado y esta ley se representa matemáticamente con la ecuación mostrada en Kilicoglu & Mehmetcik (2021). La productividad de los autores según esta ley, explica la relación entre el número de autores y el número de artículos (Lotka, 1926). La figura 5 muestra tres niveles de acuerdo al campo de estudio de la presente investigación, estos son: los keywords (en la izquierda), los autores (en el medio), y las instituciones a las que pertenecen los autores (derecha), algunas de las palabras clave que resaltan son: sport teaching, physical education, sport pedagogy, instructional models, models-based practice, exercise, self determination theory, motivation, pedagogical models, sport education, teaching, physical activity, coaching, youth sport, sport, sport education models, teaching models, evaluation models. Se resalta que physical education, sport pedagogy, instructional models y models-based practice fueron las palabras clave más utilizadas por los autores. Se resalta que "Porto" hace referencia a investigadores que tienen como filiación a la Universidad de Porto, y “Portugal” a autores que demostraron solamente filiaciones de diferentes grupos de investigación vinculados a la Universidade do Porto, Instituto Politécnico de Viana do Castelo, Instituto Politécnico de Santarém, Universidade de Trás-os-Montes e Alto Douro, Universidade de Beira Interior y Universidade Técnica de Lisboa. Figura 3. Autores con la mayor producción científica Para la presente investigación el 90% de los autores con- - 938 - Figura 5. Diagrama de tres campos: relación entre las principales palabras clave, los principales autores y las principales revistas. Retos, número 50, 2023 (4º trimestre) 2023, Retos, 50, 936-942 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (https://recyt.fecyt.es/index.php/retos/index) Al identificar a Portugal con un protagonismo significativo, se cree que la reforma propiciada por el Proceso de Bolonia pudo haber estimulado la internacionalización de las instituciones de enseñanza e investigación más allá de las fronteras europeas, ampliando y profundizando las relaciones de sus universidades con las instituciones y sistemas latinoamericanos (Sobrinho, 2007). Cabe señalar que el protagonismo de Portugal también puede darse por la congruencia del idioma de la lengua portuguesa, hecho que posibilita la conexión y el desarrollo de investigaciones y proyectos con investigadores, docentes y profesionales de diferentes países de América Latina. Las publicaciones científicas pueden desarrollarse de manera individual o colaborativa, ello se debe ver reflejado en los mapas de colaboración de autoría entre países de un mismo continente. En la figura 6 se ilustra el número de publicaciones de países que publicaron individualmente o en colaboración, China lidera el ranking con un total de 72 documentos, España con un total de 31 documentos, 6 realizados de manera colaborativa, y 25 de manera individual; USA con un total de 20, 2 realizados de manera colaborativa, y 18 de manera individual. Se cree que la apertura política de China y el creciente desarrollo de la industria del deporte, sustentada en la fortaleza intrínseca de un país portador de la cultura milenaria de la actividad física y el deporte, la favorecen como líder en el marco de la producción científica. Figura 7. Revístas más citadas Figura 8. Países más citados Figura 9. Afiliaciones más relevantes Figura 6. País del autor de correspondencia Nota. SCP: Single Country Publications – MCP: Multiple Country Publications En la figura 7 se muestra el ranking de revistas con mayores aportes significativos en el periodo de búsqueda. Entre las revistas más relevantes se muestran: Retos, Physical education and sport pedagogy, Scientific Journal of National Pedagogical Dragoma. El número de las citaciones de cada país, se muestra en la figura 8. Los que superan las 400 citas son Reino Unido (416) y España (437). Las que se encuentran en un rango de citaciones de publicaciones mayor de 200 y menor que 400, son Australia y USA. Las instituciones más resaltantes son: Universidad de Granada, German Sport University, University of Newcastle (figura 9). El alto número de citas de investigaciones realizadas en Reino Unido puede estar asociado a su papel pionero en investigar y proponer modelos de enseñanza - eg. TGfU (Bunker & Thorpe, 1982). Países como España, Australia y USA han contribuido a la reformulación de modelos de enseñanza, la difusión y consolidación de investigaciones en esta línea de investigación. - 939 - Figura 10. Red de colaboración de acuerdo con las coautorías Análisis de colaboración La figura 10 exhibe la red de autores que han investigado sobre sports and teaching models, cada color representa un grupo de colaboración de autores, y cuanto más grande es el circulo mayor es el número de citas que ha recibido este autor, las líneas representan los vínculos y la distancia está asociada con la fuerza de la relación. Se observa una gran Retos, número 50, 2023 (4º trimestre) 2023, Retos, 50, 936-942 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (https://recyt.fecyt.es/index.php/retos/index) cantidad de grupos y que no están colaborando en una mayor proporción. Se observan grupos de dos, tres y a lo mucho cinco autores. Otro punto estudiado en el análisis bibliométrico es la colaboración entre países, en la figura 11, se observa una ligera colaboración entre países como España, Chile, Portugal y Brazil (color rojo); Estados Unidos, Reino Unido y Australia para el color azul y para el color verde Norway and Ireland. Los resultados apuntan a una red de colaboración entre diferentes países y continentes, hecho que refuerza la preocupación y el compromiso de la comunidad científica con la propuesta, implementación y evaluación de prácticas basadas en modelos (Fernandez-Rio & Iglesias, 2022). Figura 11. Red de colaboración según países La agenda investigativa ha aportado importantes evidencias sobre la aplicación de diferentes modelos que contrastan con el modelo técnico-tradicional, como el Modelo de Educación Deportiva (Burgueño & Medina-Casaubón, 2020; Lucca, Impolcetto & Ginciene, 2022; Molina, Campo, Domínguez & Hopper, 2020; Muñoz, Marco, Gajardo & Oliva, 2022; Ojeda, Maxera, Giménez & Navarrete, 2019), el Modelo de Enseñanza para la Comprensión (Peña, Serrano, Catalán, Remacha & González, 2022), el Modelo de Enseñanza Modelo de Responsabilidad Personal y Social (Guillén & Sandoval, 2022; Pozo Rosado, GraoCruces, Ayllon-Negrillo & Pérez-Ordás, 2022), el Modelo de Aprendizaje Cooperativo (Martínez, Blanco, Valero, 2021; Sánchez, Grau & Vall-Llovera, 2023). Además de la diversidad de modelos instruccionales, algunos estudios revelan que la combinación de modelos o la adopción de un modelo híbrido ha demostrado ser atractivo y con mayores beneficios en el aprendizaje de niños y jóvenes (Buendía, Martínez, Izquierdo & Mármol, 2022; Costa, Nascimento & Vieira, 2016; Espinosa, Godoy, Molina & Galatti, 2017; Costa, et al., 2018; Merino & Mora, 2022; Uria-Valle & Gil-Arias, 2022). Al considerar el proceso de enseñanzaaprendizaje como un sistema complejo y no lineal, los modelos contemporáneos se basan en los constreñimientos del practicante, el entorno y la tarea (Caldeira, Paulo, Infante & Araújo, 2019). El enfoque se centra en la influencia que ejercen los constreñimientos introducidos en una tarea (Rodríguez & Macías, 2021), así como la modificación de normas, el tamaño del área de juego o el material utilizado (Gómez-Criado & Valverde-Esteve, 2021). - 940 - Limitaciones El análisis bibliométrico no utilizo otras bases indexadoras, concentrando su atención a partir de artículos publicados en el año de 2002 con los términos y criterio de búsqueda referidos a los modelos de enseñanza. Conclusiones Existe un incremento en cuanto a las publicaciones, se destaca que el último periodo de estudio, periodo III (20192021) con un total de 139 documentos. Los autores con mayor producción científica son Gil Arias, Kirk y David. Los autores corresponsales que más publicaron sobre la temática de modelos enseñanzas basados en la práctica son de nacionalidad China y de Estados Unidos. Los países que poseen una mayor cantidad de citas son United Kingdom, Australia y USA. Las publicaciones en colaboración se concentran con autores vinculados a los países de España, Portugal y Australia. Las afiliaciones más relevantes fueron: Universidad de Granada, German Sport University, University of Newcastle. Las colaboraciones científicas se establecen entre investigadores vinculados a grupos de investigaciones y universidades portugués y españoles. Se espera que esta investigación ayude a comprender mejor la dinámica de colaboración y análisis de autores. Referencias Aria, M., & Cuccurullo, C. (2017). bibliometrix: An Rtool for comprehensive science mapping analysis. Journal of Informetrics, 11(4), 959–975. https://doi.org/10.1016/J.JOI.2017.08.007 Backes, A. F., Ramos, V., Brasil, V. Z., Ristow, L., Alcoser, S. D. I., Arantes, L. C., & Nascimento, J. V. D. (2023). Pedagogical principles of constructivist-oriented teaching practices in team sports. Journal of Physical Education, 34, 1-14. https://doi.org/10.4025/jphyseduc.v34i1.3405. Borges, E. D. P. 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https://openalex.org/W4389212036	https://www.researchprotocols.org/2024/1/e48601/PDF	English	null	Understanding Employee Voice Behavior Through the Use of Digital Voice Channel in Long-Term Care: Protocol for an Embedded Multiple-Case Study	JMIR research protocols	2,024	cc-by	11,985	Abstract Background: Specific challenges in the health care sector, such as hierarchical structures, shortages of nursing staff, and high turnover of nursing staff, can be addressed by a change process of organizational culture into shared governance. Data from business organizations show that the use of digital voice channels provides employee voice. This approach makes concrete the opportunity for employees to raise their voices by answering surveys and making comments in an anonymous forum, which subsequently positively influences staff turnover and sick leave. Since there is no clear understanding of how a digital voice channel can be used in long-term care to address employee voice, a research gap has been identified. Objective: The purpose of ADVICE (Understanding Employee Voice Behavior; the acronym for this study) is to understand how the use of a digital voice channel performs in long-term care (residential long-term care and home care facilities). The aim of this study is to understand how the digital voice channel can support staff in making their voices heard and to see what managers need to use the voice channel to change the work environment. Methods: An embedded multiple-case study will be used to explore the experiences of 2 health care providers who have already implemented a digital voice channel. ADVICE is organized into two main phases: (1) a scoping review and (2) an embedded multiple-case study. For this purpose, focus group interviews with employees, discursive-dialogical interviews with managers, meeting protocols, and data from the digital voice channel will be analyzed. First, all units of analysis from every embedded unit will be separately analyzed and then comprehensively analyzed to obtain a case vignette from every embedded unit (within-analysis). In the second stage, the analyzed data from the embedded units will be compared with each other in a comparative analysis (cross-analysis). Results: The results will provide insight into how digital voice channels can be used in long-term care to address employee voice. We expect to find how the digital voice channel can empower nurses to speak up and, consequently, create a better work environment. Data collection began in August 2023, and from a current perspective, the first results are expected in summer 2024. Conclusions: In summary, the results may help to better understand the use of a digital voice channel in the health care sector and its transformative potential for leadership. Anja Kepplinger1,2, BSc, MSc; Alexander Braun3,4, MA, MSc, Prof Dr; André Fringer1,5, Prof Dr, PhD; Martina Roes1,6, RN, Prof Dr, PhD Anja Kepplinger1,2, BSc, MSc; Alexander Braun3,4, MA, MSc, Prof Dr; André Fringer1,5, Prof Dr, PhD; Martina Roes1,6, RN, Prof Dr, PhD Anja Kepplinger1,2, BSc, MSc; Alexander Braun3,4, MA, MSc, Prof Dr; André Fringer1,5, Prof Dr, PhD; Martina R 1,6 RN P f D PhD 1Department of Nursing Science, Faculty of Health, Witten/Herdecke University, Witten, Germany 2Institute Nursing Science, Department of Health Sciences, IMC University of Applied Sciences Krems, Krems, Austria 3Institute Health Management, IMC University of Applied Sciences Krems, Krems, Austria 4Institute for Management and Economics in Healthcare, UMIT Tyrole, Hall, Austria 5Institute of Nursing, School of Health Sciences, Zurich University of Applied Sciences ZHAW, Winterthur, Switzerland 6Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Witten, Germany Corresponding Author: Anja Kepplinger, BSc, MSc Institute Nursing Science, Department of Health Sciences IMC University of Applied Sciences Krems Piaristengasse 1 Krems, 3500 Austria Phone: 43 2732 802 161 Email: anja.kepplinger@fh-krems.ac.at Protocol Understanding Employee Voice Behavior Through the Use of Digital Voice Channel in Long-Term Care: Protocol for an Embedded Multiple-Case Study Anja Kepplinger1,2, BSc, MSc; Alexander Braun3,4, MA, MSc, Prof Dr; André Fringer1,5, Prof Dr, PhD; Martina Roes1,6, RN, Prof Dr, PhD 1Department of Nursing Science, Faculty of Health, Witten/Herdecke University, Witten, Germany 2Institute Nursing Science, Department of Health Sciences, IMC University of Applied Sciences Krems, Krems, Austria 3Institute Health Management, IMC University of Applied Sciences Krems, Krems, Austria 4Institute for Management and Economics in Healthcare, UMIT Tyrole, Hall, Austria 5Institute of Nursing, School of Health Sciences, Zurich University of Applied Sciences ZHAW, Winterthur, Switzerland 6Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Witten, Germany Corresponding Author: Kepplinger et al JMIR RESEARCH PROTOCOLS JMIR RESEARCH PROTOCOLS Kepplinger et al https://www.researchprotocols.org/2024/1/e48601 JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 1 (page number not for citation purposes) Background To promote high-quality, person-centered care in the context of challenges related to nursing turnover as well as the different needs and requirements of different generations, health care providers need to invest in creating a productive work environment [4]. To sustainably change the culture in an organization, the interplay of transformational leadership, shared governance, and action processes is needed [5]. In particular, transformational leadership is presented as a leadership style that is action-oriented and enables development and change through shared governance. Shared governance is seen as a management approach in the sense of enabling all stakeholders and employees to have a voice in decision-making processes [5,6]. The PTE-LTC program and the “Relationship-Centered Team Nursing Model for Care Delivery in Nursing Homes” emphasize the importance of empowering frontline staff so that in practice they can have a participatory voice that aims to influence decisions, communicate ideas, communicate residents’ needs and requirements, and initiate change [4,7]. This participatory element of workplace democracy is known as employee voice. Employee voice refers to all organizational structures, mechanisms, or practices in which employees participate and try to influence their work and the performance of their organization [12,13]. The term employee voice has a long tradition in organizational research and is variously defined in the literature [14]. A summary by Dundon et al [15] describes the concept of employee voice in terms of four characteristics: (1) addressing dissatisfaction, opinions, or suggestions; (2) expressing a collective organizational culture; (3) managing decision-making processes; and (4) demonstrating a cooperative relationship between management and employees in an organization [15]. Elizabeth Wolfe Morrison, a professor of management and organization at the Stern School of Business, New York University, defines employee voice as “informal and discretionary communication of ideas, suggestions, concerns, problems, or opinions about work-related issues, with the intent to bring about improvement or change” [14]. Recommendations to implement a change process in organizations using transformational leadership and shared governance are supported by the positive impact of the Pathway to Excellence in Long-Term Care (PTE-LTC) program. This is a special Magnet Recognition Program of the American Nurses Credentialing Center for long-term care. The PTE-LTC program is based on 6 pathway standards: professional development, shared decision-making, leadership, well-being, quality, and safety. The goal is to empower and give frontline staff a voice, promote staff participation in improving outcomes, create a sustained culture, strengthen interprofessional collaboration, and ensure the well-being of employees [7]. Overview The International Council of Nurses points out that there is a need to reduce the dropout rate in nursing and promote the loyalty of employees to their health care provider [1]. Data from the Austrian Work Climate Index that are related to working conditions in the nursing professions describe that 65% of nurses intend to leave their current position, and 15% have plans to change their profession [2]. Furthermore, according to the NORDCARE report on working conditions in long-term care in Austria [3], the working conditions perceived by nurses have declined in recent years. As justifications, the respondents noted the increase in work demands, staff shortages, and lack of time for caring activities. A low level of autonomy, especially among nurses, is highlighted by the collected data. Nursing assistants and home assistants state that they feel left alone with the responsibility for people in need of care. The NORDCARE report [3] findings are corroborated by data from the Austrian Work Climate Index [2], indicating that 44% of employees lack or have inadequate chances to participate in work activities, which may negatively impact on-job satisfaction. The “Relationship-Centered Team Nursing Model for Care Delivery in Nursing Homes” is a nursing care delivery model that attempts to address workforce challenges in long-term care. The model describes 4 assumptions. The first is to skillfully manage a multilevel team with a focus on care coordination and communication; the second is to empower and develop staff and delegate appropriately and responsibly; the third is the requisite preparation of nursing staff to deliver person-centered care; and the fourth is nursing leadership. In conclusion, the model addresses an evidence-based approach, clear communication, and staff empowerment [4]. Clear communication means, according to Siegel et al [4], open, clear, and timely communication between staff, residents, their families, and the involved structures. KEYWORDS program are that the PTE-LTC program includes a person-centered model of care and unlicensed assistive personnel such as nurse assistants [7]. Research indicates that the focus in health care organizations on a better work environment and higher engagement in shared governance decreases rehospitalization and lowers nurse burnout and job dissatisfaction levels [8-10]. A systematic review shows that certified magnet hospitals, compared with nonmagnet hospitals, have higher job satisfaction levels, less burnout, lower turnover, lower rates of nurse shortages, higher quality of care, and better patient outcomes [11]. International Registered Report Identifier (IRRID): PRR1-10.2196/48601 (JMIR Res Protoc 2024;13:e48601) doi: 10.2196/48601 International Registered Report Identifier (IRRID): PRR1-10.2196/48601 (JMIR Res Protoc 2024;13:e48601) doi: 10.2196/48601 International Registered Report Identifier (IRRID): PRR1-10.2196/48601 https://www.researchprotocols.org/2024/1/e48601 JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 2 (page number not for citation purposes) Abstract At the organizational level, research can help to improve the attractiveness of the workplace by understanding how to give employees a voice. JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 1 (page number not for citation purposes) XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al Background Lainidi et al [36] argue that understanding the concepts of voice and silence from both organizational and employee perspectives is crucial, as is the need for different solutions for different contexts. This means that individual approaches and evaluations are needed to gain a deeper understanding of employee voice, especially in the long-term care sector. h i i i i l 3 h h bli h d The literature on employee voice addresses promotive and prohibitive factors of voice, such as how supervisors respond to that behavior and the effects that voice has. For example, Holland et al [16] and Kee et al [17] show that mechanisms that address the voice of employees can reduce burnout, promote employee well-being [16], and influence organization development through structures that employees are encouraged to explore and give feedback [17]. However, according to Morrison [14], there is a need to consider how informal employee voice behavior is affected by formal opportunities and channels for voicing, both individually and collectively. Recent developments in digital and communication technology have opened new opportunities to address employee voice [18-20]. For example, the social and economic scientist and sociologist Ellmer and Reichel [19] describe the possibility of a company mirror or digital voice channel. This digital communications technology enables voice by allowing employees to periodically report their work satisfaction through minisurveys and make anonymous comments in miniforums. Ellmer and Reichel [19] show in their case study that employees are encouraged to speak up and discouraged from an affordance perspective. In this context, the affordance perspective refers to the definitions of Gibson [21] and Norman [22] and means analyzing the possibilities for action that an artifact offers to an actor, as well as the constraints. The possibilities that an artifact can support or hinder a person in achieving a goal. These results indicate that the interplay of material and social aspects, that is, the characteristics of the voice channel and the responses of the management embedded in the respective organizational context, lead to employees perceiving the channel as either promoting or restricting speech. This further leads to whether employees feel encouraged or discouraged to speak up or not speak up [19]. Background Key differences between the Magnet Recognition Program and the PTE-LTC JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 2 (page number not for citation purposes) XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al personal reflection as well as individual coaching, nursing assistants are able to influence organizational structures and initiate change. As a result, it is necessary to understand the wider context of social processes in an organization to address the voice of employees. For example, to know that psychological safety and commitment-based safety management have an impact on whether nurses are willing to raise concerns and be aware that psychological safety is a social-cognitive variable that varies between individuals within the same work context [27-29]. Research also exists regarding the content and structure of simulation training on speaking up in health care organizations [30,31]. It should be noted that the evidence on simulation training has mainly focused on inpatient and emergency settings, operating theaters, and intensive care units and not on long-term care. Martin et al [32] suggest that relying solely on formal channels may discourage some individuals from speaking up, hence the importance of complementing with informal methods. It is recommended to use a combination of both to encourage a greater variety of voices to be heard. Further studies have examined the implementation of a new role called “guardian” to promote voice in the organization. The research indicates that this role should not only focus on formal acts of voice such as support and advice. Instead, a relationship role between the guardian and colleagues is needed, as well as a well-defined role between the existing channels for voice [33]. As Jones et al [34] and O’Donovan and McAuliffe [35] noted, it is unlikely to find a 1-size-fits-all approach to creating a culture where health care professionals can raise their voice. In summary, it must be mentioned that in the health sector, the term employee voice is not used consistently. The existing literature on the use of voice in health care focuses on the use of voice in the context of patient safety and error reporting. In summary, the current review by Lainidi et al [36] points to the lack of a comprehensive theory of employee well-being and communication in the health care sector, which leads to heterogeneous mechanisms and an unclear understanding of the phenomenon of employee voice in health care. JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 3 (page number not for citation purposes) https://www.researchprotocols.org/2024/1/e48601 Background In summary, a digital voice channel can have a positive impact on the identification and attachment of the employees with the company, leading to a reduction in turnover and sick leave, which depends on job satisfaction [16], perception of autonomy, the influence of organizational context [23], and the perception of safety [24] and dependencies [19]. These results indicate that a digital voice channel is not sufficient on its own; it must be seen as a part of an overall approach to shared governance [19]. Based on these findings, due to the increasing challenges related to nursing turnover and the different needs and requirements of different generations, new measures are needed to meet the increasing demand for nurses to ensure high-quality, person-centered care. In their systematic review, Mair et al [37] note that the published literature focuses on organizational aspects yet neglects the broader social framework that needs to be considered when introducing new technologies. More recently, Krick et al [38] have recommended more evaluations of digital technologies in a real-life setting. It is not about what is technically possible; rather, it is about how new digital technologies in the daily routine can be designed to consider staff needs and preferences [20,39]. Ong et al [40] address this research gap and analyze the implementation of a digital patient feedback system using normalization process theory (NPT) from May and Finch [41] and May et al [42]. NPT is a middle-range theory that provides a sociological framework to understand social processes. The theory was developed to understand how a practice becomes or does not become routinely embedded in social contexts. A total of 4 key components of the theory are of relevance: coherence, To date, the use of a digital voice channel in the health care sector has received little attention. Improvements in the health care structure, IT, organization, and legislation are needed to ensure that health care is error-free, efficient, and effective. The Health & Care Expert Council recommends investing in the promotion of digital health literacy, that is, empowering people to deal competently with the digitalization of health care [25]. Available data indicate that in the health care sector, it can be difficult for employees to have a voice due to hierarchical structures [17,26]. For example, Kee et al [17] research how nursing assistants can develop voice behavior that transcends hierarchical levels. Overview To answer the research questions, an embedded multiple-case study will be chosen. Case studies can be used to analyze a phenomenon from multiple perspectives in a real-life context [44] to gain a deeper understanding [45]. The case study approach is especially appropriate when there is little understanding of a phenomenon in a certain context [44]. According to Yin [44], case study research is described by 5 components: questions, propositions, cases, data collection, and data analysis. Propositions in ADVICE will result from the scoping review and from NPT. The contexts are 2 health care providers that have already implemented a digital voice channel. These 2 long-term care providers describe the context in which several residential long-term care and home care facilities (cases) operate. Both providers use the same digital voice channel. This study will fill a gap in knowledge because it aims to understand how staff and managers use a digital voice channel in the social context of health care providers who offer long-term care services, to subsequently understand how employee voice behavior is affected by the digital voice channel, and to explore what opportunities arise for the employees and for the organization through a digital voice channel. The digital voice channel is a browser-based software-as-a-service system that organizations can purchase on a subscription basis. The founders of the digital channel provider aim to empower employees to actively participate in the improvement and decision-making processes in their workplace by giving them the opportunity to (1) periodically report on their job satisfaction through minisurveys and (2) post anonymous comments in a miniforum. On the user interface, a dashboard with a graph shows the progression of a sentiment metric over time, allowing analysis of current and past sentiment levels in the organization. The sentiment metric is based on responses to anonymous minisurveys covering 6 categories: well-being, health, collaboration, potential development, work activity, and organizational culture. The standard pool of questions can be supplemented by various additional pools. Employees can select from a Likert scale ranging from 1 to 7 (1=does not apply at all to 7=completely true). To gather votes, a link to minisurveys (ranging from 3 to 10 questions) is sent to all employees through email at specified intervals (weekly or monthly), depending on the organization’s preference. Managers are alerted when new votes or anonymous comments are available. Overview This information, combined with the dashboard, can be used to identify and act on trends and dynamics in the team early on, allowing decisions to be made based on this information. To analyze the social processes related to the use of a digital voice channel in health care providers, multiple data sources are necessary to create dense descriptions [44,46]. For this purpose, focus group interviews with employees, discursive-dialogical interviews [47] with managers (plus field notes), meeting protocols, and data from the digital voice channel will be analyzed (units of analysis). The qualitative data and comments in the survey will be analyzed by a deductive-inductive content analysis approach developed by Schreier [48]. Data from the digital voice channel include response rates and frequency of the mentioned topics. These quantitative data are displayed in the digital voice channel and provided by the health care provider. In summary, a phenomenon (digital voice channel) will be analyzed in a real context (2 health care providers) to achieve a deeper understanding of how a digital voice channel becomes a daily routine, that is, normalized [49]. It should be noted that the case definition or units of analysis, as well as other aspects of the research design in a case study, may be revised during data collection [50]. Background The study results show that by training in XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al about generating new knowledge to make the new techniques usable and normalized in long-term care. The overall question is as follows: How does the use of a digital voice channel support opportunities to affect employee voice behavior in long-term care? cognitive participation, collective action, and reflexive monitoring. These 4 components define the process of normalization. Coherence refers to the fact that the practice is an ensemble of beliefs, behaviors, and acts that are defined by a set of ideas about its meaning, uses, utility, and socially defined and organized competencies. The nature of embedding depends on the work that defines and organizes a practice as a cognitive and behavioral ensemble and requires that actors collectively invest meaning in it. Cognitive participation means that normalization depends on the work that defines and organizes the actors involved in a practice. Collective action includes the chain of interactions, that is, a place of mental and material work that deals with organizing and enacting a practice. Reflexive monitoring means the continuous formal and informal evaluation of the patterns of collective action and their outcomes by all participants [41,42]. Collectively, NPT provides an appropriate conceptual framework to understand issues relating to routinization of new technology [37,43]; it will therefore be used in the ADVICE (Understanding Employee Voice Behavior; the acronym for this study) study as the theoretical lens. For the planned scoping review, the following question is relevant: Which opportunities are mentioned in the literature to address employee voice in health care providers? For the embedded multiple-case study, further questions are relevant: What are the experiences in long-term care with the use of a digital voice channel? How is the normalization process described in long-term care, and how do they differ? https://www.researchprotocols.org/2024/1/e48601 develop the search components, the question is integrated into the PICO (population, interest, and context) schema for qualitative studies [52], as shown in Textbox 1. The scoping review will be conducted using the PRISMA ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) [53]. https://www.researchprotocols.org/2024/1/e48601 JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 5 (page number not for citation purposes) Phase 1: Scoping Review Phase 1: Scoping Review The scoping review [51] aims to provide an overview of the literature on employee voice in health care providers and the use of opportunities or interventions such as voice channels, assessments, tools, feedback instruments, feedback training, or meetings to promote having a voice in the organization. To Textbox 1. PICO schema. P: population • Health care provider I: interest • Opportunities like digital voice channel Co: context • Employee voice Textbox 1. PICO schema. Aim of This Study The aims of the study are to investigate the phenomenon of the digital voice channel in a real-life context and to explore opportunities and capture experiences to enable a systematic and professional approach in the future. This means that it is ADVICE is organized into two main phases: (1) the completion of a scoping review and (2) the conduction of an embedded multiple-case study, as shown in Figure 1. JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 4 (page number not for citation purposes) https://www.researchprotocols.org/2024/1/e48601 XSL•FO RenderX Kepplinger et al JMIR RESEARCH PROTOCOLS Overview of the research phases of the ADVICE (Understanding Employee Voice Behavior) study. Figure 1. Overview of the research phases of the ADVICE (Understanding Employee Voice Behavior) study. Figure 1. Overview of the research phases of the ADVICE (Understanding Employee Voice Behavior) study. Exclusion criteria • Other settings similar to business areas • Summaries and discussions • Other languages Covidence is a software for managing and simplifying reviews. The program Covidence supports the review process after the removal of duplicates and enables the screening of the literature by 2 reviewers independently [55]. Specifically, the titles and abstracts will be reviewed against the inclusion and exclusion criteria to identify appropriate studies. In the next step, the full texts of the relevant studies will also be reviewed by 2 reviewers independently. Any disagreements about the eligibility of a study will be resolved by discussion in the research group. For data extraction, a standardized data extraction form will be developed. The form will be tested with the first 3 results and discussed in the research group. The items included in the description of each article will be selected following the Template for Intervention Description and Replication (TIDieR) [56] and Criteria for Reporting and Evaluation of Complex Interventions (CReDECI 2) in health care [57]. Based on the iterative character of the scoping review, all changes will be documented in the protocol [51]. The results of the scoping review will be used to develop propositions and the interview guide used in phase 2 of the ADVICE study. Provider 1 is a health care provider with approximately 1000 employees in different residential long-term care facilities in Austria. A digital voice channel has been used there since 2020. From the current perspective, this health care provider is defined by 3 cases through 3 long-term care facilities. Provider 2 is a health care provider of several residential long-term care and home care facilities with approximately 5000 employees in Austria. The health care provider has been using a digital voice channel in a pilot phase in a few facilities since 2021. From the recent perspective, this health care provider is defined by 3 cases, namely, 1 residential long-term care and 2 home care facilities. In this case, a long-term residential care facility is an institution where people who need care can live or stay for a period of time under the responsibility of professional health care workers. Home care facilities provide professional care to patients in their own homes. The research team is aware of the different settings (residential long-term care and home care facilities) in the embedded units, which represents a limitation. Exclusion criteria The use of the different samplings can be argued by the fact that the focus of the analysis is the use of a digital voice channel, as well as employee voice behavior, in long-term care providers. Thus, it is not about the setting of health care providers per se; rather, the commonality is the same setting of long-term care, the same digital voice channel, and therefore, the same organizational culture with different characteristics. Additionally, based on the different settings, additional data, such as individual characteristics of the specific areas and differences, can be generated. The concrete number of employees and number of beds in the embedded units cannot yet be described, as the recruitment phase is only just beginning. Overview Based on the results of the scoping review, this research phase aims to go into the field and immerse itself in the day-to-day work of the cases to understand the social processes that involve the promotion of employee voice. A detailed description of the components of Yin’s [44] case study research design follows. Casing As shown in Figure 1, the ADVICE sample includes 6 different cases that are situated in 2 different contexts (providers 1 and 2). After Sandelowski [50], the cases for a case study are made, not found, through an iterative and theoretical process called casing. The boundary of the cases can be determined by the phenomenon of the use of the digital voice channel and the same scope of long-term care (content boundary). In these cases, the temporal boundary refers to the time of digital voice channel implementation. Starting with the time of implementation in provider 1, (2020) and provider 2 (2021), the end time describes the end of the data collection from the current point of view (2024) [44]. Textbox 1. PICO schema. P: population • Health care provider • Opportunities like digital voice channel Co: context The search process took place in the period ranging from September 2022 to April 2023 in the MEDLINE through PubMed, CINHAL through EBSCO, Embase, Wiley Online Library, and Cochrane Library databases. Furthermore, the reference lists of relevant results will be screened and identified through Google Scholar. The following search terms will be used for the search: digital voice channel, feedback tool, company mirror, feedback, assessment, tool, voice of employee, employee participation, health care provider, long-term care, and home care facilities. Furthermore, the search terms and their The search process took place in the period ranging from September 2022 to April 2023 in the MEDLINE through PubMed, CINHAL through EBSCO, Embase, Wiley Online Library, and Cochrane Library databases. Furthermore, the reference lists of relevant results will be screened and identified through Google Scholar. The following search terms will be used for the search: digital voice channel, feedback tool, company mirror, feedback, assessment, tool, voice of employee, employee participation, health care provider, long-term care, and home care facilities. Furthermore, the search terms and their synonyms will be linked to search strings using Boolean operators dependent on the databases, as well as MeSH (Medical Subject Headings) terms. After a sensitive search, the research team will consult with each other and adapt the search string. To support the search, the researchers will use the manual for literature search in specialized databases, namely, RefHunter [54]. The inclusion and exclusion criteria shown in Textbox 2 have been derived from the objective and research questions. There will be no restrictions regarding the currency of the literature included. JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 5 (page number not for citation purposes) https://www.researchprotocols.org/2024/1/e48601 XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Textbox 2. Inclusion and exclusion criteria for scoping review. • Qualitative and quantitative studies (all scientific studies) https://www.researchprotocols.org/2024/1/e48601 JMIR RESEARCH PROTOCOLS Kepplinger et al Textbox 2. Inclusion and exclusion criteria for scoping review. Inclusion criteria • Health care provider, residential long-term care, and home care facilities • Digital voice channel, digital feedback tools, feedback tools, evaluation, assessment, and tools • Employee voice, employee participation, and team learning • Qualitative and quantitative studies (all scientific studies) • English and German Exclusion criteria • Other settings similar to business areas • Summaries and discussions • Other languages Textbox 2. Inclusion and exclusion criteria for scoping review. Inclusion criteria • Health care provider, residential long-term care, and home care facilities • Digital voice channel, digital feedback tools, feedback tools, evaluation, assessment, and tools • Employee voice, employee participation, and team learning • Qualitative and quantitative studies (all scientific studies) • English and German Exclusion criteria • Other settings similar to business areas • Summaries and discussions • Other languages JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 6 (page number not for citation purposes) Overview Based on the recommendation of Yin [44], data collection will take place using multiple sources of evidence. Consequently, data will be collected in the form of focus group interviews, discursive-dialogical interviews [47], meeting protocols, and data from the digital voice channel (anonymous survey data and free comments). During the whole research process, field notes and written memos concerning methodological, personal, and case-related issues will be taken by the first author [58]. JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 6 (page number not for citation purposes) XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al These different data are, according to Yin [44], the embedded units of analysis. nurses in residential long-term care facilities to have different general practitioners to contact because residents bring their own general practitioners with them. This applies to other health care professionals like physiotherapy, occupational therapy, and speech and language therapy. In summary, this indicates that physicians and health care professionals are not employed in the health care providers and therefore are not using the digital voice channel. Specifically, data from the digital voice channel will be searched concerning the key mechanism of reflexive monitoring drawn from NPT [41,42], for example, how benefits or problems with the digital voice channel are identified or measured. The Interviews The interviews will include managers, nurses, and social care professionals in each case. Based on the Austrian Nursing Practice Act, nurses are defined as registered nurses with a diploma and a bachelor’s degree (full-time training, 3 years), nursing specialist assistants (full-time training, 2 years), and nursing assistants (full-time training, 1 year). In addition to nurses, social care professionals also work in residential long-term care and home care facilities. There are 3 levels of qualification based on the Social Care Profession Act (home assistant, social care workers, and certified social care workers). Home assistant complete 200 hours of theoretical course and 200 hours of practical training. Social care workers complete a 1200-hour theoretical course and 1200 hours of practical training. Certified social care workers have an 1800-hour theoretical course and 1800 hours of practical training. In addition, managers in different positions will be interviewed. The inclusion and exclusion criteria used for the interview participants are summarized in Textbox 3. The interviews will focus on the following key NPT mechanisms: coherence, cognitive participation, and collective action [41]. In the context of ADVICE, coherence means asking about sense-making both as an individual component and as a team component. For example, do the participants see a sense in the use of the digital voice channel or not? Do they see a benefit for themselves and for the team? Cognitive participation includes topics aiming to understand the initiation, promotion, and legitimation of participation. Social norms and formal or informal rules in the team play a role in this key mechanism. For example, are facilitators involved in which role they either have or do not have, and is there a collective commitment in the digital voice channel or not? The collective action mechanism refers to all measures taken to use the digital voice channel. Examples include competencies for workability, such as knowledge work, which is necessary to build trust, and digital competencies [41,59]. It should also be noted that medical care in residential long-term care in Austria varies greatly according to the laws of the individual federal states. In most facilities, it is provided by general practitioners. This means that general practitioners are not employed by the health care provider; they provide medical care by visiting their patients. For example, it is possible for Textbox 3. Inclusion and exclusion criteria for interviews. https://www.researchprotocols.org/2024/1/e48601 The Interviews Inclusion criteria • Women, men, and diverse • Nurse (registered nurse, nursing specialist assistant, and nursing assistant) • Home assistant, social care workers, and certified social care workers • Manger (nursing director, nursing manager, department manager, deputy manager, head of nursing department, and head of nursing unit o nursing home) • Employed for at least 6 months in 1 of the 2 health care providers that uses a digital voice channel • German or English Exclusion criteria • Employees who do not work in 1 of the 2 health care providers or who are retired nurses or nursing students • Employed for less than 6 months in 1 of the 2 health care providers that uses a digital voice channel • Other languages Textbox 3. Inclusion and exclusion criteria for interviews. Textbox 3. Inclusion and exclusion criteria for interviews. • Employed for at least 6 months in 1 of the 2 health care providers that uses a digital voice channel Exclusion criteria During the focus group interviews, nurses will have the opportunity to discuss the use of the digital voice channel. The aim here is to obtain a picture of what nurses need to be able to give a voice and the impact of the voice channel on their voice behavior. Focus group interviews will be conducted with nurses who work in one of the cases of provider 1 or 2 and do not have a management position. Approximately 6 focus groups will be conducted, each with at least 5-6 individuals in each group. Heterogeneity will be considered in the recruitment process. With attention to the professional qualification mix, every professional nursing group will be represented. The focus group interviews will be moderated by the first author with the help of an interview guide based on the results of the scoping review and NPT. During the focus group interviews, nurses will have the opportunity to discuss the use of the digital voice channel. The aim here is to obtain a picture of what nurses need to be able to give a voice and the impact of the voice channel on their voice behavior. Focus group interviews will be conducted with nurses who work in one of the cases of provider 1 or 2 and do not have a management position. Approximately 6 focus groups will be conducted, each with at least 5-6 individuals in each group. Heterogeneity will be considered in the recruitment process. With attention to the professional qualification mix, every The single interviews will be conducted by the first author in a dialog-discursive interview form based on the interview guide and on the results of the focus group interviews. The reason for choosing dialog-discursive interviews with the help of a semistandardized interview guide is that interviews are JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 7 (page number not for citation purposes) XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al to get to know each other directly. Participation will be strictly voluntary. The providers and work councils (employee representation in the health care provider) will provide informed consent before the data collection step. conducted openly on the one hand, while on the other hand, targeted exploratory questions can be asked regarding specific topics. Provider 1 Case 2.1: home care facility JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 8 (page number not for citation purposes) Exclusion criteria A semistandardized interview guide is intended to be of assistance, whereby the order of the questions, as well as their wording, are flexibly adapted to the interview situation [47]. The semistandardized interview guide will be developed based on the research results of the scoping review and NPT [41,42] using the SPSS principle, according to Helfferich [60]. The number of interviews is based on the fact that at least 1 interview will be conducted at each management level, for approximately 14 interviews in total. It should be mentioned that the data collection will be determined by an iterative approach. Participants will be recruited by the first author through written information as well as through participation in staff meetings All interviews will be recorded using a voice recorder. The transcriptions will be performed by the first author, and after the transcriptions, the audio files will be deleted. The transcription of the interviews will follow the content-semantic transcription process word-for-word. Statements in dialect will be, if possible, translated word-for-word into standard German [61]. Before transcription, the interviews will be stripped of any identifiers so that the participants remain anonymous. Table 1 below shows the coding rules used as an example. Table 1. Exemplary coding rules transcription. Code Interview Health care providers and embedded units Provider 1 P1CL1.1F1 Focus group interview 1 Case 1.1: long-term care P1CL1.2S2 Single interview 2 Case 1.2: long-term care Provider 2 P2CH2.1S4 Single interview 4 Case 2.1: home care facility Table 1. Exemplary coding rules transcription. Data From the Digital Voice Channel The principle of data analysis in the case study ADVICE follows the rule of playing with the data and searching for promising patterns, insights, or concepts [44]. Therefore, first, before data analysis, all qualitative and quantitative data will be transferred into MAXQDA Analytics pro software (version 22.2.1; Udo Kuckartz, VERBI software GmbH), where they will be analyzed. Data collection and analysis will be conducted partially in parallel because the case study is an iterative process. The data analysis will be carried out in 2 stages based on the analysis of the individual cases within-case analysis and a subsequent cross-case analysis, as shown in Figure 2. Relevant for ADVICE are the response rate, the frequency of topics mentioned, reactions or responses to comments, and, above all, the consistency of the topics mentioned in the interviews. Furthermore, the digital voice channel data ought to corroborate interview statements, for example, indicating infrequent usage of the free comment fields or the absence of any response. It is important to note that this is an iterative process, using different data to get deeper and deeper in understanding the case. https://www.researchprotocols.org/2024/1/e48601 XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al Figure 2. Data analysis. Figure 2. Data analysis. will be determined in consultation with the research team (ie, sample coding). The category system will then be additionally extended by inductive codes. For example, further categories will be formed that are not represented by the coding guide but are relevant to the understanding of the case. Inductive coding will be performed by the first author and selectively verified with other researchers. In summary, a qualitative content analysis with a theoretical deductive framework will be conducted, that is, a deductive-inductive content analysis [48]. will be determined in consultation with the research team (ie, sample coding). The category system will then be additionally extended by inductive codes. For example, further categories will be formed that are not represented by the coding guide but are relevant to the understanding of the case. Inductive coding will be performed by the first author and selectively verified with other researchers. In summary, a qualitative content analysis with a theoretical deductive framework will be conducted, that is, a deductive-inductive content analysis [48]. First, in the within-case analysis, the individual cases will be analyzed as independent units, and patterns and peculiarities will be identified. Data From the Digital Voice Channel The aim is to obtain an in-depth understanding of the specificities of each case. Therefore, all data from every embedded unit will be comprehensively analyzed. For this step, every unit of analysis (focus group interviews, single interviews, protocols, and data from the digital voice channel) will be repeatedly read and reread by the first author. In this iterative process, first, all focus group interviews will be analyzed in light of these results, and the first author will pursue further data collection within the single interviews. Before the interviews are conducted, the interview guide will be updated based on the results from the focus group interviews. Then, all single interviews from every embedded unit will be interpreted. Afterward, a case description will be written for each case with the results of the interviews (plus data from the digital voice channel). This within-analysis is oriented toward qualitative content analysis with regard to Schreier [48]. First, in the within-case analysis, the individual cases will be analyzed as independent units, and patterns and peculiarities will be identified. The aim is to obtain an in-depth understanding of the specificities of each case. Therefore, all data from every embedded unit will be comprehensively analyzed. For this step, every unit of analysis (focus group interviews, single interviews, protocols, and data from the digital voice channel) will be repeatedly read and reread by the first author. In this iterative process, first, all focus group interviews will be analyzed in light of these results, and the first author will pursue further data collection within the single interviews. Before the interviews are conducted, the interview guide will be updated based on the results from the focus group interviews. Then, all single interviews from every embedded unit will be interpreted. Afterward, a case description will be written for each case with the results of the interviews (plus data from the digital voice channel). This within-analysis is oriented toward qualitative content analysis with regard to Schreier [48]. In the subsequent second stage of the cross-case analysis, the analyzed data from the embedded units will be compared with each other in a comparative analysis, and possible distortions caused by individual cases will be identified. The results of the within-analysis will provide content for the case vignettes and serve as a basis for the cross-analysis [44]. In this analysis step, the stacking comparable strategy will be used. https://www.researchprotocols.org/2024/1/e48601 JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 9 (page number not for citation purposes) Data From the Digital Voice Channel This strategy includes case-oriented and variable-oriented cross-case analysis strategies. Each case is analyzed with a standard set of variables (based on the results of the within-analysis). After each case is understood in itself (the results will be the case vignettes), a matrix will be used to analyze each case in depth. From this, patterns and differences will be deduced. The case vignettes will include a case study report title, case description summary Based on the research questions, the first author will derive the categories based on the body of knowledge and then further add to or expand on the inductive codes. To be specific, after the first author has determined the deductive category system based on the theoretical framework, all coding units and context units XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al and managing change [11,17]. It is important to note that most of the available data on employee voice in health care focuses on 1 aspect of employee voice, namely speaking up regarding patient safety and managing errors [34-36]. This leads to an unclear understanding of the phenomenon of employee voice in health care [36]. Lainidi et al [36] argue that individual approaches and evaluations are needed to gain a deeper understanding of employee voice, especially in different settings. Recent developments in digital and communication technology have opened new opportunities to address employee voice, for example, as a company mirror or digital voice channel [18-20]. Analyses show that a digital voice channel can have a positive impact on employee identification and commitment to the organization [16]. However, it depends on the organizational culture [16,23,24]. In summary, the research gap is the understanding of employee voice in a specific setting, namely long-term care, and the analysis of the use of a digital voice channel. The NPL [41,42] is used in ADVICE to better understand how the social context influences how the digital voice channel gets used to address employee voice. based on the within-case analysis, analyst view, context information, issues, and uniqueness, among others [45,58]. The goal is to understand how the use of the digital voice channel addresses employee voice not only in every embedded single case but also on a meta-level in long-term care. The presentation of the results will follow the format (graphic, matrix, or networks) proposed by Miles et al [58]. Results The results will provide insight into how digital voice channels can be used in long-term care to address employee voice. We expect to find how the digital voice channel can empower nurses to speak up and consequently create a better work environment. Data From the Digital Voice Channel based on the within-case analysis, analyst view, context information, issues, and uniqueness, among others [45,58]. The goal is to understand how the use of the digital voice channel addresses employee voice not only in every embedded single case but also on a meta-level in long-term care. The presentation of the results will follow the format (graphic, matrix, or networks) proposed by Miles et al [58]. Ethical Considerations All participants will receive overall information about the study and be able to agree to participate and to fill out the informed consent form before the interview. Participation will only take place if the written consent form has been signed by the interviewee. The data will be processed in accordance with the data protection legislation in force, pursuant to the GDPR. The personal data collected (age, sex, professional activity, etc) are processed exclusively in an anonymous form and saved separately from the audio data (such as statements made in interviews). Ethics approval for the empirical parts of the project was given by the ethics committee of Witten/Herdecke University (S230/2022). The ethics vote was submitted under an expedited procedure in accordance with the rules of procedure of the Ethics Committee of the University of Witten/Herdecke, as it does not raise any difficult ethical or legal issues. Furthermore, no review was requested. We assume that the results will provide insight into how digital voice channels can be used in long-term care to address employee voice. We expect to find how the digital voice channel can empower nurses to speak up and, consequently, create a better work environment. Furthermore, we aim to understand how managers deal with the data from the digital voice channel, whether they use the data, and how they respond to trends and comments in the tool. In summary, the results may help to understand how employee voice behavior is affected by the digital voice channel and to identify what opportunities arise for the employees and for the organization through a digital voice channel. Strengths and Limitations The recruitment of the cases from the health care providers took place in consultation with the management and human resources managers. The decision was based, particularly in health care provider 2, on which facilities are already working with the digital voice channel. The recruitment for the focus group interviews and the single interviews began in June 2023. We are aware of the different settings, and we try to develop a case understanding of individual cases through our within-case analysis and gain a deeper understanding of employee voice in health care providers by analyzing their similarities and differences. The authors therefore also refer to the NORDCARE report [3], which includes both residential long-term care and mobile care under the term long-term care. Data collection began in August 2023, and from a current perspective, the first results are expected in summer 2024. The strength of the method proposed herein lies in the fact that the case study approach addresses the individual experiences of every employee and is oriented to the social processes in the daily routine to understand the use of a new approach to communication (digital voice channel) [44]. These are precisely the aspects that are addressed in the calls for future research on the use of new digital technologies [20,38,39] and employee voice [36]. Potential challenges can be encouraging employees to participate in interviews since these will take up working time, which is very limited due to staff shortages. This challenge can be addressed by attempting to allow the interviews to be completed during duty hours during the recruitment process in consultation with the health care providers. It must be mentioned that the generalizability of case studies is limited based on the high level of specificity and the small number of cases [62]. Furthermore, there is a possibility that, due to the way in which the scope of the data integration of the qualitative and JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 10 (page number not for citation purposes) https://www.researchprotocols.org/2024/1/e48601 Data sharing is not applicable as this is a protocol paper where no data sets were generated or analyzed. Data sharing is not applicable as this is a protocol paper where no data sets were generated or analyze Authors' Contributions All authors made significant contributions to the research protocol, contributed to editing, and approved the final version of the manuscript. The authors attest that there was no use of generative artificial intelligence technology in the generation of text, figures, or other informational content of this manuscript. DeepL Write (free version; DeepL SE) was only used for spelling and grammar correction. Summary Finally, to avoid conflicts of interest, it must be mentioned that the case selection process is undertaken by the company that provides the digital voice channels. It should be noted that the company is not involved Importance of This Study ADVICE also attempts to address the recommendations of Lainidi et al [36] to consider organizational and employee perspectives to better understand the concepts of voice and silence in a specific context (long-term care). Of course, individual case specifics are considered in the case descriptions, but the main focus is on the digital voice channel used in residential long-term care and home care facilities. At the organizational level, research can help to improve the attractiveness of the workplace by understanding how to give employees a voice. Acknowledgments The authors are grateful to Ann Kolanowski, PhD, RN, FAAN (Professor Emerita of Nursing; Professor Emerita of Psychiatry; Penn State Hershey College of Medicine) for her constructive feedback on a draft of this manuscript. This paper is financially supported, thankfully, by the IMC Krems. The authors are grateful to Ann Kolanowski, PhD, RN, FAAN (Professor Emerita of Nursing; Professor Emerita of Psychiatry; Penn State Hershey College of Medicine) for her constructive feedback on a draft of this manuscript. This paper is financially supported, thankfully, by the IMC Krems. Conflicts of Interest None declared. Summary To retain nurses in a health care organization, it is essential that they have a voice and that their views are heard. Reports from nurses working in the Austrian long-term care sector suggest that working conditions need to be addressed [2,3]. The international PTE-LTC program addresses these challenges. Specifically, this program focuses on empowering nurses, giving them a voice to actively participate in improving outcomes for residents [7]. Employee voice is 1 way of involving employees. It means that employees are able to influence work-related decisions through their feedback [12-15]. The current literature shows that employee voice has a positive impact on reducing burnout, promoting job satisfaction [16], XSL•FO RenderX JMIR RESEARCH PROTOCOLS Kepplinger et al in the research process, and moreover, the name of the company is not explicitly mentioned to avoid conflicts of interest. quantitative data will evolve over the course of the work, the case study will also become a mixed methods case study. This may result from the fact that more attention per individual case and subcase is needed in the data integration process that will occur between the qualitative and quantitative research steps [45]. Both interview bias and selection bias must be considered. Interviewer bias is addressed through constant reflection by the research team. The selection of the health care providers is based on the fact that they are the only ones in Austria who use this digital voice channel. The choice of cases is made by the health care provider themselves. Finally, to avoid conflicts of interest, it must be mentioned that the case selection process is undertaken by the company that provides the digital voice channels. It should be noted that the company is not involved quantitative data will evolve over the course of the work, the case study will also become a mixed methods case study. This may result from the fact that more attention per individual case and subcase is needed in the data integration process that will occur between the qualitative and quantitative research steps [45]. Both interview bias and selection bias must be considered. Interviewer bias is addressed through constant reflection by the research team. The selection of the health care providers is based on the fact that they are the only ones in Austria who use this digital voice channel. The choice of cases is made by the health care provider themselves. https://www.researchprotocols.org/2024/1/e48601 https://www.researchprotocols.org/2024/1/e48601 References 1. 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[FREE Full text] [doi: 10.1186/s13063-015-0709-y] [Medline: 25935741] JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 13 (page number not for citation purposes) https://www.researchprotocols.org/2024/1/e48601 JMIR RESEARCH PROTOCOLS Kepplinger et al 58. Miles MB, Huberman AM, Saldaña J. Qualitative Data Analysis: A Methods Sourcebook. Fourth edition, International student edition. Thousand Oaks, Kalifornien, Vereinigte Staaten. SAGE; 2020. 58. Miles MB, Huberman AM, Saldaña J. Qualitative Data Analysis: A Methods Sourcebook. Fourth edition, International student edition. Thousand Oaks, Kalifornien, Vereinigte Staaten. SAGE; 2020. 59. Johnson MJ, May CR. Promoting professional behaviour change in healthcare: what interventions work, and why? A theory-led overview of systematic reviews. BMJ Open. 2015;5(9):e008592. [FREE Full text] [doi: 10.1136/bmjopen-2015-008592] [Medline: 26423853] 62. Widdowson M. Case study research methodology. Int J Trans Anal Res Pract. 2011;2(1):25-34. [FREE Full text] JMIR Res Protoc 2024 \| vol. 13 \| e48601 \| p. 14 (page number not for citation purposes) Abbreviations Abbreviations ADVICE: Understanding Employee Voice Behavior (acronym for the study) CReDECI 2: Criteria for Reporting and Evaluation of Complex Interventions MeSH: Medical Subject Headings NPT: normalization process theory PICO: population, interest, and context PRISMA ScR: Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews PTE-LTP: Pathway to Excellence in Long Term Care TIDieR: Template for Intervention Description and Replication Edited by A Mavragani; submitted 29.04.23; peer-reviewed by L Peterson, M Kolbe; comments to author 13.10.23; revised version received 02.11.23; accepted 30.11.23; published 02.02.24 Please cite as: Kepplinger A, Braun A, Fringer A, Roes M Understanding Employee Voice Behavior Through the Use of Digital Voice Channel in Long-Term Care: Protocol for an Embedded Multiple-Case Study JMIR Res Protoc 2024;13:e48601 URL: https://www.researchprotocols.org/2024/1/e48601 doi: 10.2196/48601 PMID: 38306164 ©Anja Kepplinger, Alexander Braun, André Fringer, Martina Roes. Originally published in JMIR Research Protoco (https://www.researchprotocols.org), 02.02.2024. This is an open-access article distributed under the terms of the Creati Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, an reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. Th complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as th copyright and license information must be included. ©Anja Kepplinger, Alexander Braun, André Fringer, Martina Roes. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 02.02.2024. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included. ©Anja Kepplinger, Alexander Braun, André Fringer, Martina Roes. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 02.02.2024. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. 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https://openalex.org/W4210857920	https://phsreda.com/e-articles/10356/Action10356-100973.pdf	Russian	null	Popularization of the Traditional Culture of the Peoples of the Russian Federation as a Methodological Basis for Creating an Author’s Program of a Teacher on Moral and Patriotic Education. Project Experience: Interactive Park “Ethno-Hackathon”	Publishing house Sreda eBooks	2,021	cc-by	31,670	Муниципальное автономное общеобразовательное учреждение «Средняя общеобразовательная школа №61» муниципального образования города Чебоксары – столицы Чувашской Республики Муниципальное автономное общеобразовательное учреждение «Средняя общеобразовательная школа №61» муниципального образования города Чебоксары – столицы Чувашской Республики ПОПУЛЯРИЗАЦИЯ ТРАДИЦИОННОЙ КУЛЬТУРЫ НАРОДОВ РОССИЙСКОЙ ФЕДЕРАЦИИ КАК МЕТОДИЧЕСКАЯ БАЗА ДЛЯ СОЗДАНИЯ АВТОРСКОЙ ПРОГРАММЫ ПЕДАГОГА ПО НРАВСТВЕННО-ПАТРИОТИЧЕСКОМУ ВОСПИТАНИЮ Опыт проекта: Интерактивный парк «ЭТНО Хакатон» Гурьева, Т.Н. Николаева. – Чебоксары: ИД «Среда», 2021. – 116 с. ПОПУЛЯРИЗАЦИЯ ТРАДИЦИОННОЙ КУЛЬТУРЫ НАРОДОВ РОССИЙСКОЙ ФЕДЕРАЦИИ КАК МЕТОДИЧЕСКАЯ БАЗА ДЛЯ СОЗДАНИЯ АВТОРСКОЙ ПРОГРАММЫ ПЕДАГОГА ПО НРАВСТВЕННО-ПАТРИОТИЧЕСКОМУ ВОСПИТАНИЮ Опыт проекта: Интерактивный парк «ЭТНО Хакатон» Учебно-методическое пособие Чебоксары Издательский дом «Среда» 2021 Чебоксары Издательский дом «Среда» 2021 УДК 373.5(075.3) ББК 74.200.52я72 П58 Подготовлено в рамках федерального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Выполнено при финансовой поддержке Министерства просвещения Российской Федерации в рамках Соглашения № 073-15-2021-1032 от «07» июня 2021 г. Рецензенты: Мурзина Жанна Владимировна, кандидат биологических наук, проректор БУ ЧР ДПО «Чувашский республиканский институт образования» Минобразования Чувашии Бобовникова Валентина Борисовна, заместитель директора по УВР МАОУ «СОШ №61» г. Чебоксары Редакционная коллегия: Гурьева Наталья Михайловна, директор МАОУ «СОШ №61» г. Чебоксары Николаева Татьяна Николаевна, заместитель директора по УВР МАОУ «СОШ №61» г. Чебоксары Дизайн обложки: Фирсова Надежда Васильевна, дизайнер П58 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию. Опыт проекта: Интерактивный парк «ЭТНО- Хакатон» : учебно-методическое пособие / редкол.: Н.М. Гурьева, Т.Н. Николаева. – Чебоксары: ИД «Среда», 2021. – 116 с. УДК 373.5(075.3) ББК 74.200.52я72 П58 УДК 373.5(075.3) ББК 74.200.52я72 П58 Подготовлено в рамках федерального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Выполнено при финансовой поддержке Министерства просвещения Российской Федерации в рамках Соглашения № 073-15-2021-1032 от «07» июня 2021 г. Подготовлено в рамках федерального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Выполнено при финансовой поддержке Министерства просвещения Российской Федерации в рамках Соглашения № 073-15-2021-1032 от «07» июня 2021 г. Подготовлено в рамках федерального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Выполнено при финансовой поддержке Министерства просвещения Российской Федерации в рамках Соглашения № 073-15-2021-1032 от «07» июня 2021 г. Рецензенты: Мурзина Жанна Владимировна, кандидат биологических наук, проректор БУ ЧР ДПО «Чувашский республиканский институт образования» Минобразования Чувашии Бобовникова Валентина Борисовна, заместитель директора по УВР МАОУ «СОШ №61» г. Чебоксары Редакционная коллегия: Гурьева Наталья Михайловна, директор МАОУ «СОШ №61» г. Чебоксары Николаева Татьяна Николаевна, заместитель директора по УВР МАОУ «СОШ №61» г. Чебоксары Дизайн обложки: Фирсова Надежда Васильевна, дизайнер Рецензенты: Мурзина Жанна Владимировна, кандидат биологических наук, проректор БУ ЧР ДПО «Чувашский республиканский институт образования» Минобразования Чувашии Бобовникова Валентина Борисовна, заместитель директора по УВР МАОУ «СОШ №61» г. Чебоксары Редакционная коллегия: Гурьева Наталья Михайловна, директор МАОУ «СОШ №61» г. Чебоксары Николаева Татьяна Николаевна, заместитель директора по УВР МАОУ «СОШ №61» г. Чебоксары Дизайн обложки: Фирсова Надежда Васильевна, дизайнер Фирсова Надежда Васильевна, дизайнер П58 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию. Опыт проекта: Интерактивный парк «ЭТНО- Хакатон» : учебно-методическое пособие / редкол.: Н.М. ISBN 978-5-907411-96-8 Данное учебно-методическое пособие является частью серии учебно- методических пособий, созданных в рамках федерального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование», содержит описание паспорта проекта «Интерактивный парк «ЭТНО-Хакатон», а также методическую разработку, содержащую конкретные материалы в помощь по проведению уроков и внеурочных мероприятий по русскому языку, сочетающую описание последовательности действий при преподавании определенной темы, с методическими советами по организации занятия. ISBN 978-5-907411-96-8 DOI 10.31483/a-10356 © МАОУ «СОШ №61» г. Чебоксары, 2021 © Издательский дом «Среда», оформление, 2021 ISBN 978-5-907411-96-8 DOI 10.31483/a-10356 © МАОУ «СОШ №61» г. Чебоксары, 2021 © Издательский дом «Среда», оформление, 2021 ISBN 978-5-907411-96-8 DOI 10.31483/a-10356 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» СОДЕРЖАНИЕ 4 Введение 4 Часть 1. Паспорт проекта «Интерактивный парк «ЭТНО- Хакатон»…………………………………………………………… 6 Часть 2. Таратина Е.Г. Трудные случаи русской орфографии и пунктуации…………………………………………………………… 9 О – Ё после шипящих и Ц ............................................................. 9 Написание Ь.. ................................................................................ 21 Правописание непроизносимых согласных ............................... 25 Правописание согласных на стыке корня и суффикса…… ..... 29 Правописание согласных в корнях слов .................................... 31 Правописание двойных согласных в корне ............................... 34 Правописание непроверяемых безударных гласных в корне...36 Правописание проверяемых безударных гласных в корне ...... 44 Правописание чередующихся гласных в корне………………..52 Правописание безударных окончаний имен существительных ... 57 Правописание безударных личных окончаний глаголов……. 67 Правописание суффиксов имен существительных ................... 74 Правописание суффиксов имен прилагательных……………...81 Правописание глагольных суффиксов ....................................... 83 Н и НН в разных частях речи ...................................................... 84 Правописание сложных слов с пол- и полу- ............................... 89 Правописание частиц ................................................................... 90 Слитное и раздельное написание НЕ с разными частями речи.. .. 93 Частицы НЕ и НИ ....................................................................... 101 Вместо заключения……………………………………………….. 110 Список литературы……………………………………………….. 113 3 ВВЕДЕНИЕ Данное учебно-методическое пособие подготовлено в рамках реализации федерального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образо- вание». В 2021 году муниципальное автономное общеобразовательное учреждение «Средняя общеобразовательная школа № 61» города Чебоксары стало победителем конкурсного отбора заявок субъек- тов Российской Федерации, организованного Министерством просвещения Российской Федерации по направлению «Проведе- ние всероссийских творческих мероприятий, направленных на популяризацию традиционной культуры народов Российской Фе- дерации». Предлагаем читателям познакомиться с опытом реализации проекта Интерактивный парк «ЭТНО-Хакатон» в соответствии с грантовым конкурсом федерального проекта «Патриотическое вос- питание граждан Российской Федерации» национального проекта «Образование» и приглашаем заинтересованные стороны к обсуж- дению итогов его реализации в самых различных форматах как он- лайн, так и ходе стажировок, на которые мы приглашаем всех же- лающих. Для реализации потенциала участников проекта и развития их компетенций в ходе реализации проекта группа участников (обуча- ющиеся и педагоги) прошла обучение по программе, которая вклю- чала в себя обучение принципам программирования и создания кроссплатформенного приложения с мобильной версией. В ходе реализации проекта участники создавали информационные про- дукты и наполняли данное приложение своими наработками. Дан- ный продукт стал главным продуктом проекта, который был пре- зентован на итоговой конференции. Все продукты создавались в ходе образовательных экспедиций по 5 субъектам Российской Фе- дерации (Чувашия, Татарстан, Нижний Новгород, Марий-Эл, Мор- довия). Основной идей проекта стало развитие системы использования технологий взаимообмена педагогическими идеями, методиками и 4 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 4 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Введение проектами, в том числе с моделированием образовательных собы- тий, между педагогами и детьми в целях формирования понимания значимости основных концептуальных основ федерального про- екта «Патриотическое воспитание граждан Российской Федера- ции» национального проекта «Образование», а также развития цифровых навыков (skills) и современного представления форми- рования и решения социальных проблем детей и молодежи. проектами, в том числе с моделированием образовательных собы- тий, между педагогами и детьми в целях формирования понимания значимости основных концептуальных основ федерального про- екта «Патриотическое воспитание граждан Российской Федера- ции» национального проекта «Образование», а также развития цифровых навыков (skills) и современного представления форми- рования и решения социальных проблем детей и молодежи. 6 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию ВВЕДЕНИЕ На базе школы создан ресурсный центр по развитию и форми- рованию активной гражданской позиции жителей Чувашской Рес- публики и детей и молодежи из не менее 5 субъектов Российской Федерации, их социальной активности, духовности и развития важ- нейших патриотических качеств личности посредством изучения и раскрытия темы популяризации традиционной культуры народов Российской Федерации. Все мероприятия проекта проходили под брендом национального проекта «Образование». В рамках реализации проекта создана сеть партнерских школ, с которыми заключено соглашение о сетевом взаимодействии в це- лях повышения качества образования и формирования республи- канской сети инновационных школ. Данное пособие является частью серии учебно-методических пособий, созданных в рамках проекта, содержит описание паспорта проекта «Интерактивный парк «ЭТНО-Хакатон», а также методи- ческую разработку Таратиной Елены Геннадьевны, учителя рус- ского языка и литературы МАОУ «СОШ №61» г. Чебоксары, Чу- вашская Республика, содержащую конкретные материалы в по- мощь по проведению уроков и внеурочных мероприятий по рус- скому языку, сочетающую описание последовательности действий, при преподавании определенной темы, с методическими советами по организации занятия. 5 5 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Российской Федерации» национального проекта «Образование» 6 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога ЧАСТЬ 1. ПАСПОРТ ПРОЕКТА «ИНТЕРАКТИВНЫЙ ПАРК «ЭТНО-ХАКАТОН» Наименование проекта Интерактивный парк «ЭТНО-Хакатон» 1 2 Сроки реализации проекта 2021-2024 годы Статус проекта Федеральный Цель проекта Цель: Создание на территории Чувашской Респуб- лики устойчивой и эффективной структуры в целях предоставления условий для проведения всероссий- ских творческих мероприятий, направленных на по- пуляризацию традиционной культуры народов не менее 5 субъектов Российской Федерации для фор- мирования у обучающихся (детей и молодежи) ак- тивной гражданской позиции, социальной активно- сти, духовности и важнейших патриотических ка- честв личности, направленных на укрепление госу- дарства и защиту его интересов. Конкретная цель. Создание на базе МАОУ «Сред- няя общеобразовательная школа № 61» г. Чебоксары условий для формирования понимания значимости основных концептуальных основ федерального про- екта «Патриотическое воспитание граждан Россий- ской Федерации» национального проекта «Образова- ние», а также развития цифровых навыков (skills) и современного представления формирования и реше- ния социальных проблем детей и молодежи Задачи проекта 1. Создание на базе организации-заявителя ресурс- ного центра по развитию и формированию активной гражданской позиции жителей Чувашской Респуб- лики и детей и молодежи из не менее 5 субъектов Российской Федерации, их социальной активности, духовности и развития важнейших патриотических качеств личности посредством изучения и раскры- тия темы популяризации традиционной культуры народов Российской Федерации. 6 Часть 1. Паспорт проекта «Интерактивный парк «ЭТНО-Хакатон» Часть 1. Паспорт проекта «Интерактивный парк «ЭТНО-Хакатон» р р Продолжение таблицы 1 2 2. Организация и проведение фестивалей, конкурсов, творческих акций и событий, направленных на попу- ляризацию традиционной культуры народов Россий- ской Федерации с целью развития творческой актив- ности, приобщения к базовым национальным ценно- стям российского общества, общечеловеческим цен- ностям в контексте формирования целостной кар- тины мира и гражданской идентичности. 3. Разработка и внедрение комплекса организаци- онно-педагогических материалов по сопровождению деятельности педагогов, направленных на повыше- ние мотивации детей и молодежи в развитии совре- менных цифровых навыков через изучение и попу- ляризацию традиционной культуры народов Россий- ской Федерации. 4. Разработка и создание апробированных, тиражи- руемых моделей и методических комплексов, для организации занятий по данной методике в образо- вательных организациях 5 субъектов Российской Федерации. 5. Предоставление школьникам, благоприятных условий и ресурсов для реализации их потенциала, развитие компетенций школьников различного воз- раста, с применением новых форм и методов работы по патриотическому воспитанию граждан. 6. Содействие повышению квалификации педагогов в мотивации школьников к развитию их знаний и компетенций. 7. Внедрение современных цифровых технологий и онлайн ресурсов при организации взаимодействия педагогов, родителей, общественности по различ- ным аспектам воспитания, консолидация усилий всех заинтересованных сторон общества в повыше- нии качества воспитания. 8. Установление устойчивых партнерских отноше- ний с организациями системы общего образования, дополнительного образования, высшего образова- ния, партнерами социальной сферы и бизнес- Продолжение таблицы Прод 1 2 2. Организация и проведение фести творческих акций и событий, напр ляризацию традиционной культуры ской Федерации с целью развития ности, приобщения к базовым наци стям российского общества, общеч ностям в контексте формирования тины мира и гражданской идентич 3. Разработка и внедрение комплек онно-педагогических материалов п деятельности педагогов, направлен ние мотивации детей и молодежи в менных цифровых навыков через и ляризацию традиционной культуры ской Федерации. 4. Разработка и создание апробиро руемых моделей и методических к организации занятий по данной ме вательных организациях 5 субъект Федерации. 5. Предоставление школьникам, бл условий и ресурсов для реализации развитие компетенций школьников раста, с применением новых форм по патриотическому воспитанию г 6. Содействие повышению квалиф в мотивации школьников к развити компетенций. 7. Внедрение современных цифров онлайн ресурсов при организации педагогов, родителей, общественн ным аспектам воспитания, консоли всех заинтересованных сторон общ нии качества воспитания. 8. Установление устойчивых партн ний с организациями системы общ дополнительного образования, выс ния, партнерами социальной сферы 3. Часть 1. Паспорт проекта «Интерактивный парк «ЭТНО-Хакатон» Разработка и внедрение комплекса организаци- онно-педагогических материалов по сопровождению деятельности педагогов, направленных на повыше- ние мотивации детей и молодежи в развитии совре- менных цифровых навыков через изучение и попу- ляризацию традиционной культуры народов Россий- ской Федерации. 4. Разработка и создание апробированных, тиражи- руемых моделей и методических комплексов, для организации занятий по данной методике в образо- вательных организациях 5 субъектов Российской Федерации. 5. Предоставление школьникам, благоприятных условий и ресурсов для реализации их потенциала, развитие компетенций школьников различного воз- раста, с применением новых форм и методов работы по патриотическому воспитанию граждан. 7. Внедрение современных цифровых технологий и онлайн ресурсов при организации взаимодействия педагогов, родителей, общественности по различ- ным аспектам воспитания, консолидация усилий всех заинтересованных сторон общества в повыше- нии качества воспитания. 7 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Окончание таблицы 1 2 партнерами в целях пополнения банка данных лучших практик в области патриотического воспитания граж- дан и обеспечение публичности (популяризации в СМИ) диссеминации позитивных результатов проекта Краткая идея проекта Создание условий для формирования понимания значимости основных концептуальных основ феде- рального проекта «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование», а также развития цифровых навыков (skills) у детей и молодежи через популяри- зацию традиционной культуры народов Российской Федерации и формирование у обучающихся (детей и молодежи) активной гражданской позиции, социаль- ной активности, духовности и важнейших патриоти- ческих качеств личности с изучением этно-ресурсов не менее 5 субъектов Российской Федерации. Проект имеет название Интерактивный парк «ЭТНО-Хакатон». Суть проекта в том, что на базе ШКОЛЫ будет создана инновационная структура- лаборатория, на базе которой будет развернут хака- тон, где группы участников проекта – специалисты из разных областей будут сообща работать над ре- шением создания кроссплатформенного приложе- ния, доступного с ПК и мобильных устройств в сети Интернет для создания цифрового ресурса, включа- ющего разноплановые модули (подсистемы), рас- крывающие (отображающие) основные направления культуры, образования, народных традиций и пр. ма- лых народов Российской Федерации (5 субъектов: Чувашия, Татарстан, Марий Эл, Мордовия, Нижний Новгород), имеющих этноязыковые особенности. Основной инновационной идеей проекта будет яв- ляться сочетание традиционных проектных форм с формированием компетенций участников проекта в сфере инновационных решений в области информа- ционных технологий Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 8 8 Часть 2. Трудные случаи русской орфографии и пунктуации ЧАСТЬ 2. ТРУДНЫЕ СЛУЧАИ РУССКОЙ ОРФОГРАФИИ И ПУНКТУАЦИИ Знаете ли Вы, незнание каких правил влечёт за собой наиболь- шее количество ошибок? Ответом на данные вопросы и обуслов- лено написание данного раздела пособия. В разделе Вы познакомитесь с наиболее трудными случаями ор- фографии и пунктуации и теоретическими аспектами правил рус- ского языка. Теоретические аспекты сопровождаются наглядными примерами вызывающих трудность написаний, а также упражне- ниями, при составлении которых основная задача автора – включе- ние выработки практических навыков, развития речи и обогащение лексикона обучающихся. *** О – Ё после шипящих и Ц 1. Ожегся, словно на крапиве. 2. И дрова без поджогу не горят. Упражнение 1 Упражнение 1 1. Вспомните орфограмму «Буквы О и Ё после шипящих». Чтобы вспомнить, можете пройти по ссылке: http://www.gramota.ru/class/coach/tbgramota/45_73 или воспользо- вавшись QR-кодом 2. Прочитайте русские народные пословицы. Найдите слова, со- держащие орфограмму «О/Ё после шипящих» и объясните их напи- сание. Образец ответа: ремешок – в суффиксе имени существи- тельного ремешок под ударением пишется О 2. Прочитайте русские народные пословицы. Найдите слова, со- держащие орфограмму «О/Ё после шипящих» и объясните их напи- сание. Образец ответа: ремешок – в суффиксе имени существи- тельного ремешок под ударением пишется О 1. Ожегся, словно на крапиве. 9 9 9 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 3. У богатого черт детей качает. 3. У богатого черт детей качает. 3. У богатого черт детей качает. 4. Шёлк не рвется, булат не сечётся, красное золото не ржа- веет 5. Баба, что глиняный горшок: вынь из печки, он пуще шипит. 6. Вяжись, лычко с лычком, ремешок с ремешком. 7. Чужой собаке на селе житья нет. 8. То не пьян ещё, коли шапка на голове. 9. Чин чина почитай, а меньшой садись на край! 10. В чужом доме не будь приметлив, а будь приветлив. 11. Петь бы ещё, да на животе тощо. 12. Добрый пастух не о себе печётся, о скотине. 13. Этого днём со свечою поискать. 14. Вот тебе ершок, свари ухи горшок. 15. Долг платежом красен. 16. У грошового товару не наживешь рубля. 17. Продает с барышом, а ходит нагишом. 18. Чёрен мак, да бояре едят. 18. Чёрен мак, да бояре едят. 19. Из печёного яйца живого цыпленка высидит. 20. Его поздно учить: распашонка на нем уже не сойдется. 2. Уточните значение следующих слов по толковому словарю: булат, пуще, лычко, коли, чин, приметлив, тощо, уха, барыш, рас- пашонка Для этого пройдите по ссылке: https://slovarozhegova.ru/ или отсканируйте QR-код: 2. Уточните значение следующих слов по толковому словарю: булат, пуще, лычко, коли, чин, приметлив, тощо, уха, барыш, рас- пашонка Для этого пройдите по ссылке: https://slovarozhegova.ru/ или отсканируйте QR-код: NB! Чтобы найти слово в толковом словаре, сначала нужно поставить его в начальную форму 10 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 10 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 10 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Упражнение 2 р 1. Спишите русские народные пословицы, вставляя пропущен- ные буквы. Графически объясните свой выбор. Образец выполне- ния: пчёлка – им. сущ., пчела (Ё//Е) Часть 2. Трудные случаи русской орфографии и пунктуации 4. Как Вы понимаете выражение «красное золото»? 4. Как Вы понимаете выражение «красное золото»? 5. По толковому словарю определите лексическое значение слова добрый. В каком значении это слово употреблено в выраже- нии добрый пастух не о себе печется, а о скотине? р у 6. Составьте и запишите предложения со словами поджог, под- жёг, ожог, ожёг. Упражнение 2 Часть 1 1. На хороший цветок и пч…лка летит. 1. На хороший цветок и пч…лка летит. 2. Хоть нагиш…м, да с палаш…м. 3. Высок каблуч…к, да подломился на боч…к. 4. Хочется пирож…к съесть, да не хочется в подполье лезть. 5. Ладил мужич…к челноч…к, а свёл на уховёртку. 6. Ел бы пирог, да в печи сж…г. 7. Всегда жди беды от больш…й воды. 8. Я его калач…м, а он меня в спину кирпич…м. 9. Сверч…к тьму тараканов победил. 10. Денежка рубль береж…т, а рубль голову стереж…т. 11. Молодой друж…к что вешний ледок. 12. Рубашка беленька, да душа ч…рненька. 13. Ш…л бы в пир, да не рад мир. 13. Ш…л бы в пир, да не рад мир. 14. Щ…голь ходит, живот поджав. 14. Щ…голь ходит, живот поджав. 15. Расти больш…й, да не будь лапш…й. 15. Расти больш…й, да не будь лапш…й. 16. Правды в суч…к не засунешь. 16. Правды в суч…к не засунешь. 17. Счастье без ума нипоч…м. 17. Счастье без ума нипоч…м. 18. Уж… вот тебя баба-яга в ступе унесет. 18. Уж… вот тебя баба-яга в ступе унесет. 19. Был бы снеж…к, так скатаем в комок. 19. Был бы снеж…к, так скатаем в комок. 20. Есть и медок, да засеч…н в ледок. 20. Есть и медок, да засеч…н в ледок. 21. Душ…ю божьи, телом княжьи. 21. Душ…ю божьи, телом княжьи. 22. Чей посол, тому и поч…т. 22. Чей посол, тому и поч…т. 23. Для сч…ту и у нас голова на плечах. 23. Для сч…ту и у нас голова на плечах. 24. Поди в сундуч…к, подай пятач…к. 24. Поди в сундуч…к, подай пятач…к. 25. И рад бы душ…й, да хлеб-то чуж…й. 25. И рад бы душ…й, да хлеб-то чуж…й. 11 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2 1. Хорош… жить на почёте у миру, да ответ больш…й. 2. Взял лычко, а отдай ремеш…к. 2. Взял лычко, а отдай ремеш…к. 3. Не в сч…те деньги, а в цене. 4. Торговал и кирпич…м, да остался ни при ч…м. 5. На леч…ной кобыле далеко не уедешь. 6. Пусть не будет муки в закроме, не переводился бы печ…ный еб. По горизонтали 2. Рубящее и колющее абордажное холодное оружие с прямым и длинным клинком; 4. Бывает мельничный, ручной, каменный; 5. Военное: команда, по которой допускается некоторая свобода в строю; 6. Прост. когда-нибудь потом, позже; 8. Неугомонный ры- царь ночи, живущий у дедушки за печкою; 10. У печи в углу стою, за порядком я гляжу, не боюсь огня и жара, чтобы не было пожара, быстро уголь разгребу, от беды вас всех спасу, я – железная нога, называюсь … хлеб. 7. Чуж…й дурак – ха-ха! а свой дурак – ох-ох! 8. Барч…нок горя не вкусит, пока своя вошь не укусит. 9. Баба, что меш…к: что положишь, то несёт. 9. Баба, что меш…к: что положишь, то несёт. 10. Вольно и ч…рту о своем болоте орать. 10. Вольно и ч…рту о своем болоте орать. 11. Знай, сверч…к, свой шесток. 12. Изжила свой век за холщ…вый мех. 13. Пойду в баню ч…рным, из бани выйду – красным. 14. В чуж…м глазу суч…к видишь, а в своем и бревна не заме- аешь. 14. В чуж…м глазу суч…к видишь, а в своем и бревна не заме- аешь. 15. Толокном Волгу не замесишь, озера соломой не зажж…шь. 16. На чуж…й стороне и весна не красна. 17. У неё языч…к намелет на пятач…к. 18. Ни Богу свечка, ни ч…рту кочерга. 19. Хороший ж…рнов всё мелет. 20. Больш…му кораблю больш…е и плавание. 21. Есть пирож…к, есть и друж…к. 22. Лучш… своё отдать, чем чуж..е взять. 23. На хорош…й лошадке по дороге, на худой – по стороне. 24. Ум хорош…, а два лучш…. 24. Ум хорош…, а два лучш…. 25. Коли нет ума за кож…й, поверх кожи не пришьешь. 26. Куй железо, пока горяч…. 2. Разгадайте кроссворд (подсказка: слова-ответы есть в этом упражнении😊) 12 Часть 2. Трудные случаи русской орфографии и пунктуации русской орфографии и пунктуации 9 7 3 5 1 2 6 11 4 10 8 По горизонтали 2. Рубящее и колющее абордажное холодное оружие с прямым и длинным клинком; 4. Бывает мельничный, ручной, каменный; 5. Военное: команда, по которой допускается некоторая свобода в строю; 6. Прост. когда-нибудь потом, позже; 8. Неугомонный ры- царь ночи, живущий у дедушки за печкою; 10. У печи в углу стою, за порядком я гляжу, не боюсь огня и жара, чтобы не было пожара, быстро уголь разгребу, от беды вас всех спасу, я – железная нога, называюсь … По вертикали русской орфографии и пунктуации 9 7 3 5 1 2 6 11 4 10 8 9 7 3 5 1 2 6 11 4 10 8 П 8 По вертикали 1. Мальчик из барской семьи; 3. Особая форма удлинённых су- шек, а также суффиксальное образование от слова, имеющего зна- чение «лодка»; 5. Трад.-поэт. Весенний (о явлениях природы); 1. Мальчик из барской семьи; 3. Особая форма удлинённых су- шек, а также суффиксальное образование от слова, имеющего зна- чение «лодка»; 5. Трад.-поэт. Весенний (о явлениях природы); 13 13 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» р р Упражнение 3 Пользуясь программой, обозначьте условия выбора написания О или Ё после шипящих Программа: А – ё – глагол А – ё – отглагольное образование Б – ё – возможно чередование о//е Б – о- невозможно чередование о//е В – о – под ударением В – е – без ударения Г – о – под ударением Г – е – без ударения Г – о – под ударением Г – е – без ударения Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 6. Настоящая звезда мира членистоногих. От одного её вида визжит любая девчонка, а сама она собирает несчётное количество оваций в виде смачных шлепков газетой, стоит ей выйти в народ; 7. Сши- тый из холста; 9. Приготовить какое-нибудь количество чего-ни- будь посредством размола; 11. «Зверь родится… Почему именно …?» (Н.В. Гоголь) 6. Настоящая звезда мира членистоногих. От одного её вида визжит любая девчонка, а сама она собирает несчётное количество оваций в виде смачных шлепков газетой, стоит ей выйти в народ; 7. Сши- тый из холста; 9. Приготовить какое-нибудь количество чего-ни- будь посредством размола; 11. «Зверь родится… Почему именно …?» (Н.В. Гоголь) ( ) 3. Составьте диалог между учителем и учеником, используя не- сколько пословиц этого упражнения. Разыграйте этот диалог в аудитории 4. Затранскрибируйте предложения; не в сч…те деньги, а в цене; хочется пирож…к съесть, да не хочется в подполье лезть. Какие фонетические процессы происходят при произнесении этих фраз? Характерны ли эти процессы для вашего родного языка? Упражнение 3 Д – местоимение, отместоименное образование Часть 1 14 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 1. Цвет пч…лкам, а мёд ж…нкам 2. По привычке ч…рт в болоте живет 3. Нужда из лычка кроит ремеш…к 4. Хотя горш…к один, сам себе господин 5. Правда твоя, мужич…к, а полезай-ка в меш…к 6. Этим калач…м меня не заманишь 7. Ч…рного кобеля не вымоешь добела 8. Бедный по чуж…му тужит 9. Мой грех до меня дош…л 10. Сам себя сж…г француз, сам и поморозил 14 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 1. Цвет пч…лкам, а мёд ж…нкам 2. По привычке ч…рт в болоте живет 3. Нужда из лычка кроит ремеш…к 4. Хотя горш…к один, сам себе господин 5. Правда твоя, мужич…к, а полезай-ка в меш…к 6. Этим калач…м меня не заманишь 7. Ч…рного кобеля не вымоешь добела 8. Бедный по чуж…му тужит 9. Мой грех до меня дош…л 10. Сам себя сж…г француз, сам и поморозил 14 Часть 2. Трудные случаи русской орфографии и пунктуации 11. Дадут – в меш…к, не дадут – в другой 12. Нет рож…ного ума, не дашь и уч…ного 13. Глупый ищет больш…го места, а умного и в углу знают 15. Щ…голь Матрёшка: полтора рубля застёжка 16. Сер мужич…к, да сердит на работу 17. Ш…л бы воевать, да лень саблю вынимать 18. Нет друга – ищи, а наш…л – береги 18. Нет друга – ищи, а наш…л – береги 19. Меньш…му сыну отцовский двор, старш…му – новоселье 20. Хорош… чужими руками жар загребать 20. Хорош… чужими руками жар загребать 21. На всяк час не убереж…шься 22. Пош…л на охоту, да засосало в болоте 23. Маленькая берешь лучш… больш…го барыша 24. На брюхе ш…лк, а в брюхе-то щ…лк 25. Хорош…, как гороху купишь, да плохо, коли весь растря- сёшь, домой не донесёшь 26. Он доколачивает последнюю тысч…нку Часть 2 26. Он доколачивает последнюю тысч…нку Часть 2 1. Не отвернешь головы ключ…м, не будешь богач…м 2. Богатого ложка ковш…м, убогого ложка веселком 3. Кому кистень, а кому ч…тки 4. Под лежачий камень и вода не теч…т 5. Еж…вая голица учить мастерица 11. Живем не мотаем, а пустых щей не хлебаем: хоть сверч…к горш…к, а все с наваром бываем 12. Ничто нипоч…м: был бы Ерофеич с калач…м 13. Это ещ… цветочки, а ягодки потом будут 14. За наш… добро да нам же рож…н в ребро 15. Попало зёрнышко под коварный ж…рнов 16. Он тебя угостит, что нагиш…м домой уедешь 17. Снеж…к подпал, и следок застлал 18. Не хватило легких, так заговорил печ…нкой 15 матрёшка, барыш, веселко, кистень, чётки, голица, бич, коче- ток, арба, мотать. Укажите стилистическую принадлежность этих слов; если слово имеет стилистическую окраску, то приведите его нейтральный синоним матрёшка, барыш, веселко, кистень, чётки, голица, бич, коче- ток, арба, мотать. Укажите стилистическую принадлежность этих слов; если слово имеет стилистическую окраску, то приведите его нейтральный синоним NB! Чтобы найти слово в толковом словаре, сначала нужно поставить его в начальную форму Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 19. Деньги сч…том крепки 20. Мир крещ…ный, да меш…к холщ…вый: под одним окном вы- просит, под другим съест 21. Не корми калач…м да не бей в спину кирпич…м 22. Дорожка вместе, табач…к пополам 23. Чаще сч…т крепче дружбы 24. Уш…л воз и лошадку увез 3. Уточните значение следующих слов по толковому словарю, пройдя по ссылке: https://slovarozhegova.ru/ или отсканировав QR-код: 19. Деньги сч…том крепки 19. Деньги сч…том крепки 20. Мир крещ…ный, да меш…к холщ…вый: под одним окном вы- просит, под другим съест 20. Мир крещ…ный, да меш…к холщ…вый: под одним окном вы- просит, под другим съест 21. Не корми калач…м да не бей в спину кирпич…м 22. Дорожка вместе, табач…к пополам 23. Чаще сч…т крепче дружбы 24. Уш…л воз и лошадку увез NB! Чтобы найти слово в толковом словаре, сначала нужно поставить его в начальную форму 3. Составьте предложения со словами, данными в пункте 2 4. Как вы понимаете значение выражения сер мужич…к, да сердит на работу? Придумайте и запишите историю, в которой было бы уместно употребление этой поговорки. 1. Повторите орфограмму «Буквы О и Е после Ц» (можете вос- пользоваться ссылкой: https://licey.net/free/4-russkii_yazyk/39- kurs_russkogo_yazyka_fonetika__slovoobrazovanie__morfologiya_i_ orfografiya/stages/639-113_pravopisanie_glasnyh_posle_c.ht, пункт 1.13.3) 6 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 16 Часть 2. Трудные случаи русской орфографии и пунктуации 2. Спишите, вставляя пропущенные буквы 1. Сделайся овц…й – волки готовы 2. Была под венц…м – дело с конц…м 3. На дубу свинья гнездо свила, а овца пришла, яйц… снесла 4. Не прикидывайся овц…ю – волк съест 5. Гарц…вал пан, да с коня спал 6. Было сельц…, да сменял на кольц…, было кольц…, да за же- нину ласку сменял на коляску 7. Крадет, а конц…в хоронить не умеет 8. Кольц…м в ворота бьют, а мужик с печи отзывается 9. . Далеко куц…му (дворняжке) до зайца 11. Ты конц…м, а он кольц…м; ты кольц…м, а он конц…м 12. Не учи дубц…м, поучи рублем 13. Девушка гуляй, а дельц… помни 3. Выполните следующие разборы: Фонетический разбор: отзывается Морфемный разбор: кольц…, сельц…, яйц…, конц…ов, дельц… Морфологический разбор: крадет, поучи Синтаксический разбор: Не прикидывайся овц…ю – волк съест 4* Есть ли в этом упражнении слова с нулевыми морфемами (окончаниями и суффиксами)? Докажите свою точку зрения 2. Спишите, вставляя пропущенные буквы 2. Спишите, вставляя пропущенные буквы 1. Сделайся овц…й – волки готовы 2. Была под венц…м – дело с конц…м 3. На дубу свинья гнездо свила, а овца пришла, яйц… снесла 4 Н д й 1. Сделайся овц…й – волки готовы 2. Была под венц…м – дело с конц…м 5. Гарц…вал пан, да с коня спал 5. Гарц…вал пан, да с коня спал 6. Было сельц…, да сменял на кольц…, было кольц…, да за же- нину ласку сменял на коляску 7. Крадет, а конц…в хоронить не умеет 8. Кольц…м в ворота бьют, а мужик с печи отзывается 9. . Далеко куц…му (дворняжке) до зайца 11. Ты конц…м, а он кольц…м; ты кольц…м, а он конц…м 12. Не учи дубц…м, поучи рублем 13. Девушка гуляй, а дельц… помни 3. Выполните следующие разборы: Фонетический разбор: отзывается Морфемный разбор: кольц…, сельц…, яйц…, конц…ов, дельц… Морфологический разбор: крадет, поучи Синтаксический разбор: Не прикидывайся овц…ю – волк съест 4* Есть ли в этом упражнении слова с нулевыми морфемами (окончаниями и суффиксами)? Докажите свою точку зрения 17 окончание глагол имя сущ. Под уда- ре- нием Без уда- рения П у р н Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Прочитайте русские народные пословицы и поговорки. Поду- майте, какие буквы следует написать вместо пропусков. Распреде- лите слова с пропущенными буквами по колонкам таблицы 1. Удастся лён, так ш…лк, не удастся, так щ…лк 2. Щ…голь собака: что ни год, то рубаха, а портам смены нет 3. Горш…к чугуну не товарищ 4. Хорош… тому жить, у кого бабушка ворожит 5. Мужич…к с ноготок, а борода с локоток 6. Суч…к в кулачке сожмет так, что вода пойдет 7. Чуж…го не жалеет, а своего не теряет 8. В драке богатый лицо береж…т, а убогий кафтан 9. Тут меш…к, а в мешке-то еж…к 10. Был бы покос, да приш…л мороз 11. Где ш…л, там след, где был, там нет 12. Насеч…шь тяпкой, не сотрёшь тряпкой 13. Был бы меш…к, а деньги будут 13. Был бы меш…к, а деньги будут 14. Ещ… как-то перемелется и какова-то мука будет 15. И больш…й бадьёй реки не вычерпать 16. Собака обж…ра, а кошка сластена 17. Хоть бы в щ…ку бил, да щ…голь был 18. Где ч…рт не пахал, там и сеять не станет 19. Кто в кони пош…л, тот воду и вози 20. Испекли лепешку во всю щ…чку 21. Крут береж…к, да рыбка хороша 22. Пока травка подрастет, воды много утеч…т 23. Нет таких трав, чтоб знать чуж…й нрав 24. Не криви душ…й: кривобок на тот свет не уйдешь 25. Старич…к с кувшин, борода с аршин 3. Уточните значение следующих слов п пройдя по ссылке: https://slovarozhegova.ru/ 3. Уточните значение следующих слов по толковому словарю, пройдя по ссылке: https://slovarozhegova.ru/ 19 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 4. Найдите слова, в которых происходит оглушение звуков и вы- полните фонетический разбор двух из этих слов 5. Какие морфологические способы словообразования вам из- вестны? Выпишите по одному-два слова, образованных разными способами. 6. Что такое аршин? Вспомните и запишите фразеологизмы, в которых упоминаются древнерусские меры длины, веса, денежные единицы. Укажите значения этих фразеологизмов фр Упражнение 6 Упражнение 6 Упражнение 6 1. Спишите пословицы, вставляя пропущенные буквы. Выпол- ните орфографический комментарий по образцу: клочок – в суф- фиксе имени сущ. под ударением пишется О 1. Бумажки клоч…к в суд волоч…т 2. Трусливому каждый ш…рох – беда 3. Кто толч…т, тот и хлеб печ…т 3. Кто толч…т, тот и хлеб печ…т 4. На добрый ж…рнов что ни засыпь, всё смелется 5. Мастерство везде в поч…те 6. В гостях хорош…, а дому лучше 7. Дураку, что больш…му чину, везде простор 8. Худ торж…к, да не пуст горш…к 9. Рот нараспашку, язык на плеч… 10. Полюби нас в ч…рненьких, а в беленьких всяк полюбит 11. У всякого плута свой расч…т 12. За спасибо мужич…к в Москву сходил да ещ… полспасиба домой принес 13. Брюхо не меш…к, в запас не поешь 14. Мелет и ж…рнов, и язык 15. Не пляшет, так прищ…лкивает 16. Расчистил бы чащ…бу, да одолела трущ…ба 17. Нечет с нечетом тот же ч…т 18. Ож…гшись на молоке, станешь дуть и на воду 19. Не все бич…м да хлыстом, ино можно и свистом 20. Хлеб ч…рствый – обед честный 21. Плохое дерево не рубить, а выкорч…вывать 22. И калина с калач…м ему нипоч…м 23. С голоду не мрут, только пухнут, а с обж…рства лопаются 24. Жить бы еще, да в животе стало тощ… 20 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 2. Из подчеркнутых слов сначала выпишите слова с мягким зна- ком в три столбика соответственно роли Ь в них: 2. Из подчеркнутых слов сначала выпишите слова с мягким зна- ком в три столбика соответственно роли Ь в них: 2. Из подчеркнутых слов сначала выпишите слова с мягким зна- ком в три столбика соответственно роли Ь в них: 2. Из подчеркнутых слов сначала выпишите слова с мягким зна- ком в три столбика соответственно роли Ь в них: Для обозначения мягкости предшествующего согласного Для обозначения грамматической формы слова В качестве разделительного знака После этого выпишите слова без Ь и объясните его отсутствие. 1. Дай грош да пусти поросенка в рожь – будешь хорош. 2. Семь деревень, а лошадка одна. 3. Купил бы крестьян, да в деньгах изъян. 4. Горячее едят подьячие, голодные едят холодное. 5. Не поймал карася – поймаешь щуку. 6. Не радуйся нашедши, не плачь потерявши. 7. На богатого ворота настежь. 8. Грамоте учиться – всегда пригодится. 9. Ученья корень горек, а плод сладок. После этого выпишите слова без Ь и объясните его отсутствие. 1. Дай грош да пусти поросенка в рожь – будешь хорош. После этого выпишите слова без Ь и объясните его отсутствие. 8. Грамоте учиться – всегда пригодится. 9. Ученья корень горек, а плод сладок. 10. Не испортив дела, мастером не будешь. 11. На что нам ружье, коли стрелять не умеем. 12. Слезами моря не наполнишь, кручиною поля не изъездишь 13. Пока баба с печи летит, семьдесят семь дум передумает. 14. На ветер надеяться – без помолу быть. 15. Живешь – не с кем покалакать, помрешь – некому попла- кать. Часть 2. Трудные случаи русской орфографии и пунктуации 2. Разберите по составу следующие слова: ож…гшись, торж…к, засыпь, нипоч…м, нараспашку р р у 3. Подчеркните основы всех сложных предложений. Опреде- лите типы сложных предложений р 4. Выпишите слова, которыми вы можете прокомментировать исторические чередования в области согласных SOS! Вспомнить про исторические чередования согласных вы можете, пройдя по ссылке: https://docs.google.com/document/d/ 1PcKPShx6wW8bqHkfVNTa3kfvva86v9XB/edit или воспользовавшись QR-кодом: *** Написание Ь Упражнение 1 1. Вспомните, какие функции может выполнять буква Ь в слове. Вам поможет ссылка: https://www.yaklass.ru/p/russky-yazik/10- klass/orfografiia-10541/razlichiia-v-upotreblenii--i--10553/re- 6a0e85fd-7d86-4518-a06b-97b80247fa7e или QR-код: 21 кать. 16. Бабья вранья и на свинье не объедешь. 17. Как ни беречься, а видно ожечься. 18. Денег наживешь – без нужды проживешь. 19. Хоть пей, хоть ешь, хоть ножом режь, хоть только по- скреби, да прочь пойди. 20. Красна речь поговоркою. Часть2 1. Весною день упустишь – годом не вернешь 2. Семь раз примерь, а один отрежь 3. В каждом селенье свое заведенье 4. Без инструмента и вошь не убьешь 5. Не чванься, горох, перед бобами – будешь сам под ногами 6. Как ни жаться, а в правде признаться 22 2 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 7. Промеж худых и хорошему плохо 8. Хоть на час, да вскачь 9. Хорош соболек, да измят 10. Тишь да лад, да божья благодать 11. Всюду вхож, как медный грош 13. Ешь – не кроши, а съешь – не проси 14. Ближе живешь – уреживаешь; дальше живешь – учащи- ваешь 15. На своих именинах пирога не съешь, не съешь и на чужих 16. Плачь не плачь, а есть-пить надо 16. Плачь не плачь, а есть-пить надо 17. У голодного волка кости из зубов не вырвешь 18. Проголодаешься, так догадаешься 19. Ешь, пока рот свеж, а завянет – никто в него не заглянет 4. Просклоняйте словосочетание семьдесят семь дум, выде- лите во всех словах окончания. 5. Выполните разные виды разборов: Фонетический разбор: жаться, догадаешь Морфемный разбор: все деепричастия Синтаксический разбор: Ешь, пока рот свеж, а завянет – ни- кто в него не заглянет. Постройте схему предложения. 6. Укажите части речи всех слов в предложении Горячее едят подьячие, голодные едят холодное. Каким способом образованы подчеркнутые слова? Приведите примеры слов, образованных ана- логичным способом. Упражнение 2 1. Спишите, вставляя там, где нужно, Ь. Объясните свой выбор 1. Я крестит…ся, что не спит…ся? Погляжу, ан не ужинавши ежу. 2. Хоть пой, хоть плач…; хоть вплав…, хоть вскач….. 3. Уж… солнышко на ели, а мы еще не ели. 4. Как пирог с грибами, так все с узлами; а как плеть с узлом, так и проч… с кузлом. 5. Матушка-рож… кормит всех дураков сплош…, а пшеничка по выбору. 6. Хвощ… – деревенский овощ…. 6. Хвощ… – деревенский овощ…. 7. Кяхтинский чай да муромский калач… – полдничает богач… 23 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 8. Хорош… и квасок, коли шибает в носок. 8. Хорош… и квасок, коли шибает в носок. 9. Мешай дело с бездельем, проживеш… век с весельем. 10. Против ветру не надуеш…ся. 10. Против ветру не надуеш…ся. 11. Ключ… сильнее замка. 12. Поехал бы вскач…, ан сиди да плач… . 13. Вын… да полож… . 13. Вын… да полож… . 14. Видно, уж Дунай с Волгой не сойдут…ся. 15. Рубля у рубля не достанеш…, а копейку сплош…. 16. Нельзя кузнецу не ожеч…ся. 17. Калач… калача стоит: охотник оба съест. 18. Кряхти да гнись: упреш…ся – переломиш…ся. 19. Взялся за гуж…, не говори, что не дюж… . 20. Ни на службу вскач…, ни от службы проч… . 21. Служи сто лет, а не выслужиш… ста реп. 2. Обратите внимание на употребление Ь в словах: одинаковы или различны его роли? (см. предыдущее упр.) 3. Объясните написание подчеркнутых слов в предложении Уж… солнышко на ели, а мы еще не ели. Как называется такая игра слов? Приведите похожие примеры использования омоформ. 3. Объясните написание подчеркнутых слов в предложении Уж… солнышко на ели, а мы еще не ели. Как называется такая игра слов? Приведите похожие примеры использования омоформ. 4. Выполните синтаксический разбор всех бессоюзных сложных предложений этого упражнения. Объясните постановку знаков препинания в них. Чтобы выполнить это задание, можете восполь- зоваться ссылкой: https://foxford.ru/wiki/russkiy-yazyk/znaki- prepinaniya-v-bessoyuznom-slozhnom-predlozhenii или QR-кодом: 24 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 5. Подготовьте презентацию на тему «История буквы Ь» . 5. Подготовьте презентацию на тему «История буквы Ь» . 5. Подготовьте презентацию на тему «История буквы Ь» . 24 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 24 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 24 Часть 2. Трудные случаи русской орфографии и пунктуации Правописание непроизносимых согласных * Правописание непроизносимых согласных Упражнение 1 1. Вспомните орфограмму «Правописание непроизносимых со- гласных». В приведенных ниже пословицах и поговорках найдите слова с этой орфограммой и объясните их написание 1. Вспомните орфограмму «Правописание непроизносимых со- гласных». В приведенных ниже пословицах и поговорках найдите слова с этой орфограммой и объясните их написание 1. То не лестно, что известно 2. Много – сытно, мало – честно 3. Кто поздно встает, у того хлеба недостает 7. Язык коснееет, а перо не робеет 9. Кашица постная, да еще и без крупы 10. Сестры при братьях не вотчинницы 11. Без расчистки и лес не стоит 12. Будет и на нашей улице праздник 13. На всякое чиханье не наздравствуешься 14. Сердце матери греет лучше солнца 15. И тот вор, кто лестницу вору подставляет 16. От хорошего братца ума набраться, от плохого братца рад отвязаться 17. Что в сердце творится, то в лице не утаится 18. Праздность и старых повреждает, а молодым немалый вред бывает 19. Против притчи не поспоришь 20. Солнце – князь земли, а луна – княжна 21. Богаты, так здравствуйте, убоги, – так прощайте 22. Всугонь гостя не употчуешь 23. Суженые яства ряженому ясти 24. Это невестке в отместку 25. В осень любого гостя потчуют молоком, а нелюбого – медом 2. Найдите в приведенных предложениях устаревшие слова, уточните их значение (в этом вам поможет ссылка: https://slovarozhegova.ru/ 25 25 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» ), разделите слова на две группы: арха- измы и историзмы (освежить знания по этой теме вы можете, пройдя по ссылке: https://foxford.ru/wiki/russkiy-yazyk/arhaizmu- istorizmu или отсканировав QR-код: 3. Как вы понимаете выражение и тот вор, кто лестницу вору подставляет? Приведите пример, подтверждающий актуальность этой поговорки 4. Вспомните, как называются словари, дающие сведения о происхождении слов? Обратившись к этим словарям, уточните происхождение слов ровесница, праздник, притча, убогий, суже- ный. Подберите к ним исторически родственные слова Упражнение 2 1. Спишите, вставляя там, где необходимо, пропущенные буквы. Объясните свой выбор 1. Вместе тес…но, а розно грус…но 2. Мудрость старости чес…нее 3. Хорошо и лунный свет, как со…нца на небе нет 4. Сер…це с перцем, а душа с чесночком 5. Напусклив, что волк, пакос…лив, что кот 6. Кабы знать, где упасть, так соломки б подос..лал 7. Прес…на мякинка, да соли нет 26 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 8. Кресты да перс…ни – те ж деньги 9. Дошла ве…чинка до лычка 10. Хвас…ливого с богатым не разберешь 11. Скупой запирает крепко, а по…чует редко 12. Привет за привет, любовь за любовь, а завис…ливому – хрену да перцу, и то не с нашего стола 13. Я…ство сладенько, да ложка маленька 14. Что свис…нуло, то и гаркнуло 15. Скатер…кой трясет, а хлеб вон несет 2. Выпишите все служебные части речи, укажите их частереч- ную принадлежность 3. Как вы понимаете значение пословицы что свис…нуло, то и гаркнуло? Подберите к этому выражению синонимичные посло- вицы. Приведите пословицы со схожим смыслом из вашего род- ного языка. 4. Пользуясь этимологическими словарями, уточните этимоло- гию слов ве…чина, по…чует, скатер…ка. Подберите к ним истор- чески родственные слова и запишите их. 27 чески родственные слова и запишите их. Упражнение 3 1. Прочитайте пословицы и поговорки. Если есть непонятные слова, уточните их значение по толковому словарю (в этом вам по- может ссылка: https://slovarozhegova.ru/ ) 1. Худ Матвей, не умеет потчевать гостей 2. Кашица постная, да еще без круп 3. У праздника два невольника: одному хочется пить, да не на что купить, а другого потчуют, да пить не хочется 4. Не поглядев в святцы, да бух в колокол 5. У нашего молодца нет забавам конца р Упражнение 3 1. Прочитайте пословицы и поговорки. Если есть непонятные слова, уточните их значение по толковому словарю (в этом вам по- может ссылка: https://slovarozhegova.ru/ 3. У праздника два невольника: одному хочется пить, да не на что купить, а другого потчуют, да пить не хочется 4. Не поглядев в святцы, да бух в колокол 5. У нашего молодца нет забавам конца 27 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 6. Хотел брыснуть, а пришлось свистнуть 6. Хотел брыснуть, а пришлось свистнуть 7. Азия Африки честнее, август марта теплее 8. Кабы знал, где упасть, так бы соломки подостлал 9. Иного употчуешь кусом, а другого не употчуешь и гусем 10. Наша невестка все трескат 11. Брюзжит, словно деревянной пилой пилит 12. За эту песню и по боку свиснут 13. Суженые яства ряженому ясти 14. Дай плетью острастку, станет слушаться вожжей 15. С хорошим попутчиком и дорога короче 16. На женский норов нет угадчика. 2. Выпишите слова с орфограммой «Непроизносимые соглас- ные в корнях слов». Объясните написание этих слов 3. Что такое суженые яства, кому они предназначены? Почему слова суженые, ряженому написаны с одной Н? 4. Подберите синонимы к словам потчевать, постный, острастка р 5. Выполните следующие разборы: фонетический разбор слова брюзжит морфемный разбор слова поглядев морфологический разбор слова теплее 6. Какая поговорка представляет собой сложноподчиненное предложение с последовательным подчинением придаточных? Вы- пишите его, сделайте синтаксический разбор и постройте его схему р р р у 7. Что такое норов? Как называется фонетическая черта, при- сущая этому слову и позволяющая отнести его к русизмам, а не к старославянизмам? Как это слово звучит на старославянском языке? Сохранился ли в современном русском языке старославян- ский вариант? Приведите примеры старославянских по происхож- дению слов, сохранившихся в современном русском языке. Ука- жите в приведенных вами словах признаки старославянизмов (при- знаки старославянизмов вы можете вспомнить, пройдя по ссылке: https://docs.google.com/document/d/1PcKPShx6wW8bqHkfVNTa3kf vva86v9XB/edit или отсканировав QR-код (раздел «Чередования позднего общеславянского периода»): Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 28 Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Правописание согласных на стыке корня и суффикса Упражнение 1 Упражнение 1 1. Прочитайте пословицы и поговорки, проанализируйте напи- сание выделенных слов и сформулируйте правила написания со- гласных на стыке морфем. Попробуйте составить алгоритм исполь- зования этого правила на практике 1. Прочитайте пословицы и поговорки, проанализируйте напи- сание выделенных слов и сформулируйте правила написания со- гласных на стыке морфем. Попробуйте составить алгоритм исполь- зования этого правила на практике 1. Шапочное знакомство не в потомство. 2. Один приказчик – один вор, два приказчика – два вора. 3. Друг сердечный, а как зовут, не знаю. 4. На женский норов нет угадчика. 5. День дню не указчик. 6. Вестовщик да переносчик, что у реки перевозчик: надобен на час, а там и не знай нас. 7. Артель расходчика кормит. 8. Ищет вчерашний день, а он уже прошел. 9. От копеечной свечи Москва зажглась. 0. И перекати-поле на виновного доносчик. 10. И перекати-поле на виновного доносчик. 11. Приставить кому горчичник (досадить). 12. Лучше в обиде быть, нежели в обидчиках. 13. За здоровье тамошних и здешних и всех наших присер- дечных. 14. Калачник не табачник: не даст рожка понюхать 15. Игрушки да смешки, шутки да потешки 16. Туги орешки Терешке 29 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 17. Не ворожея, да отгадчик 18. Который час? После давишнего (прошлого) первый 2. Как называется словарь, в котором даются сведения о пра- вильном произношении слов? Как пользоваться этим словарем? Обращаясь за справками к этому словарю, сделайте транскрипцию следующих слов: шапочный, копеечный, горчичник, калачник, та- бачник (вам поможет ссылка: http://slovana.ru/slovari/reznichenko/ orfoepicheskij-slovar-reznichenko_0931.htm ) 3. Какие значения имеет слово сердечный? Меняется ли его про- изношение в зависимости от значения? (чтобы ответить на вопрос, можете воспользоваться ссылкой: http://slovana.ru/slovari/ reznichenko/orfoepicheskij-slovar-reznichenko_0931.htm) или QR- кодом: Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 17. Не ворожея, да отгадчик 17. Не ворожея, да отгадчик Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 17. Не ворожея, да отгадчик 18. Который час? После давишнего (прошлого) первый 2. Как называется словарь, в котором даются сведения о пра- вильном произношении слов? Как пользоваться этим словарем? Обращаясь за справками к этому словарю, сделайте транскрипцию следующих слов: шапочный, копеечный, горчичник, калачник, та- бачник (вам поможет ссылка: http://slovana.ru/slovari/reznichenko/ orfoepicheskij-slovar-reznichenko_0931.htm 30 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию orfoepicheskij-slovar-reznichenko_0931.htm ) 3. Какие значения имеет слово сердечный? Меняется ли его про- изношение в зависимости от значения? (чтобы ответить на вопрос, можете воспользоваться ссылкой: http://slovana.ru/slovari/ reznichenko/orfoepicheskij-slovar-reznichenko_0931.htm) или QR- кодом: ) 3. Какие значения имеет слово сердечный? Меняется ли его про- изношение в зависимости от значения? (чтобы ответить на вопрос, можете воспользоваться ссылкой: http://slovana.ru/slovari/ reznichenko/orfoepicheskij-slovar-reznichenko_0931.htm) или QR- кодом: 3. Какие значения имеет слово сердечный? Меняется ли его про- изношение в зависимости от значения? (чтобы ответить на вопрос, можете воспользоваться ссылкой: http://slovana.ru/slovari/ reznichenko/orfoepicheskij-slovar-reznichenko_0931.htm) или QR- кодом: Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 30 30 Упражнение 1 1. Спишите, вставляя пропущенные буквы. Объясните их напи- сание (вам может помочь ссылка: https://licey.net/free/4- russkii_yazyk/75-russkii_yazyk_v_nachalnoi_shkole/stages/4485- pravopisanie_parnyh_soglasnyh_v_korne_slova.html или QR-код: 1. Людям на поте…ку, белому свету на диво 1. Людям на поте…ку, белому свету на диво 6. Рад бы дру…ку пиро…ка, да у самого ни куска 7. Один с со…кой, а семеро с ло…кой 8. Кто с яры…кой поводится, без рубахи находится 9. Черная коро…ка дает белое молоко 10. Знайка доро…кой бежит, а незнайка на печке лежит 11. Иной стреляет ре…ко, да попадает ме…ко 12. Берут зави…ки на чужие пожи…ки 13. Девушка не тра…ка, не вырастет без сла…ки 14. Для милого дру…ка и сере…ку из у…ка 15. Попы за кни…ки, а миряне за пы…ки 15. Попы за кни…ки, а миряне за пы…ки 16. На дворе мороз, а в кармане дене…ки тают 17. Свашенька, свашенька, попляши, у тебя но…ки хороши! 18. Мужчина, коли хоть немно…ко казистее черта, – красавец 19. В своей сермя…ке никому не тя…ко 19. В своей сермя…ке никому не тя…ко 20. Красна яго…ка, да на вкус горька 20. Красна яго…ка, да на вкус горька 21. По росту одё…ку подбирают 21. По росту одё…ку подбирают 31 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» д р ц ц р р 2. Разгадайте кроссворд (подсказки: 1) слова-ответы (некоторые с уменьшительно-ласкательными суффиксами) есть в этом упраж- нении😊) 5 1 7 9 2 3 8 6 4 2. Разгадайте кроссворд (подсказки: 1) слова-ответы (некоторые с уменьшительно-ласкательными суффиксами) есть в этом упраж- нении😊) 5 1 7 9 2 3 8 6 4 2. Разгадайте кроссворд (подсказки: 1) слова-ответы (некоторые с уменьшительно-ласкательными суффиксами) есть в этом упраж- нении😊) 2. Разгадайте кроссворд (подсказки: 1) слова-ответы (некоторые с уменьшительно-ласкательными суффиксами) есть в этом упраж- нении😊) 2. Разгадайте кроссворд (подсказки: 1) слова-ответы (некоторые с уменьшительно-ласкательными суффиксами) есть в этом упраж- нении😊) 32 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 5 1 7 9 2 3 8 6 4 По горизонтали: 2. Устар. низший служитель полиции, для рассылки, прислуги и исполненья разных приказаний; 4. Женщина, занимающаяся свата- ньем, устройством браков; 6. Мелкое имущество, домашние вещи, скраб; 8. Маленькая птица, птичка; 9. Совокупность предметов (из ткани, меха, кожи), которыми покрывают, одевают тело 32 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации По горизонтали: 2. Устар. низший служитель полиции, для рассылки, прислуги и исполненья разных приказаний; 4. Женщина, занимающаяся свата- ньем, устройством браков; 6. Мелкое имущество, домашние вещи, скраб; 8. Маленькая птица, птичка; 9. Совокупность предметов (из ткани, меха, кожи), которыми покрывают, одевают тело 2. Устар. низший служитель полиции, для рассылки, прислуги и исполненья разных приказаний; 4. Женщина, занимающаяся свата- ньем, устройством браков; 6. Мелкое имущество, домашние вещи, скраб; 8. Маленькая птица, птичка; 9. Совокупность предметов (из ткани, меха, кожи), которыми покрывают, одевают тело 2. Устар. низший служитель полиции, для рассылки, прислуги и исполненья разных приказаний; 4. Женщина, занимающаяся свата- ньем, устройством браков; 6. Мелкое имущество, домашние вещи, скраб; 8. Маленькая птица, птичка; 9. Совокупность предметов (из ткани, меха, кожи), которыми покрывают, одевают тело 2. Устар. низший служитель полиции, для рассылки, прислуги и исполненья разных приказаний; 4. Женщина, занимающаяся свата- ньем, устройством браков; 6. Мелкое имущество, домашние вещи, скраб; 8. Маленькая птица, птичка; 9. Совокупность предметов (из ткани, меха, кожи), которыми покрывают, одевают тело 32 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 32 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 32 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 32 По вертикали: 1. Уменьшительно-ласкат. от слова, обозначающее кафтан гру- бого некрашеного крестьянского сукна; 3. Малина – это …, клуб- ника – это …, смородина – …; 5. Дыра на одежде или другом пред- мете из ткани; 7. Примитивное земледельческое орудие для вспа- хивания земли; 8. Забава, веселье, развлеченье 3. Выполните словообразовательный разбор следующих слов: смолоду, незнайка, завидки, забава 4. В этом упражнении встретились пословицы и поговорки с именами прилагательными, обозначающими цвет. Каково симво- лическое значение белого, черного, красного цветов в русской тра- диционной культуре? Употребляются ли в пословицах и поговор- ках вашего народа имена прилагательные, обозначающие цвет? Приведите примеры. Каково символическое значение цветовых прилагательных в культуре вашего народа? р Упражнение 2 1. Спишите, вставляя пропущенные буквы. Выполните орфогра- фический комментарий по образцу: похлёбки – похлебок 1. Из щец похлё…ки не сваришь. 1. Спишите, вставляя пропущенные буквы. Выполните орфогра- фический комментарий по образцу: похлёбки – похлебок 1. Из щец похлё…ки не сваришь. 2. Лу…ше раз в году родить, чем день-деньской бороду брить 3. Богатый и не тужит, да брю…ит 4. Дене…ка рубля стережет, а рубль голову бережет 5. Ло…ку за окошко даром не выкинешь 6. Нет такого дру…ка, как родная матушка 7. Мирошка, голенькие но…ки, живет понемно…ку 8. Были бы бума…ки, будут и милашки 9. В своей теремя…ке никому не тя…ко 10. Соболье одеяльце в ногах, да потонули поду…ки в слезах 11. Язык не лопа…ка, знает, где сла…ко 12. Садит в печь пиро…ки, а вынимает покры…ки да гор…ки 13. Где пичу…ка ни летала, а в нашу сеть попала 14. В приказ ворота широки, да из него – у…ки 15. Июль – макушка лета, декабрь – ша…ка зимы 16. Дене…ка доро…ку прокладывает 17. Куры да амуры, да гла…ки на сала…ках 18. Вместе ску…но, а розно то…но 18. Вместе ску…но, а розно то…но 19. Кому что по душе, а цыгану яи…ница 19. Кому что по душе, а цыгану яи…ница 33 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Правописание двойных согласных в корне Упражнение 1 * Правописание двойных согласных в корне *** Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 2. Сделайте транскрипцию следующего предложения: Язык не лопа…ка, знает, где сла…ко. Какие фонетические процессы про- исходят при произнесении этой фразы? 2. Сделайте транскрипцию следующего предложения: Язык не лопа…ка, знает, где сла…ко. Какие фонетические процессы про- исходят при произнесении этой фразы? р р фр 3. Вспомните, что такое односоставные предложения (вам мо- жет помочь ссылка: https://foxford.ru/wiki/russkiy-yazyk/vidy- odnosostavnyh-predlozheniy или QR-код: Приведите примеры односоставных предложений из приведен- ных выше предложений 4. В каком значении употреблено слово приказ в предложении В приказ ворота широки, да из него – у…ки? Какие еще значения может иметь это слово? Составьте и запишите предложения с этим словом в разных значениях 34 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию ** Правописание двойных согласных в корне Упражнение 1 1. Повторите правила написания удвоенных согласных (можете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy-yazyk/ dvojnye-soglasnye или QR-кодом: ). Упражнение 1 р 1. Повторите правила написания удвоенных согласных (можете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy-yazyk/ dvojnye-soglasnye или QR-кодом: 34 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию ). Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 34 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 34 1. Т…р…ватому бог подает, а у скупого черт отбирает 2. На словах, что на гуслях, а на деле, что на б…л…лайке 4. Др…гун бежит, земля дрожит, а оглянешься – в грязи ле- ит 5. Знай наших: последняя к…пейка ребром 6. Полы в г…лунах, а к…рманы в дырах 7. Чванства на сто рублей, а животов на три п…лушки 8. В хваленой капусте много гнилых к…чней 9. Нанизал бы тебя на ож…релья, да носил бы в воскресенье 10 д 7. Чванства на сто рублей, а животов на три п…лушки 8. В хваленой капусте много гнилых к…чней 9. Нанизал бы тебя на ож…релья, да носил бы в воскресенье 10. …рехи – девичьи потехи 7. Чванства на сто рублей, а животов на три п…лушки 8. В хваленой капусте много гнилых к…чней 9. Нанизал бы тебя на ож…релья, да носил бы в воскресенье 10. …рехи – девичьи потехи 10. …рехи – девичьи потехи 11. Красная девка в х…р…воде, что маков цвет в ог…роде 12. Зародился Никита на в…л…окиту 13. Дочку в к…лыбельку – приданое в к…робейку 14. Выходя на сватовство, связывают вместе к…чергу и по- ло Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Какому принципу русской орфографии подчиняется это пра- вило? Докажите свою точку зрения. Какому принципу русской орфографии подчиняется это пра- вило? Докажите свою точку зрения. Спишите, раскрывая скобки. 1. Озера соломой не за(жж/ж)ёшь 2. Кому кнут да во(жж/ж)и в руки, кому хомут на шею 3. Люди (сс/с)орятся, а воеводы кормятся 4. Хороша дочь А(нн/н)ушка, коли хвалят мать да бабушка. 5. Обо(жж/ж)ешься на молоке – станешь дуть и на воду 6. Счастье бедному алтын, богатому – ми(лл/л)ион 7. Его копейка нищему руку про(жж/ж)ет 8. Крестьянский а(пп/п)етит никогда не претит 9. Сегодня Са(вв/в)ы, завтра Варвары 10. Хочется бабе сусла, да губы (жж/ж)ет; хочется дуть, да не дают 11. Два кума А(вв/в)акума, две кумы Авдотьи 12. Проживет Фа(дд/д)ей и без затей 13. Са(вв/в)а съел сало, уперся, заперся, сказал: не видал 14. Возьми у Са(вв/в)ки на лавке 15. Всю неделю говорил: а с ь, а в су(бб/б)оту сказал: ч т о 2. Выпишите слова, которые входят в ваш пассивный словарь, и слова, значения которых вы не знаете. По толковому словарю уточ- ните значения этих слов (можете воспользоваться ссылкой: https://slovarozhegova.ru/ или QR-кодом: Составьте и запишите с этими словами словосочетания. Составьте и запишите с этими словами словосочетания. 3. Какими примерами из текста можно проиллюстрировать пра- вописание: а) безударных гласных в корне; б) окончаний 4. Объясните знаки препинания во всех предложениях. 35 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Упражнение 1 р 1. Спишите, вставляя пропущенные буквы. В случае затруд- нения обращайтесь к орфографическому словарю (например, к этому: https://gufo.me/dict/orthography_lopatin мело 15. За к…мпанию и м…нах удавился 16. Доброе братство милее б…гатства 17. Жалеть м…шка – не видать дружка 18. Стоя вместе у к…лодца, и ведро с ведром столкнется 18. Стоя вместе у к…лодца, и ведро с ведром столкнется 6 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 36 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации русской орфографии и пунктуации 2. По словарю (вспомните, какому) уточните происхождение следующих слов: балалайка, сковорода, копейка, карман, кочан, кочерга, богатство. Получив эту информацию, попробуйте объяс- нить правописание непроверяемых гласных в корнях этих слов 3* Заполните таблицу примерами полногласия/ неполногласия из упражнения. К каждому выписанному вами слову подберите со- ответствующую пару Примеры полногласия Примеры неполногласия 4. Что такое омонимы? Какие виды омонимов вы знаете? (мо- жете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy- yazyk/omonimy или QR-кодом: 2. По словарю (вспомните, какому) уточните происхождение следующих слов: балалайка, сковорода, копейка, карман, кочан, кочерга, богатство. Получив эту информацию, попробуйте объяс- нить правописание непроверяемых гласных в корнях этих слов 2. По словарю (вспомните, какому) уточните происхождение следующих слов: балалайка, сковорода, копейка, карман, кочан, кочерга, богатство. Получив эту информацию, попробуйте объяс- нить правописание непроверяемых гласных в корнях этих слов р р р р 3* Заполните таблицу примерами полногласия/ неполногласия из упражнения. К каждому выписанному вами слову подберите со- ответствующую пару Примеры полногласия Примеры неполногласия Примеры неполногласия Примеры неполногласия 4. Что такое омонимы? Какие виды омонимов вы знаете? (мо- жете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy- yazyk/omonimy или QR-кодом: 4. Что такое омонимы? Какие виды омонимов вы знаете? (мо- жете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy- yazyk/omonimy или QR-кодом: Чем отличается кОмпания от кАмпании? Какой вид омонимов представляет эта пара? За кОмпанию или за кАмпанию удавился монах (см. пословица 15) 1. Спишите, вставляя пропущенные буквы. В случае затруд- нения обращайтесь к орфографическому словарю (например, к этому: https://gufo.me/dict/orthography_lopatin 1. Спишите, вставляя пропущенные буквы. В случае затруд- нения обращайтесь к орфографическому словарю (например, к этому: https://gufo.me/dict/orthography_lopatin 37 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 1. С кем познаешься, тем и в…личаешься 2. Нашей …ртели прибыло 8. Хлебушка к…л…чу дедушка 9. За обедом с…л…вей, а после обеда в…р…бей 10. На рабочего дело найдется, на г…л…дного кус сыщется 11. Сыт конь – б…гатырь, голоден – с…рота 12. Где …ладьи – там и ладно; где блины, тут и мы 13. Дядя – тр…буха, съел корову да быка 14. У Ипата к пирогам борода с л…пату, а увидел дубину – и борода клином 15. На к…лбасах штаны проел 16. Стол – божья л…донь: кормит. 2. Выпишите по 2-3 примера словосочетаний с разными видами подчинительной связи (если вы забыли о видах подчинительной связи, можете воспользоваться ссылкой: https://foxford.ru/wiki/ russkiy-yazyk/vidy-podchinitelnoj-svyazi-v-slovosochetaniyah или QR-кодом: а) согласование а) со ласо а е б) управление в) примыкание 3. В древности слово л…донь выглядело как длань. Какие уста- ревшие названия частей тела вам еще известны? Вспомните и запи- шите фразеологизмы, в состав которых входят названия частей тела человека как устаревших, так и современных. 3. В древности слово л…донь выглядело как длань. Какие уста- ревшие названия частей тела вам еще известны? Вспомните и запи- шите фразеологизмы, в состав которых входят названия частей тела человека как устаревших, так и современных. 38 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 38 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Упражнение 3 1. Спишите, вставляя в пословицы пропущенные слова (см. ма- териал для справок). Запомните правописание этих слов 1. Не наша еда …, есть их иному 2. Дворянское кушанье – два гриба на … 3. Наш Тарас на перегудки … . 4. … – не цепом молотить 5. Не жалей …, валяй трепака! 6. Съешь и …, коли яблочка нет 7. … мочале не порвать, горшку с котлом не биться 8. Слоны трутся, меж себя …давят 9. … с медведем тягалась, один хвост да грива остались 10. Чем долго …, ступай лучше ко дну 11. На запретный товар весь … кидается 12. Есть в горшке …, да голова не лезет 13. Растил бы …, да изнял голод 14. Пошел бы к соседу по …, да на двор не пустят 15. … нитка, да игла за собой потянула 16. Виноват коноват перед … 17. Терпи, казак, …будешь 18. Идти …, да доставать копейку 19. Бодливой … бог рог не дает Материал для справок: Материал для справок: Лимоны, каблука, в коробейку, солод, корове, горазд, таращи- лась, бархатником, хороводить, капусту, молоко, барахтаться, морковку, атаманом, кобыла, базар, ремня, комаров, на тарелочке. Лимоны, каблука, в коробейку, солод, корове, горазд, таращи- лась, бархатником, хороводить, капусту, молоко, барахтаться, морковку, атаманом, кобыла, базар, ремня, комаров, на тарелочке. р у 2. Выполните разборы: Фонетический разбор: кидается, съешь Морфемный разбор: все глаголы этого упражнения 3. Как известно, слово капуста является общеславянским за- имствованием из латинского языка; оно содержит в себе латинский корень caput – «голова». Какие слова русского языка также содер- жат этот латинский корень? Запишите их, определите их значение, составьте и запишите с ними предложения. 39 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 40 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Упражнение 4 Упражнение 4 1. Спишите, вставляя пропущенные буквы. В случае затруд- нения обращайтесь к орфографическому словарю (например, к этому: https://gufo.me/dict/orthography_lopatin ) 1. Заяц от лисицы, а л…гушка от зайца бежит 2. Ч…репашка с зайцем навыпередки пошла 3. Какова спина широка есть, столько р…мня и ляжет 4. Не бывать к…лине м…линой. 5. Кабы на кр…пиву не мороз, с нею и ладов б не было 6. Знает и кр…стьянин, что поп не б…ярин 7. Забрякали дугами – не улежить и х…мутам 8. Без клиньев и к…фтана не сделаешь 9. И велики к…рманы, а всего свету не уберешь 10. С…лдат не крадет, а под полою, случается, унесет 11 Л д б 11. Лук с ч…сноком – родные братья 11. Лук с ч…сноком – родные братья 12. Одного сукна еп…нча 12. Одного сукна еп…нча 13. Нет, не гнед мерин, а с…врас мерин, а все тот же мерин 14. Оборотись, п…рося, в рыбу к…рася 14. Оборотись, п…рося, в рыбу к…рася 15. Без к…пейки не рубль 15. Без к…пейки не рубль 16. Слышно, что с…ловей свистит, а что в...рона каркает 17. Иди в город, там с к…локольным звоном встречают 18. На хороший цветок летит и м…тылек 19. Пей, гусь, воду, не б…ярского роду! 2. Вспомните, как называется толковый словарь, составленный В.И. Далем? Как вы думаете, каким словам В.Т. Даль в своем сло- варе дает следующие толкования? (! подсказка: эти слова есть в пословицах данного упражнения) 40 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 40 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Конская масть; светлогнедой с желтизной; Верхнее, долгополое мужское платье разного покроя; Часть конской упряжи Широкий безрукавный плащ, бурка; Смиренный жеребец С Составьте словосочетания с этими словами с разными видами связи (согласованием, управлением, примыканием). Чтобы вспом- нить виды связи в словосочетании, можете воспользоваться ссыл- кой: https://foxford.ru/wiki/russkiy-yazyk/vidy-podchinitelnoj-svyazi- v-slovosochetaniyah или QR-кодом: 42 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 3. Как вы думаете, каково значение слова навыпередки? К какой части речи оно относится? Докажите свою точку зрения. Разберите это слово по составу. 3. Как вы думаете, каково значение слова навыпередки? К какой части речи оно относится? Докажите свою точку зрения. Разберите это слово по составу. у 4. Разберите по членам предложение Нет, не гнед мерин, а с…врас мерин, а все тот же мерин. Какой частью речи являются слова гнед, саврас? Всегда ли краткие прилагательные в предложениях являются сказуемыми? Почему? Приведите примеры употребления кратких форм имен прилагательных в роли других членов предложения (не в роли сказуемого) Упражнение 5 Всегда ли краткие прилагательные в предложениях являются сказуемыми? Почему? Приведите примеры употребления кратких форм имен прилагательных в роли других членов предложения (не в роли сказуемого) Упражнение 5 1. Спишите, вставляя пропущенные буквы. Значения незнако- мых слов уточните по словарю (например, такому: https://slovarozhegova.ru/ или такому: https://www.slovardalja.net/; можно воспользоваться QR-кодами: 41 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» и 1. Баба с возу – к…быле легче 2. Смех – в…лынка: надул, поиграл, да и кинул 3. Хороши шутки, б…л…мутки 4. По барыне г...вядина, по х…рчевне едок 5. Не все в…рчать, надо и помолчать 6. Ты стал на пень, а он на к…рягу 7. Не бей мужика дубиной, попробуй п…лтиной 8. Не все такать, ино и пок…лякать 9. Дай ему потачки, так и сам от него на к…рачках 10. Играть на кожаной скрыпке в два смычка без к…н…фоли 11. С этого леща надо бы ч…шую поскрести 12. Я ему влеплю в бороду р…пей 13. Не грози попу к…дилом: им же кормится 14. Не боится огонь к…черги 15 П и 1. Баба с возу – к…быле легче 2. Смех – в…лынка: надул, поиграл, да и кинул 3. Хороши шутки, б…л…мутки 4. По барыне г...вядина, по х…рчевне едок 5. Не все в…рчать, надо и помолчать 6. Ты стал на пень, а он на к…рягу 7. Не бей мужика дубиной, попробуй п…лтиной 8. Не все такать, ино и пок…лякать 9. Дай ему потачки, так и сам от него на к…рачках 10. Играть на кожаной скрыпке в два смычка без к…н…фоли 11. С этого леща надо бы ч…шую поскрести 12. Я ему влеплю в бороду р…пей 13. Не грози попу к…дилом: им же кормится 14. Не боится огонь к…черги 10. Играть на кожаной скрыпке в два смычка без к…н…фоли 11. С этого леща надо бы ч…шую поскрести 12. Я ему влеплю в бороду р…пей 13. Не грози попу к…дилом: им же кормится 14. Не боится огонь к…черги 15. По в…таге ат…ман, по овцам пастух 16. Кусают и к…мары до поры 17. Каков м…л…бен, такова и плата 2. По словарю (вспомните, какому) уточните происхождение слов баламутки, говядина, харчевня, коряга, калякать, кочерга, ва- тага, комар. Подберите к ним и запишите исторически однокорен- ные слова, корни выделите 3. Разберите по членам все сложные предложения этого упраж- нения. Определите их вид (сложносочиненные, сложноподчинен- ные, бессоюзные). Свой ответ аргументируйте (вам может помочь ссылка: https://russkyaz.ru/predlozheniya/slozhnye-predlozheniya/ vidy-slozhnyh-predlozhenij.html или QR-код😊) 42 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 42 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 42 Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 4. Слова кадило, молебен относятся к церковной лексике (кстати, каково значение этих слов?). Вспомните и запишите по- словицы, поговорки и фразеологизмы, включающие в себя церков- ную лексику. Упражнение 6 р 1. Соедините начало пословицы с её концом. Выпишите слова с непроверяемым гласным в корне, подберите и запишите к ним по три однокоренных слова, выделите в них корни По жильцу квартира над одним рядовым По Сеньке и шапка тот и снимает Два медведя в одной берлоге так не будь вислоух Кто венец надевает по Ереме колпак Без атамана добычи не уживутся Семеро капралов по горшку и покрышка У нашего командира там голова мочало Где хвост начало служи и панихиды Пошел в попы ни шляпы, ни мундира Надел треух не делят 1. Соедините начало пословицы с её концом. Выпишите слова с непроверяемым гласным в корне, подберите и запишите к ним по три однокоренных слова, выделите в них корни 2. Найдите среди воссозданных вами пословиц синонимичные. Как вы понимаете их значение? Есть ли в фольклоре вашего народа пословицы со схожим значением? Если есть, запишите их. Переве- дите их дословно на русский язык. Придумайте ситуацию, в кото- рой уместно употребление этих пословиц. 2. Найдите среди воссозданных вами пословиц синонимичные. Как вы понимаете их значение? Есть ли в фольклоре вашего народа пословицы со схожим значением? Если есть, запишите их. Переве- дите их дословно на русский язык. Придумайте ситуацию, в кото- рой уместно употребление этих пословиц. 3. Во многих пословицах, поговорках и фразеологизмах упо- требляются имена собственные. Вспомните и запишите их. Со- ставьте с ними простые осложненные предложения, разберите эти предложения по членам. (Если вы подзабыли, что такое простое осложненное предложение, то пройдите по ссылке: https://foxford. ru/wiki/russkiy-yazyk/prostoe-oslozhnennoe-predlozhenie или отска- нировав QR-код:) 43 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Правописание проверяемых безударных гласных в корне Упражнение 1 Упражнение 1 1. Выпишите слова с безударной проверяемой гласной в корне, рядом напишите проверочное слово 1. Выпишите слова с безударной проверяемой гласной в корне, рядом напишите проверочное слово 1. Отдал убогий нищему последний пятак, а сам от богатого ушел и так 2. Полынь после меду горче самой себя 3. Хвалилась кобыла, что с возом горшки побила 4. Слепец слепцу глаза колет, а оба зги не видят 5. Все равны бобры, один я соболек 6. Тебя Бог умом наделил, так не нас же обделил 8. Тянулся с барами, да распрощался с анбарами 9. Держи девку в тесноте, а деньгу а темноте 10. Не стоит гроша Пахом, а смотрит пятаком 11. Кабы люди не сманили, и теперь бы любила 12. Когда стала любовь сознавать, тогда стал и милый от- ставать 13. Люблю, как клопа в углу: где увижу, тут и задавлю 14. Княжна хороша, и барыня хороша, а живет красна и наша сестра 15. Покрасуйся, девушка, до святой воли батюшкиной 16. Настя-Настенька, шубейка красненька: сама черноброва, опушка боброва у р 17. Держи деньги в темноте, а девку в тесноте 18. Веселая голова, не ходи вкруг моего двора 19. Благословение лучше проклятия 19. Благословение лучше проклятия 44 44 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 2. Постройте схемы всех сложноподчиненных предложений этого упражнения. Определите типы придаточных. В этом вам по- может ссылка: https://foxford.ru/wiki/russkiy-yazyk/vidy- pridatochnyh-predlozhenij или QR-код: 2. Постройте схемы всех сложноподчиненных предложений этого упражнения. Определите типы придаточных. В этом вам по- может ссылка: https://foxford.ru/wiki/russkiy-yazyk/vidy- pridatochnyh-predlozhenij или QR-код: 3. Какие пословицы и поговорки, на ваш взгляд, наиболее акту- альны сегодня? Придумайте и опишите ситуации, в которой будет уместно употребление этих пословиц 3. Какие пословицы и поговорки, на ваш взгляд, наиболее акту- альны сегодня? Придумайте и опишите ситуации, в которой будет уместно употребление этих пословиц 4. Какова начальная форма слова (с) барами в пословице №8? В чем особенность склонения этого существительного? Приведите примеры существительных, имеющих аналогичные особенности склонения. 45 4 . Какова начальная форма слова (с) барами в пословице №8? В чем особенность склонения этого существительного? Приведите примеры существительных, имеющих аналогичные особенности склонения. Упражнение 2 1. Спишите предложения, вставляя пропущенные буквы. В скобках запишите проверочные слова. Значения незнакомых слов уточните про словарю: для этого пройдите по ссылке: https://slovarozhegova.ru/ или воспользуйтесь QR-кодом: 1. В добрый час молвить, в худой пром…лчать! 2. Живы своими гр…хами, вашими м…литвами 3. За постой деньги платят, а пос…делки даром 4. Милости пр…шу к нашему шал…шу: я пир…гов покр…шу и откушать попр…шу 5. Ж…нился, сам себе под…вился Упражнение 2 Упражнение 2 1. Спишите предложения, вставляя пропущенные буквы. В скобках запишите проверочные слова. Значения незнакомых слов уточните про словарю: для этого пройдите по ссылке: https://slovarozhegova.ru/ или воспользуйтесь QR-кодом: 1. Спишите предложения, вставляя пропущенные буквы. В скобках запишите проверочные слова. Значения незнакомых слов уточните про словарю: для этого пройдите по ссылке: https://slovarozhegova.ru/ или воспользуйтесь QR-кодом: 1. В добрый час молвить, в худой пром…лчать! 2. Живы своими гр…хами, вашими м…литвами 3. За постой деньги платят, а пос…делки даром 4. Милости пр…шу к нашему шал…шу: я пир…гов покр…шу и откушать попр…шу 5. Ж…нился, сам себе под…вился 45 15. Зв…нят бубны хор…шо, да плохо кормят Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 6. Не заламывай р…бинку не вызревшу; не сватай девку не выз- навши 6. Не заламывай р…бинку не вызревшу; не сватай девку не выз- навши 7. Ж…нился богатый на с…нливой: оба счастливы 8. Не ж…нись для тёщи, не выдавай для свекра 9. Не сошлись обыч…ем, не бывать дружбе 9. Не сошлись обыч…ем, не бывать дружбе 10. С другом дружись, а за саблю д…ржись 11. В поле пш…ница годом р…дится, а добрый человек всегда приг…дится 11. В поле пш…ница годом р…дится, а добрый человек всегда приг…дится 12. Дружбу в…дить – себя не щ…дить 13. У нас и с…хи св…лись вместе (дружные соседи) 14. Близ гр…ницы не строй св…тлицы 15. Просим пр…щенья за наше уг…щенье: к нашему кр…ще- нию, р…ждества похл…бать, масленицы отведать 16. С…рдись, бр…нись, дерись, а за хлебом-солью сх…дись 17 З 6. С…рдись, бр…нись, дерись, а за хлебом-солью сх…дись 7. За совет и в пост м…соед р , р , р , 17. За совет и в пост м…соед 17. За совет и в пост м…соед 17. За совет и в пост м…соед 18. Умей у людей пог…стить, и к себе запр…сить, до ворот пров...дить и опять вор…тить 18. Умей у людей пог…стить, и к себе запр…сить, до ворот пров...дить и опять вор…тить 19. Хоть хлеба кр…юшка да пшена ч…тв…ртушка, от ласко- вого хозяина и то уг…щенье 19. Хоть хлеба кр…юшка да пшена ч…тв…ртушка, от ласко- вого хозяина и то уг…щенье 20. На чужой обед надейся, а сам прип…сай 21. Дорожный с собою н…члега не возит 2. Сделайте транскрипцию предложения за совет и в пост м…соед. Какие фонетические процессы происходят при его произ- несении? Встречаются ли эти процессы в вашем родном языке? Если встречаются, приведите примеры. 3. Согласны ли вы с выражением не сошлись обыч…ем, не бы- вать дружбе? Почему? Оформите свой ответ в виде небольшого сочинения-рассуждения. 4. Определите части речи всех слов в предложении Не заламы- вай р…бинку не вызревшу; не сватай девку не вызнавши. Разберите все слова по составу. Если это возможно, подберите ко всем словам этого предложения однокоренные с историческими чередовани- ями. Прокомментируйте эти чередования (происхождение, регу- лярность, отличия по славянским языкам). Можете воспользо- ваться ссылкой: https://docs.google.com/document/d/1PcKPShx6w W8bqHkfVNTa3kfvva86v9XB/edit или QR-кодом: 46 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 47 Упражнение 3 1. Спишите, вставляя пропущенные буквы и указывая в скобках проверочные слова. Значения незнакомых слов уточните по сло- варю, пройдя по ссылке https://slovarozhegova.ru/ или отсканировав QR-код: Часть 1 1. Родится р…ток – р…дится и кусок 2. Любо брюху, что глаза на кашу гл…дят 3. И пес перед хлебом см…ряется 4. Хлеб возит, а м…киной кормят 5. Не ел, так обомлел; наелся, так вовсе пов…лился 6. Чуть з…вает, а брюхо наб…вает 7. Дай нас…саться п…явке – сама отвалится 8. Без хлеба св…того все приестся 9. Хлеб да м…кина – Самсона сила 10. Мать наша – гречневая каша: не перцу чета, не прорвет ж…вота 11. Нап…кла, нав…лила, хоть отца с матерью жени 12. Дв…рянский сын с погл…денья сыт 13. Лаком, что македонская кн…гиня: со щучки одни щечки ку- шает 14. Скоморохова жена всегда в…с…ла 15. Зв…нят бубны хор…шо, да плохо кормят Упражнение 3 Упражнение 3 1. Спишите, вставляя пропущенные буквы и указывая в скобках проверочные слова. Значения незнакомых слов уточните по сло- варю, пройдя по ссылке https://slovarozhegova.ru/ или отсканировав QR-код: Часть 1 1. Родится р…ток – р…дится и кусок 2. Любо брюху, что глаза на кашу гл…дят 3. И пес перед хлебом см…ряется 4. Хлеб возит, а м…киной кормят 5. Не ел, так обомлел; наелся, так вовсе пов…лился 6. Чуть з…вает, а брюхо наб…вает 7. Дай нас…саться п…явке – сама отвалится 8. Без хлеба св…того все приестся 9 Х б д С 7. Дай нас…саться п…явке – сама отвалится 8. Без хлеба св…того все приестся 9. Хлеб да м…кина – Самсона сила 10. Мать наша – гречневая каша: не перцу чета, не прорвет ж…вота 11. Нап…кла, нав…лила, хоть отца с матерью жени 12. Дв…рянский сын с погл…денья сыт 13. Лаком, что македонская кн…гиня: со щучки одни щечки ку- шает 14. Скоморохова жена всегда в…с…ла 14. Скоморохова жена всегда в…с…ла 15. Зв…нят бубны хор…шо, да плохо кормят 47 Часть 2 9. При дороге жить – всех не уг…стить 10. На всех не уг…дишь 11. В одну руку всего не загр…бешь 12. Чем долго в…зиться, так пол…зай в мешок 13. Чужой рот – не св…и ворота, не затв…ришь 2. Выполните разборы: Фонетический разбор: гречневую, благородное, угостить Морфемный разбор: все глаголы в форме прошедшего вре Морфологический разбор: п…явке, македонская, в…ляй 3. Объясните постановку тире во всех предложениях, где стоит этот знак (вам может помочь ссылка: http://new.gramota.ru/ biblio/readingroom/rules/158-tire или QR-код 4. Как вы понимаете значение выражения Чужой рот – не св…и ворота, не затв…ришь? Подберите синонимичные русские посло- вицы и пословицы вашего народа. Придумайте и запишите диалог, в котором было бы уместно употребление этой пословицы Упражнение 4 1. Спишите предложения, вставляя пропущенные буквы. Объяс- ните орфограммы. 1. Спишите предложения, вставляя пропущенные буквы. Объяс- ните орфограммы. 48 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 48 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 1. Ехал бы д…лече, да б…лят плечи 2. Хоть падать, да не л…жать 3. Зам…рился бы с туркой, так царь не велит 4. Не умеешь петь, в зап…валы не суйся 5. И не хочет коза на б…зар, да в…дут за р…га 6. Заставил пл…сать под свою дудку 7. Т…ж…ло молоту, т…ж…ло и нак…вальне 8. Хоть к обедне не поспеть, а походки не пот…рять 9. Хоть ноги изл…мать, а двери выставить 10. Научат добрые люди реш…том воду н…сить б б 1. Ехал бы д…лече, да б…лят плечи 2. Хоть падать, да не л…жать 3. Зам…рился бы с туркой, так царь не велит 4. Не умеешь петь, в зап…валы не суйся 5. И не хочет коза на б…зар, да в…дут за р…га 6. Заставил пл…сать под свою дудку 7. Т…ж…ло молоту, т…ж…ло и нак…вальне 8. Хоть к обедне не поспеть, а походки не пот…рять 9. Хоть ноги изл…мать, а двери выставить 10. Научат добрые люди реш…том воду н…сить 11. Не распл…статься лопате, чтоб ёмче быть 12. По голому нечего щ…пать 12. По голому нечего щ…пать 13. Желай по силам, т…нись по достатку 14. И велик л…зун дал бог корове, да г…в…рить не в…лел 15. Хорошо зат…нул (песню), да не вынес 16. С…дит на р…ду, г…в…рит: не могу 17. Св…я рука да над св…им д…бром – владыка 18. Нал…тела шина на щ…бенку – быть ей съеденной 19. И баба смекает, как ребенка к…чает 20. Эко диво – у свиньи п…таком рыло 2. Найдите в упражнении стилистически окрашенные слова, подберите к ним нейтральные синонимы. Какой словарь может вам помочь при выполнении этого задания? 3. Найдите односоставные предложения, определите их вид. Ар- гументируйте свой ответ. Чтобы выполнить это задание, вы можете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy-yazyk/vidy- odnosostavnyh-predlozheniy или QR-кодом: 49 50 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Для выполнения этого задания можете воспользоваться ссыл- кой: https://cross.highcat.org/ru_RU/ или QR-кодом р 1. Соедините начало пословицы с её концом. Выпишите слова с проверяемым гласным в корне, подберите и запишите к ним по три однокоренных слова, выделите в них корни Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 4. Как вы понимаете значение выражения Хоть падать, да не л…жать? Приведите примеры из литературных произведений, где герои придерживаются этого принципа и где герои, наоборот, пред- почитают «лежать», зато «не падают». А к каким героям вы отно- сите себя? Упражнение 5 1. Прочитайте пословицы и поговорки. Выпишите слова с про- веряемыми гласными в корне, объясните их написание. 1. Кто долго живет, тот и стариком слывет 2. Мелко плавать – дно задевать 3. Верстой ближе, пятаком дешевле 4. Кто от солнца убегает, тот и озябает 5. И большой бадьей реки не вычерпать 10. Хоть до Кракова, так все одинаково 11. Не все едино, что хлеб, что мякина 12. Та же опара, да другой кисель 13. Иной любит попа, другой попадью, а третий попову дочку 14. Закорми чушку, станет жаловаться, что бока отлежала 15. Хорошие щи без соли хлебаю, а в худые и соли не теряю 16. Хорошее не лизать, а худое не тесать 17. У наших казаков обычай таков: где просторно, тут и спать ложись 18. Лег – свернулся, встал – встряхнулся 19. Шутил Мартын, да и свалился под тын 20. Овцу стригут, баран дрожит 21. Журба твоя часовая, гульба моя вековая 22. Такую баню задали, что небо с овчинку показалось 23. Назвался груздем – полезай в кузов 2. Составьте кроссворд из следующих слов: верста, пятак, ба- дья, ненастье, Краков, мякина, опара, казак, тын, журба, овчинка, груздь, кузов 50 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 50 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 50 50 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Составьте и запишите предложения с парами слов: парадиро- вать – пародировать, полоскать – поласкать, слезать – слизать, развиваться – развеваться, примирять – примерять, обежать – обижать, частота – чистота, запивать – запевать. К каждому слову в паре подберите проверочное. 4. Заполните пропуск: если лошадь, то пристяжная, если во- ротник, то __________. Как называются слова, которые звучат одинаково, но пишутся по-разному? Приведите примеры таких пар слов. Составьте и запишите предложения с парами слов: парадиро- вать – пародировать, полоскать – поласкать, слезать – слизать, развиваться – развеваться, примирять – примерять, обежать – обижать, частота – чистота, запивать – запевать. К каждому слову в паре подберите проверочное. NB! Не забудьте дать толкования этим словам (ссылку на толковый словарь и QR-код см. выше) NB! Не забудьте дать толкования этим словам (ссылку на толковый словарь и QR-код см. выше) 3. Найдите сложные предложения с разными видами связи. Под- черкните их основы, определите, чем выражены их главные члены. Постройте схемы предложений. Объясните постановку знаков пре- пинания. При необходимости воспользуйтесь ссылкой: https://foxford.ru/wiki/russkiy-yazyk/slozhnye-predlozheniya-s- razlichnymi-tipami-svyazi или QR-кодом 3. Найдите сложные предложения с разными видами связи. Под- черкните их основы, определите, чем выражены их главные члены. Постройте схемы предложений. Объясните постановку знаков пре- пинания. При необходимости воспользуйтесь ссылкой: https://foxford.ru/wiki/russkiy-yazyk/slozhnye-predlozheniya-s- razlichnymi-tipami-svyazi или QR-кодом 4. Объясните смысл пословицы Мелко плавать – дно задевать. Согласны ли вы с этим утверждением? Приведите примеры из ис- тории, литературы, кино, подтверждающие вашу точку зрения 4. Объясните смысл пословицы Мелко плавать – дно задевать. Согласны ли вы с этим утверждением? Приведите примеры из ис- тории, литературы, кино, подтверждающие вашу точку зрения 1. Соедините начало пословицы с её концом. Выпишите слова с проверяемым гласным в корне, подберите и запишите к ним по три однокоренных слова, выделите в них корни 51 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Дай волю на ноготок хотят щуку в озере утопить Не козырист там ему и голову сломить Покорное слово коли волк – пастух Высоко замахнулся шестым поперхнулся Повадился кувшин по воду ходить от своего языка погибает Стали щуке грозить: так и тачать станешь Глядит, будто пятерых живьем проглотил а в корень не годится Плохо овцам да умилосердится И сердится а от службы не отрекайся Как скроишь гнев укрощает Всякая сорока да низко стегнул На пристяжи бежит а он возьмет на весь локоток На службе не набивайся, да мастист Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 2. Выпишите все местоимения и выполните их морфологиче- ский разбор 3 К й б 3. Как известно, в троечной запряжке средняя, наиболее силь- ная лошадь, запряженная в оглобли, называется коренной, а ло- шади, расположенные рядом с ней, называются пристяжными. В какой пословице упоминается русская тройка? Каков иносказа- тельный смысл этой пословицы? 3. Как известно, в троечной запряжке средняя, наиболее силь- ная лошадь, запряженная в оглобли, называется коренной, а ло- шади, расположенные рядом с ней, называются пристяжными. В какой пословице упоминается русская тройка? Каков иносказа- тельный смысл этой пословицы? 4. Заполните пропуск: если лошадь, то пристяжная, если во- ротник, то __________. Как называются слова, которые звучат одинаково, но пишутся по-разному? Приведите примеры таких пар слов. Правописание чередующихся гласных в корне Упражнение 1 1. Вспомните, какие корни называются чередующимися? От ка- ких условий зависит выбор безударного гласного в чередующихся корнях? Выпишите слова с безударными гласными в чередую- щихся корнях, объясните их написание. При выполнении этого за- дания вы можете воспользоваться ссылкой: http://gramota.ru/class/ coach/tbgramota/45_93 или QR-кодом: 52 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 52 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 1. Скупые что пчелы: мед собирают, да сами умирают 2. Выше себя не вырастешь 3. Владеет городом, а помирает голодом 4. На прохожей дороге трава не растет 5. Поклониться – голова не отвалится 6. Не в похвальбу сказать, не в укор помянуть 7. Твоими молитвами, как клюкой, подпираюсь 8. Добру расти, а худу по норам ползти 1. Скупые что пчелы: мед собирают, да сами умирают 2. Выше себя не вырастешь 3. Владеет городом, а помирает голодом 4. На прохожей дороге трава не растет 5. Поклониться – голова не отвалится 6. Не в похвальбу сказать, не в укор помянуть 7. Твоими молитвами, как клюкой, подпираюсь 8. Добру расти, а худу по норам ползти 9. Жену выбирай не глазами, а ушами 10. Кто на борзом коне жениться поскачет, тот скоро по- плачет 11. Люди хвастают – не перелезешь; сват схвастает – на коне не перескочишь 12. Сваха чужие грехи на душу принимает 13. В нашем жите хорош росток 14. Больше той любви не бывает, как друг за друга умирает 15. И от доброй собаки блох наберешься 15. И от доброй собаки блох наберешься 16. Скачет баба задом и передом, а дело идет своим чередом 17. И куслива собака, да хвост поджимает 17. И куслива собака, да хвост поджимает 18. Помянули волка, а он и тут 19. Не откормить коня сухопарого, не отрастить дерева су- ховерхого р 20. На Руси никто с голоду не умирал 2. Выполните синтаксический разбор предложения Скупые что пчелы: мед собирают, да сами умирают. Объясните постановку знаков препинания. В каких случаях перед союзом ЧТО запятая не ставится? Если затрудняетесь ответить на вопрос, воспользуйтесь ссылкой: https://russkiiyazyk.ru/punktuatsiya/vsegda-li-stavitsya- zapyataya-pered-chto.html или QR-кодом: 53 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 3. Какой частью речи и каким членом предложения является вы- деленное слово в предложении На прохожей дороге трава не рас- тет? Правописание чередующихся гласных в корне А в предложении Дон Жуан сердито посмотрел вслед про- хожей, потом повернулся, куда было сказано? Как называются слова, которые совпадают по звучанию и написанию, но относятся к разным частям речи? Приведите примеры подобных слов. Каким способом образованы выделенные слова? 3. Какой частью речи и каким членом предложения является вы- деленное слово в предложении На прохожей дороге трава не рас- тет? А в предложении Дон Жуан сердито посмотрел вслед про- хожей, потом повернулся, куда было сказано? Как называются слова, которые совпадают по звучанию и написанию, но относятся к разным частям речи? Приведите примеры подобных слов. Каким способом образованы выделенные слова? 4. Какой конь имеется в виду в пословице Кто на борзом коне жениться поскачет, тот скоро поплачет? Можно ли сказать, что слово борзосмысл, употребленное в названии древнерусского па- мятника «Повесть о Дмитрии Басарге и сыне его Борзосмысле» (XV век), является однокоренным к слову борзый? Подберите не менее 2 этимологические родственных слов к прилагательному борзый, употребите их в соответствующем контексте, объясните их значение. Упражнение 2 Упражнение 2 Упражнение 2 1. Спишите, вставляя пропущенные буквы. Графически объяс- ните выбор гласного 1. Молитву тв…ри, а муку в квашню клади 2. Голодному не стать время разб…рать 2. Голодному не стать время разб…рать 3. Покуда жив человек, голодною смертью пом…рать не станет 4. В Москве калачи, как огонь, г…рячи 5. Русак как свеча предо мною заг…релся 6. Из двух зол выб…рай меньшее 7. Ярко желают, да руки подж…мают 8. Задор б…рет, да мочи нет 9. Со всего свету не соб…решь цвету 10. Выше лба уши не р…стут 54 54 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 11. Где ск…кать хочется, там и тормозят 12. Умел раств…рить, так умей и замесить 13. Наск…чила подкова на булыжник 14. Где что ведется, там то и б…рется 15. Куда голова кл…нилась, туда и повалилась 16. На битой дороге трава не р…стет, а и р…стет, да спорыш 17. Разл…жи воробья на двенадцать блюд 18. Не поймаешь ск…куху за уши Скакуха – лягушка 2. Верно ли утверждение, что слова горячий, загорелся, гора, горло, гарь, угорелый. горница являются однокоренными? Дока- жите свою точку зрения 3. Верно ли заполнена таблица? Докажите свою точку зрения Слово Количество звуков Загорелся 9 Ярко 5 Б…рет 5 Соб…решь 8 Б…рется 8 Двенадцать 9 Поймаешь 8 4. Какой частью речи является выделенное слово в предложе- нии Голодному не стать время разб…рать? Формой каких еще ча- стей речи может быть это слово? Приведите примеры Часть 2. Трудные случаи русской орфографии и пунктуации 11. Где ск…кать хочется, там и тормозят 12. Умел раств…рить, так умей и замесить 13. Наск…чила подкова на булыжник 14. Где что ведется, там то и б…рется 15. Куда голова кл…нилась, туда и повалилась 16. На битой дороге трава не р…стет, а и р…стет, да спорыш 17. Разл…жи воробья на двенадцать блюд 18. Не поймаешь ск…куху за уши Скакуха – лягушка 2. Верно ли утверждение, что слова горячий, загорелся, гора, горло, гарь, угорелый. горница являются однокоренными? Дока- жите свою точку зрения 3. Верно ли заполнена таблица? Докажите свою точку зрения Слово Количество звуков Загорелся 9 Ярко 5 Б…рет 5 Соб…решь 8 Б…рется 8 Двенадцать 9 Поймаешь 8 11. Где ск…кать хочется, там и тормозят 12. Умел раств…рить, так умей и замесить 4. Какой частью речи является выделенное слово в предложе- нии Голодному не стать время разб…рать? Формой каких еще ча- стей речи может быть это слово? Приведите примеры 5. Подчеркните основы всех односоставных предложений этого упражнения. Определите их тип. При выполнении этого задания можете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy- yazyk/vidy-odnosostavnyh-predlozheniy или QR-кодом 55 Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Упражнение 3 Упражнение 3 1. Выпишите слова с безударными гласными в чередующихся корнях, распределив их на группы в зависимости от условия напи- сания. Сколько групп у вас получилось? 1. Выпишите слова с безударными гласными в чередующихся корнях, распределив их на группы в зависимости от условия напи- сания. Сколько групп у вас получилось? 1. Выше лба уши не растут 2. На затылок очков не приберешь 3. В кулаке все пальцы равны 3. В кулаке все пальцы равны 4. Не отпадет голова, вырастет и борода 5. Кабы на Тарасовой голове да капуста росла, так был бы ого- род, а не плешь 6. Будто и съел, что в рот положил 6. Будто и съел, что в рот положил 7. Каяться кайся, да опять за то же не принимайся 8. Все черти равны, все те ж бобры 9. Хоть козла деру, да сердце тешу 10. В лесу бог лесу не уравнял, в народстве – людей 11. Не на том поле трава выросла 12. Угорела барыне в нетопленой горнице 13. Кто ковши разбирает, у того знать еще в горле не пере- сохло 14. Кость на кость наскочила 15. Кого слова не берут, с того шкуру дерут 16. Сделав добро, не кайся, и не поминай, и не попрекай 17. Чем лаптю кланяться, так уж поклонюсь сапогу 18. По чьей реке плыть, того и волю творить 19. За дело побьют – повинись да ниже поклонись 20. Не поминай бани: есть веники и про тебя 21. Малый вырастет – все выместит 22. Переминаясь с ноги на ногу, не раскалякаешься 23. Несть милости не сотворившему милости 24. На ряду жить, рядовую творить 25. Муж на службе, а жена в нужде – оба равны 2. Как вы понимаете значение выражения По чьей реке плыть, того и волю творить? Приведите синонимичные русские пого- ворки и поговорки вашего народа. 3. Сделайте исторический комментарий орфограммы «Право- писание корней с чередующимися гласными» (задание выполня- ется по группам) 56 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 56 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию *** Правописание безударных окончаний имен существительных Упражнение 1 р 1. Вспомните орфограмму «Правописание безударных окон- чаний имен существительных». При необходимости пройдите по ссылке: http://new.gramota.ru/biblio/readingroom/textbooks/gramota/ ortho/324-45-101 или отсканируйте QR-код: Прочитайте пословицы. Объясните написание окончаний имен существительных. у Часть 1 Часть 1 Часть 1 1. Рабочий конь на соломе, а пустопляс – на овсе. 2. И дурак ездит в карете, и с умом ходит пешком. 3. Живет в Туле, да ест дули. 4. Счастье – не лошадь: не везет по прямой дорожке. 5. Коли не по коню, так по оглобле. 6. Одна голова на плечах, и та на ниточке. 7. Есть в мошне, так будет и в квашне. 8. Одна рука в меду, другая в патоке. 9. Два веника в коробке да мышь в подполье. 10. На безлюдье и Фома дворянин. 11. Собирай по ягодке – наберешь кузовок. 12. Он в стороне, а ты в бороне. 13. Какова пелена, такова и цена. По барыне и говядина. 14. Дураки о добыче спорят, а умные её делят. 15. Волк по утробе вор, а человек по зависти. 57 57 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 16. Не из корысти собака кусает, из лихости. 16. Не из корысти собака кусает, из лихости. 17. Тише кричи, барин на печи. 18. По старой памяти, как по грамоте. 19. От ветоши молодой траве ходу нет. Часть 2 1. Семь в тебе душ, да не в одной пути нет. 2. Ни по старости не мрут, ни по молодости не живут. 3. Не купи у попа лошади, не бери у вдовы дочери. 4. Одна жена плачет от жалости, другая от лести. 5. Лучше в утлой ладье по морю ездить, чем жене тайну по- верить. 6. Наказуй детей в юности, успокоят тя в старости. 7. У лихой свекрови и сзади глаза. 8. Не рад больной и золотой кровати 9. Во многословии не без пустословия. 10. Память в темени, мысль во лбу, а хотение в сердце. 11. Не до обедни, коли много бредней. 12. Толки воду до мелкой пыли. 13. После хлеба, после соли отдохнуть часок, так завернется сала кусок да лени мешок 14. Коротать молодость – не видать старости. 15. Кто красен дочерьми да с сыновьями в почете, тот в бла- годати. 16. Без болезни и здоровью не рад. 17. Держи язык на привязи. 18. За безручье по головке не погладят. 19. Баба с возу – кобыле легче. 20. У нашего соседа и в затылке глаза. 2. Разгадайте кроссворд (подсказка: 1) слова-ответы есть в этом упражнении😊) 2. Разгадайте кроссворд (подсказка: 1) слова-ответы есть в этом упражнении😊) Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 58 Часть 2. Трудные случаи русской орфографии и пунктуации русской орфографии и пунктуации русской орфографии и пунктуации 1 5 3 4 2 6 13 7 11 12 8 9 10 По горизонтали: 1. Несерьёзный, легкомысленный человек; праздный гуляка. 2. Судно и шахматная фигура. 4. Все старое, от- жившее (книжн.) 7. Непрочный, некрепкий, ненадёжный. 8. Одна из двух жердей, укреплённых концами на передней оси повозки и служащих для запряжки лошади. 10. Устаревший синоним покры- вала. 12. Мешок для денег; кошелёк По вертикали: 3. Верхняя часть головы, верх черепа между лобными, затылочными и височными костями. 5. Густое, тягучее сладкое вещество – продукт неполного осахаривания крахмала. 6. … с маком что значит «ничего, совершенно ничего». 7. Внутрен- няя часть живота человека или животного. 9. Вздор, чушь. 11. Сель- скохозяйственное орудие для мелкого рыхления вспаханной земли. 13. Забродившее тесто, опара 1 5 3 4 2 6 13 7 11 12 8 9 10 1 5 3 4 2 6 13 7 11 12 8 9 10 По горизонтали: 1. Несерьёзный, легкомысленный человек; праздный гуляка. 2. Судно и шахматная фигура. 4. Все старое, от- жившее (книжн.) 7. Непрочный, некрепкий, ненадёжный. 8. Одна из двух жердей, укреплённых концами на передней оси повозки и служащих для запряжки лошади. 10. Устаревший синоним покры- вала. 12. Мешок для денег; кошелёк По вертикали: 3. Верхняя часть головы, верх черепа между лобными, затылочными и височными костями. 5. Густое, тягучее сладкое вещество – продукт неполного осахаривания крахмала. 6. … с маком что значит «ничего, совершенно ничего». 7. Внутрен- няя часть живота человека или животного. 9. Вздор, чушь. 11. Сель- скохозяйственное орудие для мелкого рыхления вспаханной земли. 13. Забродившее тесто, опара 59 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 3. Как вы понимаете слово безручье? Разберите это слово по со- ставу. Каким способом образовано это слово? Приведите примеры слов, образованных аналогичным способом. 3. Как вы понимаете слово безручье? Разберите это слово по со- ставу. Каким способом образовано это слово? Приведите примеры слов, образованных аналогичным способом. 4. Какой частью речи является выделенное слово в предложении Наказуй детей в юности, успокоят тя в старости. Определите его грамматические признаки. Что вы можете рассказать об энклитиче- ских формах местоимений? (чтобы узнать о них побольше, можете пройти по ссылке: https://pravoslavie.ru/docs/marsheva_old_slavonic_ pronoun-7cb02a.pdf или отсканировать QR-код ) Упражнение 2 Упражнение 2 Упражнение 2 1. Составьте алгоритм применения правила «Правописание без- ударных окончаний имен существительных». Спишите, вставляя пропущенные буквы. Объясните выбор гласного в окончаниях имен существительных, пользуясь вашим алгоримтом. Не забудьте уточнить по толковому словарю значения незнако- мых слов: https://slovarozhegova.ru/ или отсканируйте QR-код: 1. Улька Акульк… не хуже. 2. Яблочко от яблон… недалеко падает. 3. Ярост… берегись, она безумна. 4. Живет в рядк…, ходит в однорядк…. 5. В добром жить… сами кудри вьются, в худом секутся. 6. Голодной кум… хлеб на ум…. 60 0 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 3. Установите, какой морфемой (частью слова) или частью мор- фемы является выделенная буква О в следующих словах: яблочкО, недалекО, золотО, былО, никтО, семерО, ктО, тогО? Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 3. Установите, какой морфемой (частью слова) или частью мор- фемы является выделенная буква О в следующих словах: яблочкО, недалекО, золотО, былО, никтО, семерО, ктО, тогО? 3. Установите, какой морфемой (частью слова) или частью мор- фемы является выделенная буква О в следующих словах: яблочкО, недалекО, золотО, былО, никтО, семерО, ктО, тогО? , , , , р , , 4. Подберите синонимы к слову кручина. Какими оттенками значений они различаются? Составьте с каждым из них предложе- ние и запишите. Вам может помочь словарь синонимов, который вы найдете по ссылке: https://synonymonline.ru/ или QR-коду: 4. Подберите синонимы к слову кручина. Какими оттенками значений они различаются? Составьте с каждым из них предложе- ние и запишите. Вам может помочь словарь синонимов, который вы найдете по ссылке: https://synonymonline.ru/ или QR-коду: Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 7. До времен… все с рук сходит. 8. В тихом омут… черти водятся. 9. Золото и в гряз… блестит. 10. Воришк… зевает, а вор ничему не спускает. 11. Держи рыльц… огнивц…м, а глаза – буравц…м. 12. В город… не пляшут, да калачи едят. 13 Н б б б 12. В город… не пляшут, да калачи едят. 13. На безрыбь… и рак рыба 14. На чужой сторонушк… рад своей воронушк…. 15. Девушка – не травушка, не вырастет без славушк..: девушка на ноженьки, а слава по дороженьк…. 15. Девушка – не травушка, не вырастет без славушк..: девушка на ноженьки, а слава по дороженьк…. 16. Набирайся ума в учень…, храбрости – в сражень…. 17. Это было при дедушк… Мирошк…, когда денег было трошки. 17. Это было при дедушк… Мирошк…, когда денег было трошки. 18. Будь в голубятн… корм, голуби слетятся. 19. Не в воле счасть…, а в дол…. 20. В подполь…, в подполь… лежит пирог с морковью: есть хо- чется, да лезть не хочется. 20. В подполь…, в подполь… лежит пирог с морковью: есть хо- чется, да лезть не хочется. 21. Этой нитк… конца не будет. 22. Середка на половин… – никто не в обид…. 23. Семеро лежат в куч.., а один всех растаскает. 24. Всё на свет… к лучшему. 24. Всё на свет… к лучшему. 25. Нет в карман…, да есть в амбар…. 26. Кто в радост… живет, того кручина неймет. 2. Что такое литературный язык? Какие варианты нелитератур- ного языка вы знаете? Что такое диалект? Что такое просторечие? Из данного упражнения приведите примеры слов, не относящихся к литературному языку. При выполнении задания можете восполь- зоваться словарем лингвистических терминов, например, по ссылке: http://rus-yaz.niv.ru/doc/linguistic-terms/index.htm#196 или по QR-коду: 61 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 5. Какой частью речи является выделенное слово в предложе- нии Семеро лежат в куч.., а один всех растаскает? Какие омо- формы имеет это слово? Приведите примеры. (Уточнить что такое омоформы, вы можете по ссылке: http://rus-yaz.niv.ru/doc/linguistic- terms/fc/slovar-206-2.htm#zag-969 или по QR-коду: Упражнение 3 1. Прочитайте пословицы. Выпишите все имена существитель- ные с орфограммой «Безударные гласные в окончаниях имен суще- ствительных», определите их склонение и падеж. Вставьте пропу- щенные буквы и выделите окончания 1. Что брови-то нависли: аль я тебе не по мысл…? 2. В печал… бесконечно не будешь. 2. В печал… бесконечно не будешь. 3. Зачем к бабк… Варвар…, как свое в карман…? Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 62 2 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 4. В кабак… без денег дневать – скучно, в торговой бан… ноче- вать – душно. у 5. Купил бы село, да в карман… голо; завел бы вотчину, да ку- пило покорчило. 6. У ленивого Емел… семь воскресений на недел…. 7. В благополучи… человек сам себя забывает. 8. На обух… рожь молотит, из мякины кружево плетет. 9. У хорошего хозяина нет плохой лошад…. 10. От худого семен… не жди доброго племен…. 11. Подальше от кузен поменьше копот…. 12. От свиньи визгу много, а шерст… нет. 13. Живем в нег…, а ездим на телег... 14. Свет в храмин… от свечи, а в душ… от молитвы. 15. Не бей по рож…, себе дорож…. 16. Слепой куриц… все пшеница. Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Российской Федерации» национального проекта «Образование» Для выполнения этого задания можете воспользоваться ссыл- кой: https://cross.highcat.org/ru_RU/ или QR-кодом Часть 2 1. Как дитя вдоль лавочки ляжет, так учить поздно. 1. Как дитя вдоль лавочки ляжет, так учить поздно. 2. Шельма на шельм… сидит, шельмой погоняет. 2. Шельма на шельм… сидит, шельмой погоняет. 3. Щебечет соловушк…, забыв своих деток. 4. В своей водиц… и лягушка певица. 5. Вовремя смех не грех, а не ко времен… и молитва ни к чему. 6. Кому до чего, а кузнецу до наковальн…. 7. Держи язык на привяз…. 8. На новосель… всегда живет веселье. 9. Без имен… и овца баран. 10. Грозит мышка кошк…, да издалече. 11. Было б корытц… да было б в корытц…, а свиньи найдутся 12. Сердце тят… к ласковому дитяти лежит. 13. Дома нет, а домищ… будет. Д щ у 14. Не одно пузищ… смышляет о пищ…: и тонкий живот без еды не живет. 14. Не одно пузищ… смышляет о пищ…: и тонкий живот без еды не живет. 15. Родилось чадушк…, старше бабушк… 15. Родилось чадушк…, старше бабушк… 16. Нужда из Сызран… в Москву пешком пришла 2. Составьте кроссворд из следующих слов: вотчина, обух, нега, обедня, шельма, тятя, Сызрань, храмина, мякина, наковальня, при- вязь, пища. 2. Составьте кроссворд из следующих слов: вотчина, обух, нега, обедня, шельма, тятя, Сызрань, храмина, мякина, наковальня, при- вязь, пища. 63 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» NB! Не забудьте дать толкования этим словам (ссылка на толковый словарь и QR-код: https://slovarozhegova.ru/ или: ) 3. Найдите и выпишите слова с суффиксами субъективной оценки. Суффиксы выделите. Какой оттенок значения они придают словам? Как определять род существительных, имеющих суф- фиксы субъективной оценки? (за справкой можете пройти по ссылке: https://www.sites.google.com/site/4actipe4i/dla-studentov/ ima-susestvitelnoe/kategoria-roda-imeen-susestvitelnyh или отскани- ровать QR-код: ) 3. Найдите и выпишите слова с суффиксами субъективной оценки. Суффиксы выделите. Какой оттенок значения они придают словам? Как определять род существительных, имеющих суф- фиксы субъективной оценки? (за справкой можете пройти по ссылке: https://www.sites.google.com/site/4actipe4i/dla-studentov/ ima-susestvitelnoe/kategoria-roda-imeen-susestvitelnyh или отскани- ровать QR-код: ). Составьте с ними словосочетания сущ. лаг. ). Составьте с ними словосочетания сущ. (главное слово) + прилаг. (главное слово) + прилаг. 64 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 64 64 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 4. В чем особенность склонения слов семя, племя, время? Какие еще существительные имеют аналогичные особенности склоне- ния? Каковы причины возникновения этих особенностей? (мо- жете прочитать об этом по ссылке: http://elibrary.sgu.ru/uch_ lit/1725.pdf или отсканировав QR-код: 4. В чем особенность склонения слов семя, племя, время? Какие еще существительные имеют аналогичные особенности склоне- ния? * Каковы причины возникновения этих особенностей? (мо- жете прочитать об этом по ссылке: http://elibrary.sgu.ru/uch_ lit/1725.pdf или отсканировав QR-код: Упражнение 4 1. Вместо многоточия вставьте подходящее по смыслу имя су- ществительное (см. слова для справок). Выделите все падежные окончания этих существительных и укажите, в какой форме упо- треблено слово. 1. Вместо многоточия вставьте подходящее по смыслу имя су- ществительное (см. слова для справок). Выделите все падежные окончания этих существительных и укажите, в какой форме упо- треблено слово. р 1. Что … лопаточкой сгребал, то сынок тросточкой рас- швырял. 1. Что … лопаточкой сгребал, то сынок тросточкой рас- швырял. 2. Худенький …, да свой. 3. Скучно … на чужой … . 4. У злой … все люди канальи. 5. В одном кармане вошь на …, в другом блоха на цепи. 6. Живет на …, а хлеба ни … . 7. Слава богу, не без …: хлеба нету, так дети есть. 8. Нашему … все огарки. 8. Нашему … все огарки. 9. Дали голодной … оладьи, а она говорит: «Испечены не адно». 10. Не по … желуди, не по чину шелуди. Слова для справок: Сторонушке, Макарке, корки, чушке, Афонюшке, батюшка, Маланье, доли, горке, Натальи, умишко, аркане. р у , р , р , у , ф , , Маланье, доли, горке, Натальи, умишко, аркане. 2. Для иллюстрации каких орфографических правил можно ис- пользовать пословицы этого упражнения? Придумайте задания на основе этих пословиц. 3. Во всех предложениях подчеркните сказуемые и определите Маланье, доли, горке, Натальи, умишко, аркане. 2. Для иллюстрации каких орфографических правил можно ис- пользовать пословицы этого упражнения? Придумайте задания на основе этих пословиц. 3. Во всех предложениях подчеркните сказуемые и определите их вид. 65 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Можете воспользоваться подсказкой, пройдя по ссылкам или отсканировав QR-коды: 1) простое глагольное сказуемое: https://foxford.ru/wiki/russkiy- yazyk/osnovnye-tipy-skazuemyh-prostoe-glagolnoe-skazuemoe 2) составное глагольное сказуемое: https://foxford.ru/wiki/ russkiy-yazyk/sostavnoe-glagolnoe-skazuemoe 3) составное именное сказуемое: https://foxford.ru/wiki/russkiy- yazyk/sostavnoe-imennoe-skazuemoe 5. Какие из приведенных поговорок характеризуют человека? Какого человека? Вспомните и запишите не менее 10 поговорок и фразеологизмов, дающих оценку человеку? Оформите свой ответ в виде таблицы: Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 66 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации ру у русской орфографии и пунктуации Упражнение 1 Упражнение 1 1. Вспомните, что такое личное окончание глагола? Что такое спряжение? Дополните алгоритм правила «Правописание безудар- ных личных окончаний глаголов»: 1. Проспрягай глагол (без приставок и постфиксов) 1. Проспрягай глагол (без приставок и постфиксов) Окончание ударное Окончание безударное Пиши то, что слышишь 2. Смотри на инфинитив на -ИТЬ не на -ИТЬ NB! Не забудьте про исключения: брить, стелить, зиждиться, зыбиться – это глаголы 1 спряжения терпеть, вертеть, обидеть, зависеть, ненавидеть, видеть, смотреть; дышать, держать, слышать, гнать – это глаголы 2 спряжения При выполнении этого задания вы можете воспользоваться ссылкой: http://new.gramota.ru/biblio/textbooks/gramota/ortho/321- 45-91 или QR-кодом: 1. Проспрягай глагол (без приставок и постфиксов) Окончание ударное Окончание безударное Пиши то, что слышишь 2. Смотри на инфинитив на -ИТЬ не на -ИТЬ NB! Не забудьте про исключения: Окончание безударное Окончание ударное 2. Смотри на инфинитив Пиши то, что слышишь NB! Не забудьте про исключения: брить, стелить, зиждиться, зыбиться – это глаголы 1 спряжения терпеть, вертеть, обидеть, зависеть, ненавидеть, видеть, смотреть; дышать, держать, слышать, гнать – это глаголы 2 спряжения При выполнении этого задания вы можете воспользоваться ссылкой: http://new.gramota.ru/biblio/textbooks/gramota/ortho/321- 45-91 или QR-кодом: 67 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 2. Пользуясь алгоритмом, объясните правописание безударных личных окончаний глаголов. 2. Пользуясь алгоритмом, объясните правописание безударных личных окончаний глаголов. 1. Мать плачет, что река льется; жена плачет, что ручей те- чет; невеста плачет, как роса падает: взойдет солнце – росу вы- сушит 1. Мать плачет, что река льется; жена плачет, что ручей те- чет; невеста плачет, как роса падает: взойдет солнце – росу вы- сушит 2. Отдашь деньги руками, а ходишь за ними ногам 3. Как аукнется, так и откликнется 3. Как аукнется, так и откликнется 4. Что имеем, не храним, потерявши – плачем 5. На что ни взглянет – все вянет 5. На что ни взглянет – все вянет 6. Как душа черна, так и мылом не смоешь 7. Хорош тот, кто поит да кормит, а вдвое того, кто хлеб- соль помнит 8. И собака на того не лает, чей хлеб ест 9. Прытью людей не удивишь, а себя утомишь 10. К нашему берегу не привалит хорошее дерево 11. Хорошего человека употчуешь кусом, а худого не употчу- ешь и гусем 68 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 11. Хорошего человека употчуешь кусом, а худого не употчу ешь и гусем 12. Ино горько проглотишь, да сладко выплюнешь 13. Оттерпимся, и мы люди будем 14. По третьему разу всегда вырубишь огня 15. Худое валит пудами, хорошее каплет золотниками 16. Все перемелется, мука будет 17. Прошлого поминаем, грядущего чаем 18. Не живи, как хочется, а живи, как можется 19. Куда натянешь, там и крыто 20. Чужие куры ростятся, а наши только топорщатся 21. Счастливый скачет, бессчастный плачет 22. Бог найдет и в люди выведет 23. Что было, то видели, что будет, то увидим 24. Худой солдат, который не надеется быть генералом 25. И правда тонет, коли золото всплывает 26. За ватагу нищих одного богача не выменяешь 27. Богатство полюбится, и ум расступится 28. Деньгами души не выкупишь 29. Денег нет, так подушка под головой не вертится 30. Не с работы тощает, а с жиру бесится 12. Ино горько проглотишь, да сладко выплюнешь 13. Оттерпимся, и мы люди будем 14. По третьему разу всегда вырубишь огня 68 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 3. Как вы думаете, есть ли орфографическая ошибка в посло- вице Чужие куры ростятся, а наши только топорщатся? Прочитайте словарную статью, посвященную глаголу ро- ститься: 3. Как вы думаете, есть ли орфографическая ошибка в посло- вице Чужие куры ростятся, а наши только топорщатся? Прочитайте словарную статью, посвященную глаголу ро- ститься: 3. Как вы думаете, есть ли орфографическая ошибка в посло- вице Чужие куры ростятся, а наши только топорщатся? Прочитайте словарную статью, посвященную глаголу ро- ститься: Роститься – 1. Кудахтать перед тем, как снести яйцо (о ку- рице) … Куры ростятся — скоро нестись станут Влад, 1853 Новг, Арх, Онеж КАССР, Твер, Калин, Моек, Иван, Вят , Киров Ростится [курица], когда яичком хвастает, положилась когда Волог, Яросл, Костром. Сесть на яйца — клохчет, снесла яйцо — кудахчет, кри- чит, распевается, хочет нестись — ростится Куйбыш Перм, Свердл, Урал, Зауралье, Кемер Курица снесет яйцо, а потом опять ростится Когда курица кластись начинает, так она уже ростится Новосиб Ир- кут, Забайкал, Том, Тобо , Краснояр… • О гусыне Гусиха ростится, наговаривает, нестись хочет Новосиб, 1979 \|\| Протяжно и громко кудахтать после того, как снесено яйцо (о курице) Серая курица растится, видно, яйцо снесла Буйск Костром, 1905-1921 Новосиб \|\| 0 ласточках: щебетать Я люблю, как ласточки-те ростятся Кадн Волог, 1854. 2 ' Роститься Высиживать яйца (о птицах) Курица, утка сидят на яйцах — ростятся Свердл, 1965 \|\| 0 курице, которая водит цыплят Яросл, 1988 \|\| Собираться сесть на яйца (о курице) Том, 1959 Из какого словаря данная словарная статья? Каков принцип рас- положения информации? Для чего, по вашему мнению, предназна- чены такие словари? В чем их ценность? р Упражнение 2 Упражнение 2 Упражнение 2 1. Спишите, вставляя пропущенные буквы. Объясните свой выбор 1. Мало ль чего хоч…тся, да в кармане кол…тся 2. Чего хоч…шь, того и прос..шь 3. Кто поднос…т, тот сам не прос…т 4. Дождь вымоч…т, солнышко высуш…т, буйны ветры голову асчеш…т 4. Дождь вымоч…т, солнышко высуш…т, буйны ветры голову асчеш…т 5. Есть не сыщ…м, так посвищ…м 6. Конь о четырех ногах, да спотыка…тся 7. На бедного Макара и шишки вал…тся 8. Бог и слыш…т, да не скоро скаж…т 8. Бог и слыш…т, да не скоро скаж…т 9. Не слушай, где куры кудахч…т, а слушай, где Богу мол…тся 10. Придет счастье и с печи сгон…т 69 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 11. Жена не гусли: поиграв, на стенку не повес…шь 1. Жена не гусли: поиграв, на стенку не повес…шь 12. Жена мел…т, а муж спит. Жена пряд…т, а муж пляш…т 13. Дал Бог отца, что и родного сына не слушается 14. Не учил отец, а дядя не выуч…т 14. Не учил отец, а дядя не выуч…т 15. Сынок – свой горбок, зятек – покупной щеголек; теща хло- поч…т, тесть кропоч…т 15. Сынок – свой горбок, зятек – покупной щеголек; теща хло- поч…т, тесть кропоч…т 16. Его ласка не коляска: не сяд…шь да не поед…шь 17. От лихого не услыш…шь доброго слова 18. Хорошо того бить, кто плач…т, а учить, кто слуша…тся 19. Много захоч…шь – пораньше вскоч…шь 20. Скаж…шь – не ворот…шь, напиш…шь – не сотрёшь, от- руб…шь – не пристав…шь 2. В словах мышь – мажь все согласные одинаковые, что легко доказать, затранскрибировав эти слова: мышь – [мыш], мажь – [маш]. Найдите и выпишите из упражнения слова, в которых все согласные звуки одинаковые. Докажите свою точку зрения. Приве- дите примеры слов из вашего родного языка с одинаковыми соглас- ными звуками. 3. Выпишите сначала все сочинительные союзы, а потом подчи- нительные. Укажите значения союзов. Определите, для чего они служат в каждом предложении. Если вы подзабыли, какие значения могут иметь союзы, вам поможет ссылка: https://foxford.ru/wiki/ russkiy-yazyk/sochinitelnye-i-podchinitelnye-soyuzy или QR-код: 4. Определите лексическое и грамматическое значение слова куры в пословице Не слушай, где куры кудахч…т, а слушай, где Богу мол…тся. 4. Определите лексическое и грамматическое значение слова куры в пословице Не слушай, где куры кудахч…т, а слушай, где Богу мол…тся. Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 70 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 70 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» чередованием гласных _________________________ (2 примера) и с чередованием согласных ______________________ (2 примера). 7. Форма куря относилась в древности к иному склонению, нежели слово кур. При изменении слово куря в косвенных падежах появлялся суффикс -ат- (И.п. куря, Р.п. куряте, Д.п. куряти...). Ука- жите, какое значение имеет суффикс -ат- у современных суще- ствительных, и переведите на русский язык фрагмент чадо мое ми- лое, куря..., учитывая смысл контекст чередованием гласных ____ с чередованием согласных _ р ______________________ ( р р ) 7. Форма куря относилась в древности к иному склонению, нежели слово кур. При изменении слово куря в косвенных падежах появлялся суффикс -ат- (И.п. куря, Р.п. куряте, Д.п. куряти...). Ука- жите, какое значение имеет суффикс -ат- у современных суще- ствительных, и переведите на русский язык фрагмент чадо мое ми- лое, куря..., учитывая смысл контекст ур у Упражнение 3 72 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Попробуйте выполнить задание, посвященное этому слову (Это задание было предложено школьникам на региональном этапе ВсОШ по русскому языку в 2015-2016 учебном году) Прочитайте отрывок из «Сказания о куре и лисице» XVII век «И под то древо, к тому седящему на древе к велегласному х куру пришла к нему ласковая лисица и стала ему говарить лест- ными своими словами, глядя на то высокое древо. Рече же х куру лисица: «Чадо моё милое, куря, душеполезная моя словеса слышав, давно бы ты сошёл ко мне на покаяние». Кур же, на древе сидя, прослезися горько, слышав же душеполезная словеса от преподоб- ныя жены лисицы, поминаючи грехи своя окаянныя, и почел спу- щатца к лисице на землю... И взяла его лисица в кохти и згнела его крепко и завопил кур великим гласом: «О мати моя лисица, то ли мне от тебя праведное покаяние?» («Сказание о куре и лисице» цитируется по изданию «Русская демократическая сатира XVII века» под ред. В. П. Адриановой-Пе- ретц. М., 1977; издание не сохраняет оригинальную графику. Вопросы и задания: («Сказание о куре и лисице» цитируется по изданию «Русская демократическая сатира XVII века» под ред. В. П. Адриановой-Пе- ретц. М., 1977; издание не сохраняет оригинальную графику. Вопросы и задания: 1. Кто такой кур, упоминающийся в тексте? Аргументируйте Ваш ответ с точки зрения словообразования. 1. Кто такой кур, упоминающийся в тексте? Аргументируйте Ваш ответ с точки зрения словообразования. 2. В каких устойчивых выражениях это слово используется в со- временном русском языке (приведите один пример)? 3. Опишите фонетическое явление в сочетании х куру. Укажите в тексте ещё одно слово с подобным процессом. Приведите два ана- логичных примера на данное явление в современном русском языке. 4. Переведите сочетание згнела его крепко на современный рус- ский язык. Опишите фонетическое явление в слове сгнела, подбе- рите к данному глаголу 2 родственных слова в русском языке. 5. Укажите, какое значение слово лестный имеет в современ- ном языке, и определите его значение в данном контексте, имея в виду, что оно такое же, как и в выражении брань славна лутчи есть мира лестнаго. Переведите выражение на русский язык. 6. В некоторых древнерусских текстах в выражении брань славна лутчи есть мира лестнаго вместо слова лестнаго встре- чается другой вариант: студнаго. Переведите это слово на русский язык. Приведите слова данного корня в русском языке с 71 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» ур у Упражнение 3 1. Выпишите все глаголы, поставьте их в начальную форму, определите их спряжение. Выделите окончания глаголов. Обра- зуйте от этих глаголов, если это возможно, действительные прича- стия настоящего времени, запишите их, выделите суффиксы. 1. Выпишите все глаголы, поставьте их в начальную форму, определите их спряжение. Выделите окончания глаголов. Обра- зуйте от этих глаголов, если это возможно, действительные прича- стия настоящего времени, запишите их, выделите суффиксы. 1. Собака умней бабы: на хозяина не лает 2. Убогого докука, богатого скука одолевает 3. Худой сватается, хорошему путь кажет 4. Крестом любви не свяжешь 5. Не видишь – душа мрет, увидишь – с души прет 6. Девка не курица, парень не кочет; не жениться им, где кто захочет 7. Девка пляшет, сама себя красит 8. Оденем нагих, обуем босых; накормим алчных, напоим жаж дых; проводим мертвых – заслужим небесное царство 9. Пусти черта в дом, не вышибешь его и лбом (т.е. молитвой 10. Всяк хвалит добро, да не всех хвалит оно 11. Не смейся, горох, не лучше бобов; размокнешь, надуешься – лопнешь 12. Кто сам собою не управит, тот и других не наставит 13. И сам тонет и других топит 13. И сам тонет и других топит 14. Бочка меду, ложка дегтю – все испорит 14. Бочка меду, ложка дегтю – все испорит 15. Любовь ни зги не видит 2. Установите соответствие между словами, встречающимися в данном упражнении, и толкованиями, которые дал им В.И. Даль в своем словаре (кстати, как он называется? Почему у него именно такое название? Докажите свою точку зрения) 72 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 72 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 72 Часть 2. Трудные случаи русской орфографии и пунктуации Слово Его толкование убогий петух докука темь, потемки, темнота зга Смолистая и пригорелая жидкость, выгоняемая из бересты огнем кочет Просьба, ходатайство, хлопоты просителя лоб бедный, неимущий, нищий дёготь молитва 3. Восстановите слово по звукам Звуки этого слова вы узнаете, взяв ударный гласный в слове банты, второй согласный звук в слове ёлка, первый звук в слове чудо, последний звук в слове поклон, последний гласный звук в слове базарный, последний согласный звук в слове красивая. Напишите, какое слово у вас получилось. Каково значение этого слова? Подберите к нему синонимы. Найдите в упражнении посло- вицу, в которую входит это слово. Как вы понимаете смысл посло- вицы, в которую входит этого слово? ур у Упражнение 3 Звуки этого слова вы узнаете, взяв ударный гласный в слове банты, второй согласный звук в слове ёлка, первый звук в слове чудо, последний звук в слове поклон, последний гласный звук в слове базарный, последний согласный звук в слове красивая. Напишите, какое слово у вас получилось. Каково значение этого слова? Подберите к нему синонимы. Найдите в упражнении посло- вицу, в которую входит это слово. Как вы понимаете смысл посло- вицы, в которую входит этого слово? 4. Во всех предложениях подчеркните подлежащие, укажите, чем они выражены. При выполнении этого задания вам поможет ссылка: https://foxford.ru/wiki/russkiy-yazyk/glavnye-chleny-predlozheniya или QR-код: Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 73 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Упражнение 1 1. Вспомните правила написания суффиксов имен существи- тельных. Для этого пройдите по ссылке: https://licey.net/free/4- russkii_yazyk/39-kurs_russkogo_yazyka_fonetika__ slovoobrazovanie__morfologiya_i_orfografiya/stages/666-325_ pravopisanie_suffiksov_i или отсканируйте QR-код: Заполните таблицу: Правописание суффиксов имен существительных правило пример В качестве примеров, иллюстрирующих правила, используйте Правописание суффиксов имен существительных р уфф у правило пример В качестве примеров, иллюстрирующих правила, используйте слова этого упражнения В качестве примеров, иллюстрирующих правила, используйте слова этого упражнения 1. Бор сожгли, а соловушек по гнезду плачет. 1. Бор сожгли, а соловушек по гнезду плачет. 1. Бор сожгли, а соловушек по гнезду плачет. 2. Брал жену денечек, а плакал годочек. 3. Будет дождик, будут и грибки, а будут грибки, будет и кузовок 4. Был бы покос, да пришёл морозец. 4. Был бы покос, да пришёл морозец. 5. Была у двора Масленица, да в избу не зашла. 6. Были бы козоньки, будут и козлята. 7. Было времечко, едали и мы семечки. 8. В марте курица пьет из лужицы. 9. В умницы не попал, а из дураков не вышел. 10. В чужих людях без кнутика учат, без солнышка сушат. 11. Вкус вкусу не указчик: кто любит арбуз, а кто свиной хрящик. 74 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 12. Войдешь в дом с поклончиком – уйдешь с гостинчиком. 13. Вор на вора не челобитчик. 13. Вор на вора не челобитчик. 14. Горькому горькая песенка поется. 15. Куда падает пшеничка, туда летит и птичка. 16. Море любой камешек точит. 17. Кукуй, кукушечка, пока молода. 18. На простака хитрецы падки. 19. Чужая тетенька по головке не погладит. 20. Не петь было рано пташечке, не съела б кошечка. 21. Не смейся, братец, чужой сестрице: своя в девках сидит. 22. Детинка с сединкой везде пригодится. 23. Работнику полтина, нарядчику рубль. 24. Душа крепкая, раздумьице великое. 25. Стало плохо: указчиком Аноха. 26. На всякого мудреца довольно простоты. 27. В чужом глазу порошинка велик сучец, в своем и бревна не видать. 27. В чужом глазу порошинка велик сучец, в своем и бревна не видать. 28. Хозяюшка в дому – оладушек в меду. 29. Доносчику первый кнут. 30. Жива душа калачика хочет. 30. Жива душа калачика хочет. 31. Молчок, старичок: старушка денежку даст. 32. С гривенки на гривенку ступает, полтиною ворота запи- рает 2. Что такое хрящик, челобитчик, простак, порошинка, сучец, гривенка? Разберите эти слова по составу. Подберите к ним одно- коренные и одноструктурные слова (о том, что такое однострук- турные слова, вы можете прочитать по ссылке: https://foxford.ru/wiki/russkiy-yazyk/morfema-minimalnaya-znachimaya- edinitsa-yazyka-korni-i-affiksy или отсканировав QR-код: 75 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 3. Выполните различные виды разборов: 3. Выполните различные виды разборов: 3. Выполните различные виды разборов: р р р Фонетический разбор: раздумьице, доносчику, с гостинчиком, поется Фонетический разбор: раздумьице, доносчику, с гостинчиком, поется Фонетический разбор: раздумьице, доносчику, с гостинчиком, поется Морфологический разбор: свиной(хрящик), горькая (песенка), крепкая (душа), великое (раздумьице) Морфологический разбор: свиной(хрящик), горькая (песенка), крепкая (душа), великое (раздумьице) Морфологический разбор: свиной(хрящик), горькая (песенка), крепкая (душа), великое (раздумьице) Синтаксический разбор: Вкус вкусу не указчик: кто любит ар- буз, а кто свиной хрящик. Жива душа калачика хочет. Синтаксический разбор: Вкус вкусу не указчик: кто любит ар- буз, а кто свиной хрящик. Жива душа калачика хочет. Синтаксический разбор: Вкус вкусу не указчик: кто любит ар- буз, а кто свиной хрящик. Жива душа калачика хочет. 4. Используя слова данного упражнения, попробуйте составить 5 тестовых заданий в формате заданий ОГЭ / ЕГЭ для проверки усвоения правила «Правописание суффиксов имен существитель- ных».. 5. Как вы понимаете смысл пословицы На всякого мудреца до- вольно простоты? Вспомните и запишите пословицы вашего народа со схожим значением.  У великого русского драматурга А.Н. Островского есть коме- дия с таким же названием. Как, во-вашему, соотносятся название произведения и его содержание? р Упражнение 2 Упражнение 2 1. Спишите, вставляя пропущенные буквы. Объясните свой выбор 1. На родной сторонушке и камеш…к знаком. 1. Спишите, вставляя пропущенные буквы. Объясните свой выбор 1. Спишите, вставляя пропущенные буквы. Объясните свой выбор 1 На родной сторонушке и камеш к знаком 1. На родной сторонушке и камеш…к знаком. 2. Либо дожд…к, либо снег, либо будет, либо нет. 3. Курочка по зерн…шку клюет, да сыта бывает. 4. Ты от дела на пяд…ньку, оно от тебя на саженку. 5. Тугой лук – коромысл…це, калены стрелы – веретень…ца. 6. Есть все, что угодно душ…ньке. 7. Солом…нкой не подопрешь хором…ны. 8. Середа да пятница хозяину в доме не указ…ица. 9. Не ворожея, да отгад…ик. 10. Лазут…ик, что отопок: обносил да и бросил. 11. Холоп на господина не доказ…ик. 12. Черт монаху не попут…ик. 13. Не все коту Маслен…ца, будет и Великий Пост. 14. Суда божьего окол…цей не объедешь. 15. На том свете каждая щедринка обратится в жем- чуж…нку. 15. На том свете каждая щедринка обратится в жем- чуж…нку. 16. Судит Бог обид…ика, а человек прощает. 76 76 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 76 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 17. Перинка неж…нке горька. Часть 2. Трудные случаи русской орфографии и пунктуации 17. Перинка неж…нке горька. 18. Сама собой миленька, лич…ком беленька. 19. Кто копе…к не считает, рубля не сберегает. 20. С хитр…цом водится – в оба глаза глядеть. 21. Земля – кормил…ца, а и та есть просит. 22. От доброго сем…чка – добрый всход. 23. Ни в кладу кладуш…чки, ни в столе краюш…чки, ни в ошне полуш…чки. 24. Наша горн…ца с Богом не спорится. 25. От миру челобит…ик, а сам никому не обид…ик. 26. Птица радуется весне, а младен…ц – матери. 2. Разгадайте кроссворд (подсказка: в некоторых случаях отве- тами будут производные слова существительных этого упражне- ния😊) 1 4 2 6 3 9 7 5 8 10 11 1 4 2 6 3 9 7 5 8 10 11 77 77 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» По горизонтали: 3. Изношенный, стоптанный башмак. 5. Про- явление щедрости. 8. Предмет для ношения двух вёдер на плече — толстая изогнутая деревянная планка с крючками или выемками на концах. 9. В старину мелкая медная монета в четверть копейки. 11. Старинная русская мера длины, равная расстоянию между раз- двинутыми большим и указательным пальцами у у По вертикали: 1. Старинная русская мера длины, равная рас- стоянию между раздвинутыми большим и указательным пальцами. 2. В Древней Руси: человек, находящийся в зависимости, близок к рабству; в крепостнической России: крепостной крестьянин, слуга. 4. Устар. Проситель, истец 6. В старину: изгородь вокруг деревни, при въезде в деревню; ворота или проход в такой изгороди. 7. В старину на Руси: большой жилой дом богатого владельца. 10. Ста- рая русская мера длины, равная трем аршинам (2,13 м). 3. Разберите слова по составу: саженку, Маслен…ца, неж…нке, кормил…ца, сем…чка, попут…ик, хором…ны, перинка, пяд…ньку, полуш…чки, отоп…к. 4. Образуйте и запишите формы множественного числа имени- тельного и родительного падежа следующих слов: веретено, воро- жея, коромысло, мошна, холоп, личико, весна 5. Как известно, в древнерусском языке существовали редуци- рованные (сверхкраткие) гласные Ъ и Ь, которые, если находились в сильной позиции, перешли в гласные полного образования О и Е соответственно, а если находились в слабой позиции – исчезли (этот процесс называется падением редуцированных; об этом, а также о последствиях этого процесса вы можете прочитать, пройдя по ссылке: https://docs.google.com/document/d/1PcKPShx6wW8bq HkfVNTa3kfvva86v9XB/edit (пункт 4) или отсканировав QR-код: 78 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию ) Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 78 Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Приведите примеры из предложений упражнения, иллюстриру- ющие различные последствия падения редуцированных. р Упражнение 3 1. Спишите пословицы и поговорки, вставляя пропущенные буквы и графически объясняя орфограммы. Значения незнако- мых слов уточните по толковому словарю (https:// slovarozhegova.ru/ или: 1. Спишите пословицы и поговорки, вставляя пропущенные буквы и графически объясняя орфограммы. Значения незнако- мых слов уточните по толковому словарю (https:// slovarozhegova.ru/ или: ) 1. Гладенька головка отцу-матери не кормил…ц. 2. От избытка и стар…ц келью строит. 3. Рассказ…ики не годятся в приказ…ики. 4. Молч…к – сто рублей. 5. Выйдем на дол…нку, сядем под ряб…нку. 6. Хороша книга, да начет…ики плохие. 7. Голова не колыш…к: не шапку на неё вешать. 8. Женатому учиться – врем…чко ушло. 9 Д Б д д 1. Гладенька головка отцу-матери не кормил…ц. 7. Голова не колыш…к: не шапку на неё вешать. 8. Женатому учиться – врем…чко ушло. 9. Дал Бог денеч…к, даст и кусоч…к. 10. Какова дворн…ца, такова ей и горн…ца. 11. Умная умн…ца – светлая пугов…ца. 12. Что вашему пригож…ству до нашего убож…ства? 13. Чтобы плать…це тонело, а хозяюшка его добрела. 14. Люди тонут и за солом…нку хватаются. 15. Было време…ко, целовали нас в тем…чко, а ныне в уста, да и то ради Христа. 15. Было време…ко, целовали нас в тем…чко, а ныне в уста, да и то ради Христа. 2. Объясните написание буквы О в корне слова дол…нку. Под- берите однокоренные слова и дайте их толкование. 2. Объясните написание буквы О в корне слова дол…нку. Под- берите однокоренные слова и дайте их толкование. 3. Какого рода слово умница? Что вы можете рассказать о су- ществительных общего рода? Чем они отличаются от слов, обозна- чающих профессии? (вам может помочь ссылка: https://foxford.ru/ wiki/russkiy-yazyk/sushchestvitelnye-obshchego-roda или QR-код: 79 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 4. Вставьте слова вместо пропусков (подсказка: одно из пропу- щенных слов есть в тексте упражнения😊) Общеславянское по своему происхождению название дерева _________(1) было дано по пестрому облику его соцветий. Ведь древним значением однокоренного прилагательного _________(2) и было «пестрый, пятнистый». Цветущее дерево выглядело именно пёстрым в отличие, скажем, от белой вишни или яблони. Интересно отметить, что в этимологическом родстве с суще- ствительным находятся такие наименования птиц, как ________(3) и сказочная курочка- _____________ (4). Лесная птица семейства тетеревиных получила свое название по пестрому оперению. Что касается курочки, то она была пестрая по своему перу, т.е. __________ (5). Часть 2. Трудные случаи русской орфографии и пунктуации Интересно отметить, что в этимологическом родстве с суще- ствительным находятся такие наименования птиц, как ________(3) и сказочная курочка- _____________ (4). Лесная птица семейства тетеревиных получила свое название по пестрому оперению. Что касается курочки, то она была пестрая по своему перу, т.е. __________ (5). Кудрявое и пестрое цветение дерева было выделено древними славянами как основное, оно легло в основу названия самого рас- тения, любимого в народе и воспетого во многих фольклорных произведениях. Упражнение 4 Упражнение 4 80 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию р 1. В пословицы вставьте пропущенные слова (см. материал для справки), выделите в них суффиксы и объясните их написание 1. Мастеровой ____________, как паровой _________ – цветет, цветет, да и повянет. 2. Поговорка –____________, а _________– ягодка 3. Придет солнышко и к нашим _____________ 4. Горькому ____________ горькая и песенка 5. Не давай мое белое __________ чужим людям на __________ 6. Бог даст, батюшка ________ продаст, а _________ купит 7. Мастер один, а __________ десять 1. В пословицы вставьте пропущенные слова (см. материал для справки), выделите в них суффиксы и объясните их написание 1. В пословицы вставьте пропущенные слова (см. материал для справки), выделите в них суффиксы и объясните их написание 80 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию справки), выделите в них суффиксы и объясните их написание 1. Мастеровой ____________, как паровой _________ – цветет, цветет, да и повянет. 2. Поговорка –____________, а _________– ягодка 3. Придет солнышко и к нашим _____________ 4. Горькому ____________ горькая и песенка 5. Не давай мое белое __________ чужим людям на __________ 6. Бог даст, батюшка ________ продаст, а _________ купит 7. Мастер один, а __________ десять 80 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию люби и _ 8. Люби ___________, люби и _________ 9. Дедушка и не ведает, где обедает __________ 10. Положи, господь, камешком, подыми ___________ Материал для справок: Пер…шком, двор…к, послов…ца, оском…нку ,внуч…к, пору- гань…це, цветоч…к, ряб…нку, рукодель…це, окош…чкам, го- лубч…к, огурч…к, Куз…ньке, смород…нку, поднос…иков, ба- лала…чку, 9. Дедушка и не ведает, где обедает ___ 10. Положи, господь, камешком, подыми _ Материал для справок: Пер…шком, двор…к, послов…ца, оском…нку ,внуч…к, пору- гань…це, цветоч…к, ряб…нку, рукодель…це, окош…чкам, го- лубч…к, огурч…к, Куз…ньке, смород…нку, поднос…иков, ба- лала…чку, Пер…шком, двор…к, послов…ца, оском…нку ,внуч…к, пору- гань…це, цветоч…к, ряб…нку, рукодель…це, окош…чкам, го- лубч…к, огурч…к, Куз…ньке, смород…нку, поднос…иков, ба- лала…чку, 2. Выпишите не менее 7 слов, в которых не совпадает количе- ство звуков и букв. Объясните причины такого несовпадения. 2. Выпишите не менее 7 слов, в которых не совпадает количе- ство звуков и букв. Объясните причины такого несовпадения. 3. Во всех предложениях подчеркните основы и объясните по- становку знаков препинания. 4. Как вы понимаете значение пословицы Придет солнышко и к нашим _____________? Подберите к ней синонимичные русские пословицы и пословицы вашего народа. Составьте и запишите диа- лог между друзьями, в котором было бы уместно употребление этой пословицы. 8. Люби 2. На место пропусков вставьте подходящие по смыслу прила- гательные (см. материал для справок). Упражнение 1 1. Вспомните правила написания суффиксов имен прилагатель- ных и заполните таблицу (вам может помочь ссылка: https://licey.net/free/4-russkii_yazyk/39-kurs_russkogo_yazyka_ fonetika__slovoobrazovanie__morfologiya_i_orfografiya/stages/672- 335_pravopisanie_suffiksov_imen_prilagatelnyh.html или QR-код: 81 81 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» суффикс условие пример -ив- -ев- -лив- -чив- -ий -ов-, -оват-, -овит- -ев-, -еват-, -евит- -оньк- -еньк- -чат- -енск- -инск- -ск- -к- 2. На место пропусков вставьте подходящие по смыслу прила- гательные (см. материал для справок). 2. На место пропусков вставьте подходящие по смыслу прила- гательные (см. материал для справок). 1. Нужда _________, голь ______________. 2. __________ парень: на крутой каше распоясался 3. Хлеб да вода – ____________ еда 4. Ешь щи – будет шея бела, голова __________ . 5. Пирог_____________, с рыбой ___________ . 6. Гусли __________ думку за горы заносят, из-за гор выносят 7. ____________ сердце не ________ . 8. Родился ____________, женился ____________ . 9. Светится одно солнце на небе, а царь ___________ на земле 10. Бог __________, а царь ______________. 11. Вались народ от _________ворот! 12. И крута гора, да __________; и лиха беда, да ____________. 82 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 82 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 82 Часть 2. Трудные случаи русской орфографии и пунктуации Материал для справок: сбывчива, вежлива, бурлацкая, кудре- вата, догадлива, молодецкое, Яузских, милостив, звончатые, до- гадлив, арзамасский, русский, малешенек, уклончивое, глупешенек, жалостлив, забывчива, астраханской. р 3. Выделите суффиксы имен прилагательных и объясните их написание. 3. Выделите суффиксы имен прилагательных и объясните их написание. 4. Выполните морфологический разбор следующих слов: бела (шея), (на) крутой (каше), лиха (беда). 5. Составьте схему последнего предложения, объясните поста- новку знаков препинания. 5. Составьте схему последнего предложения, объясните поста- новку знаков препинания. Правописание глагольных суффиксов Упражнение 1 11. Ещё бы во…вал, да пищаль потерял. 3. Чего не покуп…вали, того и не нашивали. 14. Не знаешь песни, так не затяг…вай. 14. Не знаешь песни, так не затяг…вай. 15. Покряк…вает, да покашл…вает, жмется да морщится. 16. Лупи, не сказ…вай, а облупишь – не показ…вай. 17. Занузд…вать его бечевкой, да припасти на него кнут с пу- овкой. 17. Занузд…вать его бечевкой, да припасти на него кнут с пу- овкой. 18. Видали мы, слыхали мы, и сами едали, и других кармл…вали. 19. Не велят в окно загляд…вать, а в двери не пускают. 20. Вавила, утирай рыло, провал…вай мимо. 21. Ты сударь, и я сударь, а кто же присудар…вать станет? 22. Зарубай, сглаж…вай, да никому не сказ…вай. 2. Какая зависимость между этой 1 лица единственного числа и выбором гласной в глагольных суффиксах? Объясните выбор глас- ных в суффиксах глаголов. 2. Какая зависимость между этой 1 лица единственного числа и выбором гласной в глагольных суффиксах? Объясните выбор глас- ных в суффиксах глаголов. 3. От всех глаголов образуйте все формы прошедшего времени, поставьте в них ударение. 3. От всех глаголов образуйте все формы прошедшего времени, поставьте в них ударение. 4. Какой морфемой / частью морфемы является буква И в сле- дующих словах: пляши, любить, и, (не знаешь) песни, едали, при- пасти, облупишь? Определите, если это возможно, грамматиче- ское значение этих слов 5. Как вы понимаете значение выражения Пляши, да не за- пляс…вайся? Подберите синонимичные русские пословицы и по- говорки и вашего родного фольклора. Какому литературному ге- рою вы могли бы дать этот совет? Почему? Упражнение 1 р 1. Заполните таблицу, вписав глаголы с орфограммой в суф- фиксе в соответствующую колонку. Если глагол употреблен в форме НЕ 1 лица ед.ч., образуйте ее и запишите в соседней ко- лонке; если глагол употреблен в форме 1 лица ед.ч., то образуйте форму инфинитива и тоже запишите ее в соседней колонке (см. об- разец) НЕ 1 лицо единственного числа 1 лицо единственного числа ночевать ночую 1. Заполните таблицу, вписав глаголы с орфограммой в суф- фиксе в соответствующую колонку. Если глагол употреблен в форме НЕ 1 лица ед.ч., образуйте ее и запишите в соседней ко- лонке; если глагол употреблен в форме 1 лица ед.ч., то образуйте форму инфинитива и тоже запишите ее в соседней колонке (см. об- разец) НЕ 1 лицо единственного числа 1 лицо единственного числа ночевать ночую 1. Всю ночь просижу, а ноч…вать не стану. 1. Всю ночь просижу, а ноч…вать не стану. 2. Как заварили, так и расхлеб…вайте. 3. Кого боюсь, поскорее ложусь; кого не боюсь, поворач…ваюсь. 4. Спот…кается и конь, да поправляется. 5. Любить-то хоть не люби, да почаще взгляд…вай. 6. День дн…вать, ночь коротать. 7. И муха набив…вает брюхо. 8. Скоморох с Пресни наигр…вал песни. 9. И мы вид…вали, да и не завид…вали. 10. Пляши, да не запляс…вайся. 11. Ещё бы во…вал, да пищаль потерял. 8. Скоморох с Пресни наигр…вал песни. 9. И мы вид…вали, да и не завид…вали. 10. Пляши, да не запляс…вайся. 11. Ещё бы во…вал, да пищаль потерял. 83 Н и НН в разных частях речи * 12. Хоть и рано (говорит лиса в капкане), а ноч…вать при- дется. 12. Хоть и рано (говорит лиса в капкане), а ноч…вать при- дется. 13. Чего не покуп…вали, того и не нашивали. 13. Чего не покуп…вали, того и не нашивали. Упражнение 1 Упражнение 1 1. Вспомните правило выбора Н и НН в отыменных прилага- тельных, составьте алгоритм его применения и, используя его, вставьте на место пропусков Н или НН. Суффиксы выделите. Выполнить это задание вам поможет ссылка: https://foxford. ru/wiki/russkiy-yazyk/pravopisanie-n-i-nn-v-otymyonnyh- prilagatelnyh или QR-код: 84 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 84 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Были встарь сосуды деревя…ы, попы золотые; ныне сосуды зо- лотые, попы деревянны Были встарь сосуды деревя…ы, попы золотые; ныне сосуды зо- лотые, попы деревянны 1. Окольничий Засекин сви…ым ухом подавился 1. Окольничий Засекин сви…ым ухом подавился 2. Шапка волося…ая, рукавицы своекожа…ые 3. Мать праведна – ограда каме..а 3. Мать праведна – ограда каме..а 4. Дай прокормить казе…ого воробья – без своего гуся и за стол не сядем 4. Дай прокормить казе…ого воробья – без своего гуся и за стол не сядем 5. Молода жена плачет до росы утре…й, сестрица до золота кольца, мать до веку 5. Молода жена плачет до росы утре…й, сестрица до золота кольца, мать до веку 6. Острый язык – дарованье, дли…ый язык – наказанье 7. Горе наше – ржа…ая каша, а поел бы и такой, да нет ни- какой 8. Овся…ая каша хвалилась, будто с коровьим маслом родилась 9. Небитый – серебря…ый, битый – золотой 9. Небитый – серебря…ый, битый – золотой 10. Пови…ую голову и меч не сечет 11. За тобой и на переме…ых не поспеешь 12. Казе…ое на воде не тонет, на огне не горит 13. Беззако…ый клич беззаконно и внемлется 14. Тому виднее, у кого нос дли…ее 15. Эка понесла: ни ко…ому, ни крылатому не догнать 2. Найдите и подчеркните составные именные сказуемые, ука- жите, чем они выражены. За справкой можете пройти по ссылке: https://foxford.ru/wiki/russkiy-yazyk/sostavnoe-imennoe-skazuemoe или отсканируйте QR-код: 85 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 3. Укажите в упражнении 5-6 неизменяемых слов. Как выражено их грамматическое значение? К какой части речи они относятся? 3. Укажите в упражнении 5-6 неизменяемых слов. Как выражено их грамматическое значение? К какой части речи они относятся? р р 4. Выберите наиболее приглянувшуюся вам пословицу или по- говорку и придумайте с ней мем. 4. Выберите наиболее приглянувшуюся вам пословицу или по- говорку и придумайте с ней мем. Упражнение 2 Упражнение 2 1. Как отличить отглагольные прилагательные от причастий? Как умение различать слова этих частей речи поможет вам при написании Н и НН? Чтобы ответить на эти вопросы, пройдите по ссылке: https://foxford.ru/wiki/russkiy-yazyk/n-i-nn-v-polnyh-i- kratkih-stradatelnyh-prichastiyah-i-otglagolnyh-prilagatelnyh или от- сканируйте QR-код: 1. Как отличить отглагольные прилагательные от причастий? Как умение различать слова этих частей речи поможет вам при написании Н и НН? Чтобы ответить на эти вопросы, пройдите по ссылке: https://foxford.ru/wiki/russkiy-yazyk/n-i-nn-v-polnyh-i- kratkih-stradatelnyh-prichastiyah-i-otglagolnyh-prilagatelnyh или от- сканируйте QR-код: 2. Определите части речи выделенных слов, аргументируйте свой ответ. Вставьте на место пропусков Н или НН, суффиксы вы- делите 2. Определите части речи выделенных слов, аргументируйте свой ответ. Вставьте на место пропусков Н или НН, суффиксы вы- делите 1. Не суже…ный кус изо рту валится 2. Пошел по шерсть, а воротился стриже…ый 3. Жела…ый кус не мимо уст 3. Жела…ый кус не мимо уст 6 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 6 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 86 Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 4. Не наказа…ый сын – бесчестье отцу 4. Не наказа…ый сын – бесчестье отцу 5. Сказали: беше…ых всех перевешали; неправду сказали: од- ного не связали 6. Хвале…ый пуще хая…ого 7. Сырого не ем, жаре…ого не хочу, варе…ого терпеть не могу 8. Пуга…ая ворона и куста боится 8. Пуга…ая ворона и куста боится 9. Святым кулаком да по окая…ой шее 10. Уче…ому коню лишь плеть покажи 11. За проще…ую вину и бог не мучит 12. Кушай варе…ое, да слушай говоре…ое 3. Что нужно сделать с отглагольными прилагательными, чтобы превратить их в причастия? Сделайте это, составьте с полу- ченными причастиями предложения и запишите их. 4. В этом правиле много исключений. Вот они: Невиданный Невиденный 87 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Обетованный Жданный Неожиданный Неведанный Ненадеванный Отъявленный Окаянный А также: Прощеное Воскресение, названый брат / сестра, поса- женый отец / мать, конченый человек. Составьте связный текст со всеми исключениями. Выучите эти исключения. Упражнение 3 1. Вспомните, как отличить краткое отглагольное прилагатель- ное и краткое причастие. Ответ оформите в виде таблицы, где нужно указать отличительные признаки этих частей речи (Ссылка и QR-код, которые вам могут помочь, даны в предыдущем упраж- нении). 1. Вспомните, как отличить краткое отглагольное прилагатель- ное и краткое причастие. Ответ оформите в виде таблицы, где нужно указать отличительные признаки этих частей речи (Ссылка и QR-код, которые вам могут помочь, даны в предыдущем упраж- нении). ) 2. Определите часть речи выделенных слов, свой ответ аргумен- тируйте. На место пропусков вставьте Н или НН. Выделите суф- фиксы. ) 2. Определите часть речи выделенных слов, свой ответ аргумен- тируйте. На место пропусков вставьте Н или НН. Выделите суф- фиксы. 1. Суже…а ряже…а не обойдешь и на коне не объедешь. 2. Кому луковка облупле…а, а нам тукманка не купле…а. 3. Терпи, голова, в кости скова…а. 4. У умного мужа жена выхоле…а, у глупого по будням за- таска…а. 5. Много говоре…о, да мало сказа…о. 6. Была вина, да проще…а 7. Скоро сказа…о, кабы не сдела…о 8. И то переговоре…о, что еще не сваре…о 3. Каков смысл пословицы Много говоре…о, да мало сказа…о? Придумайте ситуации, где было бы уместно употребление этой по- говорки. 3. Каков смысл пословицы Много говоре…о, да мало сказа…о? Придумайте ситуации, где было бы уместно употребление этой по- говорки. 4. Разберите по членам предложения все пословицы и пого- ворки. Определите вид всех односоставных предложений. 5. Кто такой суженый? Определите, опираясь на историю слов супруг, сустав, сутки, суженый, сугроб, какое из них явля- ется «лишним» в этом ряду? Каким словарем вы воспользуетесь, 88 88 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 88 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации чтобы выполнить это задание? Кто из лингвистов составлял по- добные словари? чтобы выполнить это задание? Кто из лингвистов составлял по- добные словари? чтобы выполнить это задание? Кто из лингвистов составлял по- добные словари? Чтобы выполнить это задание, можете пройти по ссылке: https://vasmer.slovaronline.com/ или отсканируйте QR-код: 1. Вспомните правило слитного, дефисного и раздельного напи- сания пол- и полу-. Для этого пройдите по ссылке: http://gramota.ru/class/coach/tbgramota/45_64 или воспользуйтесь QR-кодом: 2. Спишите пословицы и поговорки, раскрывая скобки. Объяс- ните написание слов, начинающихся с пол- и с полу-. 2. Спишите пословицы и поговорки, раскрывая скобки. Объяс- ните написание слов, начинающихся с пол- и с полу-. 1. Кому веселье, а мне и (пол)веселья нет 2. Денежка не бог, а (пол)бога есть 2. Денежка не бог, а (пол)бога есть 3. В полном мешке – брат родной; в (полу)мешке – двоюродный; на дне мешка – в сватовстве; а нет в мешке, нет и родни 4. Холостой – (пол)человека 5. (Пол)пятаста – те же пять девяносто 6. Один ум –(пол)ума; три ума – (пол)тора ума; два ума – ум 89 7. Голодному всегда (пол)дни 7. Голодному всегда (пол)дни 7. Голодному всегда (пол)дни 8. Сыт покуда, как съел (пол)пуда; теперь как бы проведать, не станет ли кто обедать 9. По (полу)кринке все сливки; а захочу, все в сметану оборочу 10. Кабы не деньги, так был бы в (пол)деньги 10. Кабы не деньги, так был бы в (пол)деньги 11. Не (пол)сорока, а двадцать 11. Не (пол)сорока, а двадцать 12. На (пол)третья годины барин 13. Тара-бара – и вышло ни два, ни (пол)тора 3. Какие слова можно использовать в качестве примеров для ил- люстрации правила «Правописание безударных окончаний имен существительных»? 4. Просклоняйте словосочетание полтора ума 5. Запишите цифрами: (пол)пятаста, пять девяносто, (пол)тора, (пол)пуда, (пол)сорока, (пол)третья (годины). 6. В грамоте князя Мстислава и сына ешо Всеволода Новгород скому Юрьеву монастырю (XII век) говорится: «А язъ далъ роукою своею … полъ третия десяте гривьнъ». Переведите фразу на современный русский язык. Каково значе- ние каждого из подчеркнутых слов? Дайте им грамматическую ха- рактеристику с исторической точки зрения. Переведите фразу на современный русский язык. Каково значе- ние каждого из подчеркнутых слов? Дайте им грамматическую ха- рактеристику с исторической точки зрения. * Правописание частиц * Упражнение 1 Упражнение 1 Упражнение 1 1. Вспомните, что такое частицы, какие значения частиц выде- ляют? Для того чтобы ответить на эти вопросы, пройдите по ссылке: https://www.yaklass.ru/p/russky-yazik/7-klass/chastitca- 10517/chastitca-kak-sluzhebnaia-chast-rechi-10592/re-b13043fc-4902- 4c22-aa49-2931d47a4b45 или отсканируйте QR-код: 90 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 0 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 90 Часть 2. Трудные случаи русской орфографии и пунктуации 2. Прочитайте предложения, найдите в них частицы, определите их разряд. Заполните таблицу по образцу: 2. Прочитайте предложения, найдите в них частицы, определите их разряд. Заполните таблицу по образцу: 2. Прочитайте предложения, найдите в них частицы, определите их разряд. Заполните таблицу по образцу: предложение частица разряд функция Похвали-ка ты меня, а там и я тебя похвалю -ка формообра- зующая Служит для образования повелительного наклонения 1. Похвали-ка ты меня, а там и я тебя похвалю. 2. Пришла свинья к коню, говорит: и ноги-де кривы и шерсть нехороша. 2. Пришла свинья к коню, говорит: и ноги-де кривы и шерсть нехороша. 3. Полюби-ко нас в черне, а в красне всяк полюбит. 4. Не пил бы, не ел, все б на милую глядел. 5. Ох охонюшки, тошно без Афонюшки, Иван-то тут, да уряд- то худ. у 6. Приятелей-то много, а друга нет. 7. Рада б душа посту, так тело бунтует. 8. Приняла б душа, а брюхо не прогневается. 9. Чья бы ни рычала, да твоя-то бы молчала. 10. Рад бы душой, да хлеб-это чужой. 11. Молока-то шилом, а хлеба-то вилами. 12. Спела б и рыбка песенку, когда б голос был. 13. Один пошел – полтину нашел; семеро пойдут – много ли найдут? 13. Один пошел – полтину нашел; семеро пойдут – много ли найдут? 14. Кабы бабушка не бабушка, так была б она дедушкой. 15. То же бы ты слово, да не так бы молвил. 16. Те ж кафтаны, да не те карманы. 17. Отложь-ка блины до маслены. 17. Отложь-ка блины до маслены. 18. Тоже смешно, да на то же пошло. 19. Грех-то в мех, а грешки в мешки да по подлавочью. 20. Дать-то бы ничто, да было б за что. 21. Не тебе бы говорить, да не мне бы слушать. 22. Мало ль чего говорят, да не все перенять. 3. Изучите материал по ссылке: https://www.yaklass.ru/p/russky- yazik/7-klass/chastitca-10517/pravopisanie-chastitc-10594/re- 5c1b56db-4ccb-407a-85db-aab731618829 или по QR-коду и объяс- ните написание частиц. 4. Какой частью речи являются подчеркнутые слова в предло- жении Тоже смешно, да на то же пошло? Аргументируйте свой 91 91 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» ответ. Как называются слова, имеющие одинаковой звучание и написание, но являющиеся формами разных частей речи? Приве- дите примеры таких слов. р р 5. О каком словаре идет речь? р р 5. О каком словаре идет речь? «Словарная статья содержит значение устаревшего слова, грам- матическую и стилистическую характеристику, а также иллюстра- тивный материал. Многие словарные статьи сопровождаются ри- сунками. «Словарная статья содержит значение устаревшего слова, грам- матическую и стилистическую характеристику, а также иллюстра- тивный материал. Многие словарные статьи сопровождаются ри- сунками. В Словарь включены следующие приложения: 1. Наименова- ния, отражающие родственные отношения; 2. Единицы измерения, принятые в России в XVIII – начале XX в.; 3. Табель о рангах; 4. Титулование, принятое в России; 5. Иерархия духовных лиц в рус- ской православной церкв.» Какие слова из этого упражнения могли бы войти в этот сло- варь? 6. Изучите словарную статью из этого словаря: 6. Изучите словарную статью из этого словаря: Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 6. Изучите словарную статью из этого словаря: « БАТА´ЛИЯ и (прост.) БАТА´ЛЬЯ, -и, ж. Битва, сраже- ние. – Ну, что? – сказала комендантша. – Каково идет б а т а л ь я? Пушкин. Капитанская дочка. БАТА´ЛЬНЫЙ, -ая, -ое. Прилаг. к баталия; военный. ◊ Бата´льный ого´нь. Беспрерывная стрельба из пушек и ружей. Как я увидал, ваше сиятельство, что первый батальон расстроен, я стал на дороге и думаю: «Пропущу этих и встречу б а т а л ь н ы м о г н е м»; так и сделал. Л. Толстой. Война и мир. р [От франц. bataille – сражение, битва]. 92 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию [ фр ц р , ] » По аналогии составьте и запишите словарную статью для 1-2 слов из этого упражнения. » По аналогии составьте и запишите словарную статью для 1-2 слов из этого упражнения. По аналогии составьте и запишите словарную статью для 1-2 слов из этого упражнения. 92 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 92 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 17. (Не)дорог конь, дорог заяц 16. Похвальба (не)крестьянское дело Упражнение 1 Упражнение 1 1. Вспомните правила слитного и раздельного написания НЕ с именами прилагательными. Для этого пройдите по ссылке: https://foxford.ru/wiki/russkiy-yazyk/slitnoe-i-razdelnoe-napisanie-ne- s-prilagatelnymi или отсканируйте QR-код: 2. Спишите пословицы и поговорки, раскрывая скобки. Объяс- ните слитное и раздельной написание частицы НЕ р 1. (Не)зрел виноград – невкусен, а молод человек – (не)искусен. 2. Взялся за гуж – не говори, что (не)дюж 3 (Н ) б д 2. Взялся за гуж – не говори, что (не)дюж р щ ( ) 6. Пришла свинья (не)корыстна, съела зернышко пшенично 7. (Не)мудра голова, да кубышка полна 8. (Не)богатый кормит, а тороватый 9. Где нет доли, тут и счастье (не)велико 10. (Не)хитер парень, да удачлив; (не) казист, да таланен 11. Любит жена и старого мужа, коли (не)ревнив 12. Все мужья добры, покупили женам бобры, а мой муж не)уклюж: невидаль, корову купил 13. Перед злой женой сатана – младенец (не)порочный 14. (Не)милостивому мил не будешь 15. (Не) прав волк, что овцу съел, (не)права и овца, что в лес пошла 16. Похвальба (не)крестьянское дело 17. (Не)дорог конь, дорог заяц 93 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 18. Я (не)архиерейский зять, с меня нечего взять 19. (Не)силен – не борись, (не)богат – не сердись 20. (Не)ломлив бы парень, да в гости не зовут 21. И (не)рад хрен терке, да по ней боками пляшет 22. Неволя бьет Еромола: Ермол и (не)виноват, да нельзя ми- новать 3. Найдите в упражнении краткие прилагательные, подчерк- ните их как члены предложения. 4. Выполните синтаксический разбор бессоюзных сложных предложений. Объясните постановку в них знаков препинания. 5. Вспомните русские пословицы, поговорки и фразеологизмы, в которых есть имена собственные. Объясните их значение. Есть в вашем родном языке и фольклоре вашего народа устойчивые выра- жения с именами собственными? Если есть, запишите их. Упражнение 2 1. Какое (какие) правило (правила) можно проиллюстрировать, использовав в качестве примеров следующие пословицы и пого- ворки? 1. Все кузни исходил, а (не)кован воротился 2. Сам поет соловьем, (не)зная о чем 3. (Не)поздоровится от таких похвал 4. (Не)накормлен конь – скотина, (не) пожалован молодец – сиротина 5. Индюшки от воробья (не) распознает 6. Без мужа голова (не)покрыта; без жены дом (не)крыт 7. У кого детей много, то (не) забыт от бога 8. Счастью (не)верь, а беды (не)пугайся 9. Ехал, да (не)доехал: опять поедем – авось доедем 10. Наелся, как бык, и (не)знает, как быть 11. Миленек – и (не)умыт беленек 12. (Не)скроив, (не)сошьешь 13. Хочется, да (не)можется 2. Спишите, раскрывая скобки и объясняя свой выбор. При вы- полнении этого задания можете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy-yazyk/slitnoe-i-razdelnoe-napisanie-ne- s-raznymi-chastyami-rechi-zadanie-13-ege или отсканируйте QR-код: 94 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 94 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 3. Сделайте транскрипцию предложения Хочется, да (не)мо- жется, выполните фонетический разбор всех слов этого предло- жения 3. Сделайте транскрипцию предложения Хочется, да (не)мо- жется, выполните фонетический разбор всех слов этого предло- жения 4. Выпишите все причастия, выполните их морфологический разбор 4. Выпишите все причастия, выполните их морфологический разбор 5. Про кого так говорят: Сам поет соловьем, (не)зная о чем? Вспомните героев литературных произведений, которым подходит эта характеристика. Вспомните и запишите фразеологизмы, в которых упоминаются названия птиц и которые характеризуют человека. Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Ответ оформите в виде таблицы: Фразеологизм О ком так говорят Гусь лапчатый хитроумный, изворотливый человек, способный выйти из ситуации с личной выгодой Вспомните и запишите фразеологизмы, в которых упоминаются названия птиц и которые характеризуют человека. Ответ оформите в виде таблицы: Фразеологизм О ком так говорят Гусь лапчатый хитроумный, изворотливый человек, способный выйти из ситуации с личной выгодой Упражнение 3 1. Вспомните правило «Слитное и раздельное частицы НЕ с именами существительными». Для этого пройдите по ссылке: https://foxford.ru/wiki/russkiy-yazyk/slitnoe-i-razdelnoe-napisanie-ne- s-sushchestvitelnymi или воспользуйтесь QR-кодом: 95 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Российской Федерации» национального проекта «Образование» 2. Прочитайте пословицы, объясните слитное и раздельное написание НЕ с существительными 1. Кукушка не ястреб, а неуч не мастер 2. То не дьячество, коли не бывал в подьячестве 3. Ружье да ранец не тяга, а крылья 4. Не все ненастье, проглянет и красно солнышко 5. Добро не лихо; бродит о мир тихо 6. Счастье не дворянство, не родом ведется 7. Деньги не голова: наживное дело 8. Богатый не золото ест, а бедный не камень гложет 9. Перерод хуже недорода 10. Недоля пудами, доля золотниками 11. Бояться несчастья – счастья не будет 12. Жена не седло: со стены не сымешь 13. Не наряд жену красит – домостройство 14. Доброй жене домоводство не мука 15. Давно то было баяно, что жена не барыня 16. Счастье не батрак: за вихор не притянешь 3. Найдите предложения, в которых подлежащее и сказуемое выражены именами существительными в именительном падеже. Объясните постановку тире или его отсутствие. (Вспомнить пра- вила постановки тире между подлежащим и сказуемым вы можете, пройдя по ссылке: https://foxford.ru/wiki/russkiy-yazyk/tire-mezhdu- podlezhaschim-i-skazuemym или отсканировав QR-код: 2. Прочитайте пословицы, объясните слитное и раздельное написание НЕ с существительными 2. Прочитайте пословицы, объясните слитное и раздельное написание НЕ с существительными 1. Кукушка не ястреб, а неуч не мастер 2. То не дьячество, коли не бывал в подьячестве 7. Деньги не голова: наживное дело 8. Богатый не золото ест, а бедный не камень гложет 9. Перерод хуже недорода 10. Недоля пудами, доля золотниками 11. Бояться несчастья – счастья не будет 12. Жена не седло: со стены не сымешь 13. Не наряд жену красит – домостройство 14. Доброй жене домоводство не мука 15. Давно то было баяно, что жена не барыня 16. Счастье не батрак: за вихор не притянешь 3. Найдите предложения, в которых подлежащее и сказуемое выражены именами существительными в именительном падеже. Объясните постановку тире или его отсутствие. (Вспомнить пра- вила постановки тире между подлежащим и сказуемым вы можете, пройдя по ссылке: https://foxford.ru/wiki/russkiy-yazyk/tire-mezhdu- podlezhaschim-i-skazuemym или отсканировав QR-код: ) ) ) 4. Разберите слово баяно по составу, определите часть речи. Подберите однокоренные, в том числе и этимологически родствен- ные слова, дайте их толкование, составьте и запишите с ними пред- ложения 4. Разберите слово баяно по составу, определите часть речи. Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Подберите однокоренные, в том числе и этимологически родствен- ные слова, дайте их толкование, составьте и запишите с ними пред- ложения 96 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 96 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации 5. Что такое антонимы? Языковые антонимы? Контекстуальные антонимы? В каких предложениях данного упражнения употреб- лены антонимы? Определите их тип. С какой целью употреблены антонимы? Приведите примеры русских пословиц и пословиц ва- шего народа, в которых употреблены антонимы. Вспомнить, что такое антонимы и каких видов они бывают, с какой целью употребляются в тексте, вы можете, пройдя по ссылке: https://lingvotech.com/styleanti или отсканировав QR-код: Упражнение 4 1. Спишите предложения, раскрывая скобки. Объясните слит- ное и раздельное написание частицы НЕ с именами существитель- ными 1. Наш Андрей никому (не)злодей 2. Сердцем копья у (не)друга не переломишь 3. Пень (не)околица, глупая речь (не)пословица 4. Охота пуще (не)воли 5. (Не)довольство, а охота человека тешит 6. Одна игра (не)потеха 7. Счастье (не)кляп: в руки не возьмешь 8. (Не)Стенька: на ковре по Волге не поплывешь 9. За (не)волю хода, коли ноги болят 10. За (не)имением гербовой пишут и на простой 11. (Не)солнышко: всех не обогреешь 12. Один в поле (не)воин 13. Золотая клетка соловью (не)(потеха 14. Бедность (не)грех, а (не)воля (не)смех 15. Богатый (не)золото ест, а бедный (не)камень гложет 2. В пословице За (не)имением гербовой пишут и на простой пропущено существительное, от которого зависят прилагательные гербовой и простой. Что это за существительное? Как называются 97 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» предложения с пропуском того или иного члена, легко восстанав- ливаемого из контекста или речевой ситуации? Как отличать такие предложения от односоставных предложений? Прочитать о таких предложениях вы можете, пройдя по ссылке: https://foxford.ru/wiki/ russkiy-yazyk/polnye-i-nepolnye-predlozheniya# или отсканировав QR-код: Поставьте ударение в словах гербовой, гербовый. Составьте с ними предложения, осложненные обособленными определениями, объясните постановку знаков препинания. 3. Что такое синонимы? Языковые синонимы? Контекстуаль- ные? Для чего синонимы нужны в речи? Подберите синонимы к следующим словам: потеха, пуще, золотая, смех, недруг, неволя. К одной из пословиц этого упражнения подберите синонимич- ную из предыдущего упражнения. Каково их значение? Составьте и запишите диалог, использовав обе пословицы. К одной из пословиц этого упражнения подберите синонимич- ную из предыдущего упражнения. Каково их значение? Составьте и запишите диалог, использовав обе пословицы. * Бывают ли синонимичные морфемы (приставки, суффиксы)? Если да, то приведите примеры. * Бывают ли синонимичные морфемы (приставки, суффиксы)? Если да, то приведите примеры. Прочитать про синонимы и освежить свои знания вы можете по ссылке: https://ebooks.grsu.by/pustoshilo_lang/8-sinonimiya-tipy- sinonimov-sinonimicheskij-ryad.htm или отсканировав QR-код: Прочитать про синонимы и освежить свои знания вы можете по ссылке: https://ebooks.grsu.by/pustoshilo_lang/8-sinonimiya-tipy- sinonimov-sinonimicheskij-ryad.htm или отсканировав QR-код: 98 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 98 Часть 2. Трудные случаи русской орфографии и пунктуации 4. Постройте «обратную» словообразовательную цепочку от ко- нечного производного до первоначального исходного. Определите способ образования производных. В случае крайнего затруднения 3. Объясните слитное и раздельное написание НЕ с наречиями Часть 2. Трудные случаи русской орфографии и пунктуации 4. Как вы понимаете значение выражения (Не)довольство, а охота человека тешит? Согласны ли вы с этим утверждением? Докажите свою точку зрения. В качестве аргументов приведите примеры из литературы, истории и других областей знаний. Упражнение 5 1. Вспомните слитное и раздельное написание частицы НЕ с наречиями. Для этого пройдите по ссылке: http://rozental. gramatik.ru/pravopisanie-ne-i-ni/ss-71-pravopisanie-ne-s-narechiyami или отсканируйте QR-код: 1. Вспомните слитное и раздельное написание частицы НЕ с наречиями. Для этого пройдите по ссылке: http://rozental. gramatik.ru/pravopisanie-ne-i-ni/ss-71-pravopisanie-ne-s-narechiyami или отсканируйте QR-код: 2. Соедините начало пословиц (они даны в первой колонке таб- лицы) с их концовкой (они даны во второй колонке таблицы). 2. Соедините начало пословиц (они даны в первой колонке таб- лицы) с их концовкой (они даны во второй колонке таблицы). лицы) с их концовкой (они даны во второй колонке таблицы). Поживешь счастливо с медведем бороться Кому ничего что не туда идет И не туго а неплохо и нам, коли полон карман Стужа да нужа – нет их хуже; а нам не больше того И пастух овцу бьет недалеко уедешь И нелюбо да сбойливо Тем хорошо, другим хорошо холод да голод – не легче того Летала высоко да волк недалечко Несподручно бабе паши не лениво! И неказисто, да смейся На кнуте да упруго Сказал бы словечко а села недалеко Объясните слитное и раздельное написание НЕ с наречиями 99 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» обратитесь к словообразовательному словарю по ссылке: https://grammatika-rus.ru/wp-content/uploads/2020/02/Tihonov-A.-N.- Novyj-slovoobrazovatelnyj-slovar-russkogo-yazyka-dlya-vseh-kto- hochet-byt-gramotnym- или воспользуйтесь QR-кодом: обратитесь к словообразовательному словарю по ссылке: https://grammatika-rus.ru/wp-content/uploads/2020/02/Tihonov-A.-N.- Novyj-slovoobrazovatelnyj-slovar-russkogo-yazyka-dlya-vseh-kto- hochet-byt-gramotnym- или воспользуйтесь QR-кодом: Вспомнить, что такое словообразовательная цепочка и другие термины словообразования, вам поможет ссылка: https://foxford.ru/ wiki/russkiy-yazyk/slovoobrazovatelnaya-tsepochka или QR-код: Часть 2 Часть 2 1. Какое орфографическое правило, связанное с частицей НЕ, иллюстрируют следующие пословицы и поговорки? 1. Какое орфографическое правило, связанное с частицей НЕ, иллюстрируют следующие пословицы и поговорки? 1. Бывает добро, да не всякому равно 2. Не каждый конь ко двору приходится 3. Не все коту масленица, будет и Великий Пост 4. Не наше счастье, чтоб найти, а наше, чтоб потерять 5. Не всяку правду муж жене сказывает, а и сказывает, так обманывает 2. Выпишите все местоимения, определите их разряд. О написа- нии НЕ с местоимениями каких разрядов здесь нет примеров? При- ведите эти примеры сами. На основании всех примеров сформули- руйте правило «Слитное и раздельное написание частицы НЕ с ме- стоимениями». 100 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 100 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 3. По толковому словарю выпишите все значения слова великий. В каком значении употреблено это слово в выражении Великий Пост? 3. По толковому словарю выпишите все значения слова великий. В каком значении употреблено это слово в выражении Великий Пост? 4. В чем заключается иносказательный смысл поговорки Не все коту масленица, будет и Великий Пост? * У великого русского драматурга А.Н. Островского есть коме- дия «Не все коту масленица». Предположите, почему автор дал та- кое название своему произведению. (а теперь прочитайте комедию и проверьте, насколько вы были правы 😉) * У великого русского драматурга А.Н. Островского есть коме- дия «Не все коту масленица». Предположите, почему автор дал та- кое название своему произведению. (а теперь прочитайте комедию и проверьте, насколько вы были правы 😉) *** 2. Ни тпру, ни ну Частицы НЕ и НИ 1. Какие значения могут иметь частицы НЕ и НИ? Приведите примеры. Заполните таблицу, используя в качестве примеров при- веденные ниже пословицы и поговорки (если на какой-либо отме- ченный вами случай нет пословицы в данном упражнении, можете использовать предложения из следующих упражнений или подбе- рите пример сами). (при выполнении задания можете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy-yazyk/razlichenie-ne-i-ni-chastica-ni- soyuz-ni-ni-pristavka-ni или QR-кодом: 101 ) Различие между НЕ и НИ Чем является НЕ Чем является НИ 1. Сколько кобылке ни прыгать, а быть в хомуте 2. Ни тпру, ни ну 3. Наш Касьян на что ни взглянет, все вянет 4. Кабы на коня не спотычка, так ему бы и цены не было 101 Различие между НЕ и НИ Чем является НЕ Чем является НИ 1. Сколько кобылке ни прыгать, а быть в хомуте 2. Ни тпру, ни ну 3. Наш Касьян на что ни взглянет, все вянет 4. Кабы на коня не спотычка, так ему бы и цены не было 101 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 5. Нашему барану ни в чем нет талану 6. Кому ничего, а нам не больше того 7. Где ворона ни летала, а к ястребу в когти попала 8. Журить, бранить есть кому, а жаловать некому 9. Пропал рублик ни за копейку 10. Кабы все знал, так бы не погибал 11. От сумы да от тюрьмы никто не отрекайся 12. Что ни хвать, то ерш да еж 13. Счастье без ума нипочем 14. Не родись ни хорош, ни пригож, родись счастлив 15. Кому полтина, а кому и ни алтына 12. Что ни хвать, то ерш да еж 13. Счастье без ума нипочем 14. Не родись ни хорош, ни пригож, родись счастлив 15. Кому полтина, а кому и ни алтына 16. Каков ни будь грозен день, а вечер настанет 17. Богачи едят калачи, да не спят ни в день, ни в ночи; бедняк чего ни хлебанет, да заснет 18. Плох базар, коли хлеба купить не на что 18. Плох базар, коли хлеба купить не на что 19. Алтын Мартыну – ни сапог подшить, ни скобы подковать 20. Грех не беда – на ком не живет 21. Ни хитру, ни горазду, ни убогу, ни богату суда божьего не миновать 2. Выпишите все слова, где количество букв меньше количества звуков. Объясните это неравенство 3. Вспомните, каким членом предложения в основном являются краткие прилагательные? Найдите в упражнении краткие прилага- тельные и подчеркните их как члены предложения. Что вы заме- тили? Изменяются ли в современном русском языке краткие прила- гательные по падежам? Как вы думаете, всегда ли так было? Аргу- ментируйте свой ответ, опираясь на предложения этого упражнения. ру , р р у р 4. Выпишите все сложноподчиненные предложения и постройте их схемы. 5. Что такое талан и его производное бесталанный? Имеют ли эти слова отношение к таланту и талантливому? Упражнение 2 1. Прочитайте пословицы и поговорки. Какие из них показа- лись вам наиболее актуальными? Почему? у у 1. Так врет, что (н…) себе, (н…) людям передышки (н…) дае 2 (Н ) б ( ) ( ) д ( ) 1. Так врет, что (н…) себе, (н…) людям передышки (н…) дает 2. (Н…) себе (н…) гож, (н…) людям (н…) пригож 1. Так врет, что (н…) себе, (н…) людям передышки (н…) дает 2. (Н…) себе (н…) гож, (н…) людям (н…) пригож 102 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 3. Добрую жену взять – (н…) скуки, (н..) горя не знать 4. Бабий язык, куда (н…) завались, достанет 5. Живи так, чтоб (н…) от Бога греха, (н…) от людей стыда 6. Хоть денег (н…) гроша, да походка хороша 7. У нашего свата (н…) друга, (н…) брата 8. У доброй девки (н…) ушей, (н…) глаз 8. У доброй девки (н…) ушей, (н…) глаз 9. Как (н…) заплетай косу, девка, (н…) миновать, что распле- ать 10. Глуп совсем, кто (н…) знается (н…) с кем 11. Куда (н…) поехал, а мимо (н…) проехал 12. Сколько (н…) думай, а лучше хлеба-соли (н…) придумаешь 13. В гости едет, а из гостей (н…) с места 14. Где любят, тут (н…) учащай; а где (н…) любят, туда (н…) ногой 15. Каков (н…) есть, а просит есть 16. Тощий живот (н…) в пляску, (н…) в работу 17. Дыры в глотке (н…) чем (н…) зачинишь 18. Наг поле перейдет, а голоден (н…) с места 2. Выпишите сначала простые предложения, потом сложные определите их виды. Скобки раскройте, буквы вставьте. 3. Разберите по составу все глаголы в повелительном накло- нении 4. До XV века звуки [ж] и [ш] были мягкими. Докажите это, опираясь на слова данного упражнения 5. Один англичанин, мистер Джонс, читая сборник русских по- словиц В.И. Даля, любил делать скетчи. Одна поговорка его очень заинтересовала, он долго думал над ней и сделал такой рисунок: Какая поговорка заинтересовала мистера Джонса? Правильно ли любитель русского фольклора передал ее суть? Почему? 103 Упражнение 3 Упражнение 3 р 1. Спишите, раскрывая скобки и вставляя пропущенные буквы. Объясните свой выбор 1. Спишите, раскрывая скобки и вставляя пропущенные буквы. Объясните свой выбор 1. Спишите, раскрывая скобки и вставляя пропущенные буквы. Объясните свой выбор 1. На наше трепало что б (н…) попало – все мнет 2. (Н…) росиночки, (н…) порошиночки во рту (н…) было 3. Хлеба (н..) куска, так и в тереме тоска 4. Брюхо – глухо: словом (н…) уймешь: что (н…) говори, а корми 4. Брюхо – глухо: словом (н…) уймешь: что (н…) говори, а корми 5. Как (н…) тесно (на брюхе), а все есть место. 6. Хлеба (н…) куска, так и в тереме тоска, а хлеба край, так и под елью рай. 6. Хлеба (н…) куска, так и в тереме тоска, а хлеба край, так и под елью рай. 7. (Н…) казни, бог, (н…)чем, как капуста (н…) с чем. 8. Чем бы дитя (н…)тешилось, лишь бы (н…)плакало. 9. Худо в карты играть, а (н…)мастей, (н…)козырей (н…)знать 10. Гусаром игры (н…)заканчивать. 11. На небо (н…)влезешь, под землю (н…)уйдешь. 12. Как (н…)толкуй, а бог всех больше. 13. (Н…)воля чего (н…) делает! 13. (Н…)воля чего (н…) делает! 14. Во всем доля, да воли (н..) в чем. 15. Нищий на нищем (н…)ищет. 15. Нищий на нищем (н…)ищет. 16. Рад бы горстью, да нет (н…) щепоти. 17. Как (н…)вертись собака, а хвост позади. 18. Как (н…)мойся, белее снегу (н…)будешь. 19. Как (н…)ворочай: одно одного короче. 20. Сколько с быком (н…)биться, а молока от него (н…)до- биться. 20. Сколько с быком (н…)биться, а молока от него (н…)до- биться. 21. Который палец (н…)куси – все больно. 22. (Н…) мытьем, так катаньем. 23. Кто (н…)поп, тот и батька. 23. Кто (н…)поп, тот и батька. 24. Ты тянешь, и он тянет: кто кого (н…)перетянет, а обоим падать 25. Упрямый, что лукавый: (н…)богу свеча, (н…) черту ко- черга. р 26. Когда солдат палки не боится, (н …)в строй, (н…) в дело не годится. 26. Когда солдат палки не боится, (н …)в строй, (н…) в дело не годится. 2. В каком значении употреблено выделенное слово в посло- вице Хлеба (н…) куска, так и в тереме тоска, а хлеба край, так и 104 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 104 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации под елью рай. Приведите примеры подобного употребления имен существительных в значении наречий. под елью рай. Приведите примеры подобного употребления имен существительных в значении наречий. 3. Укажите в сложноподчиненных предложениях средства связи между главным и придаточным предложениями. Определите, что это: союзы или союзные слова? Назовите виды придаточных пред- ложений. 3. Укажите в сложноподчиненных предложениях средства связи между главным и придаточным предложениями. Определите, что это: союзы или союзные слова? Назовите виды придаточных пред- ложений. Чтобы вспомнить, чем отличаются союзы от союзных слов, мо- жете воспользоваться ссылкой: https://foxford.ru/wiki/russkiy- yazyk/podchinitelnye-soyuzy-i-soyuznye-slova-v- жете воспользоваться ссылкой: https://foxford.ru/w yazyk/podchinitelnye-soyuzy-i-soyuznye-slova-v- slozhnopodchinyonnyh-predlozheniyah или QR-кодом: slozhnopodchinyonnyh-predlozheniyah или QR-кодом: 4 Рассмотрите мем и найдите орфографическую ошибку Про 4. Рассмотрите мем и найдите орфографическую ошибку. Про- комментируйте ее: 4. Рассмотрите мем и найдите орфографическую ошибку. Про- комментируйте ее: 105 ру 105 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Упражнение 4 Упражнение 4 1. Рассмотрите алгоритм выбора НЕ и НИ с отрицательными ме- стоимениями и местоименными наречиями. Оформите алгоритм до конца. Впишите примеры, иллюстрирующие соответствующие пункты правила 1. Рассмотрите алгоритм выбора НЕ и НИ с отрицательными ме- стоимениями и местоименными наречиями. Оформите алгоритм до конца. Впишите примеры, иллюстрирующие соответствующие пункты правила НЕ и НИ с отрицательными местоимениями и наречиями НЕ и НИ с отрицательными местоимениями и наречиями 1. Поставьте ударение Падает Не падает НЕ Наречия Местоимения Всегда в одно слово Есть ли предлог 06 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию НЕ и НИ с отрицательными местоимениями и наречиями 1. Поставьте ударение Падает Не падает НЕ Наречия Местоимения Всегда в одно слово Есть ли предлог 1. Сидеть одному, как нет никого в дому 2. Идти было в гости, да никто не зовет 3. Есть о чем тужить, как не с кем рожь молотить 4. Чего нет, того негде взять 5. Верно служу – ни по чем не тужу 6. И то бывает, что ничего не бывает 7. Что плохо, что худо, что никуда не годится 8. Кулак не сласть, а без него – ни шасть 9. Все ну, да ну, а тпрукнуть-то и некому Не падает Падает Местоимения Наречия Всегда в одно слово Есть ли предлог 8. Кулак не сласть, а без него – ни шасть 9. Все ну, да ну, а тпрукнуть-то и некому 06 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 10. Крепкое молчанье ни в чем не ответ 10. Крепкое молчанье ни в чем не ответ 10. Крепкое молчанье ни в чем не ответ 11. Все в старостах будем – некому будет и шапки перед нами сымать 12. Живет – не тужит, никому не служит 2. Подчеркните все местоимения и местоименные наречия как члены предложения 3. Выпишите примеры слов (не менее трех) с нулевым оконча- нием, без окончания, состоящие только из одной морфемы 4. Выполните словообразовательный разбор слова тпрукнуть. Приведите примеры слов, образованных по такой же словообразо- вательной модели (уточнить, что такое словообразовательная мо- дель вы можете по ссылке: https://foxford.ru/wiki/russkiy- yazyk/slovoobrazovatelnaya-model или по QR-коду: 5. Прочитайте текст и заполните пропуски (одно из пропущен- ных слов встречается в пословицах этого упражнения, все осталь- ные пропущенные слова являются его однокоренными). Слова данного словообразовательного гнезда представлены в русском языке в нескольких фонетических вариантах. Этот корень есть в прилагательном _____________ (1) со значе- нием «имеющий приятный вкус, свойственный сахару или меду». Это прилагательное является заимствованным. А исконно русский вариант этого корня есть в устаревшем, не сохранившемся до наших дней прилагательном _________ (2) и в существительном мужского рода со значением «продукт из проросших и смолотых зерен хлебных злаков, употребляется при изготовлении пива, кваса, спиртных напитков, дрожжей» ________ (3). Этот корень есть в прилагательном _____________ (1) со значе- нием «имеющий приятный вкус, свойственный сахару или меду». Это прилагательное является заимствованным. А исконно русский вариант этого корня есть в устаревшем, не сохранившемся до наших дней прилагательном _________ (2) и в существительном мужского рода со значением «продукт из проросших и смолотых зерен хлебных злаков, употребляется при изготовлении пива, кваса, спиртных напитков, дрожжей» ________ (3). В форме среднего рода это прилагательное может субстантиви- роваться, то есть употребляться как существительное со значением «десерт, третье блюдо» ___________ (4). 107 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Однокоренные существительные, употребляющиеся, как пра- вило, во множественном числе, обозначают одно и то же: «конди- терские изделия». Одно существительное _____________(5) обще- употребительное, имеет тот же фонетический вариант корня, что и прилагательное, от которого образовано. А другое существитель- ное ___________ (6) чаще употребляется в разговорной речи. ___________ ( ) у р р р р О чрезмерно ласковом, приторно-нежном голосе скажут, что он (какой?) ___________ (7). О чрезмерно ласковом, приторно-нежном голосе скажут, что он (какой?) ___________ (7). Однокоренной глагол ______________ (8) мы используем, когда испытываем удовольствие от чего-либо, восторгаемся чем-нибудь, радуемся. А однокоренные наречия _____________ (9) употреб- ляем, когда поедим сытно, вкусно, с удовольствием или сделаем что-то до полного удовлетворения. Однокоренной глагол ______________ (8) мы используем, когда испытываем удовольствие от чего-либо, восторгаемся чем-нибудь, радуемся. А однокоренные наречия _____________ (9) употреб- ляем, когда поедим сытно, вкусно, с удовольствием или сделаем что-то до полного удовлетворения. Упражнение 5 Упражнение 5 1. К пословицам из левой колонки подберите синонимичные из правой колонки Ни то, ни се кипело, да и то пригорело Каков ни есть, а хочет есть Этого тебе видом не видать, слыхом не слыхать Гол, да не вор Денег ни гроша, да слава хороша Не сохранит Господь града, не сохранит ни стража, ни ограда Сколько ни мудри, а воли божьей не перемудришь Как ни ворочай, а все блин блином Как был квас, так не было вас; а как не стало ни квасины, так и вас разносило Не видать тебе этого, как своих ушей Каков ни будь урод, а хлеб несет в рот Есть кус, так гостя нет; нет ни корки, а гости с горки Лягушке волом не быть, сколько воды ни пить Ни то, ни се клевало, да и то сорвалось Куда ни кинь, всюду клин Сколько утка не бодрись, а лебедем не быть К пословицам из левой колонки подберите синонимичные из ой колонки 108 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 108 Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию 108 Часть 2. Трудные случаи русской орфографии и пунктуации Часть 2. Трудные случаи русской орфографии и пунктуации Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» 1. «Грамота.ру» Если вы хоть немного интересуетесь русским языком, то навер- няка слышали про этот сайт. Здесь можно задать любой вопрос по орфографии, пунктуации или стилистике «Справочному бюро», воспользоваться электронным словарём, а ещё почитать заметки и статьи лучших лингвистов. Из них вы узнаете, например, откуда появилось выражение «дойти до ручки» и есть ли связь между Тму- тараканью и тараканами. В разделе «Азбучные истины» редакция сайта развенчивает по- пулярные мифы о языке, а «Письмовник» поможет правильно со- ставить резюме и не ошибиться при склонении сложной фамилии. Если хочется немного взбодриться – на сайте можно проверить себя, написав интерактивный диктант: их здесь очень много! ВМЕСТО ЗАКЛЮЧЕНИЯ Предлагаем немного позаниматься: представляем для вас позна- вательные лекции, онлайн-курсы и сайты о русском языке, с кото- рыми точно не соскучишься! Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию Часть 2. Трудные случаи русской орфографии и пунктуации 2. Объясните написание частиц НЕ и НИ 3. Выпишите слова, словооизменение и словообразование ко- торых сопровождается историческим чередованием согласных и гласных (освежить свои знания по историческому чередованию вы можете, пройдя по ссылке: https://docs.google.com/document/ d/1PcKPShx6wW8bqHkfVNTa3kfvva86v9XB/edit или отсканировав QR-код: 3*. Выпишите слова, словооизменение и словообразование ко- торых сопровождается историческим чередованием согласных и гласных (освежить свои знания по историческому чередованию вы можете, пройдя по ссылке: https://docs.google.com/document/ d/1PcKPShx6wW8bqHkfVNTa3kfvva86v9XB/edit или отсканировав QR-код: 109 4. Онлайн-курс «Арзамаса» «Русский язык от „гой еси“ до „лол кек“» – так называется курс о русском языке на сайте издания «Арзамас». Здесь есть небольшой видеоролик об истории языка – с красивой графикой, картинками и закадровым голосом. Кроме того, сюда входит подкаст о языке, где эксперты обсуж- дают, почему нельзя всё-таки писать «жи-ши» с «Ы», сколько в России диалектов и как появились запрещённые русские корни (упс!). А в завершение авторы курса предлагают пройти тест «Пой- мёте ли вы древнерусских?». 3. Подкаст «Розенталь и Гильденстерн» 3. Подкаст «Розенталь и Гильденстерн» Тем, кому ближе аудиоформат, точно понравится этот научно- популярный подкаст о русском языке и лингвистике. Темы подкаста совершенно разные, но всегда интересные. В од- ном выпуске ведущие могут говорить с филологами и лингвистами об истории русской орфографии или лингвистической экспертизе, а в другом – рассуждать, имеют ли право на жизнь «авторки» и «блогерки». Новый эпизод выходит каждый понедельник. Даже если вы включите подкаст уже сегодня, можете быть уверены, что успеете прослушать все выпуски, пока сидите дома. Просто потому, что оторваться будет сложно! 2. Онлайн-курсы «Тотального диктанта» «Тотальный диктант» – ежегодная акция в форме доброволь- ного диктанта, которая уже охватила не только Россию, но и другие страны. Обычно диктант проходит очно, а тексты читают актёры, музыканты, писатели и другие известные люди. Здесь работает он- лайн-школа по подготовке к диктанту. Занятия проходят каждый четверг, а за пропущенные уроки можно не переживать: все они со- браны здесь в удобном видеоформате и в слайдах. Кроме того, на сайте можно пройти курс по истории русской ор- фографии со смешным названием «Мыш кродёться» или большой курс по орфографии и пунктуации русского языка «Никогда не пиши „ни когда“». А ещё здесь есть задания, придуманные специ- ально для иностранцев, которые изучают русский язык! 110 Вместо заключения 7. Приложение «Глазарий языка» «Глазарий языка» – приложение-словарь, где собраны неболь- шие статьи о структуре и истории русского языка, терминах и нор- мативном употреблении слов и выражений. Все статьи здесь удобно разбиты по блокам: фонетика, морфология, стилистика, правописание и так далее. 6. Лекции факультета гуманитарных наук ВШЭ На сайте гуманитарного факультета Высшей школы эконо- мики – множество видеолекций по лингвистике и филологии от вы- дающихся учёных. Здесь можно послушать лекции о «бандитской» речи 90-х и современном русском языке, речевом этикете. Из лекций можно узнать, когда и как в русском языке появилась буква «Ё», и о вариативности ударения. 8. «Грамотность на „Меле“» Каждую неделю в «Грамотности на „Меле“» разбирается новая тема. На сайте рассказывается про неправильные ударения в словах и критикуются знаменитости за ошибки в соцсетях, мы избавля- емся от канцеляризмов и лишних запятых, объясняем, что такое па- ронимы и как изменять сложные слова по падежам. Регулярно про- веряются читатели в весёлых тестах по грамотности! 112 5. «Культура письменной речи» «Культура письменной речи» – сайт о русском языке и литера- туре, где каждый найдёт для себя что-то полезное. Как пишут ав- торы проекта, «сайт создаёт группа энтузиастов» – это два препо- давателя русского языка и литературы и технический специалист. Как и на «Грамоте», можно задать вопрос в разделе «Справка», а школьники найдут на сайте всё о ЕГЭ и ОГЭ по русскому языку и литературе. Для учителей тоже есть целый раздел – с методиче- скими рекомендациями, ссылками на нужные законы и образова- тельные стандарты. А в «Зоне отдыха» можно почитать перлы из сочинений школьников и абитуриентов. 111 Федеральный проект «Патриотическое воспитание граждан Российской Федерации» национального проекта «Образование» СПИСОК ЛИТЕРАТУРЫ 1. Губанов А.Р. Обусловленность как лингвистическая онтоло- гия верхнего уровня / А.Р. Губанов, Ю.Н. Исаев, О.В. Свеклова, Г.Ф. Губанова // Вестник Чувашского университета. – 2017. – № 4. – С. 271-278. 1. Губанов А.Р. Обусловленность как лингвистическая онтоло- гия верхнего уровня / А.Р. Губанов, Ю.Н. Исаев, О.В. Свеклова, Г.Ф. Губанова // Вестник Чувашского университета. – 2017. – № 4. – С. 271-278. 1. Губанов А.Р. Обусловленность как лингвистическая онтоло- гия верхнего уровня / А.Р. Губанов, Ю.Н. Исаев, О.В. Свеклова, Г.Ф. Губанова // Вестник Чувашского университета. – 2017. – № 4. – С. 271-278. 2. Исаев Ю.Н. Анималистическая когниция. Метафора и срав- нение / Ю.Н. Исаев, Н.В. Николаева, В.И. Сергеев. – Чебоксары, 2016. 2. Исаев Ю.Н. Анималистическая когниция. Метафора и срав- нение / Ю.Н. Исаев, Н.В. Николаева, В.И. Сергеев. – Чебоксары, 2016. 3. Исаев Ю.Н. Словообразовательный и семантический анализ флористической терминологии языков различных систем / Ю.Н. Исаев. – Чебоксары, 2010. 3. Исаев Ю.Н. Словообразовательный и семантический анализ флористической терминологии языков различных систем / Ю.Н. Исаев. – Чебоксары, 2010. 4. Исаев Ю.Н. Фитонимы как составная часть языковой кар- тины мира / Ю.Н. Исаев // Вестник Чувашского университета. – 2012. – № 1. – С. 229-231. 4. Исаев Ю.Н. Фитонимы как составная часть языковой кар- тины мира / Ю.Н. Исаев // Вестник Чувашского университета. – 2012. – № 1. – С. 229-231. 5. Исаев Ю.Н. Язык: внутренняя структура и функционирова- ние / Ю.Н. Исаев. – Чебоксары, 1999. 5. Исаев Ю.Н. Язык: внутренняя структура и функционирова- ние / Ю.Н. Исаев. – Чебоксары, 1999. 6. Мурзина Ж.В. Анализ эффективности форматов проведения Всероссийской научно-практической конференции «Тенденции развития образования: педагог, образовательная организация, об- щество – 2018» / Ж.В. Мурзина // Тенденции развития образования: педагог, образовательная организация, общество – 2018: матери- алы Всероссийской научно-практической конференции / под ред. Ж.В. Мурзиной. – 2018. – С. 11-13. 7. Мурзина Ж.В. Итоги реализации федерального проекта по ранней профессиональной ориентации учащихся 6–11-х классов общеобразовательных организаций Чувашской Республики «Билет в будущее» / Ж.В. Мурзина, Л.А. Степанова, А.В. Штыкова // Об- разование и педагогика: теория, методология, опыт. – Чебоксары, 2020. – С. 8-30. 8. Студенникова С.Л. Патриотическое воспитание молодежи - залог успешного развития общества и государства [Электронный ресурс]. – Режим доступа: https://cyberleninka.ru/article/n/ patrioticheskoe-vospitanie-molodezhi-zalog-uspeshnogo-razvitiya- obschestva-i-gosudarstva 8. Студенникова С.Л. Патриотическое воспитание молодежи - залог успешного развития общества и государства [Электронный ресурс]. – Режим доступа: https://cyberleninka.ru/article/n/ patrioticheskoe-vospitanie-molodezhi-zalog-uspeshnogo-razvitiya- obschestva-i-gosudarstva 113 Для заметок 114 Для заметок 115 Учебное издание Учебное издание Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию. Опыт проекта: Интерактивный парк «ЭТНО-Хакатон» Учебно-методическое пособие Ответственные редакторы Н.М. Гурьева, Т.Н. Николаева Компьютерная верстка Л.С. Миронова Подписано в печать 08.12.2021 г. Дата выхода издания в свет 10.12.2021 г. Формат 60×84/16. Бумага офсетная. Печать офсетная. Гарнитура Times. Усл. печ. л. 6,7425. Заказ К-927. Тираж 500 экз. Издательский дом «Среда» 428005, Чебоксары, Гражданская, 75, офис 12 +7 (8352) 655-731 info@phsreda.com https://phsreda.com Отпечатано в Студии печати «Максимум» 428005, Чебоксары, Гражданская, 75 +7 (8352) 655-047 info@maksimum21.ru www.maksimum21.ru Отпечатано в Студии печати «Максимум» 428005, Чебоксары, Гражданская, 75 +7 (8352) 655-047 info@maksimum21.ru www.maksimum21.ru Популяризация традиционной культуры народов Российской Федерации как методическая база для создания авторской программы педагога по нравственно-патриотическому воспитанию. Опыт проекта: Интерактивный парк «ЭТНО-Хакатон» Ответственные редакторы Н.М. Гурьева, Т.Н. Николаева Компьютерная верстка Л.С. Миронова Издательский дом «Среда» 428005, Чебоксары, Гражданская, 75, офис 12 +7 (8352) 655-731 info@phsreda.com https://phsreda.com Отпечатано в Студии печати «Максимум» 428005, Чебоксары, Гражданская, 75 +7 (8352) 655-047 info@maksimum21.ru www.maksimum21.ru
https://openalex.org/W4220834631	https://nottingham-repository.worktribe.com/file/7566146/1/The%20Formulation%20of%20the%20Quadratic%20Failure%20Criterion%20for%20Transversely%20Isotropic%20Materials%3A%20Mathematical%20and%20Logical%20Considerations	English	null	The Formulation of the Quadratic Failure Criterion for Transversely Isotropic Materials: Mathematical and Logical Considerations	Journal of composites science	2,022	cc-by	11,377	Citation: Li, S.; Xu, M.; Sitnikova, E. The Formulation of the Quadratic Failure Criterion for Transversely Isotropic Materials: Mathematical and Logical Considerations. J. Compos. Sci. 2022, 6, 82. https:// doi.org/10.3390/jcs6030082 Academic Editor: Stelios K. Georgantzinos Received: 8 February 2022 Accepted: 1 March 2022 Published: 7 March 2022 Keywords: quadratic failure function; Tsai–Wu criterion; failure envelope; strength; transverse shear strengths; transversely isotropic materials Citation: Li, S.; Xu, M.; Sitnikova, E. The Formulation of the Quadratic Failure Criterion for Transversely Isotropic Materials: Mathematical and Logical Considerations. J. Compos. Sci. 2022, 6, 82. https:// doi.org/10.3390/jcs6030082 Article The Formulation of the Quadratic Failure Criterion for Transversely Isotropic Materials: Mathematical and Logical Considerations Shuguang Li * , Mingming Xu and Elena Sitnikova Shuguang Li * , Mingming Xu and Elena Sitnikova Faculty of Engineering, University of Nottingham, Nottingham NG8 1BB, UK; mingming.xu@nottingham.ac.uk (M.X.); elena.sitnikova@nottingham.ac.uk (E.S.) * Correspondence: shuguang.li@nottingham.ac.uk Abstract: The quadratic function of the original Tsai–Wu failure criterion for transversely isotropic materials is re-examined in this paper. According to analytic geometry, two of the troublesome coefﬁ- cients associated with the interactive terms—one between in-plane direct stresses and one between transverse direct stresses—can be determined based on mathematical and logical considerations. The analysis of the nature of the quadratic failure function in the context of analytic geometry enhances the consistency of the failure criterion based on it. It also reveals useful physical relationships as intrinsic properties of the quadratic failure function. Two clear statements can be drawn as the outcomes of the present investigation. Firstly, to maintain its basic consistency, a failure criterion based on a single quadratic failure function can only accommodate ﬁve independent strength properties, viz. the tensile and compressive strengths in the directions along ﬁbres and transverse to ﬁbres, and the in-plane shear strength. Secondly, amongst the three transverse strengths—tensile, compressive and shear—only two are independent. 1. Background Ever since Tsai and Wu proposed their failure criterion [1] based on a quadratic failure function, the polynomials employed to construct failure criteria have been mostly kept to the second order. Whilst one could legitimately argue for higher orders of polynomials along the line, as was suggested in [2], or to partition the failure function according to the failure modes, as was attempted in [3], many are on the verge of abandoning all such theories developed on a phenomenological basis, having been discouraged by their unsatisfactory performance. However, a sensible question does not seem to have ever been asked: ‘Has the quadratic failure function been understood well enough before increasing or abandoning any further efforts along this line of development?’ It is true that quadratic functions are well understood in analytic geometry as a branch of mathematics, and yet it will be revealed in the present paper that the established mathematics has not been properly utilised in the important subject of composite failure. The well-established conclusions of analytic geometry have simply not been appropriately recognised in the formulations of failure criteria for composites based on quadratic functions. Therefore, before this aspect has been appropriately examined and evaluated, it would surely be a premature decision to propose more complicated arrangements for the failure function or to ditch every account of phenomenological criteria based on macroscopic stresses or strains completely. Academic Editor: Stelios K. Georgantzinos Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). A thorough re-examination of popular existing failure criteria for modern compos- ites [4] by two of the authors of this paper suggested that whilst the predictions of these theories fell short of the expectation of the users, none of them had been formulated consis- tently enough at a basic level. Phenomenological approaches based on macroscopic stresses https://www.mdpi.com/journal/jcs J. Compos. Sci. 2022, 6, 82. https://doi.org/10.3390/jcs6030082 J. Compos. Sci. 2022, 6, 82 2 of 17 2 of 17 or strains are bound to have their limitations. However, blaming the phenomenological nature alone for the deﬁciencies in the existing criteria is a far too quick and easy escape. 1. Background Once one moves into the micromechanics of composites, there will be many more compli- cations involved due to the consideration of both mechanics and materials, let alone the necessary mathematics and logic. As an example, representative volume elements and unit cells as basic and also necessary means of micromechanical investigations were subjected to a serious and systematic examination in [5]. It was revealed that their formulation and implementation are full of pitfalls and any oversight leads to an erroneous interpretation of genuine material behaviours. If the mathematics and logic cannot be sorted out at a macroscopic level, where there are only six macroscopic stresses to deal with, it is hard to imagine that the study of composites failure can be accomplished at a microscopic scale, where one can easily be swamped with new parameters, sometimes of little physical meaning. Whilst researchers should not be discouraged from undertaking explorations into micromechanics, it is worthwhile to re-examine the aspect of the rationality of conventional and phenomenological failure criteria, not aiming to generate any new criterion, but to tidy up the basic mathematics and logic underlying existing theories and to eliminate any irrational elements before presenting them on a footing of a basic level of consistency. By then, one could make an objective decision on how applicable such rationally formulated failure criteria are and how far such a phenomenological theory can be used reliably. This may even lay a ﬁrm basis for more sophisticated developments, as mentioned above. 2. A Critical Review of the Tsai–Wu Criterion Restrictions were introduced in [1] on the ranges of their values as: 2 F23, F13 and F12 have been left somewhat loose. Restrictions were introduced in [1] on the ranges of their values as: F2 23 < F22F33 F2 13 < F11F33 F2 12 < F11F22. (3) (3) Given the inequality form of (3), these restrictions are insufﬁcient to fully quantify F23, F13 and F12. However, at this point, certain mathematically inaccurate statements were made in [1], and they need to be corrected, since the clariﬁcation of this matter will eventually lead to the development that will be presented in this paper. In general, the failure envelope is deﬁned in a six-dimensional stress space. If (1) is re-written as: F1σ1 + F2σ2 + F3σ3 + F11σ2 1 + F22σ2 2 + F33σ2 3 + 2F23σ2σ3 + 2F13σ1σ3 + 2F12σ1σ2 = 1 − F44τ2 23 + F55τ2 13 + F66τ2 12 = K (4) (4) it can be considered that the presence of any non-vanishing shear stress can be equivalently considered as a reduction in the value of the constant term in (1) from 1 to a smaller value, denoted as K, which could be negative under some stress states (K = 0 implies that failure is due to shear stresses alone). The nature of the failure function can be sufﬁciently comprehensively discussed within the three-dimensional subspace of direct stresses. p y p It was claimed in [1] that the conditions of (3) were to keep the failure envelope closed on the basis that materials should exhibit ﬁnite strengths. This was misleading on two accounts. Firstly, the conditions of (3) do not ensure the closeness of the failure envelope. According to analytic geometry [9], each of the three conditions ensures that the intersection of the failure envelope with the respective coordinate plane as a locus is an ellipse. Take coordinate plane 1–2 for example. In this case, the locus is obtained when σ3 = 0 as: F1σ1 + F2σ2 + F11σ2 1 + F22σ2 2 + 2F12σ1σ2 = 1 −F66τ2 12. (5) (5) The condition F2 12 < F11F22 ensures that (5) is an ellipse in the 1–2 plane [9]. The same argument can be made for the remaining two conditions in (3). 2. A Critical Review of the Tsai–Wu Criterion A reasonably comprehensive literature review on the subject of the Tsai–Wu failure criterion is given in a recent publication [6] and will be waived in this paper. The criterion was originally proposed as [1]: F1σ1 + F2σ2 + F3σ3 + F11σ2 1 + F22σ2 2 + F33σ2 3 + 2F23σ2σ3 + 2F13σ1σ3 + 2F12σ1σ2 +F44τ2 23 + F55τ2 13 + F66τ2 12 −1 = 0 (1) (1) in the context of generally orthotropic materials. The left-hand side is a quadratic failure function. A careful examination of (1) suggests that many terms, such as linear and bilinear expressions of shear stresses, are missing from the failure function for it to be a complete quadratic polynomial of six stress components. These terms have been dropped because the failure function must be even for each shear stress as a basic requirement of objectivity. Therefore, the failure function in the form of a quadratic function as given in (1) is complete as far as homogeneous orthotropic materials are concerned. The coefﬁcients F1, F11, F44, etc., in (1) should be determined by the strengths of the material obtained through standard or special experiments. In fact, most of them have been explicitly expressed in terms of conventional strengths. For instance, those coefﬁcients that are of particular relevance to the present discussion are listed below as: F1 = 1 σ∗ 1t − 1 σ∗ 1c , F2 = 1 σ∗ 2t − 1 σ∗ 2c , F11 = 1 σ∗ 1tσ∗ 1c , F22 = 1 σ∗ 2tσ∗ 2c , F44 = 1 (τ∗ 23) 2 and F66 = 1 (τ∗ 12) 2 (2) (2) where σ∗ 1t and σ∗ 1c are the tensile and compressive strengths of the material in the direction along the ﬁbres, respectively; σ∗ 2t and σ∗ 2c are the tensile and compressive strengths of the material in the direction transverse to the ﬁbres, respectively; and τ∗ 23 and τ∗ 12 are the shear strength transverse and parallel to the ﬁbres, respectively. These conventional strength properties of the composite should be obtained when appropriate specimens are loaded under uniaxial stress states or pure shear stress states in their material’s principal axis according to available standards [7,8]. However, the coefﬁcients of the interactive terms J. Compos. Sci. 2022, 6, 82 3 of 17 F23, F13 and F12 have been left somewhat loose. 2. A Critical Review of the Tsai–Wu Criterion The precise physical interpretation of (3) is that strength is ﬁnite under any plane stress condition in each of the principal planes of the material, which is a reasonable observation amongst all available experimental data, as was also argued in [6]. However, the conditions of (3) do not exclude the possibility of the failure envelope being open in the 3D space. Consider a real composite, for example, T300/PR-319, which was one of the composites involved in WWFE-II [10] and was also employed in [6] as one of the cases studied. With F12 given as: F12 = −1 2 p F11F22 (6) (6) which is the same as that introduced in [11], and the measured transverse shear strength as τ∗ 23 = 45 MPa, the failure envelope (4) can be proven to be an ellipsoid in the subspace of direct stresses. It is well known that no strength properties can be practically obtained without experimental error, although clear indications of such errors are not usually pro- vided in the published data. A rare example of published raw data for strength properties can be found in [12], where the variability was as high as 30%, although the authors used a different type of composite (AS4/8552). Suppose that there was a 10% experimental error in τ∗ 23 and its value dropped to 40.5 MPa whilst satisfying (3) perfectly well. Then, the failure envelope would turn into an hourglass-like hyperboloid. As will become clear later in this paper, logically, the conditions in (3) serve as a set of necessary conditions for the failure envelope to be closed, whilst the sufﬁcient conditions provided later in this paper are more restrictive [9]. The second issue is that the claim made in [1] prohibited the openness of the failure envelope or any inﬁnite strength. On one hand, a claim of an inﬁnite strength under some speciﬁc conditions can be equally as scientiﬁcally unfounded as a claim of a ﬁnite strength J. Compos. Sci. 2022, 6, 82 4 of 17 4 of 17 under any condition; on the other hand, either claim could be a reasonable assumption under the appropriate considerations. If a material under a speciﬁc stress state could sustain a stress level much higher than its strength under any uniaxial stress state, it would not be unreasonable to assume the strength under that special condition was practically inﬁnite. 2. A Critical Review of the Tsai–Wu Criterion Realistically, the available means for testing materials under multiaxial stress states only allow for stress within a limited range. Without loading the material to failure, one cannot be certain whether the strength is inﬁnite. In view of this, a more meaningful question to ask is not whether a claim of ﬁnite or inﬁnite strength is correct, but whether a claim has been made consistently within the theoretical framework. y In [11], Tsai and Hahn introduced (6), and their justiﬁcation was that the Tsai–Wu criterion should reproduce the von Mises criterion for isotropic materials of equal tensile and compressive strengths. If so, the claim of a ﬁnite strength under all conditions would lead to a logical contradiction, because the failure envelope for the von Mises criterion is in fact open [13], since it is a cylinder in the space of three principal stresses intersecting a coordinate plane in closed ellipses. Its axis corresponds to the hydrostatic stress state under which the material is assumed to have inﬁnite strength. An attempt to reconcile the contradictive issues described above was made in [6]. Without violating the conditions in (3), it was concluded that an elliptic paraboloidal envelope offers a reasonable compromise for logical consistency. Mathematically, there exists a unique elliptic paraboloid sitting on the borderline between the ellipsoids and the elliptic hyperboloids. Employing an elliptic paraboloid as the failure envelope, given its open appearance, allows inﬁnite strength, but only under a unique stress ratio [6] in triaxial compression. There appears to be two ways to lock the failure envelope in an elliptic paraboloid. One method is to abandon (6) and to employ the expression of F12 as was obtained in [6]: F12 = −1 2 √ δ p F11F22 (7) δ = 4 −F44 F22 = 4 −σ∗ 2tσ∗ 2c τ∗ 23 2 . (8) (7) where where 3. The Quadratic Failure Function for Transversely Isotropic Materials 3. The Quadratic Failure Function for Transversely Isotropic Materials Similar to [6], as well as in [1,3,14–16], the present paper will be conﬁned only to trans- versely isotropic materials, which cover a large class of unidirectionally ﬁbre-reinforced composites. In this case, the transverse isotropy offers the following relationships: F2 = F3, F22 = F33, F13 = F12, F44 = 2(F22 −F23) or F23 = F22 −1 2 F44 and F55 = F66. (9) (9) The Tsai–Wu criterion reduces to [1,6,11]: The Tsai–Wu criterion reduces to [1,6,11]: F1σ1 + F2(σ2 + σ3) + F11σ2 1 + F22 σ2 2 + σ2 3 + 2F23σ2σ3 + 2F12σ1(σ3 + σ2) +2(F22 −F23)τ2 23 + F66 τ2 13 + τ2 12 −1 = 0 (10) (10) and there are seven seemingly independent coefﬁcients F1, F2, F11, F22, F23 (or F44), F12 and F66 to be determined from experimental data. The relationships in (9) are a natural consequence of the transverse isotropy. However, a follow-up question would have a far-reaching effect. The third relationship in (9) suggests that F13 and F12 make equal contributions to the failure function as material properties, regardless of the stress state. The justiﬁcation for this is, of course, the identical strength characteristics of the material in directions 2 and 3. In general, given the anisotropy of the material, F12 and F23 are not expected to be the same. However, should the difference between F12 and F23 be quantitatively associated with the degree of anisotropy? This is a question that has never been asked properly in the literature to the best of our knowledge, let alone has it been addressed. An attempt will be made and justiﬁed in this paper. Having achieved what was presented in [6], the objective of the present paper is to address some intrinsic relationships in the quadratic failure function that will have pro- found implications for the understanding of the strength of transversely isotropic materials in general. The consequence is that a quadratic failure function can only accommodate ﬁve independent strength properties, and two out of the seven coefﬁcients as involved in (10) can be expressed in terms of the remaining ﬁve in general, provided that the material is homogeneous and transversely isotropic, and the failure function is a single quadratic function. where δ = 4 −F44 F22 = 4 −σ∗ 2tσ∗ 2c τ∗ 23 2 . (8) (8) This leads to a method of determining F12 in terms of known strength properties, eliminating the need for an additional strength measured under combined stress states. However, a critical drawback of this approach is that δ as deﬁned in (8) can be negative for some materials, as shown in [6]; based on this, it was concluded that the Tsai–Wu criterion was inapplicable to these materials, which remained an unsatisfactory aspect of this development. p Alternatively, an elliptic paraboloidal failure envelope can also be achieved by the slight modiﬁcation of transverse strengths, given the wide variability in experimental data. This consideration results in the development that will be presented in this paper, leading to the determination of the transverse shear strength whilst delivering the necessary logical consistency. There have been a number of other approaches proposed to determine the transverse shear strength from the transverse tensile and/or compressive strengths, e.g., in [14–16], which will be discussed later in this paper, as well as those summarised in [6]. It is now clear that the conditions in (3) should be satisﬁed; however, the satisfaction of (3) is not enough. It should also be noted that the three conditions in (3) are equally critical in term of necessity, because violating any of them will allow an open locus in the respective coordinate plane, implying an inﬁnite strength under a plane stress condition. The conditions in (3) are also equally restrictive for F23, F13, and F12 in specifying their ranges. J. Compos. Sci. 2022, 6, 82 5 of 17 4. Characteristics of the Quadratic Failure Function for Transversely Isotropic Materials 4.1. Necessary and Sufﬁcient Conditions for an Elliptic Paraboloid 4.1. Necessary and Sufﬁcient Conditions for an Elliptic Paraboloid As was argued in Section 2, as well as in [6], the failure envelope should be allowed to be an elliptic paraboloid to maintain a basic level of consistency in the logic behind the Tsai–Wu criterion. The failure envelope for transversely isotropic materials in the three-dimensional subspace of direct stresses is given as: F1σ1 + F2(σ2 + σ3) + F11σ2 1 + F22 σ2 2 + σ2 3 + 2F23σ2σ3 +2F12σ1(σ3 + σ2) = K. (11) (11) This deﬁnes a quadric surface in the σ1 −σ2 −σ3 space in general, of which the following invariants can be introduced [9]. A = F11 F12 F12 1 2 F1 F12 F22 F23 1 2 F2 F12 F23 F22 1 2 F2 1 2 F1 1 2 F2 1 2 F2 −K (12) J = F11 F12 F12 F22 + F22 F23 F23 F22 + F22 F12 F12 F11 = 2 F11F22 −F2 12 + F2 22 −F2 23 (13) A = F11 F12 F12 1 2 F1 F12 F22 F23 1 2 F2 F12 F23 F22 1 2 F2 1 2 F1 1 2 F2 1 2 F2 −K (12) (12) J = F11 F12 F12 F22 + F22 F23 F23 F22 + F22 F12 F12 F11 = 2 F11F22 −F2 12 + F2 22 −F2 23 (13) (13) J. Compos. Sci. 2022, 6, 82 6 of 17 D = F11 F12 F12 F12 F22 F23 F12 F23 F22 = F11F222 + 2F23F122 −2F22F122 −F11F232 = (F22 −F23)∆ (14) (14) where ∆= F11(F22 + F23) −2F2 12. (15) (15) In general, quadric surfaces can be a range of different shapes, and for complete categorisation a few more invariants [9] are required. The above conditions are sufﬁ- cient to determine if the surface is an elliptic paraboloid. Most quadric surfaces, such as paraboloidal cylinders and various hyperboloids, lead to physically impermissible be- haviours, e.g., possible inﬁnite strength under a plane stress condition, or inﬁnite strengths at an inﬁnite number of different stress conditions, some involving tensile stresses, etc., as elaborated in [6]. For instance, the elliptic cylinder deﬁnes the failure envelope for a class of materials that show inﬁnite strength under a triaxial stress state at a certain stress ratio both in tension and compression—a typical ductile behaviour. 4. Characteristics of the Quadratic Failure Function for Transversely Isotropic Materials It is inapplicable to modern composites, which are mostly brittle, i.e., the material failure characteristics show signiﬁcant differences under tension and compression. The permissible quadric surfaces are limited to an ellipsoid and an elliptic paraboloid. As argued earlier and will be further elaborated later, an ellipsoid as a failure envelope for (11) cannot be the right choice. The elliptic paraboloid, which is open at only one end and allows inﬁnite strength only at one speciﬁc triaxial compressive stress ratio, remains the only viable choice. The conditions that are both necessary and sufﬁcient for a quadratic function to be an elliptic paraboloid are [9]: (16) A < 0, J > 0 and D = 0. (16) Whilst the inequalities are discriminants, the satisfaction of which will be shown later, the equation, i.e., D = 0, offers an additional relationship purely from a mathematical point of view, which will be exploited below. Given (14), the ﬁrst condition in (3) rules out the possibility of F22 −F23 = 0; therefore, D = 0 is equivalent to ∆= 0, i.e., F2 12 = 1 2 F11(F22 + F23) (17) (17) which agrees with what was obtained in [6]. This means that F23 and F12 are not both independent. Given the relationship between F23 and F12 shown in (17), one could be tempted to shed the remaining responsibility of the full determination of F23 and F12 to experiments, i.e., to have one of these values measured experimentally. Unfortunately, there are no reliable experimental means to directly determine either of them. The best one can do is to determine the transverse shear strength, from which F44 can be evaluated, and then obtain F23 using the fourth relationship in (9). Then, F12 can be determined using (17), which is in line with [6]; however, as discussed in Section 2, this approach is inefﬁcient. On the other hand, the available resources in terms of mathematics and logic have not been exhausted. The consideration of mathematical and logical consistency will offer another much-desired condition, and will be pursued in the next subsection. 4.2. Determination of F12 and F23 4.2. Determination of F12 and F23 Equation (17) shows F12 as a function of F23, which is a parabola on the F23-F12 plane, as sketched in Figure 1. The domain of the function can be obtained from the ﬁrst condition in (3). For transversely isotropic materials, it becomes: F2 23 < F2 22. (18) (18) In regard to the domain, the range of the function happens to coincide with the third condition in (3), i.e., F2 12 < F11F22, given that F11 and F22 are both positive and, hence, F23 < F22. The permissible values of F23 and F12 can only be within the green rectangle J. Compos. Sci. 2022, 6, 82 7 of 17 abola, to be 7 of 17 abola, to be shown in Figure 1, with the parabola passing its two corners on the right and touching its side on the left. theorem [9], there exists an unbiased point where the failure criterion will be equally sen- sitive to the perturbations in F12 and F23. The question now is how to determine it logically. shown in Figure 1, with the parabola passing its two corners on the right and touching its side on the left. theorem [9], there exists an unbiased point where the failure criterion will be equally sen- sitive to the perturbations in F12 and F23. The question now is how to determine it logically. Figure 1. The relationship between F12 and F23 and their permissible ranges. Figure 1. The relationship between F12 and F23 and their permissible ranges. Figure 1 The relationship between F12 and F23 and their permissible ranges Figure 1. The relationship between F12 and F23 and their permissible ranges. The parabola shown in Figure 1 will be subject to further examination, as its intricacy has yet to be decoded. F12 as a function of F23 is apparently not single valued. The top and bottom halves of the parabola both provide a single valued branch. Following the relevant discussion in [6] on the sense of F12, for the quadratic form (11) to be an elliptic paraboloid with an opening on the triaxial compression side, the value of F12 must be negative; there- fore, it is the lower half of the parabola in Figure 1 that should be considered as the per- missible branch for p to fall upon. 4.2. Determination of F12 and F23 For this branch, the legitimate range of F12 is (0, −ඥܨଵଵܨଶଶ) as F23 varies within its range of (–F22, F22), covering the zone shaded in Figure 1. Both F12 and F23 can now be normalised with respect to the breadths of their single valued ranges as follows: ܨ෨ଵଶ= ிభమ ඥிభభிమమ and ܨ෨ଶଷ= ிమయ ଶிమమ. (19) ܨ෨ଵଶ varies from 0 to –1 over a non-dimensional unit distance, while ܨ෨ଶଷ takes its value from −1/2 to 1/2 also covering a non dimensional unit distance Thus both F12 and F23 are Any point on the parabola in Figure 1 satisﬁes (17). To determine F12 and F23 com- pletely means to ﬁx a point on the parabola, referred to as point P. This requires one additional consideration that can be obtained by examining (17) in the context of its physi- cal implications. The coefﬁcients F12 and F23 are expressed one way or another in terms of strength properties, which are obtained experimentally; hence, errors are inevitable. Any perturbation due to an experimental error in one of the coefﬁcients will have to be met by a variation in the other in order to keep (17) satisﬁed. If P is selected close to the tip of the parabola, where dF12/dF23 approaches inﬁnity, it would require a large variation in F12 to correct a small perturbation in F23, making the failure criterion extremely sensitive to small errors in the evaluation of F23. Similarly, if P is placed close to the open end of the parabola, although the derivative is neither inﬁnite nor zero, the failure criterion is bound to be more sensitive to F12 than to F23. Between these two extremes, according to the mean-value theorem [9], there exists an unbiased point where the failure criterion will be equally sensitive to the perturbations in F12 and F23. The question now is how to determine it logically. from −1/2 to 1/2, also covering a non-dimensional unit distance. Thus, both F12 and F23 are The parabola shown in Figure 1 will be subject to further examination, as its intricacy has yet to be decoded. F12 as a function of F23 is apparently not single valued. The top and bottom halves of the parabola both provide a single valued branch. 4.2. Determination of F12 and F23 Following the relevant discussion in [6] on the sense of F12, for the quadratic form (11) to be an elliptic paraboloid with an opening on the triaxial compression side, the value of F12 must be negative; therefore, it is the lower half of the parabola in Figure 1 that should be considered as the permissible branch for p to fall upon. For this branch, the legitimate range of F12 is (0, −√F11F22) as F23 varies within its range of (−F22, F22), covering the zone shaded in Figure 1. Both F12 and F23 can now be normalised with respect to the breadths of their single valued ranges as follows: eF12 = F12 √F11F22 and eF23 = F23 2F22 . (19) (19) eF12 varies from 0 to –1 over a non-dimensional unit distance, while eF23 takes its value from −1/2 to 1/2, also covering a non-dimensional unit distance. Thus, both F12 and F23 are J. Compos. Sci. 2022, 6, 82 8 of 17 normalised in a consistent and unbiased manner. Accordingly, the expression of ∆as given in (15) can also be normalised as: normalised in a consistent and unbiased manner. Accordingly, the expression of ∆as given in (15) can also be normalised as: e∆= ∆ 2F11F22 = 1 2 + eF23 −eF2 12. (20) (20) For e∆to be equally sensitive to eF12 and eF23 in their unnormalized form, it is reasonable to expect that: For e∆to be equally sensitive to eF12 and eF23 in their unnormalized form, it is reasonable to expect that: e e ∂e∆ ∂eF12 = ∂e∆ ∂eF23 . (21) (21) From (20), one obtains: ∂e∆ ∂eF12 = −2eF12 and ∂e∆ ∂eF23 = 1 (22) (22) Thus, (21) leads to: eF12 = −1 2 or F12 = −1 2 p F11F22 as given in (6). (23) (23) Since ∆= 0, its nondimensionalised form e∆as deﬁned in (20) should also vanish. Given (23), the relationship in (17) leads to: Since ∆= 0, its nondimensionalised form e∆as deﬁned in (20) should also vanish. Given (23), the relationship in (17) leads to: eF23 = −1 4 or F23 = −1 2 F22. (24) (24) Thus, both F12 and F23 are determined as shown above. Whilst the relationship in (17) represents a rigorous consideration from analytic geometry, the relationship in (21) results from the logical consideration of the unbiased sensitivity of the failure criterion to F12 and F23. 4.2. Determination of F12 and F23 The normalisations made to F12 and F23 in (19) ensure that they can be compared as like with like. From (24), given the fourth condition of (9) and the expressions of F44 and F22 in (2), a natural consequence is: F44 = 2(F22 −F23) = 3F22 or τ∗ 23 = r σ∗ 2tσ∗ 2c 3 . (25) (25) This suggests that amongst the three transverse strengths—tensile, compressive and shear—of a transversely isotropic material, only two are independent as long as the failure criterion is based on a single quadratic function. Therefore, based on the elaboration presented above, the fully rationalised Tsai–Wu criterion can be delivered as follows: F1σ1 + F2(σ2 + σ3) + F11σ2 1 + F22 σ2 2 + σ2 3 −σ2σ3 + 3τ2 23 −√F11F22σ1(σ3 + σ2) + F66 τ2 13 + τ2 12 = 1 (26) (26) where the coefﬁcients F1, F2, F11, F22 and F66 are determined by conventional strength properties, as given in (2). It is clear that under triaxial compression at the following stress ratio: s σ1 : σ2 : σ3 = − s F22 F11 : −1 : −1 (27) (27) whilst all shear stresses vanish, criterion (26) will never be satisﬁed, i.e., failure will never take place, implying an inﬁnite strength under this special and unique stress state. In fact, the position holds even in presence of shear stresses. The triaxial compression at the ratio J. Compos. Sci. 2022, 6, 82 9 of 17 given in (27) has a reinforcing effect on the shear strengths. This can be seen clearly if (26) is re-written under this particular stress state as: 3F22τ2 23 + F66 τ2 13 + τ2 12 = 1 + F1 s F22 F11 + 2F2 ! σ (28) (28) where σ is the magnitude of the transverse stresses (i.e., non-negative) of the triaxial compressive stress state. For 2D stress states in the 1–2 plane, (26) is simpliﬁed to: For 2D stress states in the 1–2 plane, (26) is simpliﬁed to: F1σ1 + F2σ2 + F11σ2 1 + F22σ2 2 − p F11F22σ1σ2 + F66τ2 12 = 1. (29) (29) An important observation to make is that to predict the failure of a material under a 3D stress state from (26), the same set of strength properties is required as that for a 2D stress state from (29). 4.3. Veriﬁcation and Discussion The point on the parabola given by (23) and (24) is special in multiple ways. It was argued above that it renders failure criterion (10) to be equally sensitive to F12 and F23 under the condition that the failure envelope is an elliptic paraboloid. The slope to the parabola at this point happens to be equal to the slope of the secant between the two extremes of the single valued branch concerned, i.e., the tip of the parabola and the bottom right end. The tangent and secant are marked in Figure 1 by solid and dashed red lines, respectively. Therefore, this is the point whose existence has been asserted by the mean- value theorem [9]. In order to interpret the results above and to verify the normalisations introduced in (19), all the terms in the failure criterion (10) are normalised as follows, which is similar to procedure used in [11]. eF1eσ1 + eF2(eσ2 + eσ3) + eσ2 1 + eσ2 2 + eσ2 3 −eσ2eσ3 −eσ1(eσ3 + eσ2) + eτ2 23 + eτ2 13 + eτ2 12 = 1 (30) where eσ1 = √F11σ1, eσ2 = √F22σ2, eσ3 = √F22σ3, eτ23 = √F44τ23 = p 2(F22 −F23)τ23, eτ13 = √F66τ13, eτ12 = √F66τ12 eF1 = F1 √F11 , eF2 = F2 √F22 (31) eF1eσ1 + eF2(eσ2 + eσ3) + eσ2 1 + eσ2 2 + eσ2 3 −eσ2eσ3 −eσ1(eσ3 + eσ2) + eτ2 23 + eτ2 13 + eτ2 12 = 1 (30) (30) eσ1 = √F11σ1, eσ2 = √F22σ2, eσ3 = √F22σ3, eτ23 = √F44τ23 = p 2(F22 −F23)τ23, eτ13 = √F66τ13, eτ12 = √F66τ12 eF1 = F1 √F11 , eF2 = F2 √F22 (31) (31) The values of eF12 and eF23 as obtained in (23) and (24) are also incorporated above. All terms of the similar nature tend to contribute to F in the same way, whilst the differences due to the anisotropy of the material in all second order terms are absorbed in the normalisation scheme. The contributions of F12 and F23 obtained in (23) and (24) have indeed been equalised in their normalised form. The outcomes presented in (23) and (24) rest heavily on the normalisations in (19). 4.2. Determination of F12 and F23 It should be emphasised here that the rationalisation above has to be carried out on the criterion in its 3D form before it is reduced to 2D. Although (29) is a well-known form [11], its rational justiﬁcation can be obtained only through the route of manipulating 3D stress states, as presented above. 4.3. Veriﬁcation and Discussion p As a further veriﬁcation, reducing the materials to isotropic materials of unequal tensile and compressive strengths, whilst (33) and (34) still hold: A = −27 16 F2 11F2 1 < 0 since F1 ̸= 0. (35) (35) In this case, Equation (10) leads to: In this case, Equation (10) leads to: 1 σ∗ t −1 σ∗c (σ1 + σ2 + σ3) + 1 σ∗ t σ∗c σ2 1 + σ2 2 + σ2 3 −σ2σ3 −σ1σ3 −σ1σ2 + 3τ2 23 + 3τ2 13 + 3τ2 12 = 1 (36) (36) where σ∗ t and σ∗ c are the tensile and compressive strengths of the isotropic material, re- spectively. This reproduces the Raghava–Caddell–Yeh criterion [17], for which the failure envelope is a circular paraboloid demonstrating inﬁnite strength against hydrostatic com- pression, but not hydrostatic tension. As a case of further specialisation for isotropic materials of equal tensile and compres- sive strengths, i.e., σ∗ t = σ∗ c = σ∗, (36) further reduces to the von Mises criterion [13]: σ2 1 + σ2 2 + σ2 3 −σ2σ3 −σ1σ3 −σ1σ2 + 3τ2 23 + 3τ2 13 + 3τ2 12 = (σ∗)2 (37) (37) where σ∗is the strength of the material, which the same for both tension and compression. This corresponds to the case when the condition for A > 0 does not hold. Given the expression of A in (32), the only alternative is A = 0. In this case, (10) turns into a cylindrical surface according to [9]. It can be seen that a cylindrical surface can be mathematically considered as an extreme case of an elliptic paraboloidal surface, allowing the von Mises criterion to be seen as a special case of the Tsai–Wu criterion. As a further point of discussion, (24) shows that δ = 1 according to (8). Equation (7), as obtained in [6], naturally leads to (23), reproducing (6) as Tsai and Hahn suggested in [11]. However, the only justiﬁcation given in [11] was that it allowed the Tsai–Wu criterion to reproduce the von Mises criterion for isotropic materials of equal tensile and compressive strengths, whereas in this paper, (23) and (24) were obtained simultaneously from systematic and logical deduction. In [6], it was suggested that δ was a material constant that could vary from between materials. 4.3. Veriﬁcation and Discussion If one is prepared to accept (19) as the correct normalisations, which equalise the contributions of eF12 and eF23 to the failure function as the major premise for the deductive reasoning as presented above, conclusions (23) and (24) will be the logical consequence. The existence of a relationship amongst transverse strengths, as shown in (24), can also be argued as follows. There are only two independent strength properties for isotropic materials of different tensile and compressive strengths for 3D stresses in general, as well as for any plane stress conditions as special cases, according to the established Raghava– Caddell–Yeh criterion [17], which has been more rationally formulated in [18]. When a transversely isotropic material is under a plane stress condition in its transverse plane, J. Compos. Sci. 2022, 6, 82 10 of 17 10 of 17 i.e., the 2–3 plane, the material is effectively isotropic; therefore, the Raghava–Caddell– Yeh criterion in its 2D form should be applicable. If so, it will only require two strength properties. For the Tsai–Wu criterion to reproduce the Raghava–Caddell–Yeh criterion under this condition, the third strength property cannot be independent. i.e., the 2–3 plane, the material is effectively isotropic; therefore, the Raghava–Caddell– Yeh criterion in its 2D form should be applicable. If so, it will only require two strength properties. For the Tsai–Wu criterion to reproduce the Raghava–Caddell–Yeh criterion under this condition, the third strength property cannot be independent. g p p y p With F12 and F23 as determined in (23) and (24), one can verify conditions in (16) as follows: A = −3 16 F22 p F22F1 + 2 p F11F2 2 < 0 provided −F1 √F11 ̸= 2 F2 √F22 (32) J = F22(2F11 + F22) −1 2 F11F22 −1 4 F2 22 = 3 4 F22(2F11 + F22) > 0 (33) D = (F22 −F23) F11(F22 + F23) −2F2 12 = 3 2 F22 1 2 F11F22 −1 2 F11F22 = 0 (34) (32) (33) (34) Hence, (11) deﬁnes an elliptic paraboloid. The inequality of (32) is met by most composites of practical importance and the only alternative is A = 0, which arises only under special conditions, as will be discussed later. 4.3. Veriﬁcation and Discussion Relationship (24) asserts further that δ is a universal constant, i.e., δ = 4 −σ∗ 2tσ∗ 2c (τ∗ 23) 2 = 1, for all transversely isotropic materials, brittle d l l d l l l l h l or ductile, including completely isotropic materials as a special case. This is apparently observed in isotropic materials obeying the Raghava–Caddell–Yeh criterion [17] and the von Mises criterion [13], as special cases. Any variation in δ, similar to those shown or ductile, including completely isotropic materials as a special case. This is apparently observed in isotropic materials obeying the Raghava–Caddell–Yeh criterion [17] and the von Mises criterion [13], as special cases. Any variation in δ, similar to those shown J. Compos. Sci. 2022, 6, 82 11 of 17 11 of 17 in [6], should be attributed to the variability of the experimentally measured transverse strength properties. This resembles a universal relationship amongst Young’s moduli and Poisson’s ratios, ν12E2 ν21E1 = 1, although the underlying considerations are rather different. Raw experimental data would show signiﬁcant variability as well if they were all independently measured. 4.4. Comparisons of Predicted Transverse Shear Strengths with Measured Values and Those from Other Theories 4.4. Comparisons of Predicted Transverse Shear Strengths with Measured Values and Those from Other Theories In the Puck failure criterion [14,15], the transverse shear strength is a derived quantity that is equal to the transverse tensile strength. In comparison to (25), it corresponds to the special case of σ∗ 2c = 3σ∗ 2t. The transverse shear strengths obtained according to the Puck criterion and the present derivation are compared in Table 1 over a range of composites from [10,19,20] where experimental data are available. Relative to the measured values of τ∗ 23 in the third row of Table 1, those obtained from (25) outperform those from the Puck criterion, except for S-2 glass/Epoxy 2 and G40-800/5026, as highlighted in Table 1. For these two cases, one is only marginally worse than that predicted by the Puck criterion, whilst the other is identical to that predicted from the Puck criterion, because σ∗ 2c = 3σ∗ 2t holds, although the experimental shear strength of 57 MPa is nearly 20% below q σ∗ 2tσ∗ 2c 3 for this particular material. 4.3. Veriﬁcation and Discussion q Given that q σ∗ 2tσ∗ 2c 3 is the strength of the material under pure transverse shear, as predicted from failure criterion (26), the degree of its agreement with the experimentally obtained transverse shear strength—i.e., the comparison between the measured values in the third row of Table 1—and that predicted from (25), as shown in the fourth rows of Table 1, can serve as a basic level of validation for the criterion (26). When applying (26) to problems involving general stress states, one should be prepared for discrepancies of a magnitude comparable with those between the ﬁgures in the third and fourth rows in Table 1, as an indication of the level of accuracy that criterion (26) is capable of offering. Another relationship amongst transverse strengths can be found in [16], and is given as follows:  ∗ ∗ (τ∗ 23)2 =   1 + σ∗ 2t σ∗ 2c 3 + 5 σ∗ 2t σ∗ 2c  σ∗ 2tσ∗ 2c or (τ∗ 23)2 σ∗ 2tσ∗ 2c = 1 + σ∗ 2t σ∗ 2c 3 + 5 σ∗ 2t σ∗ 2c . (38) (38) It is apparently a more complicated expression than that given in (25). The derivation in [16] was based on the following failure criterion stemming from Hashin’s matrix failure criterion [3] under a plane stress state in the transverse plane before it was split into tensile and compressive modes. It is apparently a more complicated expression than that given in (25). The derivation in [16] was based on the following failure criterion stemming from Hashin’s matrix failure criterion [3] under a plane stress state in the transverse plane before it was split into tensile and compressive modes. F2(σ2 + σ3) + F22(σ2 + σ3)2 + F44 τ2 23 −σ2σ3 = 1. (39) (39) This is an incomplete quadratic form. The contributions from ﬁbre direction as well as the interactive term associated with F12 have been excluded a priori. The interaction between the stresses represented by F12 resembles the role of Poisson’s ratios in the generalised Hooke’s law in the theory of elasticity. It might be true that in the solutions to many elastic problems, the contributions from Poisson’s ratios are limited in terms of magnitude. 4.3. Veriﬁcation and Discussion Whilst ignoring such contributions would not lead to excessive error, the absence of Poisson’s ratios would prevent the appropriate understanding of many important mechanical behaviours of materials, such as the anticlastic behaviour, free edge effects, etc. J. Compos. Sci. 2022, 6, 82 12 of 17 Table 1. Experimentally measured strengths and predicted transverse strengths according to different theories, all in MPa. Category Row No. Strength Type of Composite Considered AS4 3501-6 T300 BSL914C E-Glass LY556 E-Glass MY750 IM7 8551-7 T300 PR-319 A-SE poxy 1 S-2 Glass Epoxy 2 G40-800 5026 Experimentally measured strengths 1 σ∗ 2t 48 27 35 40 68 40 38 56.5 70 2 σ∗ 2c 200 200 114 145 185 125 150 180 210 3 τ∗ 23 55 N/A 50 50 57 45 50 40 57 Predicted τ∗ 23 from σ∗ 2t and σ∗ 2c 4 Present from Equation (25). 56.57 42.43 36.47 43.97 64.76 40.82 43.59 58.22 70.00 5 Puck [14,15], which is equal to σ∗ 2t. 48 27 35 40 68 40 38 56.5 70 6 Christenson, as shown in Equation (39) [16]. 50.93 43.82 33.50 42.56 58.59 36.91 41.90 54.48 56.12 Table 1. Experimentally measured strengths and predicted transverse strengths according t ured strengths and predicted transverse strengths according to different theories, all in MPa. J. Compos. Sci. 2022, 6, 82 13 of 17 13 of 17 Similarly, to reveal the special relationships elaborated in this paper, it is important to incorporate the interactive terms in the failure criterion. The approach to treating the interactive terms can also be viewed as a meaningful indicator of the consistency of the formulation. For instance, in the well-known Hashin criterion [3], the interactive term F12 was ignored based on the assumption that failure is determined by stresses on the failure plane, which was inherited from the Mohr criterion [21] for isotropic materials; however, this particular assumption is inapplicable to anisotropic composites, as was argued in [22]. Whilst the numerical errors could be insigniﬁcant in many cases, as Hashin also stated in [3], the exclusion of such interactions would not allow the intricate relationships obtained in this paper to be revealed. From another perspective, including one interactive coefﬁcient (F23) in the failure function, whilst excluding another (F12), as in [3], can at least be viewed as a degree of inconsistency. 4.3. Veriﬁcation and Discussion The numerical closeness of the results from a theory to experimental data in one aspect or another is important; however, we believe that the mathematical and logical consistency of the theory is even more important. g y y p The numerical values obtained from (38) [16] over the same range of materials as above are also shown in the ﬁfth row of Table 1. Relative to the experimental data in the third row of Table 1, the predictions from (25) listed in the fourth row of Table 1 outperform those from (38) for ﬁve out of eight materials. The trend is further shown graphically in Figure 2, where the ratio (τ∗ 23) 2 σ∗ 2tσ∗ 2c is plotted as a function of the ratio σ∗ 2t σ∗ 2c . The curve corresponding to (38) does not seem to be more representative than the straight line obtained according to (25), understanding the variability in measured transverse strengths as argued in [16]. The predictions are not more relevant to carbon composites (black symbols) than to glass composites (green symbols) either, as was claimed in [16]. REVIEW 13 of 17 Figure 2. Graph of ሺఛమయ ∗ሻమ ఙమ೟ ∗ఙమ೎ ∗versus ఙమ೟ ∗ ఙమ೎ ∗. 5. Retrospection and Prospection According to analytic geometry [9], the sufficient conditions for the failure envelope of transversely isotropic materials to be closed, i.e., to be an ellipsoidal surface, are: Figure 2. Graph of (τ∗ 23)2 σ∗ 2tσ∗ 2c versus σ∗ 2t σ∗ 2c . Logically, the major premise underlying (38) is failure criterion (39), which is at its best a special form of the criterion based on the full quadratic failure function from which (26) 5. Retrospection and Prospection According to analytic geometry [9], the sufficient conditions for the failure envelope Logically, the major premise underlying (38) is failure criterion (39), which is at its best a special form of the criterion based on the full quadratic failure function from which (26) 5. Retrospection and Prospection According to analytic geometry [9], the sufficient conditions for the failure envelope Logically, the major premise underlying (38) is failure criterion (39), which is at its best a special form of the criterion based on the full quadratic failure function from which (26) 5. 5. Retrospection and Prospection According to analytic geometry [9], the sufﬁcient conditions for the failure envelope of transversely isotropic materials to be closed, i.e., to be an ellipsoidal surface, are: A < 0; J > 0; D ̸= 0 and DI > 0 (40) (40) where A, J and D are given in (12)–(14), respectively, and the following is another invariant of quadratic form (11): (41) I = F11 + 2F22 (41) Since I is always positive, given the expression of D in (14), and F22 −F23 is always positive according to the fourth equation in (9) (because F44 is always positive), conditions D ̸= 0 and DI > 0 can be re-written as: ∆= 1 2 F11(3F22 −F44) > 0 i.e., 3F22 −F44 > 0 given F11 > 0. (42) (42) With F12 as given in (23), if the transverse shear strength τ∗ 23 (and hence F44) was left as an independent property, (42) could not be satisﬁed in general. An ellipsoid could be guar- anteed provided that τ∗ 23 was sufﬁciently large. In the case of isotropic materials obeying the von Mises criterion, as a special case of transversely isotropic materials, D = ∆= 0, which satisﬁes (24) or (25) under the condition of (23), but violates (42). In other words, (42) would prohibit (11) to degenerate to the von Mises criterion as a special case for isotropic materials of equal tensile and compressive strengths, because D = 0 as a necessary condition is shared by cylinders and elliptic paraboloids, but not by ellipsoids, according to analytic geometry. It is clear now that having a closed failure envelope and having the von Mises criterion as a special case are two mutually exclusive propositions. Sometimes, it only requires a very small experimental or data processing error to nudge D from positive to negative; then, the nature of the failure envelope would change dramatically from an ellipsoid to an elliptic paraboloid or a hyperboloid. Whilst the imposition of (17) alone eliminates the possibility of the failure envelope being an ellipsoid or hyperboloid, errors in measured transverse strengths could still make the elliptic paraboloid imaginary according to analytic geometry. Relationship (25), in addition to (23), not only ensures that the failure envelope is a real and unique elliptic paraboloid, but also offers a way to mitigate experimental errors amongst measured transverse strengths. 4.3. Veriﬁcation and Discussion Retrospection and Prospection According to analytic geometry [9], the sufficient conditions for the failure envelope Logically, the major premise underlying (38) is failure criterion (39), which is at its best a special form of the criterion based on the full quadratic failure function from which (26) J. Compos. Sci. 2022, 6, 82 14 of 17 14 of 17 is obtained. There is no reason to expect (39) to be more representative than (26). In [16], efforts were made to eliminate an independent strength property from (39) whilst keeping the manipulations all within the transverse plane. is obtained. There is no reason to expect (39) to be more representative than (26). In [16], efforts were made to eliminate an independent strength property from (39) whilst keeping the manipulations all within the transverse plane. 5. Retrospection and Prospection 5. Retrospection and Prospection This illustrates a perfect example of how mathematical consistency brings insight into physical problems. y g g p y p The conventional Tsai–Wu criterion could degenerate to the von Mises criterion for isotropic materials of equal tensile and compressive strengths [11] because the failure envelope in the original Tsai–Wu criterion was not limited to an ellipsoid, no matter how much it was desired; it could be ellipsoid, elliptic paraboloid and different hyperboloids depending on the value of the transverse shear strength τ∗ 23, whilst satisfying the inequalities in (3). The necessary conditions of (3) were far too loose to impose such a restriction. The controlling factor of the shape of the failure envelope rests on the transverse shear strength τ∗ 23, which affects the values of various invariants, in particular D. Narrowing the failure envelope down to an elliptic paraboloid, as proposed in this paper, can help to ﬁlter out many physically prohibitive scenarios whilst restoring the mathematical and logical consistency of the Tsai–Wu criterion. This is the spirit of rationalisation. The fully rationalised Tsai–Wu criterion for general 3D stress states is presented in (26). For this criterion, the transverse shear strength is not a required strength property, but it can be predicted from the criterion. There will inevitably be a discrepancy between the predicted value and the measured value. The available independently measured transverse shear strength should be used to correct the experimental errors in other transverse strength J. Compos. Sci. 2022, 6, 82 15 of 17 15 of 17 properties, instead of being employed as an independent strength property for the failure criterion based on a quadratic failure function. It should be employed to correct the transverse tensile and compressive strengths in order to minimise systematic errors in these measured strengths in the same way that the experimentally measured ν21 should be employed to correct ν12, E1 and E2, rather than being incorporated in the generalised Hooke’s law. A practical method of such corrections will be addressed in a subsequent publication. p The 2D version of the fully rationalised Tsai–Wu criterion is given in (29). Although it looks identical to its familiar form, as presented in [11], the transverse tensile and compressive strengths required to evaluate F22 should be understood as their corrected values using the transverse shear strength, whenever available. The rationalisation still makes a difference to the 2D Tsai–Wu criterion but in an implicit way. 5. Retrospection and Prospection The failure criterion for transversely isotropic materials based on a single quadratic failure function can now be considered fully established, with a complete understanding of the nature of a quadratic failure function having been achieved mathematically. It can be fully and logically deﬁned with ﬁve independent strength properties, namely tensile and compressive strengths in the directions along ﬁbres and transverse to ﬁbres, and the in- plane shear strength. All coefﬁcients involved in the failure function can be expressed with mathematical rigour and logical consistency. Any additional strength property employed as an independent one will compromise the consistency of the theoretical framework, even if they were experimentally measured. y p y Having established the failure criterion (26) as the fully rationalised Tsai–Wu criterion, any signiﬁcant deﬁciency or genuine discrepancy with experimental observations can now be conﬁdently attributed to the lack of representativeness of the basic assumptions underlying the criterion: (1) The transverse isotropy and homogeneity of the material; (2) Failure function being a single quadratic function. Any deviation from these ideal positions will inevitably have an effect on the outcomes and should be anticipated in assessing the accuracy of the predictions. The use of these assumptions brings convenience but also restrictions. The obtained criterion can only be as accurate as its underlying assumptions allow. The consistent use of a quadratic function for a failure criterion helps to eliminate undue errors and anomalies due to mathematical and logical oversights, but not the generic deﬁciency due to these assumptions themselves. The limitations of using a single quadratic function as the failure function could be improved whilst remaining within the framework of a phenomenological approach in one of two ways: (1) To use a higher order polynomial for the failure function; (2) To partition the stress space into subspaces and to use a quadratic function in each subspace. (2) To partition the stress space into subspaces and to use a quadratic function in each subspace. Either way, the number of independent strength properties will inevitably increase. One should always bear in mind how the newly introduced properties are to be determined, experimentally or otherwise. Abandoning phenomenological approaches completely or discontinuing their improvement before alternatives are established is partially responsible for the confused state of the art on the subject of composite failure criteria. 6. Conclusions Using a complete quadratic failure function, there is only one rational choice for the failure envelope—an elliptic paraboloid, as a single-sided open surface that allows inﬁnite strength under a unique stress ratio in triaxial compression. Any alternative would lead to contradictions one way or another. This quadratic failure function can only accommodate ﬁve independent strength properties. Seven were initially introduced in the original Tsai– Wu criterion [1] after considerations of transverse isotropy. A relationship was established between the two interactive coefﬁcients F12 and F23 based purely on analytic geometry. It ensures that the failure envelope is an elliptic paraboloid. A further relationship between these coefﬁcients can be obtained from a logical consideration that the sensitivity of the failure criterion to these two coefﬁcients should be unbiased. Thus, they can both be uniquely determined analytically as F12 = −1 2 √F11F22 and F23 = −1 2 F22. Although the former is well known, it can only be fully justiﬁed in presence of the latter, which can be re-written as 3(τ∗ 23)2 = σ∗ 2tσ∗ 2c, given the transverse isotropy of the material. This introduces a relationship amongst transverse strengths as a natural consequence of the rationalisation; the relationship is an intrinsic property of the quadratic failure function for transversely isotropic materials. Failing to comply with this relationship compromises the consistency of the criterion based on a single quadratic failure function. Due to the high variability in the measured transverse strengths, the obtained rela- tionship amongst transverse strengths may not be perfectly represented in the available experimental data. In presence of all three transverse strengths independently obtained experimentally, the transverse shear strength can be employed to validate the relationship in (25), i.e., 3(τ∗ 23)2 = σ∗ 2tσ∗ 2c. The failure criterion can only be as accurate as the agreement between τ∗ 23 and q σ∗ 2tσ∗ 2c 3 . q The fully rationalised Tsai–Wu criterion is given in (26) for 3D stress states. Its 2D version in (29) looks identical to its familiar form. Although no changes were made to (29), the rationalisation offers a ﬁrm basis for (29), and the determination of F12 no longer relies on experimental data ﬁtting or other empiricism, but on a rigorous deduction from the mathematical and logical consequences of the basic assumptions introduced instead. 5. Retrospection and Prospection With consis- tency established, one would be in a position to explore meaningful ways of determining strength properties as required for alternatives to experimental measurements, for instance, molecular dynamics as investigated in [23], although the material used in [23] was not transversely isotropic. The ﬁrst option above seems to pose formidable difﬁculties, as the understanding of the analytic geometry of polynomial functions higher than the second order in multidimen- sional spaces has been very limited and is certainly far less than that of quadratic functions. This is not an attractive direction forward practically. J. Compos. Sci. 2022, 6, 82 16 of 17 The second option is likely to offer a practical way forward. The Hashin criterion [3] can be seen as an attempt in this direction, followed by a wide range of different attempts, including those in [15,24]. However, it is the authors’ view that an appropriate rational- isation is necessary for the Hashin criterion before a fundamental breakthrough can be expected. This will be the objective of another one of our subsequent publications. In the literature, there is lack of failure criteria for genuinely orthotropic materials, apart from the maximum stress/strain criteria. The Tsai–Wu criterion was nominally pro- posed for orthotropic materials. However, without the appropriate means to determine the coefﬁcients to the interactive terms, it has never been applied seriously beyond transversely isotropic materials. This is a problem we will address in another subsequent publication. 6. Conclusions g q p The required ﬁve independent strength properties are limited to conventional and widely available tensile and compressive strengths in the directions along and transverse to ﬁbres and the in-plane shear strength for the failure predictions of both 2D and 3D stress states. The most signiﬁcant outcome of the present paper is the achievement of a thorough understanding of the nature of the quadratic failure function, with its intrinsic relationships having been revealed. Obeying these relationships ensures the self-consistency of the failure criterion. This should conclude a phase of investigations on the subject of failure criteria involved in recent publications [6,16,25] based on a quadratic failure function, as far as transversely isotropic materials are concerned. The failure criterion given in (26) can be employed in design and analysis with conﬁdence within its applicability deﬁned by its assumptions, viz. the transverse isotropy and homogeneity of the material, and the failure function being a single quadratic function. 17 of 17 J. Compos. Sci. 2022, 6, 82 17 of 17 Author Contributions: Conceptualization, S.L.; methodology, S.L. and E.S.; validation, S.L., M.X. and E.S; formal analysis, S.L.; investigation, S.L.; writing—original draft preparation, S.L.; writing— S.L. and E.S.; visualization, S.L. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: There is no additional data. Data Availability Statement: There is no additional data. Acknowledgments: The second author wishes to acknowledge the ﬁnancial support from CSC, China and the scholarship from the Faculty of Engineering, the University of Nottingham. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W3161138157	https://ojmo.centre-mersenne.org/item/10.5802/ojmo.4.pdf	English	null	Exact makespan minimization of unrelated parallel machines	Open journal of mathematical optimization	2,021	cc-by	13,983	Abstract We study methods for the exact solution of the unrelated parallel machine problem with makespan minimization, generally denoted as R\|\|Cmax. Our original application arises from the automotive assembly process where tasks needs to be distributed among several robots. This involves the solutions of several R\|\|Cmax instances, which proved hard for a MILP solver since the makespan objective induces weak LP relaxation bounds. To improve these bounds and to enable the solution of larger instances, we propose a branch–and–bound method based on a Lagrangian relaxation of the assignment constraints. For this relaxation we derive a criterion for variable ﬁxing and prove the zero duality gap property for the case of two parallel machines. Our computational studies indicate that the proposed algorithm is competitive with state-of-the-art methods on diﬀerent types of instances. Moreover, the impact of each proposed feature is analysed. Digital Object Identiﬁer 10.5802/ojmo.4 Keywords unrelated parallel machine problem, makespan, variable ﬁxing, binary knapsack, Lagrangian 1 Introduction We study the unrelated parallel machine problem (e.g., [29, 31]), which is to assign n tasks to m machines, where the processing time of an assignment depends on both task and machine, and the goal is to minimize the makespan (i.e., the ﬁnish time of the last machine); see [23]. By the three-ﬁeld notation from scheduling theory, cf. [20], this problem is denoted R\|\|Cmax, where R denotes that the processing times are unrelated, \|\| denotes that there are no additional constraints on the tasks, and Cmax denotes that the makespan is to be minimized. The R\|\|Cmax problem is a classical problem in the ﬁeld of scheduling and it can be utilized when scheduling tasks on, e.g., production lines or multiprocess computers. A previous study, considering an application from the automotive manufacturing industry, concerned the makespan minimization of several industrial robots completing a set of welding tasks without entering each other’s workspaces; it also included sequences of tasks, robot routing, and path planning; see [1, 3]. The requirement of not intersecting each other’s workspaces while working on the robots’ respective assigned tasks resulted in an extension of the R\|\|Cmax with a few set packing side-constraints, the extension was iteratively solved in order to provide candidate robot–task assignments. Each of these candidate assignments were later analysed in order to ﬁnd improved sequences of the tasks as well as the corresponding motions of the robots. The main reasons to perform the study resulting in this article is that our suggested model proved hard to solve with a general mixed–integer linear programming (MILP) solver; see [3], and that the issue of long computing times remained when relaxing our model to the R\|\|Cmax. Thus, this study aims to construct exact methods for the R\|\|Cmax, preferably such that can be generalized to our extension of the R\|\|Cmax, and using considerably less computing time as compared to a general MILP solver applied to the original R\|\|Cmax formulation. For the sake of generality, we consider general R\|\|Cmax instances; see, e.g., [29, 32, 42]. The main contributions of our study are a new variable ﬁxing criterion and, for m = 2, a proof of no duality gap. Related work on R\|\|Cmax is reviewed in Section 2. Inspired by the strongest Lagrangian relaxation suggested in [29], (i) the knapsack structure of the subproblems is utilized to derive a variable ﬁxing criterion, (ii) a deﬂected Volume 2 (2021), article no. Open Journal of Mathematical Optimization Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri Exact makespan minimization of unrelated parallel machines Volume 2 (2021), article no. 2 (15 pages) <http://ojmo.centre-mersenne.org/item/OJMO_2021__2__A2_0> Open Journal of Mathematical Optimization is member of the Centre Mersenne for Open Scientiﬁc Publishing www.centre-mersenne.org Open Journal of Mathematical Optimization is member of the Centre Mersenne for Open Scientiﬁc Publishing www.centre-mersenne.org Open Journal of Mathematical Optimization is member of the Centre Mersenne for Open Scientiﬁc Publishing www.centre-mersenne.org Exact makespan minimization of unrelated parallel machines Edvin Åblad Fraunhofer-Chalmers Research Centre and Chalmers University of Technology, Gothenburg, Sweden edvin.ablad@fcc.chalmers.se Fraunhofer-Chalmers Research Centre and Chalmers University of Technology, Gothenburg, Sweden edvin.ablad@fcc.chalmers.se Ann-Brith Strömberg Chalmers University of Technology and University of Gothenburg, Gothenburg, Sweden anstr@chalmers.se Ann-Brith Strömberg Chalmers University of Technology and University of Gothenburg, Gothenburg, Sweden anstr@chalmers.se Domenico Spensieri Fraunhofer-Chalmers Research Centre and Chalmers University of Technology, Gothenburg, Sweden domenico.spensieri@fcc.chalmers.se © Edvin Åblad & Ann-Brith Strömberg & Domenico Spensieri; licensed under Creative Commons License Attribution 4.0 International 2 Related work In 1975, Sandi [37] studied a mixture of R\|\|Cmax and P\|\|Cmax, where P denotes that the processing times are identical over the machines. In 1976, Horowitz and Sahni [23] were among the ﬁrst to suggest an exact solution method for the R\|\|Cmax based on dynamic programming (however, exponential in nature) and in 1990, Lenstra et al. [27] showed that no polynomial 3 2-approximation algorithm for the R\|\|Cmax can exist unless P=NP, and that the R\|\|Cmax is NP-hard even for m = 2. Hence, approximation algorithms are often suggested for the R\|\|Cmax, e.g., a classical 2-approximation algorithm based on rounding the linear programming (LP) solution; see [27] and [40, Ch. 17]. For ﬁxed values of m there exist fully polynomial-time (1 + ε)-approximation algorithms; e.g., in 2001, Jansen and Porkolab [25] presented a running time of n(m/ε)O(m). If m and n are not too large (e.g., m ≤20, n ≤200), exact algorithms are also applicable. In 1991, Velde et al. [39] suggested a branch–and–bound algorithm based on Lagrangian relaxation. In 1997, Martello et al. [29] improved the branch–and–bound algorithm by using several Lagrangian relaxations and so-called residual costs [14]. In 2002, Mokotoﬀand Chrétienne [32] suggested a cutting plane method; by assuming integer valued processing times, the value of the makespan is iteratively increased until a feasible solution exists. A branch–and–price algorithm was suggested in 2007 by Wotzlaw [42] along with a survey of 2-approximation and heuristic algorithms comprising, e.g., the one described in [16]. Local search heuristics are often applied in order to ﬁnd good feasible solutions to the R\|\|Cmax. In 1994, Glass et al. [19] deﬁned neighbourhoods as reassign a task to another machine and interchange two tasks between two machines. Wotzlaw [42] emphasized the importance of applying local search techniques in a branch–and–price implementation to allow early pruning. In 2004, Frangioni et al. [15] suggested two larger neighbourhoods, a cyclic exchange and a path exchange of tasks, and showed that the problem of verifying that these neighbourhoods do not contain an incumbent solution is NP-complete; similar but smaller neighbourhoods are employed in our suggested methodology (see Section 4). In 2010, Fanjul-Peryro and Ruiz [12] introduced an iterated greedy heuristic based on a destruction–reconstruction methodology and the local search heuristics suggested in [19]; they compared their result with a general MILP solver and found that, given a short time limit, their heuristic generally outperformed the MILP solver. 2 Related work In 2005, Ghirardi and Potts [18] suggested a so-called recovery beam heuristic for the R\|\|Cmax; it uses a so-called truncated branch–and–bound algorithm, where the lower and upper bounds in each node are computed as in [39] and [29], respectively. The R\|\|Cmax is a common relaxation of scheduling problems including additional constraints; hence, the R\|\|Cmax yields a lower bound for such problems. In 2014, such relaxations were used by Borba and Ritt [9] and Vilá and Pereira [41]: both studied precedence constraints among tasks and used lower bounds for the R\|\|Cmax in their respective branch–and–bound algorithms. In 2017, Åblad et al. [3] solved the R\|\|Cmax with additional set packing constraints using a branch–and–cut solver. Exact methods for the R\|\|Cmax subgradient method is used to maximize the dual function, (iii) the duality gap is investigated, and (iv) a branch–and–bound rule is suggested in Section 3. A local search heuristic is presented in Section 4. Some MILP approaches, test instances, and results are presented in Section 5 and conclusions are presented in Section 6. subgradient method is used to maximize the dual function, (iii) the duality gap is investigated, and (iv) a branch–and–bound rule is suggested in Section 3. A local search heuristic is presented in Section 4. Some MILP approaches, test instances, and results are presented in Section 5 and conclusions are presented in Section 6. We apply the Lagrangian relaxation suggested in [29], however, with a variable ﬁxing criterion that diﬀers from their implementation. Moreover, in [29] the focus is on the branch–and–bound root node, in which several heuristics are employed and the strong Lagrangian dual function is maximized, whereas in the child nodes only weaker Lagrangian relaxations are utilized. We, however, apply local search heuristics and spend most of the computational eﬀort on maximizing the strong dual function using a deﬂected subgradient method in every branch–and–bound node. 1 Introduction 2 Volume 2 (2021), article no. 2 2 xij = ( 1, if machine i performs task j, 0, otherwise, i ∈I, j ∈J . Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 3 Introducing the set X(z) := ( x ∈Bmn X j∈J pijxij ≤z, i ∈I ) , X(z) := ( x ∈Bmn X j∈J pijxij ≤z, i ∈I ) , z ∈R, (2a) z ∈R, (2a) I ) , z ∈R, (2a) (2a) hine must respect the makespan value z, and the set in which each machine must respect the makespan value z, and the set Xsa := ( x ∈Bmn X i∈I xij = 1, j ∈J ) , Bmn X i∈I xij = 1, j ∈J ) , (2b) (2b) in which each task is assigned to exactly one machine (semi-assignments, sa), the R\|\|Cmax is for in which each task is assigned to exactly one machine (semi-assignments, sa), the R\|\|Cmax is formulated as minimize x∈Bmn z∈R {z \| x ∈X(z) ∩Xsa} . (3) in which each task is assigned to exactly one machine (semi-assignments, minimize x∈Bmn, z∈R {z \| x ∈X(z) ∩Xsa} . (3) minimize x∈Bmn, z∈R {z \| x ∈X(z) ∩Xsa} . We also let S := {(x, z) ∈Bmn × R \| x ∈X(z) ∩Xsa} denote the feasible set of (3). A Lagrangian relaxation of the constraints in (2a) (as suggested in [39]) yields a subproblem that is polynomially solvable, while relaxing the constraints in (2b) (as suggested by Martello et al. [29]) leads to a subproblem involving m binary knapsack problems, which are NP-hard. Moreover, as shown in [29], it leads to a stronger lower bound. We consider the Lagrangian relaxation of the constraints in (2b), which results in the expression of the Lagrangian dual function h : Rn 7→R as h(u) := X j∈J uj + minimum z≤z≤z (z −f(z, u)) , f(z, u) := X i∈I maximum xi·∈Xi(z) X j∈J ujxij, h(u) := X j∈J uj + minimum z≤z≤z (z −f(z, u)) , (4a) f(z, u) := X i∈I maximum xi·∈Xi(z) X j∈J ujxij, (4b) where Xi(z) := {xi· ∈Bn \| P j∈J pijxij ≤z} (i.e., X(z) = X1(z) × · · · × Xm(z)), u ∈Rn is the Lagrangian multiplier vector, and z and z are the current best known lower and upper bounds on the optimal objective value z∗of (3), respectively. The optimal value of the Lagrangian dual is found by maximizing h(u) over u ∈Rn. Note that the constraints z ≤z ≤z are redundant in (3), however, tight bounds can improve the lower bound h(u). Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri For example, if h(u) > z for some u ∈Rn then z := ⌈h(u)⌉, where the ceiling operation is valid due to the integrality of pij. Hence, the dual function (4a) gets redeﬁned whenever tighter bounds z and z are found, cf. Section 3.3. The lower bound z is initialized using the LP relaxation of (3) and updated using h(u) and branch–and–bound. The upper bound is provided and updated by our heuristics; see Section 4. For ﬁxed values of z and u the problem (4b) amounts to solving m binary knapsack problems. In [29] these knapsack problems are solved by a branch–and–bound algorithm; we, however, employ the classical dynamic programming approach presented in [30, p. 38]. Although a more sophisticated dynamic programming approach can be applied (see, e.g., [34, 28]), our preliminary tests indicated that the classical implementation suﬃces for our instances, where twenty years of development in computer memory size and speed might be the enabler. This classical approach also provides a direct computation of bounds to be used in the variable ﬁxing procedure speciﬁed next, whereas a more sophisticated dynamic programming approach might not provide the computation of these bounds and is thus left for future studies. The variable ﬁxing is one of our main contributions. Moreover, in Section 3.3 we emphasize the strength of the lower bound (4a) and describe how to maximize it in Section 3.2. 3 A Lagrangian relaxation of the R\|\|Cmax In this section we present a MILP model for the R\|\|Cmax and the corresponding Lagrangian relaxation of the task-assignment constraints. We then exploit the lower bound of this relaxation and the structure in the resulting subproblems to ﬁx the values of variables which result in a reduction of the total computation time. A MILP model for the R\|\|Cmax (cf. [39]) is the following: let I := {1, . . . , m} denote the set of machines, J := {1, . . . , n} the set of tasks, and pij ∈Z+ the processing time of task j ∈J on machine i ∈I. Deﬁne subproblems to ﬁx the values of variables which result in a reduction of the total computation time. A MILP model for the R\|\|Cmax (cf. [39]) is the following: let I := {1, . . . , m} denote the set of machines, J := {1, . . . , n} the set of tasks, and pij ∈Z+ the processing time of task j ∈J on machine i ∈I. Deﬁne (1) xij = ( 0, otherwise, Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 3 Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri Exact methods for the R\|\|Cmax where V i 0· := 0. As a result, for any z ∈{0, . . . , ¯z}, V i jz is the optimal value of the knapsack of size z using items {1, . . . , j}; hence, f(z, u) = P i∈I V i nz. Thus, these value matrices enable a direct evaluation of the Lagrangian dual function h(u). where V i 0· := 0. As a result, for any z ∈{0, . . . , ¯z}, V i jz is the optimal value of the knapsack of size z using items {1, . . . , j}; hence, f(z, u) = P i∈I V i nz. Thus, these value matrices enable a direct evaluation of the Lagrangian dual function h(u). A variable ﬁxing criterion is available as a by-product of the value matrices V i, i ∈I. This is accomplished by bounding f(z, u \| x¯ı¯= 1) from above, by having xi¯unrestricted for i ∈I and ensuring that a duplicate item of x¯ı¯is inserted in the ¯ı-th knapsack, resulting in V ¯ı n(z−p¯ı¯) + X i∈I\{¯ı} V i nz. (6) f(z, u \| x¯ı¯= 1) ≤u¯+ V ¯ı n(z−p¯ı¯) + X i∈I\{¯ı} V i nz. (6) f(z, u \| x¯ı¯= 1) ≤u¯+ V ¯ı n(z−p¯ı¯) + X i∈I\{¯ı} V i nz. (6) By using the inequality in (6) a lower bound on h(u \| x¯ı¯= 1) is available at low computational cost when the knapsack subproblems in (4b), are solved using the dynamic programming approach. We next show how the value matrices can be utilized to compute f(z, u \| x¯ı¯= 1) and h(u \| x¯ı¯= 1) without having to resolve the subproblem for each ¯ı ∈I, ¯∈J . By using the inequality in (6) a lower bound on h(u \| x¯ı¯= 1) is available at low computational cost when the knapsack subproblems in (4b), are solved using the dynamic programming approach. We next show how the value matrices can be utilized to compute f(z, u \| x¯ı¯= 1) and h(u \| x¯ı¯= 1) without having to resolve the subproblem for each ¯ı ∈I, ¯∈J . e The idea is to also compute the entities eV i jz by an analogous recursion as for V i jz, where the items are inserted in reversed order: j = n, . . . , 1. Exact methods for the R\|\|Cmax Hence, for each i ∈I, eV i jz is the optimal value of the i-th knapsack of size z using items {j, . . . , n}. According to Lemma 1, the value of f(z, u \| x¯ı¯= 1) can be computed by combining V i jz and eV i jz. As a result, the inequality h(u \| x¯ı¯= 1) ≥z can be eﬃciently checked; if it holds the corresponding variable x¯ı¯can be ﬁxed to zero. ▶Lemma 1. For i ∈I, ¯∈J , W i ¯z := maximumv∈{0,...,z}{V i (¯−1)v + eV i (¯+1)(z−v)} is the optimal value of the knapsack i with capacity z and the items J \ {¯}. Moreover, f(z, u \| x¯ı¯= 1) = u¯+ W¯ı ¯(z−p¯ı¯) + P i∈I\{¯ı} W i ¯z ▶Lemma 1. For i ∈I, ¯∈J , W i ¯z := maximumv∈{0,...,z}{V i (¯−1)v + eV i (¯+1)(z−v)} is the optimal value of the knapsack i with capacity z and the items J \ {¯}. Moreover, f(z, u \| x¯ı¯= 1) = u¯+ W¯ı ¯(z−p¯ı¯) + P i∈I\{¯ı} W i ¯z. Proof. For each i ∈I, consider the binary knapsack formulation where the ¯-th item is not present: Proof. For each i ∈I, consider the binary knapsack formulation where the ¯-th item is not present: Proof. For each i ∈I, consider the binary knapsack formulation where the ¯-th item max xi·∈Bn ( X j∈J \{¯} ujxij X j∈J \{¯} pijxij ≤z ) . max xi·∈Bn ( X j∈J \{¯} ujxij X j∈J \{¯} pijxij ≤z ) . (7) (7) (7) First, consider the cases ¯= 1 and ¯= n, the corresponding optimal values of (7) are eV i (¯+1)z and V i (¯−1)z, respectively. Moreover, V i 0z = eV i (n+1)z = 0, hence W i ¯z is the optimal value of (7). Second, assume ¯∈J \ {1, n} and let ¯xi· be feasible in (7) and let ¯v := P¯−1 j=1 pij ¯xij. Then (¯xi·, ¯v) is feasible in the following formulation: max v∈{0,...,z} ( max xi·∈Bn ( X j∈J \{¯} ujxij ¯−1 X j=1 pijxij ≤v, n X j=¯+1 pijxij ≤z −v )) . (8) (8) Similarly, let (bxi·, bv) be feasible in (8). By aggregating the constraints in (8) it follows that bxi· is feasible in (7). Thus, the formulations (7) and (8) are equivalent. 3.1 Variable ﬁxing using the subproblem solution Given a lower bound for a problem with a binary variable restricted to one, if this bound exceeds a known upper bound for the unrestricted problem then the variable can be set to zero; this is called variable ﬁxing. This lower bound is often deduced by the reduced costs of the LP relaxation of the problem, being a special case of residual cost, cf. [14]. However, we will compute h(u \| x¯ı¯= 1) and f(z, u \| x¯ı¯= 1), i.e., restricting x¯ı¯to one by replacing the set X¯ı(z) in (4b) by X¯ı(z) ∩{x¯ı· ∈Bn \| x¯ı¯= 1} for some ¯ı ∈I, ¯∈J . Moreover, the feasible set Xsa is respected by also restricting xi¯= 0, i ∈I \ {¯ı}, i.e., the task ¯is assigned to only machine ¯ı. Thus, if h(u \| x¯ı¯= 1) ≥z, we may ﬁx x¯ı¯= 0, since the constraint x¯ı¯= 1 does not allow for an improved upper bound. Before describing the computation of h(u \| x¯ı¯= 1) we present the classical dynamic programming approach ([30, p. 38]) for solving binary knapsack problems. For each i ∈I a value matrix V i ∈Rn×(¯z+1) + is introduced and to ﬁnd an optimal solution to the i-th binary knapsack problem, the items are recursively inserted as    Before describing the computation of h(u \| x¯ı¯= 1) we present the classical dynamic programming approach ([30, p. 38]) for solving binary knapsack problems. For each i ∈I a value matrix V i ∈Rn×(¯z+1) + is introduced and to ﬁnd an optimal solution to the i-th binary knapsack problem, the items are recursively inserted as V i jw := ( V i (j−1)w, w ∈{0, . . . , pij −1}, max{V i (j−1)w, V i (j−1)(w−pij) + uj}, w ∈{pij, . . . , ¯z}, j ∈J , (5) j ∈J , 4 Exact methods for the R\|\|Cmax For a ﬁxed value v = ev, the inner maximization problem in (8) decomposes into two knapsack problems using only the items before ¯and after ¯, respectively. The optimal values of these two knapsack problems are thus V i (¯−1)˜v and eV i (¯+1)(z−˜v), respectively. Hence, by maximizing the decomposed problem (8) for every v ∈{0, . . . , z}, the value of the knapsack without item ¯can be computed by the proposed formula for W i ¯z. By the deﬁnition of W i ¯z for i ∈I, ¯∈J , and size z, the value of knapsack ¯ı ∈I restricted by x¯ı¯= 1 (i.e., with feasible set X¯ı(z) ∩{x¯ı· ∈Bn \| x¯ı¯= 1}) equals W¯ı ¯(z−p¯ı¯) + u¯while the values of the knapsacks i ∈I \ {ı} that are restricted by xi¯= 0 (i.e., with feasible sets Xi(z) ∩{xi· ∈Bn \| xi¯= 0}) equal W i ¯z. The sum of these values then yields the proposed formula for f(z, u \| x¯ı¯= 1). ◀ ▶Remark 2. In contrast to the by-product (6), using Lemma 1 for variable ﬁxing requires computing W i jz for z ∈{max{0, z −pij}, . . . , z −pij} ∪{z, . . . , z}, i ∈I, j ∈J . However, the variable ﬁxing is done once in each branch–and–bound node, whereas ﬁnding suitable values of u (see Section 3.2) typically requires solving many subproblem instances. Our preliminary results indicated that less than 5% of the overall computation time was spent on the variable ﬁxing. ▶Remark 2. In contrast to the by-product (6), using Lemma 1 for variable ﬁxing requires computing W i jz for z ∈{max{0, z −pij}, . . . , z −pij} ∪{z, . . . , z}, i ∈I, j ∈J . However, the variable ﬁxing is done once in each branch–and–bound node, whereas ﬁnding suitable values of u (see Section 3.2) typically requires solving many subproblem instances. Our preliminary results indicated that less than 5% of the overall computation time was spent on the variable ﬁxing. ▶Remark 3. Our idea of using V i jz and eV i jz originates from the bidirectional search approach suggested in [24] for the computation of residual costs of a shortest path problem with resource constraints. Exact methods for the R\|\|Cmax Moreover, in [36] V i jz, eV i jz, and the set Xsa are also used to assign a job exactly once and to compute the similar residual costs for the generalized assignment problem. However, our approach considers minimizing the makespan. 3.2 Maximizing the Lagrangian dual (11) uk+1 := [uk + tkdk]+, tk := δk zk −h(uk) ∥dk∥2 , k = 0, 1, . . . (11) uk+1 := [uk + tkdk]+, (11) where δ0 := 1 2, and if no improvement has been made to h(uk) over the last twenty iterations then δk+1 := δk 2 ; otherwise δk+1 := δk. The algorithm (11) is terminated when δk < δ, where we use δ = 10−3, which in our preliminary tests showed to be a reasonable choice. where δ0 := 1 2, and if no improvement has been made to h(uk) over the last twenty iterations then δk+1 := δk 2 ; otherwise δk+1 := δk. The algorithm (11) is terminated when δk < δ, where we use δ = 10−3, which in our preliminary tests showed to be a reasonable choice. Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 5 We found that the variable ﬁxing is highly useful for reducing the computation time. Hence, we suggest a further variable ﬁxing similar to the dominance criterion suggested in [29]. Given a machine ¯ı ∈I and two tasks j, ¯∈J , we attempt to assign both tasks to the machine and use a bound similar to that in (6), i.e., f(z, u \| x¯ıj = x¯ı¯= 1) ≤uj + u¯+ V ¯ı n(z−p¯ıj−p¯ı¯) + X i∈I\{¯ı} V i nz. (9) (9) If the bound (9) does not allow an incumbent solution (i.e., if it implies that h(u \| x¯ıj = x¯ı¯= 1) ≥z) then the inequality x¯ıj + x¯ı¯≤1 can be introduced and the following dominance criterion can be checked. Let Ij ⊂I denote the subset of machines i available for task j, i.e., those that have not been removed by variable ﬁxing or by branching. If the inequalities p¯ıj ≤p¯ı¯and pij ≥pi¯hold for all i ∈Ij \ {¯ı}, it follows that any solution satisfying x¯ı¯= 1 and x¯ıj = 0 is dominated by a solution satisfying x¯ı¯= 0 and x¯ıj = 1, hence the variable x¯ı¯can be ﬁxed to zero; cf. [29]. We also apply the dominance criterion suggested in [29], where the inequality x¯ıj + x¯ı¯≤1 is instead ensured by the decisions in the branch-and-bound tree. Note that computing f(z, u \| x¯ıj = x¯ı¯= 1) as in Lemma 1 for each combination of ¯ı, j, and ¯, would be computationally too expensive. 3.2 Maximizing the Lagrangian dual In order to ﬁnd the best lower bound z on z∗, the Lagrangian dual function h : Rn 7→R, which is non-smooth and concave, should be maximized. Hence, we need to ﬁnd h∗:= maxu∈Rn h(u). This maximization is often done using subgradient optimization methods; e.g., Martello et al. [29] used it for the R\|\|Cmax. However, since evaluating h(u) comprises solving m binary knapsack problems (which are NP-hard) we need to limit the number of such evaluations. Note that, by the non-negativity of pij, the search can be restricted to u ≥0, since uj < 0 is dominated by uj = 0 in (4a). An issue with the classical subgradient algorithm is that it often stalls due to zigzagging; cf. [6]. We therefore employ the modiﬁed deﬂected subgradient (MDS) method by Belgacem and Amir [7], which combines, the modiﬁed gradient technique (MGT) by Camerini et al. [10] and the average direction strategy (ADS) by Sherali and Ulular [38]. The MDS method reduces the zigzagging behaviour by determining the step direction as a weighted average of a current subgradient and the previous step direction. k g g g p p Letting sk be a subgradient of h at uk and dk be the step direction, and deﬁning [x]+ := max{0, x}, we employ the formulas dk := sk + Γk MDSdk−1, Γk MDS := (1 −αk)Γk MGT + αkΓk ADS, (10a) Γk MGT := " −ηk (sk)⊤dk−1 ∥dk−1∥2 # + , Γk ADS := ∥sk∥ ∥dk−1∥ . (10b) dk := sk + Γk MDSdk−1, Γk MGT := " −ηk (sk)⊤dk−1 ∥dk−1∥2 # + , dk := sk + Γk MDSdk−1, Γk MDS := (1 −αk)Γk MGT + αkΓk ADS, (10a) Γk MGT := " −ηk (sk)⊤dk−1 ∥dk−1∥2 # + , Γk ADS := ∥sk∥ ∥dk−1∥ . (10b) Γk MDS := (1 −αk)Γk MGT + αkΓk ADS, (10a) Γk ADS := ∥sk∥ ∥dk−1∥ . (10b) (10a) (10b) wing the suggestions in [7], we let ηk := 1 2−αk and αk := h −(sk)⊤dk−1 ∥sk∥∥dk−1∥ i +. Following the suggestions in [7], we let ηk := 1 2−αk and αk := h −(sk)⊤ ∥sk∥∥ + To determine the step lengths, we follow the classical rule given by Polyak [35] and let the step length parameter δk > 0 decrease as suggested by Held et al. [22], i.e., tk := δk zk −h(uk) ∥dk∥2 , k = 0, 1, . . . Exact methods for the R\|\|Cmax We conclude that the inequalities in (15) cannot all hold, and hence that there exists a ¯k ∈¯K and an ¯ı ∈I such that the inequality P j∈J ¯k 0 p¯ıj ≤ P j∈J ¯k 2 p¯ıj holds. Thus, there exists an ¯x ∈Xsa ∩X(z¯k) such that ¯x¯ıj = 1 for j ∈J ¯k 0 , ¯x¯ıj = x¯k ¯ıj for j ∈J ¯k 1 , ¯x¯ıj = 0 for j ∈J ¯k 2 , and ¯x(3−¯ı)j = x¯k (3−¯ı)j for j ∈J . Now since z¯k = z and (¯x, z¯k) ∈S it follows that z¯k = z∗, and hence that h∗= P k∈¯K α∗ kzk = z = z∗. ◀ Then, inserting the rightmost equality of (14) in (16) yields a contradiction. We conclude that the inequalities in (15) cannot all hold, and hence that there exists a ¯k ∈¯K and an ¯ı ∈I such that the inequality P j∈J ¯k 0 p¯ıj ≤ P j∈J ¯k 2 p¯ıj holds. Thus, there exists an ¯x ∈Xsa ∩X(z¯k) such that ¯x¯ıj = 1 for j ∈J ¯k 0 , ¯x¯ıj = x¯k ¯ıj for j ∈J ¯k 1 , ¯x¯ıj = 0 for j ∈J ¯k 2 , and ¯x(3−¯ı)j = x¯k (3−¯ı)j for j ∈J . Now since z¯k = z and (¯x, z¯k) ∈S it follows that z¯k = z∗, and hence that h∗= P k∈¯K α∗ kzk = z = z∗. ◀ ▶Remark 5. A consequence of Theorem 4 is that there is no duality gap for m = 2, since either the lower bound z can be strengthened, or the bound coincides with the optimal value. ▶Remark 5. A consequence of Theorem 4 is that there is no duality gap for m = 2, since either the lower bound z can be strengthened, or the bound coincides with the optimal value. ▶Remark 6. From (13) follows that h∗≥z also for m > 2. However, if h∗= z then the lower bound z cannot be strengthened and the duality gap z∗−h∗can be non-zero. 3.3 Duality gap The strength of the Lagrangian relaxation as deﬁned in (4a) was studied in [29], where the corresponding lower bound h∗≤z∗was found to be stronger than the LP bound. In Theorem 4, we strengthen this result further and show that for m = 2, either h∗> z or h∗= z∗hold. Hence, the best known lower bound z can be increased until z = z∗holds. For convenience, we ﬁrst restate the Lagrangian dual function (4a) in its original form where the structure of the set X(z) has not yet been utilized: h(u) = X j∈J uj + minimum (x,z)∈Bmn×[z,z] ( z − X i∈I X j∈J ujxij x ∈X(z) ) . (12) (12) 6 Exact methods for the R\|\|Cmax Exact methods for the R\|\|Cmax ▶Theorem 4. Let m = 2. If h∗≤z then h∗= z∗. ▶Theorem 4. Let m = 2. If h∗≤z then h∗= z∗. Proof. By the relation between the Dantzig–Wolfe reformulation ([11, Ch. 8.2]) and Lagrangian relaxation, it is holds that h∗= minimum α≥0 X k∈K αkzk, (13a) such that X k∈K αk X i∈I xk ij = 1, j ∈J , (13b) X k∈K αk = 1, (13c) h∗= minimum α≥0 X k∈K αkzk, such that X k∈K αk X i∈I xk ij = 1, j ∈J , X k∈K αk = 1, (13a) h∗= minimum α≥0 (13b) (13c) where (xk, zk), k ∈K, denote the extreme points of the polytope conv {(x, z) ∈Bmn × [z, z] \| x ∈X(z)}; cf. (12). Let α∗be an optimal solution to the Dantzig–Wolfe master problem (13) and deﬁne the set ¯K := {k ∈K \| α∗ k > 0}. It holds that zk = z for all k ∈¯K, since otherwise the strict inequality h∗> z must hold. Moreover, let J k l := {j ∈J \| P i∈I xk ij = l}, k ∈¯K, and Kj l := {k ∈¯K \| P i∈I xk ij = l}, j ∈J , l ∈{0, 1, 2}. From (13b) and (13c) it then follows that 2 X k∈Kj 2 α∗ k + X k∈Kj 1 α∗ k − X k∈¯K α∗ k = 0 =⇒ X k∈Kj 2 α∗ k = X k∈Kj 0 α∗ k, j ∈J . (14) (14) Now, assume that the inequalities Now, assume that the inequalities X j∈J k 0 pij > X j∈J k 2 pij, k ∈¯K, i ∈I, X j∈J k 0 pij > X j∈J k 2 pij, k ∈¯K, i ∈I, (15) (15) old, scale the inequalities (15) by α∗ k and sum over k ∈¯K, for each i ∈I. Then, it follows that hold, scale the inequalities (15) by α∗ k and sum over k ∈¯K, for each i ∈I. Then, it follows that X k∈¯K α∗ k X j∈J k 0 pij > X k∈¯K α∗ k X j∈J k 2 pij =⇒ X j∈J pij X k∈Kj 0 α∗ k − X k∈Kj 2 α∗ k > 0. (16) (16) Then, inserting the rightmost equality of (14) in (16) yields a contradiction. ▶Lemma 8. If h∗≤z, where h∗= maxu∈Rn h(u), then ¯h(u) ≤h∗, ∀u ∈Rn. Proof. From (13) it is clear that h∗≥z; thus our assumption implies that h∗= z holds. Moreover, in every optimal solution α∗of (13) we have that α∗ k = 0 for all k ∈K such that zk > z. Hence, restricting (13) to the subset of extreme points satisfying zk = z does not modify the set of optimal solutions. As a consequence, the feasible set to the minimization in (4a) can be restricted to z = z, i.e., it holds that h∗= max µ∈Rn (X j∈J µj + z −f(z, µ) ) ≥ X j∈J uj + z −f(z, u), u ∈Rn. h∗= max µ∈Rn (X j∈J µj + z −f(z, µ) ) ≥ X j∈J uj + z −f(z, u), u ∈Rn. (19) (19) Now assume that ∃¯u ∈Rn : ¯h(¯u) > z, where ¯h(u) is deﬁned by (18). It follows that Now assume that ∃¯u ∈Rn : ¯h(¯u) > z, where ¯h(u) is deﬁned by (18). It follows tha Now assume that ∃¯u ∈Rn : ¯h(¯u) > z, where ¯h(u) is deﬁned by (18). It follows that X j∈J ¯uj −f(z, ¯u) > 0 =⇒ h∗> z, (20) X j∈J ¯uj −f(z, ¯u) > 0 =⇒ h∗> z, (20) X ¯uj −f(z, ¯u) > 0 =⇒ h∗> z, which contradicts our assumption that h∗≤z. Hence, ¯h(u) ≤z must hold for all u ∈Rn and the lemma follows, since the equality h∗= z holds. ◀ 3.4 Strategies for the branch–and–bound algorithm For the primal–dual pair z∗and h∗there might be a non-zero duality gap. Hence, the branch–and–bound algorithm is used in order to ﬁnd an optimal solution. In order to get a small branch–and–bound tree we choose to use the worst–ﬁrst node selection strategy, i.e., always choose the branch–and–bound node with the highest lower bound. To determine which task to branch on we use the rule suggested in [29, §4], but employ our residual costs ¯cij := h(u \| xij = 1) −h(u), i.e., ¯∈arg maxj∈J:\|Ij\|>1{m −\|Ij\| + Θ mini∈Ij ¯cij}, where Ij is the set of machines available for task j and Θ = 20, as suggested in [29]. For a selected task j, we partition the available machines into two equally sized sets by repeatedly assigning the unassigned machine having the maximum ¯cij, in an alternating fashion. The idea is to make the two branches equally promising, and preliminary results indicated that this approach is more eﬀective than branching on a single variable. We think that a more advanced partitioning rule, such as reliability branching [4], local branching [13], or machine learning based branching [5], could improve our algorithm and intend to develop these ideas in future research. In the root node, the LP dual solution is utilized as a starting point. This provides an initial lower bound z and initial values for the Lagrangian multipliers u as the corresponding LP dual variable values. In the child nodes, the Lagrangian multipliers are initialized from the parent node. Note that, as in [29] the Lagrangian relaxation of the constraints in (2a) is used to compute the LP dual solution. Exact methods for the R\|\|Cmax Consider the following counterexample for m = 3, n = 6, z = 4, z = 5, and K ⊆{1, 2}: p =   6 1 2 2 3 6 1 6 3 3 6 1 1 1 6 6 4 2  , x1 =   0 0 1 1 0 0 0 0 1 0 0 1 0 0 0 0 1 0  , x2 =   0 1 0 0 1 0 1 0 0 1 0 0 1 1 0 0 0 1  , α1 = α2 = 1 2. (17) (17) It can be veriﬁed that z1 = z2 = 4 and z∗= 5 and that α1 and α2 constitute a feasible solution to the LP master problem (13). Hence, the relations 4 = z ≤h∗≤4 < z∗hold, i.e., the duality gap is non-zero. ▶Remark 7. In Theorem 4, the Dantzig–Wolfe reformulation was used. Preliminary results indicated that this is, however, an ineﬃcient approach for computing h∗, because (i) when z was increased, most of the generated columns became infeasible and needed to be discarded from the master problem and (ii) solving the master problem was computationally expensive. Consider instead maximizing the Lagrangian dual function (4a) using a subgradient optimization method; then there is no negative computational impact of increasing the value of z. Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 7 7 In [29] it was shown that the lower bound h(u) can be improved according to ¯h(u) := minimum z∈{z,...,z} ( z X j∈J uj ≤f(z, u) ) , (18) i.e., that the inequality h(u) ≤¯h(u), holds for all u ∈Rn. The fact that f is an non-decreasing function of z was used to eﬃciently evaluate ¯h(u), unlike our approach of computing f(z, u) for every z ∈{z, . . . , z} (recall Section 3.1). Despite being the strongest bound suggested in [29], the authors stated that since ¯h(u) was cumbersome to maximize their weaker bounds were sometimes more useful. i.e., that the inequality h(u) ≤¯h(u), holds for all u ∈Rn. The fact that f is an non-decreasing function of z was used to eﬃciently evaluate ¯h(u), unlike our approach of computing f(z, u) for every z ∈{z, . . . , z} (recall Section 3.1). Despite being the strongest bound suggested in [29], the authors stated that since ¯h(u) was cumbersome to maximize their weaker bounds were sometimes more useful. Note that the maximum of ¯h(u) will not provide an improved bound compared to h∗, as shown in Lemma 8 below. However, since h∗is not always retrieved due to early termination of the subgradient algorithm, the improved bound (18) is evaluated for all values of u encountered. Moreover, we suggest not to use the improved bound (18) in the subgradient based algorithm (11) to decide the step length and direction. This motivated by Lemma 8 and because ¯h is a discrete function and thus progress becomes less frequent and less regular, resulting in a quickly diminishing step size. 8. If h∗≤z, where h∗= maxu∈Rn h(u), then ¯h(u) ≤h∗, ∀u ∈Rn. Lemma 8. If h∗≤z, where h∗= maxu∈Rn h(u), then ¯h(u) ≤h∗, ∀u ∈Rn. 5 Tests and results Our computational results were generated on a computer with an AMD Ryzen 9 3900X 12-Core 3.79 GHz and 32GB of RAM. To simplify the presentation of the results, all algorithms were limited to run on a single core, and report the CPU time. The implementations, see [2], are made in C++. The instances and the algorithms tested are presented in Section 5.1. An analysis of the features of the Lagrangian relaxation algorithm is presented in Section 5.2. An overview of the performance is given in Section 5.3, and details on the results are given by type of test instances in Sections 5.4–5.6. Exact methods for the R\|\|Cmax Figure 1 Examples of operations allowed within the two neighbourhood deﬁnitions; each dot represents a variable xij and a ﬁlled dot encodes the value 1. Figure 1 Examples of operations allowed within the two neighbourhood deﬁnitions; each dot represents a variable xij and a ﬁlled dot encodes the value 1. 3-cycle: All perturbations of a feasible solution such that one task is assigned to another machine. The receiving machine can reassign one of its tasks to a third machine; the third machine can reassign one of its tasks to any of the two previous machines. See the illustration in Figure 1a. This is thus a special case of either a cyclic exchange or a path exchange. 2-1-swap: All perturbations of a feasible solution such that two tasks, assigned to a machine with zero slack in (3), are reassigned to another machine and one task from this machine is reassigned to the ﬁrst machine. See the illustration in Figure 1b. The neighbourhoods are searched (greedily) in the following order: task reassign, task interchange, 2-1-swap, and 3-cycle. Note that the entire neighbourhoods are searched, not just partially as done in [15] for the cyclic exchange and path exchange neighbourhoods. The heuristics described in this section are used in the branch–and– bound algorithm described in Section 3.4. We found it beneﬁcial to run the heuristics every tenth iteration of the subgradient algorithm in the root node while only in the ﬁnal iteration in the child nodes. 4 Heuristics In Section 3 we seek to improve the lower bound z ≤z∗; however, its usefulness relies on a tight upper bound. As noted in Section 2, there are many heuristic algorithms that provide feasible solutions to the R\|\|Cmax (3). First, to ﬁnd a feasible solution from a solution to (4a) we use a greedy destruction–reconstruction heuristic 8 Exact methods for the R\|\|Cmax that removes machines from multiply-assigned tasks, and assign a machine to each unassigned task, always greedily minimizing the makespan. Second, we construct a fractional solution as the convex combination of all subproblem solutions xk (from the current branch–and–bound node) weighted proportionally to the step lengths tk; this forms a so-called ergodic sequence of subproblem solutions; see [26, 33] for details. To construct a binary solution each job is assigned to the machine with the highest fraction. Third, we use a local search heuristic with the two neighbourhoods 3-cycle and 2-1-swap deﬁned below; preliminary results indicated these to be suﬃciently small w.r.t. the overall computing time. These neighbourhoods include the reassign and interchange neighbourhoods from [19] and are partially included in cyclic exchange and path exchange from [15]. that removes machines from multiply-assigned tasks, and assign a machine to each unassigned task, always greedily minimizing the makespan. Second, we construct a fractional solution as the convex combination of all subproblem solutions xk (from the current branch–and–bound node) weighted proportionally to the step lengths tk; this forms a so-called ergodic sequence of subproblem solutions; see [26, 33] for details. To construct a binary solution each job is assigned to the machine with the highest fraction. Third, we use a local search heuristic with the two neighbourhoods 3-cycle and 2-1-swap deﬁned below; preliminary results indicated these to be suﬃciently small w.r.t. the overall computing time. These neighbourhoods include the reassign and interchange neighbourhoods from [19] and are partially included in cyclic exchange and path exchange from [15]. tasks, j machines, i 1 2 3 4 5 6 1 2 3 (a) 3-cycle tasks, j machines, i 1 2 3 4 5 6 1 2 3 (b) 2-1-swap Figure 1 Examples of operations allowed within the two neighbourhood deﬁnitions; each dot represents a variable xij and a ﬁlled dot encodes the value 1. tasks, j machines, i 1 2 3 4 5 6 1 2 3 (a) 3-cycle tasks, j machines, i 1 2 3 4 5 6 1 2 3 (b) 2-1-swap Figure 1 Examples of operations allowed within the two neighbourhood deﬁnitions; each dot represents a variable x and a ﬁlled dot encodes the value 1 (a) 3-cycle Figure 1 Examples of operations allowed within the two neighbourhood deﬁnitions; each dot represents a variable xij and a ﬁlled dot encodes the value 1. Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 9 We have tested our formulations and algorithms on three sets of instances denoted: (i) uncorrelated, (ii) correlated jobs, and (iii) correlated machines. All combinations of the sizes m ∈{3, 5, 10, 15, 20} and n ∈{30, 50, 80, 100, 200} were tested, with ten random instances for each combination. Uncorrelated means that the processing times possess no correlation between machines or jobs, i.e., pij is generated from a discrete uniform distribution with the support {10, 11, . . . , 100}, denoted U{10, 100}. Correlated jobs means that the processing times correlate with jobs, i.e., pij = dij + bj, where bj and dij are generated from U{1, 100} and U{1, 20}, respectively. Correlated machines means that the processing times correlate with machines, i.e., pij = ai + dij, where ai and dij are generated from U{1, 100} and U{1, 20}, respectively. Our instance settings are nearly identical to the ones used in [42], where we do not use U{1, 100} for the uncorrelated instances, since such instances are solved faster (see, e.g., [12]) and since U{10, 100} is a more common choice (see, e.g., [29, 32]). Each instance is solved by eight diﬀerent algorithms. DEF: solve the model (3) (default algorithm). CUT: solve model (3) using the aggressive cuts parameter setting in Gurobi. MOK: use the cutting plane method by Mokotoﬀ and Chrétienne [32], with two diﬀerences: we use (a) Gurobi instead of CPLEX and (b) our heuristics instead of the suggested one to initialize the upper bound. DEF+y, CUT+y, and MOK+y: include the aggregated variables and the constraints (21) in the models of the three methods, DEF, CUT, and MOK, respectively. LR: use the branch–and–bound algorithm based on the Lagrangian relaxation (4a), with the local search heuristic from Section 4. LR-F: LR with the variable ﬁxing based on the reduced costs of the LP relaxation instead of Lemma 1. The use of CUT and CUT+y are motivated by the fact that Gurobi’s parameter tuning tool detected that the aggressive cuts parameter reduced the CPU time the most. For MOK and MOK+y the tuning tool did not ﬁnd any signiﬁcant reductions as compared with the default parameters. All algorithms were given a time limit of 120 s to solve each instance (to veriﬁed optimality). The components of the LR algorithm is analysed separately in Section 5.2. 5.1 Test instances and algorithms Since we were unable to use previously published work (instances and implementations), we use the MILP solver Gurobi 9 [21] as a point of reference by solving (3). Further, to ease future comparisons our instances are available online. In addition to this, we solve an extended formulation of (3) that includes the following aggregated variables subject to integrality constraints: yi = X j∈J xij, yi ∈Z+, i ∈I. (21) The idea behind the inclusion of these aggregated variables is the ability to branch on the number of tasks assigned to a machine, which is beneﬁcial in particular when a machine contributing to the makespan is assigned a fractional number of tasks; after branching it is assigned more tasks in one of the branches and thus the makespan is likely to increase, leading to an improved quality of the lower bound. We disable presolve when using the aggregated variables to prevent them from being removed. A technique for branching based on presolve information is suggested in [17], hence presolve might not need to be disabled in other MILP solvers. 5.2 Sensitivity analysis of the Lagrangian relaxation algorithm In Sections 3 and 4 several features of the LR algorithm were presented; the impact of each of these is analysed here. The idea is to switch all of these features on and oﬀand measure the corresponding overall solution times. Moreover, when a feature is switched oﬀ, a well-known alternative is sometimes used instead. Var. ﬁx. denotes the use of the variable ﬁxing based on Lemma 1 (on) or based on the reduced Var. ﬁx. denotes the use of the variable ﬁxing based on Lemma 1 (on) or based on the reduc LP l ti f th R\|\|C (3) ( ﬀ) B th ti l th ﬁi i h b h d b Var. ﬁx. denotes the use of the variable ﬁxing based on Lemma 1 (on) or based on the reduced costs of the LP relaxation of the R\|\|Cmax (3) (oﬀ). Both options apply the ﬁxing in each branch–and–bound node. Var. ﬁx. denotes the use of the variable ﬁxing based on Lemma 1 (on) or based on the reduced costs of the LP relaxation of the R\|\|Cmax (3) (oﬀ). Both options apply the ﬁxing in each branch–and–bound node. Var. ﬁx. denotes the use of the variable ﬁxing based on Lemma 1 (on) or based on the re LP relaxation of the R\|\|Cmax (3) (oﬀ). Both options apply the ﬁxing in each branch–and LP relaxation of the R\|\|Cmax (3) (oﬀ). Both options apply the ﬁxing in each branch–and–bo P relaxation of the R\|\|Cmax (3) (oﬀ). Both options apply the ﬁxing in each branch–and–bound no Subgr. iter. denotes few (oﬀ) / many (on) subgradient iterations, to emphasize the importance of maximizing the strong dual function (4a). Few uses a maximum of 300 and 10 subgradient iterations in the root and child nodes respectively (cf. [29]). The corresponding numbers for many are 1000 and 100. Note that a small step length parameter δk also terminates the subgradient algorithm; cf. Section 3.2. Deﬂect denotes the use of the subgradient deﬂection method MDS (on) (recall Section 3.2) or to not deﬂect the subgradient sk, i.e., letting dk := sk in (11) (oﬀ). Deﬂect denotes the use of the subgradient deﬂection method MDS (on) (recall Section 3.2) or to not deﬂect the subgradient sk, i.e., letting dk := sk in (11) (oﬀ). Exact methods for the R\|\|Cmax Exact methods for the R\|\|Cmax Erg. heur. LNBH Deﬂect Subgr. iter. Var. ﬁx. 2−4 2−3 2−2 2−1 20 21 22 23 Score = Tall T Sensitivy analysis of the Lagrangian relaxation algorithm mean outliers Figure 2 The impact of not using (oﬀ, white) or using a feature (on, black). T denotes each execution time on ten uncorrelated instances and ten correlated jobs instances of size m = 10 and n = 100. The score for each instance and feature is the ratio between switching all features on (Tall) and T. The scores for the 20 instances and each feature combination are presented with boxplots (median, lower and upper quartiles, and the most extreme values in the 1.5·IQR (inter-quartile range)), outliers (grey circles), and mean (blue squares). Erg. heur. LNBH Deﬂect Subgr. iter. Var. ﬁx. 2−4 2−3 2−2 2−1 20 21 22 23 Score = Tall T Sensitivy analysis of the Lagrangian relaxation algorithm mean outliers Figure 2 The impact of not using (oﬀ, white) or using a feature (on, black). T denotes each execution time on ten uncorrelated instances and ten correlated jobs instances of size m = 10 and n = 100. The score for each instance and feature is the ratio between switching all features on (Tall) and T. The scores for the 20 instances and each feature combination are presented with boxplots (median, lower and upper quartiles, and the most extreme values in the 1.5·IQR (inter-quartile range)), outliers (grey circles), and mean (blue squares). Sensitivy analysis of the Lagrangian relaxation algorithm Sensitivy analysis of the Lagrangian relaxation algorithm Figure 2 The impact of not using (oﬀ, white) or using a feature (on, black). T denotes each execution time on ten uncorrelated instances and ten correlated jobs instances of size m = 10 and n = 100. The score for each instance and feature is the ratio between switching all features on (Tall) and T. The scores for the 20 instances and each feature combination are presented with boxplots (median, lower and upper quartiles, and the most extreme values in the 1.5·IQR (inter-quartile range)), outliers (grey circles), and mean (blue squares). especially when considering the median values. This is expected due to the strong bound it provides (recall Section 3.3). However, using many subgradient iterations have a negative impact on less demanding instances, thus aﬀecting the mean values. Exact methods for the R\|\|Cmax On the primal heuristic side, we observe that searching the larger neighbourhood (i.e., LNBH) generally pays oﬀin terms of the median values. For the instances considered in this section, there is no major improvement by using the ergodic sequence heuristic (i.e., Erg. heur.). However, for some larger correlated jobs instances in Section 5.5 these were slightly useful. Similarly, for the 20 instances in this section, the dominance criterion (9) did not have a measurable impact and is thus not illustrated in the ﬁgure; however, for some larger correlated jobs it was slightly beneﬁcial, consequently; this criterion is always applied. Another trend that is visible in Figure 2 is that usage of more features, especially var. ﬁx. and subgr. iter. decreases the spread of computing times. This more robust behaviour is expected since more computational eﬀort is made in each node aiming at decreasing the number of nodes that will need to be visited. Hence, some computationally easy instance might get an increase in computing time whereas a decrease is expected for harder instances. This manifests when employing all features except LNBH, which results in high mean and median scores, at the cost of roughly 25% of the instances having a score of a 2−1 or lower. Moreover, the deﬂected subgradient method seems dependent on using LNBH. We suspect that this is due to the smoother trajectory of the deﬂected version, which tends to improve the lower bound but also to reduce the variety of subproblem solutions; thus, the upper bound suﬀers without the LNBH. Related to the deﬂected method is that we also attempted to use the ADS deﬂection method, which had a bit slower convergence of the lower bound but found better feasible solutions faster. The overall diﬀerence between MDS and ADS was, however, not signiﬁcant. 5.2 Sensitivity analysis of the Lagrangian relaxation algorithm LNBH denotes the use of large neighbourhoods 3-cycle and 2-1-swap from Section 4 (on), or to only use the reassign and interchange neighbourhoods (oﬀ). LNBH denotes the use of large neighbourhoods 3-cycle and 2-1-swap from Section 4 (on), or to only use the reassign and interchange neighbourhoods (oﬀ). Erg. heur. denotes the use of the rounding heuristic on fractional values retrieved by the ergodic sequence (on), or to use only the destruction–reconstruction heuristic on subproblem solutions (oﬀ) (recall Section 4). The local search heuristic is used to improve the solutions in both cases (on and oﬀ). Erg. heur. denotes the use of the rounding heuristic on fractional values retrieved by the ergodic sequence (on), or to use only the destruction–reconstruction heuristic on subproblem solutions (oﬀ) (recall Section 4). The local search heuristic is used to improve the solutions in both cases (on and oﬀ). All combinations of using and not using each of the above stated features, and the resulting performance ratios Tall/T, are presented in Figure 2; where T and Tall denote the execution time for an instance using a certain combination of features and using all features, respectively. Each combination of features was executed on ten uncorrelated and correlated jobs instances, with m=10 machines and n=100 tasks. We found that the ten uncorrelated instances were solved by every feature combination whereas the ten correlated jobs instances were solved only by a few of them. The correlated machines instances were also easy to solve (see Section 5.6); hence, to get a balanced set of instances these are not included here. Note that when an instance is not solved then the time limit T = 120 s is reported and the true execution time Ttrue ≥120 s is underestimated; hence the ratio Tall/T is an overestimate. However, for each of the 20 instances Tall ≤3.47 s, hence the error introduced by the time limit is bounded by Tall T −Tall Ttrue ≤3.47 120 ≤2−5. true Figure 2 shows the consistent beneﬁt (by higher distributed scores) of using the variable ﬁxing from Lemma 1 rather than basing it on the reduced cost from an LP relaxation. The ﬁgure also shows the importance of maximizing the Lagrangian dual function, since more computational eﬀort (many subgradient iterations) pays-oﬀ, 10 5.4 Uncorrelated instances In Table 1, the average CPU times over ten instances and the number of unsolved instances for the uncorrelated instances are presented. Comparing the two ﬁrst columns it seems that the algorithm DEF+y is faster than DEF for large problem instances. Likewise, for the tuned Gurobi parameters, the third and fourth columns Table 1 The average computing time [s] for solving ten uncorrelated instances of size m × n. Within parentheses are the numbers of instances exceeding the time limit of 120 s; these instances do not contribute to the average computing times. The average number of branch–and–bound nodes and the average gap ( z−z z ) in the root node is presented for LR. Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 11 10−3 10−2 10−1 100 101 102 0% 25% 50% 75% 100% CPU time [s] % instances solved Fraction of solved instances w.r.t. CPU time. DEF DEF+y CUT CUT+y MOK MOK+y LR-F LR Figure 3 The seven algorithms applied to all the instances, uncorrelated, correlated jobs, and correlated machines. Fraction of solved instances w.r.t. CPU time. Fraction of solved instances w.r.t. CPU time. Figure 3 The seven algorithms applied to all the instances, uncorrelated, correlated jobs, and correlated machines. result, both of these hypotheses are rejected with a certainty greater than 99.9% when comparing DEF with DEF+y and MOK with MOK+y, where the average speed up (geometric mean of the ratios T A i /T B i ) are in both cases 1.5. However, the hypotheses are not rejected when comparing CUT with CUT+y and the speed up is 1.0. To conclude, the reason why the algorithms DEF+y and MOK+y solve more instances faster as compared to DEF and MOK, respectively, is very unlikely to be a coincidence. 5.3 Overall performance Figure 3 presents the fraction of solved instances by the eight algorithms described above as a function of CPU time. The ﬁgure indicates that the Lagrangian based branch–and–bound method is both the fastest and the most robust, i.e., it is able to solve the most instances within the given time limit of 120 s. Moreover, using the variable ﬁxing from Lemma 1 seems to enable the solution of more instances. Another trend that is observed (although less prominent) is that the inclusion of the aggregated variables y reduces the computing time and enables the solution of more instances, in particular within the algorithms DEF+y and MOK+y. To establish the observed utility of using the aggregated variables y, two statistical tests, cf. [8], are used. First, the McNemar’s test considers the hypothesis that two algorithms (A and B) solve equally many instances. Let sA and sB denote the number of instances solved by only algorithm A and B, respectively; if the diﬀerences are random then (sA−sB)2 sA+sB follows a χ2–distribution with one degree of freedom. Second, the one-sample Wilcoxon’s signed rank test considers the hypothesis that the logarithm of the solution time ratios, i.e., log(T A i /T B i ), originates from a distribution with zero median, i.e., that the two algorithms are equally fast. Here T A i and T B i denote the CPU times for the two algorithms, and only instances i solved by both algorithms are included. As a Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri 5.5 Correlated jobs instances In Table 2, the average CPU times over ten instances and the number of unsolved instances for the correlated jobs instances are presented. The most obvious diﬀerence from the uncorrelated instances is that the correlated jobs instances are harder to solve, and for the largest instance size none of the algorithms can solve all instances within the given time limit. Another diﬀerent property of for the correlated jobs instances is that the aggregated variables y seem to be of less use for the algorithms DEF+y and CUT+y, which for some instances require longer computing time and even exceed the time limit. For the algorithm MOK+y the result is, however, unchanged w.r.t. the uncorrelated jobs instances: this algorithm seems preferable among the ﬁrst six algorithms. The LR algorithm dominates the other algorithms on most instance sizes and it often requires the stronger variable ﬁxing (i.e., Lemma 1). One reason for LR not being able to solve some instances, is that the local search heuristics failed to ﬁnd the optimal solution. Moreover, we attempted to use a sophisticated heuristic such as the destruction–reconstruction heuristic described in [12], but this yielded no signiﬁcant improvement as compared Table 2 The average computing time [s] for solving ten correlated jobs instances of size m × n. Within parentheses are the numbers of instances exceeding the time limit of 120 s; these instances do not contribute to the average computing times. The average number of branch–and–bound nodes and the average gap ( z−z z ) in the root node is presented for LR. Exact methods for the R\|\|Cmax show that the algorithm CUT+y is faster than CUT for the largest instances. The same result holds for the algorithms MOK+y and MOK, for which the importance of the variables y seem to be the greatest. Apart from the LR algorithm, it seems that CUT+y, or possibly MOK+y, are the fastest and most robust methods for these instances. show that the algorithm CUT+y is faster than CUT for the largest instances. The same result holds for the algorithms MOK+y and MOK, for which the importance of the variables y seem to be the greatest. Apart from the LR algorithm, it seems that CUT+y, or possibly MOK+y, are the fastest and most robust methods for these instances. Moreover, the LR algorithm is able to solve every uncorrelated instance in our test-bed, and branching is necessary only for large instances, verifying the strength of the Lagrangian lower bound (4a), whereas the variable ﬁxing from Lemma 1 only marginally decreases the computing times. To get another point of reference, consider that the number of branch–and–bound nodes required by Martello et al. [29] to solve corresponding (unfortunately not identical) instances of size 10 × 80 was on average 5723, whereas the corresponding number for our algorithm is 2.2; a similar diﬀerence in magnitude holds for all large uncorrelated instances. This improvement is mainly explained by our focus to maximize the Lagrangian lower bound also in the child nodes. Note that the variable ﬁxing also reduce the computing times but it is more important for harder instances; compare LR-F with LR. 5.4 Uncorrelated instances m n DEF DEF+y CUT CUT+y MOK MOK+y LR-F LR LR nodes LR root gap [%] 3 30 0.02 0.02 0.02 0.03 0.04 0.03 0.0 0.0 1.0 0.0 50 0.02 0.02 0.03 0.03 0.04 0.03 0.0 0.0 1.0 0.0 80 0.02 0.03 0.07 0.07 0.04 0.04 0.0 0.0 1.0 0.0 100 0.03 0.04 0.13 0.16 0.07 0.06 0.0 0.0 1.0 0.0 200 0.05 0.07 0.14 0.15 0.13 0.12 0.02 0.02 1.0 0.0 5 30 0.04 0.04 0.06 0.07 0.05 0.05 0.0 0.0 1.0 0.0 50 0.06 0.08 0.09 0.11 0.1 0.09 0.0 0.0 1.0 0.0 80 0.12 0.1 0.17 0.27 0.12 0.1 0.0 0.0 1.0 0.0 100 0.11 0.16 0.29 0.32 0.17 0.14 0.02 0.02 1.5 0.02 200 0.91 1.34 2.75 1.84 1.4 0.84 0.17 0.14 2.9 0.02 10 30 0.11 0.12 0.12 0.15 0.16 0.12 0.0 0.0 1.0 0.0 50 0.43 0.25 0.24 0.54 0.66 0.26 0.01 0.01 1.0 0.0 80 12.8 0.75 2.08 2.21 8.8 (2) 0.63 0.03 0.02 2.2 0.07 100 17.1 (7) 10.3 (1) 6.37(1) 9.27 21.9 (5) 8.54 0.09 0.06 3.8 0.11 200 0.39(7) 1.07(7) 15.9 (3) 19.7 (1) 35.5 (7) 17.5 0.85 0.44 7.6 0.14 15 30 0.15 0.11 0.1 0.14 0.03 0.06 0.01 0.0 1.0 0.0 50 0.68 0.21 0.31 0.45 0.17 0.34 0.01 0.01 1.0 0.0 80 13.1 (2) 3.8 (1) 1.82 1.15 19.8 9.18 0.16 0.05 2.4 0.22 100 24.7 (3) 17.4 (3) 7.48 10.3 22.9 (1) 14.3 0.21 0.1 8.4 0.18 200 84.7 (9) 43.8 (8) 28.0 (5) 37.5 (4) 57.0 (8) 27.8 (5) 3.19 0.58 29.6 0.19 20 30 0.16 0.12 0.08 0.12 0.01 0.03 0.01 0.01 1.0 0.0 50 0.52 0.5 0.25 0.29 0.11 0.1 0.01 0.01 1.0 0.0 80 1.25(1) 2.6 0.9 0.92 7.97(2) 2.23 0.04 0.04 1.0 0.0 100 7.53(3) 3.9 (1) 3.75 2.98 34.1 (3) 22.0 (1) 0.12 0.08 1.2 0.13 200 — 80.7 (8) 65.0 (8) 38.4 (5) 28.8 (8) 62.4 (7) 17.4 1.09 95.3 0.56 12 Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri Table 3 The average computing time [s] for solving ten correlated machines instances of size m × n. Within parentheses are the numbers of instances exceeding the time limit of 120 s; these instances do not contribute to the average computing times. The average number of branch–and–bound nodes and the average gap ( z−z z ) in the root node is presented for LR. Table 3 The average computing time [s] for solving ten correlated machines instances of size m × n. Within parentheses are the numbers of instances exceeding the time limit of 120 s; these instances do not contribute to the average computing times. The average number of branch–and–bound nodes and the average gap ( z−z z ) in the root node is presented for LR. Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri to our simple local search heuristic. When an optimum was found, the veriﬁcation of optimality was usually quite immediate. Hence, for these unsolved instances the weakness is in the upper bound. Note, however, that the gap is roughly 1% or less in the root node, thus the quality of either bound is still quite good. Moreover, for the instances we were able to solve, e.g., for m = 10 we also computed the lower gap, i.e., z∗−z z∗: its value of roughly 0.05–0.1% again suggests that the issue is not the strength of the lower bound but the weakness of the upper bound. to our simple local search heuristic. When an optimum was found, the veriﬁcation of optimality was usually quite immediate. Hence, for these unsolved instances the weakness is in the upper bound. Note, however, that the gap is roughly 1% or less in the root node, thus the quality of either bound is still quite good. Moreover, for the instances we were able to solve, e.g., for m = 10 we also computed the lower gap, i.e., z∗−z z∗: its value of roughly 0.05–0.1% again suggests that the issue is not the strength of the lower bound but the weakness of the upper bound. 5.6 Correlated machines instances In Table 3, the average CPU times over ten instances and the number of unsolved instances for the correlated machines instances are presented. We observe that the three Gurobi-based algorithms beneﬁt from the inclusion of the aggregated variables; e.g., DEF+y is preferred over DEF. Furthermore, similarly to the uncorrelated instances, the algorithm MOK+y seems to be the fastest and most robust among the ﬁrst six algorithms. Unlike for the previously reported instances, it also seems to be competing with the algorithm LR. Note that the LR algorithm almost never needs to branch on these instances, since the bounds from the root node are suﬃcient to determine optimality. 5.5 Correlated jobs instances m n DEF DEF+y CUT CUT+y MOK [s] MOK+y LR-F LR LR nodes LR root gap [%] 3 30 0.02 0.04 0.06 0.09 0.04 0.03 0.0 0.0 1.0 0.0 50 0.07 0.08 0.08 0.12 0.07 0.06 0.0 0.0 1.0 0.0 80 0.08 0.09 0.18 0.2 0.12 0.05 0.01 0.01 1.0 0.0 100 0.1 0.12 0.15 0.19 0.09 0.09 0.02 0.02 1.0 0.0 200 0.08 0.06 0.1 0.17 0.06 0.07 0.09 0.1 1.0 0.0 5 30 4.31 1.65 3.52 2.42 3.72 1.21 0.0 0.0 1.0 0.0 50 12.3 8.46 9.61 13.9 8.7 3.77 0.05 0.04 3.9 0.06 80 7.18(1) 9.03 14.1 32.3 13.9 3.46 0.15 0.12 6.0 0.04 100 26.5 21.1 (1) 43.1 (1) 35.7 (3) 22.0 5.35 0.33 0.28 5.6 0.05 200 15.6 9.83(1) 36.5 14.1 (1) 15.9 4.26 2.21 1.85 16.7 0.04 10 30 33.4 (3) 40.3 (2) 22.5 (1) 20.6 (2) 34.4 (5) 24.5 0.03 0.02 4.6 0.14 50 40.6 (8) — 65.3 (9) 72.0 (8) — 27.4 (9) 0.29 0.09 13.2 0.37 80 — — 114 (9) — — 29.3 (9) 9.26 0.75 273.1 0.39 100 — — — — — — 28.6 1.8 599.0 0.42 200 — — — — — — 48.9 (8) 10.1 1611.3 0.23 15 30 3.93(1) 1.32 1.19 2.45 0.94 2.89(1) 0.01 0.01 1.0 0.0 50 — — — — — — 1.15 0.27 122.8 1.01 80 — — — — — — 28.9 3.51 1565.1 0.84 100 — — — — — — 42.7 (9) 14.5 5466.8 0.94 200 — — — — — — — 87.5 (9) 23 162.3 0.44 20 30 0.63 0.15(1) 0.42 0.89 0.01(1) 0.14 0.01 0.01 1.0 0.0 50 79.4 (9) 66.1 (8) 66.5 (9) 5.28(9) 9.77(8) 58.4 (7) 0.38 0.17 42.5 0.97 80 — — — — — — 54.0 (4) 6.46 2903.3 1.33 100 — — — — — — — 39.8 (2) 19 980.4 1.18 13 Edvin Åblad, Ann-Brith Strömberg & Domenico Spensieri m n DEF DEF+y CUT CUT+y MOK MOK+y LR-F LR LR nodes LR root gap [%] 3 30 0.01 0.02 0.03 0.03 0.02 0.02 0.0 0.0 1.0 0.0 50 0.03 0.02 0.03 0.07 0.04 0.03 0.0 0.0 1.0 0.0 80 0.05 0.03 0.08 0.05 0.07 0.05 0.0 0.0 1.0 0.0 100 0.04 0.04 0.06 0.05 0.06 0.06 0.0 0.0 1.0 0.0 200 0.06(1) 0.07 10.1 0.2 0.31 0.1 0.02 0.02 1.0 0.0 5 30 0.03 0.02 0.07 0.03 0.03 0.03 0.0 0.0 1.0 0.0 50 0.08 0.05 0.07 0.06 0.07 0.06 0.0 0.0 1.0 0.0 80 0.25(1) 0.2 1.1 0.6 0.21 0.16 0.02 0.02 1.3 0.04 100 0.63(1) 0.47 0.5 0.62 10.4 0.14 0.02 0.02 1.0 0.0 200 6.97(3) 3.98 0.83(2) 1.67 5.14(2) 0.67 0.11 0.12 1.0 0.0 10 30 0.11 0.07 0.19 0.16 0.11 0.1 0.0 0.0 1.0 0.0 50 0.24 0.09 0.21 0.21 0.15 0.14 0.01 0.01 1.0 0.0 80 0.41 0.18 0.52 0.35 5.78 0.22 0.03 0.03 1.2 0.05 100 0.54 0.19 0.48 0.35 0.48 0.33 0.02 0.02 1.0 0.0 200 1.19(3) 0.63 2.3 (1) 1.24 1.89(1) 0.93 0.61 0.38 1.2 0.01 15 30 0.22 0.1 0.17 0.16 0.07 0.06 0.0 0.01 1.0 0.0 50 0.46 0.16 0.39 0.32 0.18 0.22 0.01 0.01 1.0 0.0 80 0.74 0.46 0.44 0.48 0.3 0.24 0.02 0.02 1.0 0.0 100 1.14 0.61 0.65 0.6 0.36 0.37 0.03 0.03 1.0 0.0 200 4.66 1.28 2.1 1.47 10.6 0.97 1.63 0.57 4.3 0.03 20 30 0.29 0.16 0.27 0.26 0.07 0.07 0.01 0.01 1.0 0.0 50 0.56 0.4 0.53 0.41 0.15 0.15 0.02 0.02 1.0 0.0 80 0.97 0.72 1.22 0.72 0.48 0.46 0.05 0.05 1.0 0.0 100 1.08 1.07 1.45 0.91 0.52 0.54 0.07 0.07 1.0 0.0 200 2.67(2) 2.28 2.54 2.15 7.94 1.1 0.38 0.4 1.0 0.0 to our simple local search heuristic. When an optimum was found, the veriﬁcation of optimality was usually quite immediate. Hence, for these unsolved instances the weakness is in the upper bound. Note, however, that the gap is roughly 1% or less in the root node, thus the quality of either bound is still quite good. Moreover, for the instances we were able to solve, e.g., for m = 10 we also computed the lower gap, i.e., z∗−z z∗: its value of roughly 0.05–0.1% again suggests that the issue is not the strength of the lower bound but the weakness of the upper bound. Exact methods for the R\|\|Cmax model for R\|\|Cmax and in the model suggested by Mokotoﬀand Chrétienne [32]. These models are of special interest since they can also be applied to extensions of the R\|\|Cmax, for example with side-constraints. model for R\|\|Cmax and in the model suggested by Mokotoﬀand Chrétienne [32]. These models are of special interest since they can also be applied to extensions of the R\|\|Cmax, for example with side-constraints. For the majority of our 750 instances with sizes ranging from m = 3, n = 30 to m = 20, n = 200, our branch–and–bound algorithm based on Lagrangian bounds is superior to the other algorithms tested, including our versions of the cutting plane algorithm from [32], both with and without branching on the aggregated variables. The numbers of nodes traversed in the branch–and–bound tree are magnitudes fewer than that reported in [29]. This property indicates the strength of the Lagrangian relaxation and the high impact of our variable ﬁxing strategy. Moreover, the sensitivity analysis indicates that our proposed variable ﬁxing outperforms variable ﬁxing based on the LP reduced costs and also that a careful maximization of the Lagrangian dual function successfully reduces the computation time. Furthermore, we found that, by including the aggregated variables in the model suggested in [32], the algorithm (i.e., MOK+y) was able to solve more instances within the given time limit. The general trend is that these variables contribute to an eﬃcient partitioning of the solution space which increases the LP lower bounds. To conclude, we have identiﬁed some possible further studies to conduct. First, we have highlighted some potential improvements to our branch–and–bound method: (i) a further development of the branching strategies, Furthermore, we found that, by including the aggregated variables in the model suggested in [32], the algorithm (i.e., MOK+y) was able to solve more instances within the given time limit. The general trend is that these variables contribute to an eﬃcient partitioning of the solution space which increases the LP lower bounds. Furthermore, we found that, by including the aggregated variables in the model suggested in [32], the algorithm (i.e., MOK+y) was able to solve more instances within the given time limit. The general trend is that these variables contribute to an eﬃcient partitioning of the solution space which increases the LP lower bounds. To conclude, we have identiﬁed some possible further studies to conduct. Exact methods for the R\|\|Cmax First, we have highlighted some potential improvements to our branch–and–bound method: (i) a further development of the branching strategies, (ii) the eﬃcient solution of the subproblems, (iii) a tuning of the subgradient algorithm, (iv) a concurrent traversal of the branch–and–bound tree, (v) the possibility of branching also on the aggregated variables suggested for the MILP models. Second, it will be interesting to investigate the inclusion of set packing constraints (see Section 1) in the model to ﬁnd out if a generalization of our Lagrangian based method can still outperform a general MILP solver. Third, since the inclusion of the aggregated variables seems to be beneﬁcial for most problem instances and for both MILP models, we expect that there exist many other applications that can beneﬁt of this kind of extension. Such an extension could be a generalization of the R\|\|Cmax with additional side-constraints, or another problem with a makespan objective. Acknowledgements This work was carried out within the project Smart Assembly 4.0, supported by the Swedish Foundation for Strategic Research (SSF), project no. RIT15-0025. It is also part of the Sustainable Production Initiative and the Production Area of Advance at Chalmers University of Technology. Finally, we would like to thank the anonymous reviewers for their valuable feedback. 6 Conclusion We have developed and analysed an exact algorithm for the solution of the R\|\|Cmax, based on the Lagrangian relaxation suggested by Martello et al. [29]. We derived a variable ﬁxing strategy based on the solution of the subproblem consisting of m binary knapsack problems, in order to reduce the problem size. We showed that for m = 2 the Lagrangian relaxation has no duality gap. We performed a sensitivity analysis of our proposed algorithm features by computing their individual contribution to the overall algorithm performance. We also compared our method with several MILP formulations, e.g., including the aggregated variables in a standard 14 References 1 Edvin Åblad. Intersection-free load balancing for industrial robots. Master’s thesis, Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, 2016. Å 1 Edvin Åblad. Intersection-free load balancing for industrial robots. Master’s thesis, Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, 2016. 2 Edvin Åblad. R\|\|Cmax Solver. 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https://openalex.org/W1421255664	https://zenodo.org/record/1337087/files/9997733.pdf	English	null	Trial Development the Evaluation Method of Quantification the Feeling of Preventing Visibility by Front A Pillar	Zenodo (CERN European Organization for Nuclear Research)	2,014	cc-by	2,539	World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 Toshiya Arakawa is with the Department of Mechanical Systems Engineering, Aichi University of Technology, 50-2, Manori, Nishihasama-cho, Gamagori, Aichi, Japan (phone: +81-533-68-1135; fax: +81-533-68-0352; e-mail: arakawa-toshiya@aut.ac.jp). International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publicati Eye fixation duration near right A pillar becomes longer if driver strongly feel “obstacle feeling” because that the direction that driver recognize visually is superposed with right A pillar. In addition that, due to “obstacle feeling”, driver tends to move eye from nearby pillar to windshield by drivers’ seat and recognize visually obstacles such as an oncoming vehicle through windshield by drivers’ seat. Thus, the longer the eye fixation time near pillar becomes, the more driver feels “obstacle feeling”. Fig. 1 shows this situation. In Fig. 1, encircled part means eye fixation duration due to “obstacle feeling”, and driver seems to move his eye like orange-colored arrow under this situation. Keywords—Eye fixation, modeling, obstacle feeling, right A pillar. Trial Development the Evaluation Method of Quantification the Feeling of Preventing Visibility by Front A Pillar T. Arakawa, H. Sato II. EXPERIMENT Abstract—There are many drivers who feel right A pillar of Japanese right-hand-drive car preventing visibility on turning right or left at intersection. On the other hand, there is a report that almost pedestrian accident is caused by the delay of finding pedestrian by drivers and this is found by drivers’ eye movement. Thus, we developed the evaluation method of quantification using drivers’ eye movement data by least squares estimation and we applied this method to commercial vehicle and evaluation the visibility. It is suggested that visibility of vehicle can be quantified and estimated by linear model obtained from experimental eye fixation data and information of vehicle dimensions. Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 <Hypothesis 1> I. INTRODUCTION N fI N this paper, we show the method of quantification the feeling of preventing visibility by right A pillar of Japanese right-hand-drive car. The report shows that there are many drivers who feel right A pillar preventing visibility on turning right or left at intersection [1], [2]. On the other hand, there is a report that almost pedestrian accident is caused by the delay of finding pedestrian by drivers and this is found by drivers’ eye movement [3]. The monitor system showing the pedestrian or obstacle of blind corner is developing in order to improve the visibility on turning right or left at intersection [4]-[7], however, this device is costly that it takes long time to equip this device to commercial vehicle. Thus, it is important to design front pillar by driver’s seat quantitative and it is suggested that eye movement can estimate the feeling of preventing visibility by right A pillar and it is useful if the method of quantification by data of eye movement. Engineers may be able to design the pillar efficiently by using this data and it may be bring the efficiency of design and development a vehicle. I Fig. 1 Eye movement around right A pillar Thus, we developed the evaluation method of quantification using drivers’ eye movement data by least squares estimation and we applied this method to commercial vehicle and evaluation the visibility. Fig. 1 Eye movement around right A pillar International Scholarly and Scientific Research & Innovation 8(2) 2014 International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 In this paper, we defined the feeling of preventing visibility by right A pillar as “obstacle feeling”. We set up following two hypothesizes as “obstacle feeling”. IV. CONSIDERATION A. Correlation between the Result of Sensory Evaluation and Modeling A. Correlation between the Result of Sensory Evaluation and Modeling <Hypothesis 2> The factors affecting visibility of right A pillar are dead angle of right A pillar, the angle between straightforward and right A pillar and the angle between straightforward and meter face. Following these hypotheses, in this paper, experiment and modeling were done. scholar.waset.org/1307-6892/9997733 455 International Scholarly and Scientific Research & Innovation 8(2) 2014 World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 and 1 point if they felt visibility worst. Table II shows the result. Table II shows that the difference of score between evaluators is not larger from the point view of standard deviation. We defined “eye fixation rate” as following formula: where where ˆy =[yA, yB, …,yE]T, R=[RA, RB, …, RE]T, ˆy =[yA, yB, …,yE]T, R=[RA, RB, …, RE]T, Eye fixation rate = EFT TT (1) Eye fixation rate = EFT TT (1) (1) B=[BA, BB, …,BE]T and L=[LA, LB, …, LE]T. Coefficient sign of (2) means that estimated average eye fixation rate is get to be small and that means “obstacle feeling” is get to be reduced if dead angle of right A pillar is reduced. In addition that, estimated average eye fixation rate is get to be small and that means “obstacle feeling” is get to be reduced if the angle between straightforward and right A pillar and the angle between straightforward and meter face get to be increased. where EFT: eye fixation time to A pillar and windshield by drivers’ seat TT: the total time of turning the steering wheel on driving each curve line Table I shows “eye fixation rate” on driving the course. Furthermore, we watched the movie of driving recorded by Eye-mark recorder as frame-by-frame playback and calculated eye fixation rate. ˆy = (-2.3934)R + 5.0427B + (-0.3719)L + 48.003 (2) ˆy = (-2.3934)R + 5.0427B + (-0.3719)L + 48.003 (2) (2) We defined “eye fixation rate” as following formula: B. Experimental Procedure Experiment was done in order to measure the eye movement and eye fixation duration of drivers on driving vehicle. 3 persons were joined as subjects of this experiment. Each subject drove 5 kinds of C-segment vehicle and they wore Eye-mark recorder (EMR-9 of NAC Image Technology, Inc.) [7]. Subjects wearing Eye-mark recorder was measured their eye movement and eye fixation time. On the other hand, 3 parameters of 5 kinds of C-segment vehicle, which was used in the experiment, are the angle between straightforward and right A pillar, dead angle of right A pillar and the angle between straightforward and meter face, were measured. We verified about “obstacle feeling” from the point of correlation between 3 parameters and eye movement and eye fixation time of 5 kinds of C-segment vehicle. TABLE II RESULT OF SENSORY EVALUATION Vehicle Subject Average S. D. 1 2 3 A 2.5 2.5 2.0 2.3 0.1 B 3.0 3.0 2.5 2.8 0.1 C 1.5 2.0 1.5 1.7 0.1 D 3.5 3.5 3.0 3.3 0.1 E 4.0 4.0 3.5 3.8 0.1 B. Modeling Using Result of Experiment st ab p o w lo e th m th to su o m ti w se e F E e a o In International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 rnational Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 Dependent variable and independent variable was set as following and estimated average eye fixation rate, that is to say “obstacle feeling”, was calculated by least squared method. Here, suffix i means the number of 5 vehicles used experiment. For example, RA means the angle between straightforward and right A pillar of vehicle A. International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 Eye movement and eye fixation time were measured at winding course of Subaru Research and Development Center, located in Sano-city Tochigi Japan. 3 subjects of this experiment are instructed to wear Eye-mark recorder and drive the course in 5 kinds of C-segment vehicle and their eye movement and eye fixation time were measured. In advance of the experiment, we explained about the content of experiment to the subject of this experiment and get their consensus to be subject. In addition, we taught subjects not to move their head on driving because traffic accidents happen when drivers don’t move their head so that data of eye movement and eye fixation time were measured. <independent variable> average eye fixation rate: yi International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 wase <dependent variable> the angle between straightforward and right A pillar: Ri dead angle of right A pillar: Bi the angle between straightforward and meter face: Li The result of applying by least squared method, estimated average eye fixation rate ( ˆy ) was expressed as (2). International Science Index, Mechanical and Mechatronics Engineering V V. CONNCLUSION Fig. 3 Correl C Applying lation between estim g the Mode experimental m mated value el to Evalua measured value ate Visibility and y othe “ob rec str an ad qu inf co C- on mo In this pape bstacle feelin corder and t raightforward nd the angle ddition that, it uantified and formation of ntribute the ef -segment vehi nly 3 subjects ore subjects an er, we constr ng” by eye fix the parameter and right A p between stra t is suggested estimated b f vehicle dim fficiency desig icle in this pa are used so it nd apply this m ructed linear xation rate me rs, such as pillar, dead an aightforward d that visibilit y this linear mensions. Th gning, howev aper and the e t is future stud method to oth model that easured by eye the angle be ngle of right A and meter fa ty of vehicle r model and is result see ver, we evalua experimental d dy to acquire her segment ve means e-mark etween A pillar ace. In can be using ems to te only data of data of ehicle. A. Correlation between the Result of Sensory Evaluation and Modeling Compariso Modeling on between the Result of Experiment and International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Publication/9997733 av ob ey ra w Ri, Bi, Li is s verage eye fix btained by ex ye fixation rat ate obtained b we constructed substituted for xation rate is xperiment (Fig te have a stron by experiment seems to expr r linear equati compared to g. 3). Fig. 3 m ng correlation (R=0.9611), ress actual ey on (2) and es the eye fixati means that es n with visual f so that linear e fixation rate timated ion rate timated fixation r model e. International Science Index, Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 waset.org/Pub Fig. 4 Estimated value of eye fixation rate (vehicle A-GG) A. Correlation between the Result of Sensory Evaluation and Modeling TABLE I VISUAL FIXATION RATE OF EACH SUBJECT (UNIT: %) Subject Vehicle A B C D E 1 42.25 45.08 44.09 45.24 39.54 2 49.93 49.84 52.54 58.06 45.58 3 51.86 53.44 61.87 32.99 41.21 Fig. 2 shows the correlation between estimated average eye fixation time of 5 vehicles calculated by (2) and sensory evaluation of 3 subjects. Fig. 2 means that the linear model fits subjectivity of subjects in our case and eye fixation time seems to comparative with actual visibility. Next, each subjects evaluated subjectively the visibility around right A pillar of each vehicle. Each subjects score units of 0.5 point, and they scored 4 point if they felt visibility best scholar.waset.org/1307-6892/9997733 International Scholarly and Scientific Research & Innovation 8(2) 2014 456 Fi M av ob ey ra w ig. 2 Correlation B. Compariso Modeling Ri, Bi, Li is s verage eye fix btained by ex ye fixation rat ate obtained b we constructed n between expe ev on between substituted for xation rate is xperiment (Fig te have a stron by experiment seems to expr erimental eye fi valuation the Result r linear equati compared to g. 3). Fig. 3 m ng correlation (R=0.9611), ress actual ey ixation rate and of Experime on (2) and es the eye fixati means that es n with visual f so that linear e fixation rate d sensory nt and timated ion rate timated fixation r model e. Fig. 4 Estimated value of eye fixation rate (vehicle A-GG) World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 Fig. 2 Correlation between expe ev erimental eye fi valuation ixation rate and d sensory World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 World Academy of Science, Engineering and Technology International Journal of Mechanical and Mechatronics Engineering Vol:8, No:2, 2014 Fig. 4 Estimated value of eye fixation rate (vehicle A-GG) Fig. 2 Correlation between expe ev erimental eye fi valuation ixation rate and d sensory Fig. 2 Correlation between expe ev erimental eye fi valuation ixation rate and d sensory Fig. 2 Correlation between expe ev erimental eye fi valuation ixation rate and sensory M B. 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https://openalex.org/W4322102695	https://insep.hal.science//hal-04060129/document	English	null	Effects of Intermittent Normobaric Hypoxia on Health-Related Outcomes in Healthy Older Adults: A Systematic Review	Sports medicine - open/Sports medicine - Open	2,023	cc-by	11,486	To cite this version: Rafael Timon, Ismael Martinez-Guardado, Franck Brocherie. Effects of Intermittent Normobaric Hypoxia on Health-Related Outcomes in Healthy Older Adults: A Systematic Review. Sports medicine - Open, 2023, 9 (1), pp.19. 10.1186/s40798-023-00560-0. hal-04060129 Distributed under a Creative Commons Attribution 4.0 International License Abstract Background Aging is a degenerative process that is associated with an increased risk of diseases. Intermittent hypoxia has been investigated in reference to performance and health-related functions enhancement. This system‑ atic review aimed to summarize the effect of either passive or active intermittent normobaric hypoxic interventions compared with normoxia on health-related outcomes in healthy older adults. Methods Relevant studies were searched from PubMed and Web of Science databases in accordance with PRISMA guidelines (since their inceptions up until August 9, 2022) using the following inclusion criteria: (1) randomized con‑ trolled trials, clinical trials and pilot studies; (2) Studies involving humans aged > 50 years old and without any chronic diseases diagnosed; (3) interventions based on in vivo intermittent systemic normobaric hypoxia exposure; (4) articles focusing on the analysis of health-related outcomes (body composition, metabolic, bone, cardiovascular, functional fitness or quality of life). Cochrane Collaboration recommendations were used to assess the risk of bias. Results From 509 articles initially found, 17 studies were included. All interventions were performed in moderate normobaric hypoxia, with three studies using passive exposure, and the others combining intermittent hypoxia with training protocols (i.e., using resistance-, whole body vibration- or aerobic-based exercise). Conclusions Computed results indicate a limited effect of passive/active intermittent hypoxia (ranging 4–24 weeks, 2–4 days/week, 16–120 min/session, 13–16% of fraction of inspired oxygen or 75–85% of peripheral oxygen satura‑ tion) compared to similar intervention in normoxia on body composition, functional fitness, cardiovascular and bone health in healthy older (50–75 years old) adults. Only in specific settings (i.e., intermediate- or long-term interven‑ tions with high intensity/volume training sessions repeated at least 3 days per week), may intermittent hypoxia elicit beneficial effects. Further research is needed to determine the dose–response of passive/active intermittent hypoxia in the elderly. Trial registration. Systematic review registration: PROSPERO 2022 CRD42022338648. Keywords Normobaric hypoxia, Elderly, Bone health, Cardiovascular health, Metabolic health, Functional fitness Correspondence: Rafael Timon rtimon@unex.es 1 Sport Sciences Faculty, Universidad de Extremadura, Av/ Universidad s/n, 10004 Cáceres, Spain 2 BRABE Group. Faculty of Life and Nature Sciences, Universidad de Nebrija, Madrid, Spain 3 Laboratory Sport, Expertise and Performance (EA 7370), French Institute of Sport (INSEP), Paris, France © The Author(s) 2023. Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. HAL Id: hal-04060129 https://insep.hal.science/hal-04060129v1 Submitted on 6 Apr 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Timon et al. Sports Medicine - Open (2023) 9:19 https://doi.org/10.1186/s40798-023-00560-0 Sports Medicine - Open Open Access Open Access Effects of Intermittent Normobaric Hypoxia on Health‑Related Outcomes in Healthy Older Adults: A Systematic Review Rafael Timon1 , Ismael Martinez‑Guardado2 and Franck Brocherie3 *Correspondence: Rafael Timon rtimon@unex.es 1 Sport Sciences Faculty, Universidad de Extremadura, Av/ Universidad s/n, 10004 Cáceres, Spain 2 BRABE Group. Faculty of Life and Nature Sciences, Universidad de Nebrija, Madrid, Spain 3 Laboratory Sport, Expertise and Performance (EA 7370), French Institute of Sport (INSEP), Paris, France Key Points and degradation of HIF-1α, allowing its stabilization and entry into the cell nucleus to activate genetic tran- scription factors related to erythropoiesis, osteogenesis, angiogenesis, lipolysis, and antioxidant capacity [22–24]. For instance, short-term daily IH sessions consisting of 3–4 bouts of 5–7 min exposure to 10–12% of fraction of inspired oxygen (%FiO2) alternated with similar peri- ods of normoxia (%FiO2 21%) for at least 2–3 weeks have been shown to be beneficial for cardiovascular, respira- tory and neurological disorders [25, 26]. • Passive and active intermittent normobaric hypoxia has a limited effect on health-related outcomes in healthy older adults, compared to similar interven- tion in normoxia. • Intermediate- or long-term intermittent hypoxia interventions with high intensity/volume training sessions repeated at least 3 days per week could have a putative effect on functional fitness and fat mass loss compared to normoxia. Although previous studies have suggested that IH could have positive effects on hypertension, hemodynamics, neurodegeneration, and obesity [6, 7, 14], the current scientific knowledge is not unanimous. The physiological and metabolic adaptations induced by IH could depend on the hypoxic dose (i.e., severity, duration, and exposure time of the intervention), as well as other factors such as genetics, age of the individuals or training status [27–29]. To date, there is no systematic review in the present sci- entific literature that specifically examines whether IH has positive or negative effects on health-related out- comes in healthy older adults. We therefore summarize the effect of either passive or active IH interventions compared with normoxia on health-related outcomes in healthy older adults. • More research is needed to determine the dose– response of passive/active intermittent normobaric hypoxia in healthy older adults Page 2 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open Search Strategy A systematic review was performed following the Pre- ferred Reporting Guidelines for Systematic Reviews and Meta-analyzes (PRISMA) [30]. The systematic review was registered using the PROSPERO International data- base of systematic review protocols (Registration num- ber: PROSPERO 2022 CRD42022338648). Randomized controlled trials (RCTs), clinical trials and pilot studies were identified by electronically searching the follow- ing databases: PubMed (MEDLINE) and Web of Science (WoS), and through manual searching of reference lists of eligible studies. To optimize the identification of relevant articles, the terms “intermittent hypoxia”, “hypoxic con- ditioning”, “normobaric hypoxia”, “elderly”, “older adults”, “therapeutic” and “health benefits” were combined with Boolean operators (“AND” and “OR”) and searched from inception up until August 9, 2022. All references were extracted and imported into an open-source research tool to systematize studies. IH could be defined as repeated exposure to hypoxia interspersed with normoxia. In the clinical setting, IH has been associated with obstructive sleep apnea syndrome (OSAS), a disorder of sleep breathing characterized by nightly high frequency repetitive and prolonged periods of complete or partial obstruction of the upper airway [17, 18] that causes multiple alterations and pathologies in individuals [19, 20]. However, such chronic, severe, and repetitive IH must be differentiated from IH with controlled reduction of oxygen. OSAS causes a dysregu- lated transcription of HIF-1α and HIF-2 α, increasing HIF-1α and decreasing HIF-2α [21]. On the contrary, IH and controlled reoxygenation prevents the hydroxylation Introduction Aging is a degenerative process produced as a result of different cellular dysfunctions and tissue damages, which cause a gradual loss of physical and mental capacities [1]. This progressive deterioration has been associated with the development of age-related disorders [2]. To circum- vent them, different strategies, such as individualized socio-health care, the use of medications, exercise, main- taining healthy lifestyles or improving the psycho-social environment of people, have been proposed [3, 4]. Currently, some review studies have proposed expo- sure to hypoxic conditions, either passive or active in combination with exercise, as a promising tool to achieve health benefits [5–8], targeting hypoxia-inducible factor (HIF) and its signaling pathway as a novel therapeutic option to deal with various chronic diseases [9, 10]. On the one hand, an oxygen-deprived (hypoxia) environment impairs cell adaptation and survival [11] with chronic exposure to severe hypoxia leading to hypoxemia and cardiovascular and pulmonary complications [12, 13]. On the other hand, intermittent hypoxia (IH) has been shown to exert beneficial effects at the cardiovascular, metabolic, and cognitive levels, both in healthy and path- ological individuals [14–16].i 1. Types of studies. Articles published in peer-reviewed journals written in English. Randomized controlled Characteristics of Studies and Interventions Characteristics of Studies and Interventions Fourteen studies [29, 33, 34, 36, 37, 39–47] were classi- fied as RCTs, one study [32] as a quasi-experimental clin- ical trial, and two studies [35, 38] were described as pilot studies. Only one study [47] had a sample including solely males (n = 24), while the remaining investigations used mixed samples (n ranging 8–22 males and 4–32 females, respectively). The duration of the interventions varied widely, from short-term (4–6 weeks) [29, 38, 39], to intermediate (8–12 weeks) [36, 37, 40, 41, 45–47] and long-term duration (18–24 weeks) [32–35, 42–44], with a frequency of 2–4 sessions per week. The duration of the sessions was also very wide-ranging, from 16-min per session [35, 42–44] up to 90–120 min per session [38, 39, 47], and with intermediate durations of 30–60 min per session [29, 32–34, 36, 37, 40, 41, 45, 46]. All inter- ventions were performed under moderate normobaric hypoxia, with %FiO2 ranging 13.0–16.1% or %SpO2 rang- ing 75–85%. Among all these interventions, three stud- ies proposed the exclusive use of passive IH [32, 35, 36], but the rest of the studies (n = 14) combined IH with Risk of Bias of Included Studies According to the Cochrane Collaboration recommenda- tions, the results of the risk of bias assessment showed that 9 out of 17 articles had a low risk of bias in all domains. All articles had a low risk of allocation conceal- ment and blinding of outcomes bias. Each article was described as randomized, but the randomization method was unclear for one study [32]. Two studies revealed high risk of blinding participants and personnel bias [33, 34], and in two other studies, the information about the blinding was unclear [32, 35]. Incomplete outcome data were at a low risk of bias in all articles, except for two studies [35, 36] that were at high risk (exclusions were not defined from the analysis and lack of several relevant sta- tistics). Moreover, the results for selective reporting bias were at high risk for four studies [37–39] (conclusions were only partially supported by the findings presented). The risk of bias assessment of all included studies is dis- played in Table 2. Selection Process Two investigators (RT and FB) selected the eligible arti- cles based on title, abstract and full paper, using the inclusion criteria. Disagreements were resolved by con- sensus. Steps followed in the selection of studies were: (1) identification of potential studies, (2) duplicates removal, (3) title and abstract examination, (4) full text exploration and (5) checking of the quality of research and relevance to the purpose of the review. Search Resultsh 2. Type of participants. Articles with humans (females and males) over 50 years old who had not been diag- nosed with chronic diseases. 2. Type of participants. Articles with humans (females and males) over 50 years old who had not been diag- nosed with chronic diseases. The process of identifying eligible studies is shown in Fig. 1. Five hundred and nine records were initially iden- tified through the databases. One hundred and fifteen articles were removed for being duplicates. Of the rest of the identified manuscripts, 26 potentially eligible articles were included based on their title and abstract. After full text exploration, only 17 articles fulfilled the inclusion criteria. The characteristics of each included study are described in Table 1. 3. Types of interventions. To be included in this sys- tematic review, interventions had to use only in vivo systemic IH training. Protocols using hypobaric hypoxia, hyperoxia or blood flow restriction (or other combination) were excluded, as well as acute or sin- gle IH trials. 3. Types of interventions. To be included in this sys- tematic review, interventions had to use only in vivo systemic IH training. Protocols using hypobaric hypoxia, hyperoxia or blood flow restriction (or other combination) were excluded, as well as acute or sin- gle IH trials. 4. Types of outcomes evaluated. Articles that focused on the analysis of health-related variables (i.e., meta- bolic, body composition, bone, cardiovascular, func- tional fitness or quality of life) were included. Inclusion Criteria Timon et al. Sports Medicine - Open (2023) 9:19 Page 3 of 15 Page 3 of 15 trials (RCTs), clinical trials and pilot studies, compar- ing normobaric hypoxia vs. normoxia, were included. trials (RCTs), clinical trials and pilot studies, compar- ing normobaric hypoxia vs. normoxia, were included. trials (RCTs), clinical trials and pilot studies, compar- ing normobaric hypoxia vs. normoxia, were included. Risk of Bias Assessment Cochrane Collaboration recommendations [31] for sys- tematic reviews of interventions were used to assess the risk of bias for all articles. Investigators’ assessments were classified as ’low risk’, ’high risk’ or ’unclear risk’ of bias, referring to the following domains: random sequence generation, allocation concealment, blind- ing of participants and personnel, blinding of outcome assessments, incomplete outcome data and selective reporting. Two investigators (RT and FB) indepen- dently assessed the methodological quality of all arti- cles selected. The discrepancies were resolved with the conciliation work carried out by another investigator (IMG). Effects of IH Interventions Compared with Normoxia on Health‑Related Outcomes In the included studies, the different health-related out- comes investigated included metabolic [32, 36, 37, 40, 41, 46], body composition [29, 32, 33, 35, 37, 41, 43–47], blood pressure [32, 36, 37, 41, 45], bone [32, 35, 42], inflammatory [32, 34, 36] and hematological [29, 39] parameters, as well as functional fitness [29, 33, 38, 39, 43–45, 47] or parameters related to quality of life [38, 39]. The main findings of the studies considered for review are summarized in Table 1. Data Extraction Data extraction was completed by the lead investigator (RT) who compiled them into descriptive tables. The data extraction was subsequently checked by another investi- gator (IMG). The following data were extracted for each category: (a) author, year of publication, (b) participants, (c) experimental groups, (d) intervention, (e) duration/ frequency (f) %FiO2 or % peripheral oxygen saturation (%SpO2), (g) health-related outcomes. Page 4 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open Potenal studies idenﬁed (n = 394) PubMed (n = 377) Duplicated (n = 115) Full arcle considered (n = 26) Arcles fulﬁlled the inclusion criteria (n = 17) Excluded (n = 9) - Parcipants with chronic diseases (n = 2) - Not health-related outcomes (n = 2) - Not English language (n = 2) - Original results not available (n = 1) - Do not meet the age criteria (n = 2) Web of Science (n = 132) Records excluded on tle and/or abstract (n= 368) Fig. 1 Flowchart of the study selection PubMed (n = 377) Web of Science (n = 132) Potenal studies idenﬁed (n = 394) Arcles fulﬁlled the inclusion criteria (n = 17) Fig. 1 Flowchart of the study selection ffects of IH Interventions Compared with Normoxia exercise training, either sequentially [38, 39] or simul- taneously [29, 33, 34, 37, 40–47]. Six studies carried out resistance training programs with full-body routines (4 to 8 exercises) within the session, which were performed with strength training machines [29, 38, 40, 41] or elas- tic bands and kettlebells [33, 34]. Four studies used a sta- ble-load, submaximal-intensity aerobic cycling training program [37, 39, 45, 46]. In one study [47], participants performed aerobic exercises and resistance exercises with elastic bands within the same session. Finally, in three studies [42–44], all conducted by the same author group, whole-body vibration (WBV) training was performed on a vibrating platform at an intensity of 12.6 Hz and an amplitude of 4 mm. Effects of IH Interventions Compared with Normoxia on Health‑Related Outcomes Metabolic Parameters Six of the seventeen studies evaluated metabolic parame- ters [32, 36, 37, 40, 41, 46] such as glucose, total and HDL- cholesterol, triglycerides, glucose transporter 4 (GLUT4), insulin, insulin sensitivity index and the homeostatic Page 5 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Table 1 Characteristics of included studies Study Participants Experimental groups Intervention Duration/ Frequency FiO2 or SpO2 Health-related outcomes Allsopp et al. [40] n = 20 (12 males; 8 females) age: 60–75 yr Normoxia + exercise (n = 10) Hypoxia + exercise (n = 10) Resistance training (4 exercises at 70% 1RM with 4 × 10 reps) 8 weeks; 2 days per week; ~ 60 min per session Normobaric hypoxia 14.4% →Glucose →Insulin →HOMA-IR Allsopp et al. [41] n = 20 (12 males; 8 females) age: 60–75 yr Normoxia + exercise (n = 10) Hypoxia + exercise (n = 10) Resistance training (4 exercises at 70% 1RM with 4 × 10 reps) 8 weeks; 2 days per week; ~ 60 min per session Normobaric hypoxia 14.4% →Lean mass →Fat mass →Glucose → CHO →Systolic BP Camacho-Cardenosa et al. [44] n = 19 (8 males; 11 females) age: 65–75 yr Normoxia + exercise (n = 10) Hypoxia + exercise (n = 9) WBV training (12.6 Hz, 4 mm) (4 sets × 30 s, 60-s rest) 18 weeks; 2 days per week; 16 min per session Normobaric hypoxia 16.1% →Leg lean mass →Functional mobility (TUGT) Camacho-Cardenosa et al. [35] n = 10 age: ≥ 75 yr Control, no intervention (n = 5) Hypoxia (n = 5) Passive hypoxic exposure 18 weeks; 2 days per week; 16 min per session Normobaric hypoxia 16.1% →Body fat mass →Trunk fat mass →Femoral BMD →Trochanter BMD ↑Whole body BMD Camacho-Cardenosa et al. [43] n = 31 (11 males, 20 females) age: ≥ 65–75 yr Control, no intervention (n = 10) Normoxia + exercise (n = 11) Hypoxia + exercise (n = 10) WBV training (12.6 Hz, 4 mm) (4 sets × 30 s, 60-s rest) 18 weeks; 2 days per week; 16 min per session Normobaric hypoxia 16.1% →Body lean mass →Maximal strength →Endurance strength Camacho-Cardenosa et al. Metabolic Parameters [40] n = 30 (10 males, 20 females) age: ≥ 65–75 yr Control, no intervention (n = 10) Normoxia + exercise (n = 10) Hypoxia + exercise (n = 10) WBV training (12.6 Hz, 4 mm) (4 sets × 30 s, 60-s rest) 18 weeks; 2 days per week; 16 min per session Normobaric hypoxia 16.1% →Whole body BMD →Femoral BMD →Trochanter BMD Chacaroun et al.[37] n = 23 (19 males; 4 females) age: 54 ± 11 yr Normoxia + exercise (n = 11) Hypoxia + exercise (n = 12) Submaximal constant-load exercise in cycle ergom‑ eter (75% HRmax) 8 weeks; 3 days per week; 45 min per session Normobaric hypoxia 13% →Glucose →Insulin →HOMA2-IR →CHO →Triglycerides →HDL →Lean mass →Fat mass →Systolic BP Page 6 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open Table 1 (continued) Study Participants Experimental groups Intervention Duration/ Frequency FiO2 or SpO2 Health-related outcomes Chacaroun et al. [36] n = 35 (24 males, 11 females) age: 54 ± 9.3 yr Normoxia, sham condition (n = 11) Sustained hypoxia (n = 13) Intermittent hypoxia (n = 12) Passive hypoxic exposure 8 weeks; 3 days per week; Sustained hypoxia: 60 min per session Intermittent hypoxia: 7 cycles (5 min hypoxia + 3 min nor‑ moxia) per session Normobaric hypoxia FiO2 based on SpO2 = 75% →Systolic BP ↓Diastolic BP →Vascular function (PWV) →HOMA-IR →Glucose →Lipid profile →CRP Chobanyan-Jürgens et al. [46] n = 29 (15 males, 14 females) age: 55–75 yr Normoxia + exercise (n = 15) Hypoxia + exercise (n = 14) Submaximal bicycle exer‑ cise at 60–70% VO2max 8 weeks; 3 days per week; 30–40 min per session Normobaric hypoxia 15% →Insulin sensivity index →HOMA-IR →Waist circumference →%Fat mass →CHO →GLUT4 Hein et al. [45] n = 29 (15 males, 14 females) age: 55–75 yr Normoxia + exercise (n = 15) Hypoxia + exercise (n = 14) Submaximal bicycle exer‑ cise at 60–70% VO2max 8 weeks; 3 days per week; 30–40 min per session Normobaric hypoxia 15% →Body fat mass →VO2max →Rest BP →Exercise BP Park et al. Metabolic Parameters [34] n = 54 (22 males; 32 females) age: 65–75 yr Control, no intervention (n = 19) Normoxia + exercise (n = 18) Hypoxia + exercise (n = 17) Resistance training (9 exercises with elastic bands and kettlebells; 3 sets × 12–15 reps) 24 weeks; 3 days per week; 45 min per session Normobaric hypoxia 16.1% →CRP →Interleukin 6 →Interleukin 8 →Interleukin 10 →VCAM1 Torpel et al.[29] n = 36 (18 males; 18 females) age: 60–75 yr Normoxia + exercise (n = 17) Hypoxia + exercise (n = 19) Resistance training (circuit with 8 machine-based resistance exercises at 25–40% 1RM; 3 × 15 reps); RPE: 7 out of 10 5 weeks; 4 days per week; ~ 60 min per session Normobaric hypoxia FiO2 based on SpO2 rang‑ ing 80–85% →Fat mass →Fat-free mass →VO2max → Muscular strength →BP →Hb →Erythropoietin →Blood volume ↑ Significant increase in the study variable; ↓ Significant decrease in the study variable; → The study variable remains unchanged 1RM: one-repetition maximum; b-CTX: Beta C-terminal telopeptide of collagen; BDNF: brain-derived neurotrophic factor; BMC: bone mineral content; BMD: bone mineral density; BP: blood pressure; CHO: cholesterol; CRP: C-reactive protein; FEV1: forced expiratory volume in 1 s; FiO2: fraction of inspired oxygen, FVC: forced vital capacity; GLUT4: glucose transporter 4; Hb: hemoglobin; Hct: hematocrit; HDL: high-density lipoprotein; HOMA-IR: homeostatic model assessment-insulin resistance; HRmax: maximum heart rate; HRV: Heart rate variability; MVV: maximum voluntary ventilation; PINP: N-terminal propeptide of type I procollagen; PWV: pulse wave-velocity; RPE: rating of perceived exertion; SFT: senior fitness test; SpO2: peripheral oxygen saturation; TUGT: timed up and go test; VCAM1: vascular cell adhesion molecule 1; VO2max: maximal oxygen uptake; WBV: whole body vibration ignificant increase in the study variable; ↓ Significant decrease in the study variable; → The study variable remains unchanged M: one-repetition maximum; b-CTX: Beta C-terminal telopeptide of collagen; BDNF: brain-derived neurotrophic factor; BMC: bone mineral content; BMD: bone mineral density; BP: blood pressure; CHO: cholesterol; P: C-reactive protein; FEV1: forced expiratory volume in 1 s; FiO2: fraction of inspired oxygen, FVC: forced vital capacity; GLUT4: glucose transporter 4; Hb: hemoglobin; Hct: hematocrit; HDL: high-density lipoprotein; MA-IR: homeostatic model assessment-insulin resistance; HRmax: maximum heart rate; HRV: Heart rate variability; MVV: maximum voluntary ventilation; PINP: N-terminal propeptide of type I procollagen; PWV: pulse ve-velocity; RPE: rating of perceived exertion; SFT: senior fitness test; SpO2: peripheral oxygen saturation; TUGT: timed up and go test; VCAM1: vascular cell adhesion molecule 1; VO2max: maximal oxygen uptake; WBV: ole body vibration →Interleukin 10 Timon et al. Metabolic Parameters [47] n = 24 (men) age: 65–70 yr Normoxia + exercise (n = 12) Hypoxia + exercise (n = 12) Aerobic exercise (60–70% HRmax) on treadmill (30 min) and bicycle (30 min) + elastic resist‑ ance training (6 exercises, 3 × 10–15 reps; RPE: 6–7 out of 10; 30–45 min) 12 weeks; 3 days per week; 90–120 min per session Normobaric hypoxia 14.5% ↑Fat-free mass ↓Body fat mass ↑Physical fitness ↑FVC ↑FEV1 ↑MVV ↑HRV (high frequency) Schega et al.[39] n = 36 (19 males, 17 females) age: 60–75 yr Normoxia + exercise (n = 18) Hypoxia + exercise (n = 18) Passive hypoxia exposure (90 min) + aerobic exercise on bicycle (30 min, 70–75% HRmax) 4 weeks; 3 days per week; 120 min per session Normobaric hypoxia FiO2 based on SpO2 = 80% →BDNF →VO2max →Cognitive function (Stroop test) ↑Hb; Hct ↑Red-blood cells Schega et al.[38] n = 34 (8 males; 16 females) age: 60–70 yr Normoxia + exercise (n = 17) Hypoxia + exercise (n = 17) Passive hypoxic exposure (60 min) + full-body strength-endurance (30 min, 5 exercises at 50% 1RM with 2 × 25 reps) 6 weeks; 3 days per week; 90 min per session Normobaric hypoxia FiO2 based on SpO2 = 80% ↑Attention (d2 test) ↑Sleep quality (PSQI test) →Speed of cognitive per‑ formance (ZVT test) →Quality of life (physical and mental component) →Strength-endurance capacity Page 7 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open Table 1 (continued) Study Participants Experimental groups Intervention Duration/ Frequency FiO2 or SpO2 Health-related outcomes Timon et al. [32] n = 38 (15 males; 23 females) age: 65–75 yr Control, no intervention (n = 19) Hypoxia (n = 19) Passive hypoxic exposure 24 weeks; 3 days per week; 45 min per session Normobaric hypoxia 16.1% →Lean mass ↓Body fat mass →Whole body BMD →Whole body BMC →Systolic BP →Glucose → CHO →Triglycerides CRP ↑PINP b-CTX →Interleukin 8 →Interleukin 10 Timon et al. Metabolic Parameters [33] n = 54 (22 males; 32 females) age: 65–75 yr Control, no intervention (n = 19) Normoxia + exercise (n = 18) Hypoxia + exercise (n = 17) Resistance training (9 exercises with elastic bands and kettlebells; 3 sets × 12–15 reps) 24 weeks; 3 days per week; 45 min per session Normobaric hypoxia 16.1% →Lean mass →Fat mass →Functional fitness (SFT) →Balance →Fear of falling Timon et al. Metabolic Parameters [33] n = 54 (22 males; 32 females) age: 65–75 yr Control, no intervention (n = 19) Normoxia + exercise (n = 18) Hypoxia + exercise (n = 17) Resistance training (9 exercises with elastic bands and kettlebells; 3 sets × 12–15 reps) 24 weeks; 3 days per week; 45 min per session Normobaric hypoxia 16.1% →Lean mass →Fat mass →Functional fitness (SFT) →Balance →Fear of falling Timon et al. [34] n = 54 (22 males; 32 females) age: 65–75 yr Control, no intervention (n = 19) Normoxia + exercise (n = 18) Hypoxia + exercise (n = 17) Resistance training (9 exercises with elastic bands and kettlebells; 3 sets × 12–15 reps) 24 weeks; 3 days per week; 45 min per session Normobaric hypoxia 16.1% →CRP →Interleukin 6 →Interleukin 8 →Interleukin 10 →VCAM1 Torpel et al.[29] n = 36 (18 males; 18 females) age: 60–75 yr Normoxia + exercise (n = 17) Hypoxia + exercise (n = 19) Resistance training (circuit with 8 machine-based resistance exercises at 25–40% 1RM; 3 × 15 reps); RPE: 7 out of 10 5 weeks; 4 days per week; ~ 60 min per session Normobaric hypoxia FiO2 based on SpO2 rang‑ ing 80–85% →Fat mass →Fat-free mass →VO2max → Muscular strength →BP →Hb →Erythropoietin →Blood volume ↑ Significant increase in the study variable; ↓ Significant decrease in the study variable; → The study variable remains unchanged 1RM: one-repetition maximum; b-CTX: Beta C-terminal telopeptide of collagen; BDNF: brain-derived neurotrophic factor; BMC: bone mineral content; BMD: bone mineral density; BP: blood pressure; CHO: cholesterol; CRP: C-reactive protein; FEV1: forced expiratory volume in 1 s; FiO2: fraction of inspired oxygen, FVC: forced vital capacity; GLUT4: glucose transporter 4; Hb: hemoglobin; Hct: hematocrit; HDL: high-density lipoprotein; HOMA-IR: homeostatic model assessment-insulin resistance; HRmax: maximum heart rate; HRV: Heart rate variability; MVV: maximum voluntary ventilation; PINP: N-terminal propeptide of type I procollagen; PWV: pulse wave-velocity; RPE: rating of perceived exertion; SFT: senior fitness test; SpO2: peripheral oxygen saturation; TUGT: timed up and go test; VCAM1: vascular cell adhesion molecule 1; VO2max: maximal oxygen uptake; WBV: whole body vibration Table 1 (continued) Timon et al. Blood Pressurehf The effect of IH on blood pressure was investigated in six studies [29, 32, 36, 37, 41, 45]. In general, either passive [i.e., over 24 weeks [32]] or active IH [i.e., 5–8 weeks of resistance training [29, 41] or aerobic training [37, 45]], did not produce greater benefit than normoxic interven- tion on blood pressure values in healthy older adults. Only one study [36] using a 8-week passive hypoxia (SpO2 = 75%) intervention showed a significant decrease in diastolic blood pressure compared to the normoxia intervention. Metabolic Parameters Sports Medicine - Open (2023) 9:19 Page 8 of 15 Table 2 Risk of bias assessment of included studies Table 2 Risk of bias assessment of included studies Study Random sequence Allocation concealment Blinding of participants/ personnel Blinding of outcomes Incomplete outcome data Selective reporting Allsopp et al. [40] Low Low Low Low Low Low Allsopp et al. [41] Low Low Low Low Low Low Camacho-Cardenosa et al. [44] Low Low Low Low Low Low Camacho-Cardenosa et al. [35] Low Low Unclear Low High Low Camacho-Cardenosa et al. [43] Low Low Low Low Low Low Camacho-Cardenosa et al. [42] Low Low Low Low Low Low Chacaroun et al. [37] Low Low Low Low Low High Chacaroun et al. [36] Low Low Low Low High High Chobanyan-Jürgens et al. [46] Low Low Low Low Low Low Hein et al. [45] Low Low Low Low Low Low Park et al. [47] Low Low Low Low Low Low Schega et al. [39] Low Low Low Low Low High Schega et al. [38] Low Low Low Low Low High Timon et al. [32] Unclear Low Unclear Low Low Low Timon et al. [33] Low Low High Low Low Low Timon et al. [34] Low Low High Low Low Low Torpel et al. [29] Low Low Low Low Low Low mass, after a 24-week intervention with three sessions per week of 45 min at FiO2 of 16.1%. model assessment-insulin resistance (HOMA-IR). None of the studies showed significantly improved values after an IH intervention compared to normoxia, either using a passive [32, 36] or active [37, 40, 41, 46] paradigm. Body Composition Eleven studies included body composition parameters in their assessments, albeit using different technologies: one study [37] used whole body magnetic resonance imagery (MRI) to determine fat and lean mass, two studies [29, 47] applied bioelectrical impedance to assess fat mass and fat-free mass, two other studies [45, 46] determined body fat and fat-free mass by air-displacement plethys- mography, and six studies [32, 33, 35, 41, 43, 44] used dual-energy X-ray absorptiometry (DXA) to determine lean mass, fat mass or bone mineral content (BMC), from the whole body or specific areas. None of the active IH interventions using resistance training, aerobic training or WBV had any added benefit on lean mass, fat mass or BMC compared to similar intervention in normoxia, with the exception of the study by Park et al. [47] who observed increases in fat-free mass and decreases in body fat percentage. In this study, unlike all the others, long multimodal training sessions (90–120 min) were used, with a FiO2 of 14.5%, combining aerobic exercise on treadmill (30 min) then on bicycle (30 min) followed by elastic resistance exercises (30–45 min). Regarding the two studies that used passive IH, only one [32] obtained decreases of body fat mass, without changes in body lean Hematological Parameters In two studies [29, 39], variations in hematological parameters were evaluated after 4–5 weeks of active IH in healthy older adults. Normobaric hypoxic dose was tai- lored based on an SpO2 target of 80–85% in both experi- ments. Increases in red-blood cells, hemoglobin and hematocrit were observed after twelve sessions (120 min) that combined IH (90 min) with aerobic cycling exercise (30 min) [39]. However, when using resistance training (i.e., 20 sessions of 60 min) [29], no changes in hemo- globin, blood volume and erythropoietin were observed. Inflammatory Biomarkers Only three studies, using passive [32, 36] and active IH [34] investigated the influence of such hypoxic interven- tion on inflammatory biomarkers. Reportedly, a 24-weeks IH (%FiO2 16.1%) intervention including resistance training with elastic bands and kettlebells did not affect C-reactive protein, vascular cell adhesion molecule 1, and interleukins 6, 8 and 10 in comparison with similar inter- vention in normoxia [34]. Similarly, an 8-week interven- tion of passive hypoxia (SpO2 = 75%) also did not cause significant changes in C-reactive protein levels [36]. On the contrary, passive IH exposure at FiO2 of 16.1% (i.e., 24 weeks) induced a decrease in C-reactive protein com- pared to a normoxic control (i.e., no intervention) group [32]. Quality of Life Two studies [38, 39] evaluated the effect of IH on charac- teristics related to quality of life and cognitive function. In both cases, the level of hypoxia was based on an SpO2 target of 80%. After a 6-week intervention with 90-min sessions combining passive IH (60 min) and active IH (30 min of strength-endurance exercises), improvements were only observed in attention levels and quality of sleep compared to the normoxic intervention, but not in speed of cognitive performance or on a quality of life question- naire [38]. Similarly, after 4 weeks (3 sessions per week) of a combined intervention consisting of 90 min of pas- sive IH followed by 30 min of active IH in each session, no changes were observed in cognitive function or in brain-derived neurotrophic factor (BDNF), a key mole- cule for long-term memory and neurogenesis stimulation [39]. Bone Parameters Bone mineral density (BMD) (from whole body or spe- cific areas) was assessed in three studies [32, 35, 42], with additional dynamic biomarkers related to bone remode- ling [N-terminal propeptide of type I procollagen (PINP) and beta C-terminal telopeptide of collagen b-CTX)] evaluated in one of them [32]. Two of these studies were carried out with passive IH interventions [32, 35], while the third one used active IH WBV training [42]. Eighteen weeks of IH WBV training (%FiO2 16.1%) did not lead to significant changes compared to the normoxic group in whole body BMD, femoral BMD and trochanter BMD [42]. However, passive IH sessions of 16–45 min over 18–24 weeks at %FiO2 of 16.1% induced changes in whole Page 9 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open standing) than normoxic training in men [47]. With the exception of one study [47], no studies found that active IH interventions, either with resistance [29, 33, 38], aerobic [39, 45] or WBV [43, 44] training, had an added positive effect on the physical fitness of healthy older adults compared to similar interventions performed in normoxia. body BMD [35] and in PINP (bone formation biomarker) and b-CTX (bone resorption biomarker) in sedentary older adults [32] compared to similar intervention in normoxia. Discussionh This systematic review identified seventeen studies inves- tigating the effects of IH on health-related parameters in healthy older adults in comparison with similar inter- vention in normoxia. Three studies [32, 35, 36] proposed passive IH interventions, and all others used active IH [29, 33, 34, 37–47]. The interventions were performed in normobaric hypoxia—%FiO2 ranging 13.0–16.1% or %SpO2 ranging 75–85%—corresponding to moderate altitude level; reason why none of the studies reported any complication or serious adverse effects in the partici- pants despite their age. The most frequently investigated health-related outcomes were body composition [29, 32, 33, 35, 37, 41, 43–47], functional fitness [29, 33, 38, 39, 43–45, 47] and blood pressure [29, 32, 36, 37, 41, 45]. Functional Fitness and Pulmonary Capacity Functional Fitness and Pulmonary Capacity A total of eight studies [29, 33, 38, 39, 43–45, 47] eval- uated the functional fitness of older adults. Different physical capacities were measured using various tests. One study [47] evaluated pulmonary function [i.e., forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and maximal voluntary ventilation (MVV)] with a spirometer, two studies [29, 43] assessed muscle force parameters using an isokinetic dynamometer, three studies [29, 39, 45] measured maximal oxygen uptake (VO2max) by respiratory gas exchange systems, and four studies [33, 38, 44, 47] used functional tests (i.e., senior fitness test, timed up and go test, one leg standing, peg- board) and exercise strength (i.e., grip strength, crutches, push-ups). A 12-week IH intervention using moderate hypoxia (FiO2 14.5%) with 90–120 min of multimodal training sessions (aerobic exercise and elastic resistance training) showed greater improvement in pulmonary capacity (FVC, FEV1 and MVV) and functional fitness (i.e., chair stand, pegboard, tandem test and one leg Overall, the results of this systematic review indicate that passive or active normobaric IH would have a lim- ited positive effect on health-related outcomes in healthy older adults compared to similar intervention in nor- moxia, without any observed adverse effects in the par- ticipants. These findings differ from two recent reviews [48, 49] indicating that intermittent hypoxia-hyperoxia protocols could be effective at the cardiovascular, meta- bolic, and cognitive levels in an elderly population suf- fering from various diseases. The free radical signaling during hypoxia-hyperoxia interventions may lead to bet- ter induction of antioxidant synthesis than IH only [25]. Page 10 of 15 Page 10 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open (2023) 9:19 Moreover, patients with coronary artery diseases showed a greater trend of change in hematological parameters and an enhanced erythropoietic response than healthy older adults [50], which may result in a different hypox- emic response and oxyhemoglobin dissociation curve between healthy and pathological older adults. Likewise, it was observed that HIF-1α mRNA expression after a 3-week intervention of passive IH (%FiO2 12%) was increased in pre-diabetic individuals, but not in healthy older adults (40–70 years old) [51]. Without consider- ing possible effects of medication, this could explain the different adaptive responses to IH shown by older adults with or without diseases. Functional Fitness and Pulmonary Capacity In addition to this, previ- ous studies concluded that low doses of hypoxia (%FiO2 13–15%) might not be a sufficient stimulus to induce adaptive mechanisms [25], and it is important to note that most of the studies included in this review carried out interventions within this range. Illustratively, when pre-diabetics and healthy older adults followed a 3-week passive IH intervention, only the pre- diabetic individuals reported significant reductions in fasting glucose [51]. This may indicate that molecular regulation of glucose and lipid metabolism, as well as mitochondrial function and the physiological mechanism underlying the beneficial effects of IH, would be different in older adults with or without diseases. Additionally, our results also suggest that active IH does not lead to major additional effects on metabolic parameters. Presumably, in a group of untrained older individuals, adaptations to the delivery, transport, and intramyocellular metabolism of glucose and insulin signaling pathway may be suffi- ciently large with exercise training in normoxia [46]. Effects of IH on Blood Pressure and Hematological Parameters Blood pressure has been one of the most studied health- related outcomes among the studies included in this review. While IH has been demonstrated to provide beneficial effects on the blood pressure of hypertensive [57] and obese [58] individuals, mainly due to vasodi- lation of the arteries and a decrease in arterial stiffness concomitant with an increase in nitric oxide [59], none of the studies included in this review observed significant changes of systolic blood pressure in healthy older adults [29, 32, 36, 37, 41, 45]. Only one study from this review [36] showed a significant decrease of diastolic blood pressure in overweight individuals after an 8-week pas- sive hypoxia intervention (three 1-h sessions per week) compared to normoxia. The severity of the hypoxic dose used in this study (SpO2 = 75%) could have played an important role in lowering diastolic blood pressure. In this vein, hypoxic dose was shown to be a determining factor in achieving beneficial effects with IH, when %FiO2 ranged between 10 and 12% [25, 27]. Additionally, the fact that some older adults might be resistant to exercise- induced blood pressure reduction [60] can also influence the results obtained after an IH intervention. Effects of IH on Body Composition and Metabolic Parameters Sports Medicine - Open (2023) 9:19 Page 11 of 15 Page 11 of 15 Timon et al. Sports Medicine - Open However, in another study [36] included in this review, no change in C-reactive protein levels was observed after an 8-week intervention of passive hypoxia. Previous studies have suggested that IH protocols could exert an anti-inflammatory and tissue-protective effects [68, 69], notably via the suppression of pro-inflammatory media- tors such as TNF-α and IL-4 [70] and contribution to the production of anti-inflammatory interleukins by B cells [71]. However, when using active IH, such as resistance training with elastic bands during a 24-week interven- tion [34], no additive effect on inflammatory biomark- ers was observed. It has been stated that the myokines produced during normoxic exercise have a long-term positive anti-inflammatory effect in older people [72, 73]. Supposedly, the anti-inflammatory role of exercise could mask the beneficial effect of the IH without any observed added effect on inflammatory parameters. Nonetheless, more evidence is needed to confirm the potential anti- inflammatory effect that passive or active IH could have on healthy older adults. depend on both the total duration of the intervention and the session time [61]. In this vein, Gore et al. [62] stated that tHbmass increases by 1.1% per 100 h of hypoxic expo- sure. On the other hand, not all participants could be considered ‘good’ responders, due to the large individual variability in the erythropoietic response to hypoxia [63]. Effects of IH on Bone Parametersh Three studies focused on bone parameters [32, 35, 42]. In the analysis of bone health, it is recommended to use both static biomarkers [bone mineral content (BMC) and bone mineral density (BMD)], which provide information on long-term adaptations, and dynamic bone remod- eling biomarkers [beta C-terminal telopeptide of collagen (b-CTX) and N-terminal propeptide of type I procollagen (PINP)] that are related to the rate of bone turnover and short-term adaptations [64]. In connection with BMD, only one study [35] observed significant improvements in whole body BMD, but not in femoral or trochanter BMD after 18 weeks of passive IH (%FiO2 16.1%, two weekly 16-min sessions) in comparison with similar intervention in normoxia. However, these results must be interpreted with caution since an unclear risk of bias of blinding of participants and incomplete outcome data were detected in this study, in addition to the fact that the sample size was very small (n = 5 in the hypoxic group). In the other two studies, no change in whole body BMD was observed after 18 weeks of active IH (%FiO2 16.1%) combined with WBV [42] or after 24 weeks of passive IH (%FiO2 16.1%, three weekly 45-min sessions) [32]. In fact, previous sci- entific literature has stated that alterations in bone mass in the elderly require long-term physical exercise pro- grams maintained over time [65], in addition to appro- priate nutritional intake containing a good supply of proteins, minerals and vitamin D [66]. However, Timon et al. [32] observed both a significant increase of PINP (bone formation biomarker) and a significant decrease of b-CTX (bone resorption biomarker) after 24 weeks of passive IH intervention compared to normoxic interven- tion. The upregulation of HIF-1α that occurs under IH could activate different genes involved in bone remod- eling, such as vascular endothelial growth factor (VEGF), erythropoietin and osteoprotegerin [67]. Likewise, IH could modulate the mesenchymal stem cells differentia- tion leading to a possible inhibition of bone resorption by increasing the osteoprotegerin/receptor activator [35]. Effects of IH on Functional Fitness, Pulmonary Capacity, and Quality of Lifei and Quality of Life Eight studies evaluated physical fitness [29, 33, 38, 39, 43–45, 47], but only one of them [47] observed improve- ments in pulmonary capacity (FVC, FEV1 and MVV) and functional fitness (i.e., chair stand, pegboard, tandem test and one leg standing) in healthy older individuals when IH intervention was compared with normoxia. Park et al. [47] stated that this improvement was due to the greater aerobic and anaerobic exercise capacity and muscular function required by IH training vs. normoxic training. However, and despite the fact that some other studies have shown that IH could be beneficial for improving health-related fitness in healthy older adults [28] (albeit in the absence of comparison with a normoxic train- ing group in this study) or those with cardiopathologies [50], the majority of the active IH experiments that used either resistance exercises [29, 33, 38], aerobic exer- cise [39, 45] or WBV [43, 44], indicated that the poten- tial improvement obtained was not significantly greater than when using normoxic training. These contradictory findings may be explained by some methodological dif- ferences (i.e., design of the training program, hypoxic dose). Unlike the other interventions, Park et al. [47] used a multimodal training program that combined aerobic work (treadmill and bicycle) with full-body muscular resistance training within the same session. This type of intervention has been shown to be very useful in older populations, both in normoxic [4, 74] and in hypoxic- hyperoxic conditions [75], to maintain or improve their physical and functional capacity. Additionally, it should be noted that the intensity of exercise used in this study Effects of IH on Inflammatory Biomarkershll Effects of IH on Body Composition and Metabolic Parameters Only two studies [32, 47] out of eleven found an added effect of IH in parameters related to body composition, specifically a decrease in body fat mass [32, 47] and an increase in fat-free mass [47]. An oxygen-deprived envi- ronment leads to a decrease in body composition with larger changes occurring with higher hypoxic stress [52]. Previous studies have concluded that IH could have beneficial effects on weight loss as a consequence of an increase in the basal metabolic rate [14] and in appe- tite reduction as a consequence of an increase in leptin levels (satiety hormone) and a decrease in ghrelin levels (hunger-stimulating hormone) [53]. Likewise, the carotid body chemo-receptors under hypoxic exposure stimulate ventilation and lead to sympathetic activation increasing metabolic demands [54]. However, in most of the stud- ies included in this systematic review no changes were found. The magnitude of changes in body composition depend on various factors, such as the type of interven- tion [55], the severity of hypoxic dose (i.e., %FiO2 and duration) [52], the level of physical activity and the nutri- tional intake of the participants [56], in addition to the individual adaptive response [52]. These issues have not been carefully reported or controlled in any of the above- mentioned studies, suggesting their possible influence.i Regarding hematological parameters, contradictory results were presented in the only two studies avail- able: the first of these found increases in red-blood cells, hemoglobin and hematocrit after 12 sessions (3 sessions per week) of 120 min of IH [39], while the most recent one found no added effect of IH (i.e., 5 weeks, 4 sessions per week, 60 min of resistance training) on hemoglobin, erythropoietin and blood volume [29]. It is noteworthy that, in the absence of total hemoglobin mass (tHbmass) measurement, the changes in hematological param- eters must be interpreted with caution, as the increases reported could be the consequence of diuresis and plasma volume reduction. Previous studies have con- cluded that changes in hematological parameters will l Regarding metabolic parameters, none of the five stud- ies that investigated these variables revealed significant differences between either passive [32, 36] or active [37, 40, 41, 46] IH and similar intervention in normoxia. Given the fact that the participants included in those experiments had healthy normative values for metabolic parameters, it is plausible that IH did not affect them. Timon et al. Conclusions IH has been suggested as potentially useful in older pathological individuals. However, this systematic review indicates that passive and active moderate IH had a lim- ited effect on health-related outcomes in healthy older adults, compared to similar intervention in normoxia. No clear benefit has been evidenced on cardio-metabolic, inflammatory, hematological, and cognitive parameters, or on BMD, BMC, and lean mass, with the magnitude of changes dependent on the type of intervention and the hypoxic dose’s severity among others. Only in specific settings (i.e., intermediate- or long-term interventions with high intensity/volume training sessions repeated at least 3 days per week under moderate-to-high hypoxic stress), may IH elicit optimal HIF-1α upregulation and consequent molecular and structural adaptations. In such conditions, some putative effect has been observed on functional fitness and fat mass loss. Nevertheless, more research is needed to determine the dose–response of passive/active IH in healthy older adults, especially to elucidate which factors are more decisive in explaining the individual variability of the response to IH. if Finally, It is also noteworthy that the hypoxic dose used by Park et al. [47] represents the highest training volume of all the included studies (36 sessions, 3 sessions per week, 90–120 min, %FiO2 14.5%), which would indicate that long-term intervention of moderate IH and higher number of sessions could lead to greater benefits for functional fitness in the elderly. i Concerning quality of life and cognitive function, the results obtained are limited. Only two studies conducted by Schega et al. [38, 39] analyzed parameters related to these health-related outcomes. It was observed that 6 weeks (3 sessions per week) of 60-min passive IH fol- lowed by 30-min active IH (full-body resistance exercises at SpO2 of 80%) caused a positive added effect on atten- tion and sleep quality compared with normoxic interven- tion, but not on the speed of cognitive performance or the mental component related to quality of life [38]. Like- wise, 4 weeks (3 sessions/week) of passive IH (90 min) followed by 30 min of active IH (aerobic exercise on bicy- cle at SpO2 = 80%) also did not lead to any added benefit on cognitive function [39]. A recent systematic review has claimed that moderate hypoxia (depending on the type, severity, exposure duration and frequency) could have potential therapeutic applications for neurodegen- erative diseases, such as mild cognitive impairments or dementia [6]. However, Schega et al. Abbreviations 1RM O Abbreviations 1RM One-repetition maximum b-CTX Beta C-terminal telopeptide of collagen BDNF Brain-derived neurotrophic factor BMC Bone mineral content BMD Bone mineral density BP Blood pressure CHO Cholesterol CRP C-reactive protein DXA Dual-energy X-ray absorptiometry FEV1 Forced expiratory volume in 1 s FiO2 Fraction of inspired oxygen FVC Forced vital capacity GLUT4 Glucose transporter 4 Hb Hemoglobin Hct Hematocrit HDL High-density lipoprotein HIF Hypoxia-inducible factor HOMA-IR Homeostatic model assessment-insulin resistance HRmax Maximum heart rate HRV Heart rate variability IH Intermittent hypoxia MVV Maximum voluntary ventilation OSAS Obstructive sleep apnea syndrome Effects of IH on Inflammatory Biomarkershll The influence of IH on inflammatory parameters has been scarcely investigated. To date, a 24-week interven- tion of passive IH exposure (%FiO2 16.1%) produced a decrease in C-reactive protein levels compared to a con- trol group, with no changes in IL-8 and IL-10 levels [32]. Page 12 of 15 Page 12 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 Timon et al. Sports Medicine - Open was moderate-to-high, both in aerobic exercises (60–70% of maximal heart rate) and in resistance exercises (rating of perceived exertion-RPE of 6–7 out of 10). As suggested in previous studies [29, 76], low-to-moderate intensity or load may not be optimal to elicit molecular and structural adaptation. Nonetheless, similar absolute exercise inten- sity (e.g., exercising at 100W) implies a higher relative workload when training in hypoxia than in normoxia, leading to greater physiological and perceptual responses [77, 78]. This variable should also be taken into account when analyzing the heterogeneous results of the studies, since some research has reported that if training in nor- moxia is carried out at the same workload as in hypoxia, no significant effects are observed [79]. the domains analyzed according to the Cochrane Collab- oration recommendations. Thirdly, the included studies did not differentiate men and women, and a possible sex- related effect may have influenced the results. Fourthly, no studies concerning hypobaric hypoxia were included in this review, which may lead to different physiological responses than normobaric hypoxia. Finally, a meta-anal- ysis could not be performed because the included studies presented a high diversity of interventions and great het- erogeneity in health-related outcomes. Conclusions [38] indicate that the training effects of physical activity seem to outweigh the effect of IH which explains the ineffectiveness of IH on quality of life and cognitive performance in healthy older adults. More research is warranted to confirm this specu- lative hypothesis. Consent for publication Not applicable. 21. Prabhakar NR, Peng YJ, Nanduri J. Hypoxia-inducible factors and obstruc‑ tive sleep apnea. J Clin Invest. 2020;130(10):5042–51. Availability of data and materials Not applicable. Availability of data and materials Not applicable. 18. Franklin KA, Lindberg E. Obstructive sleep apnea is a common disorder in the population-a review on the epidemiology of sleep apnea. J Thorac Dis. 2015;7(8):1311–22. Received: 5 September 2022 Accepted: 5 February 2023 Received: 5 September 2022 Accepted: 5 February 2023 24. Avezov K, Aizenbud D, Lavie L. Intermittent hypoxia induced formation of “endothelial cell-colony forming units (EC-CFUs)” is affected by ros and oxidative stress. Front Neurol. 2018;9:447. 25. Serebrovska TV, Serebrovska ZO, Egorov E. Fitness and therapeutic potential of intermittent hypoxia training: a matter of dose. Fiziol Zh. 2016;62(3):78–91. Acknowledgements Not applicable. 12. Chen PS, Chiu WT, Hsu PL, Lin SC, Peng IC, Wang CY, et al. Patho‑ physiological implications of hypoxia in human diseases. J Biomed Sci. 2020;27(1):63. Author contributions 13. Morin R, Goulet N, Mauger JF, Imbeault P. Physiological responses to hypoxia on triglyceride levels. Front Physiol. 2021;12:730935. RT wrote the first draft of the manuscript. RT and FB conducted database searches and article identification. Data extraction was conducted by RT and subsequently checked by IMG. Methodological quality assessments were conducted by RT and FB, and then confirmed by IMG. FB and IMG reviewed the original article. All authors read and approved the final manuscript. 14. Kayser B, Verges S. Hypoxia, energy balance, and obesity: an update. Obes Rev. 2021;03(22 Suppl 2):e13192. 15. Wang H, Shi X, Schenck H, Hall JR, Ross SE, Kline GP, et al. Intermittent hypoxia training for treating mild cognitive impairment: a pilot study. Am J Alzheimers Dis Other Demen. 2020;35:1533317519896725. Ethics approval and consent to participate The research project was approved by the Bioethics Committee of the Univer‑ sity (Ref: 651/2018). 20. Guscoth LB, Appleton SL, Martin SA, Adams RJ, Melaku YA, Wittert GA. The association of obstructive sleep apnea and nocturnal hypoxemia with lipid profiles in a population-based study of community-dwelling austral‑ ian men. Nat Sci Sleep. 2021;13:1771–82. Declarations 19. Gottlieb DJ. Sleep apnea and cardiovascular disease. Curr Diab Rep. 2021;21(12):64. Competing interests 22. Zhuang Y, Zhao Z, Cheng M, Li M, Si J, Lin K, et al. HIF-1α regulates osteo‑ genesis of periosteum-derived stem cells under hypoxia conditions. Front Cell Dev Biol. 2022;10:836285. Rafael Timon, Ismael Martinez-Guardado and Franck Brocherie declare no competing interests. 23. Musutova M, Weiszenstein M, Koc M, Polak J. Intermittent Hypoxia Stimulates Lipolysis, But Inhibits Differentiation and de novo lipogenesis in 3T3-L1 cells. Metab Syndr Relat Disord. 2020;18(3):146–53. Received: 5 September 2022 Accepted: 5 February 2023 Limitationsh This systematic review is not without limitations. Firstly, only full articles written in English were reviewed, and two articles were not included in the review because they were written in Russian. Secondly, although most of the included studies (9 of 17) had a low risk of bias, several studies had a high risk or unclear risk of bias in some of Page 13 of 15 Page 13 of 15 Timon et al. Sports Medicine - Open (2023) 9:19 PINP N-terminal propeptide of type I procollagen RCT Randomized controlled trial RPE Rating of perceived exertion SFT Senior fitness test SpO2 Peripheral oxygen saturation TUGT Timed up and go test VCAM1 Vascular cell adhesion molecule 1 VEGF Vascular endothelial growth factor VO2max Maximal oxygen uptake WBV Whole body vibration 7. Millet GP, Debevec T, Brocherie F, Malatesta D, Girard O. Therapeutic use of exercising in hypoxia: promises and limitations. Front Physiol. 2016;7:224. g yp p y ; 8. Verges S, Chacaroun S, Godin-Ribuot D, Baillieul S. Hypoxic conditioning as a new therapeutic modality. Front Pediatr. 2015;3:58. as a new therapeutic modality. Front Pediatr. 2015;3:58. 9. Brocherie F, Millet GP. Hypoxic exercise as an effective nonpharmacologi‑ cal therapeutic intervention. Exp Mol Med. 2020;52(3):529–30. p p 10. Lee JW, Ko J, Ju C, Eltzschig HK. Hypoxia signaling in human diseases and therapeutic targets. Exp Mol Med. 2019;51(6):1–13. 11. 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https://openalex.org/W2168293460	https://europepmc.org/articles/pmc3906223?pdf=render	English	null	Qualichem In Vivo: A Tool for Assessing the Quality of In Vivo Studies and Its Application for Bisphenol A	PloS one	2,014	cc-by	16,119	Introduction importance of adequate reporting for informing policy and scientific practice in animal research, Kilkenny et al. [9] proposed the ARRIVE (Animals in Research: Reporting In vivo Experi- ments) guideline for reporting in vivo studies. The objective of this guideline is to allow in-depth critique of reported quality controls using a framework for including all relevant information about what was done, why and how. However, very few complete frameworks for quality assessment of in vivo toxicology studies have been proposed and/or tested. Among them, the Klimisch score [10] defines data quality by three properties: adequacy, relevance and reliability. Biomedical research has been evaluated using quality assess- ment frameworks for many years. Existing scales use between 2 and 100 criteria to assess the methodological quality of clinical trials [1]. For example, the Jadad scale provides a 7-point checklist for assessing the quality of clinical trials in pain research [2]. The proposed GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) framework evaluates the quality of evidence and strength of recommendations about therapeutic and diagnostic interventions and clinical management strategies [3–4]. Adapted to the Australian context, the FORM framework was created to formulate and grade recommendations in clinical practice guidelines [5]. Public health in Europe depends critically on effective implementation of the REACH (Registration, Evaluation, Author- isation & restriction of CHemicals) regulation, which concerns the risks of most chemicals on the market. Protection of public health will not be effective without ensuring the data submitted by industry—on which political decisions are based—is of high scientific quality. REACH uses the Klimisch score [11] to assess the quality of individual studies. However, the method used to assess a study’s adequacy and relevance leaves significant room for subjectivity. It is relatively easy to assess reliability of data arising from standardized tests—in particular OECD or national guide- Transparent and complete reporting is key for assessing the methodological quality of a study. Therefore, there are specific recommendations for reporting (for example) randomized con- trolled trials [6] and observational studies in epidemiology [7]. Quality assessment frameworks for academic and regulatory toxicology are less developed. Wandall et al. [8] made one of the first attempts to identify sources of bias in toxicology, but did not develop a quality assessment framework. Highlighting the Transparent and complete reporting is key for assessing the methodological quality of a study. Abstract In regulatory toxicology, quality assessment of in vivo studies is a critical step for assessing chemical risks. It is crucial for preserving public health studies that are considered suitable for regulating chemicals are robust. Current procedures for conducting quality assessments in safety agencies are not structured, clear or consistent. This leaves room for criticism about lack of transparency, subjective influence and the potential for insufficient protection provided by resulting safety standards. We propose a tool called ‘‘Qualichem in vivo’’ that is designed to systematically and transparently assess the quality of in vivo studies used in chemical health risk assessment. We demonstrate its use here with 12 experts, using two controversial studies on Bisphenol A (BPA) that played an important role in BPA regulation in Europe. The results obtained with Qualichem contradict the quality assessments conducted by expert committees in safety agencies for both of these studies. Furthermore, they show that reliance on standardized guidelines to ensure scientific quality is only partially justified. Qualichem allows experts with different disciplinary backgrounds and professional experiences to express their individual and sometimes divergent views—an improvement over the current way of dealing with minority opinions. It provides a transparent framework for expressing an aggregated, multi-expert level of confidence in a study, and allows a simple graphical representation of how well the study integrates the best available scientific knowledge. Qualichem can be used to compare assessments of the same study by different health agencies, increasing transparency and trust in the work of expert committees. In addition, it may be used in systematic evaluation of in vivo studies submitted by industry in the dossiers that are required for compliance with the REACH Regulation. Qualichem provides a balanced, common framework for assessing the quality of studies that may or may not be following standardized guidelines. Editor: Cecilia Williams, University of Houston, United States of America Editor: Cecilia Williams, University of Houston, United States of America Received October 14, 2013; Accepted December 29, 2013; Published January 29, 2014 Received October 14, 2013; Accepted December 29, 2013; Published January 29, 2014 Copyright: 2014 Maxim, van der Sluijs. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Laura Maxim1, Jeroen P. van der Sluijs2 nication du CNRS (UPS 3088), Centre National de la Recherche Scientifique, Paris, France, 2 Environmental Sciences, Copernicus The Netherlands 1 Institut des Sciences de la Communication du CNRS (UPS 3088), Centre National de la Recherche Scientifique, Paris, France, 2 Environmental Sciences, Copernicus Institute, Utrecht University, Utrecht, The Netherlands Abstract Funding: This work has been funded by the French Ministry of Ecology in the framework of the PNRPE 2010 programme (URL: http://www.pnrpe.fr/), as part of the project ‘‘Toolkit for uncertainty and knowledge quality analysis of endocrine disruptors’ risk assessments: the case study of Bisphenol A’’ (DICO-Risk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: laura.maxim@iscc.cnrs.fr PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 Introduction However, criteria included in frameworks such as ToxRTool or ARRIVE focus on how completely a study is reported rather than on its scientific quality. But, reporting of seemingly straightforward details such as the study species or strain chosen can be a source of scientific debate about, for example, the sensitivity of that species or strain to estrogens [13]. – normative: incorporates interpretation of raw data and conclusions about the level of evidence provided by that data. – communication: incorporates how completely and under- standably the research is reported. Compared with the current framework for evaluation that is commonly used in regulatory chemical risk assessment [11], our definition of quality covers both relevance and reliability. In addition, quality includes aspects related to the interpretation and commu- nication of the results, and to technical aspects of measurement, e.g., analytical techniques. Most importantly, the concept of quality highlights the importance of the knowledge production process—which directly influences the robustness and usefulness of scientific results used for a particular decision-making situation— instead of focusing on the results alone. Our approach aligns with previous work on knowledge quality assessment (KQA) tools, which are essential for timely and adequate policy responses in situations of risk governance [17] and for responding to the credibility crisis of science used to set controversial policy [18–19]. We draw on previous experience with the KQA tool NUSAP (Numeral, Unit, Spread, Assessment, Pedigree) [20], already used to assess the quality of estimates of NOx, SO2, NH3 [21] and volatile organic compound emissions [22] in the Netherlands and to assess health risks from tropospheric ozone [23] emissions from a waste incinerator [24]; and from electromagnetic fields from overhead power lines [25]. Existing tools fail to provide a systematic approach for assessing the quality of in vivo studies used to inform policy by institutions charged with implementing regulatory frameworks or responding to requests for policy advice. Examples of such institutions relevant to chemical risks are EFSA (European Food Safety Authority) and ECHA (European Chemicals Agency). Our paper aims to fill this methodological gap by developing and testing the Qualichem in vivo tool (or simply, ‘‘Qualichem’’). For endocrine disrupters in general and BPA in particular, the challenge is to incorporate divergent views from scientists with affiliations in industry, academia, health agencies at the national and European level, and governments by providing a synthesized view of the global quality of a study. Introduction Therefore, there are specific recommendations for reporting (for example) randomized con- trolled trials [6] and observational studies in epidemiology [7]. Quality assessment frameworks for academic and regulatory toxicology are less developed. Wandall et al. [8] made one of the first attempts to identify sources of bias in toxicology, but did not develop a quality assessment framework. Highlighting the January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org 1 January 2014 \| Volume 9 \| Issue 1 \| e87738 Qualichem In Vivo knowledge (epistemological uncertainty), and looks more broadly at the concept of quality, which also includes the following dimensions: lines and good laboratory practice (GLP). However, the definition of reliability in the Klimisch score disadvantages studies that do not follow standardized guidelines, but are nevertheless scientif- ically robust; i.e., most of the published academic literature. In REACH, the Klimisch score is often applied by industry itself, when submitting data to health agencies. Industry must assess its own studies or studies from the academic literature. Room for subjectivity in assessing studies may lead to selection bias in choosing and weighting the set of studies that is ultimately used by industry and health agencies to inform decision-making. lines and good laboratory practice (GLP). However, the definition of reliability in the Klimisch score disadvantages studies that do not follow standardized guidelines, but are nevertheless scientif- ically robust; i.e., most of the published academic literature. In REACH, the Klimisch score is often applied by industry itself, when submitting data to health agencies. Industry must assess its own studies or studies from the academic literature. Room for subjectivity in assessing studies may lead to selection bias in choosing and weighting the set of studies that is ultimately used by industry and health agencies to inform decision-making. – technical: incorporates technical errors caused by imprecise instruments or measurement methods. – technical: incorporates technical errors caused by imprecise instruments or measurement methods. – methodological: incorporates whether and how researchers use the best available scientific knowledge and practices in drafting the research protocol, make assumptions when knowledge is lacking or choose among several available methods for assessing a parameter. Recognizing the lack of precision of the Klimisch categories and the need for a more transparent, harmonized and objective framework for assessing the reliability of the toxicological data submitted under REACH, the ToxRTool was created [12]. Materials and Methods As industrial chemicals such as BPA are present in many consumer products [15], studies used to create a regulatory framework have the potential to impact the lives of millions of people; as such, assessments of them must be rigorous. Quality assessment is a key step in helping to choose which studies regulatory decisions should be based on. Introduction Previous tools like ToxRTool assume that heterogeneity in ratings is not a natural consequence of the differences among respondents (discipline, level of compe- tence on the subject, previous experience, epistemic communities, etc), and that it can be solved if the questions are framed better. This assumption does not reflect real life situations: when evaluating studies of controversial topics like endocrine disrupters, scientists from different disciplinary backgrounds and socio- economic horizons openly disagree. Literature shows that the same raw data can be interpreted differently by different experts in different contexts, which can lead to conflicting conclusions [14]. Our approach solves the problem of ToxRTool’s unrealistic assumption, as it incorporates the differences among respondents, and allows for a useful representation of the entire range of responses. The objective of Qualichem is to provide a systematic and transparent framework to assess the quality of studies used in regulatory chemical risk assessment (Materials and methods). To validate this tool, it was tested with relevant academic and health agency scientists, and its applicability checked using both short (several pages) and long (4,000 pages) studies (Results). Other objectives of this paper are to 1) compare the quality criteria addressed in our tool with those previously used by European institutions that provide expertise on the risk of BPA and by the OECD and OPPTS standardized guidelines relevant to the two BPA studies evaluated here (sections 3.2. and 3.3.), 2) examine whether some criteria hold more weight in determining the final quality of a study (section 3.4), and 3) examine whether quality assessments are influenced by the disciplinary background and publication history of the respondents (section 3.5). Furthermore, Qualichem could be used to include a wider range of studies in risk assessment in a more balanced way. Quality assessment using Qualichem would apply the same criteria to evaluate both industry studies that follow OECD or GLP guidelines and non-standardized academic studies that also provide scientific knowledge that is useful for decision-making. January 2014 \| Volume 9 \| Issue 1 \| e87738 2.1. The quality criteria: an original typology Both studies claimed to comply with regulatory guidelines, and details of the guidelines that were relevant for assessing the study may not have been reported as part of the study. For this reason, we also copied the elements of the guidelines appropriate to each criterion in the survey. We developed the typology of quality criteria (Text S1) iteratively, following the main steps of the process of knowledge production of in vivo studies; using ECHA’s guidelines for the evaluation of information [11]; analysis of study criticism expressed by scientists (e.g., [13], [26]) or safety agencies like EFSA; previous literature on reporting in vivo studies [9], [12] and on sources that look at heterogeneity in expert judgments [8], [14], authors’ personal experiences with regulatory documents and authors’ expertise in a safety agency. In these sources, we identified the criteria used to criticize, argue in favor of, or evaluate the scientific robustness of in vivo studies. We considered the various lines of argumentation identified as expressions of expert judgments about in vivo studies, and that were therefore relevant criteria to include in our typology. Each respondent assessed one of the two studies, not both. To assess the quality of each study—specifically, how well they incorporate best scientific knowledge and practices—we presented each respondent with a question related to each of the criteria included in our typology. Elicitation protocols can be provided on demand. For example, the first question of our protocol was: ‘‘Were the substance’s properties checked before and during the experiment, in accordance with best scientific practices?’’ The text from the study that refers to the check of substance properties was copied below the question. The respondent was invited to answer using a Likert scale (Table 2) and to explain his/her response (Text S7). To check the robustness of our typology and incorporate feedback from the scientists interviewed, our interview protocol contained a final question about the need to exclude criteria or to include new ones. Interviews were recorded and transcribed. We used the transcriptions to analyze the results (Results section, Text S3 and Text S4). We tested the typology with 12 scientists in academia and health agencies—a sample that is in line with the current literature on expert elicitation [27] that recommends 6 to 12 experts. A thirteenth expert validated the typology but his responses have been excluded from the Qualichem analysis. 2.4. Choice of case studies The controversy over the health risks of BPA repeatedly focuses on the quality assessment methods used in different health agencies, and on the reliance on standardized guidelines rather than academic research to select pivotal studies. The two studies used as case studies here played an important role in BPA regulation. Tyl et al. (2002) was used as a critical study for choosing the NOAEL for BPA in Europe. Stump (2009) was devised in response to divergent views about BPA neurotoxicity between three Nordic and other European countries; it has been extensively reviewed by an EFSA working group. Tyl et al. (2002) has been considered robust enough to drive regulatory decisions [30–31], while Stump (2009) has been considered invalid [32–33]. The number of criteria evolved slightly thorough the interviews, based on comments from the experts. Therefore, some experts did not use the full set of 45 criteria. Four of the 45 criteria were added after interviews with two experts, one more criterion was added after the interview with the third expert, and two additional criteria were added after interviews with the sixth expert. The six remaining experts used the full set of 45 criteria and considered it to be complete. Ethics statement This study did not involve patients, and written consent was not required. Consent to participate was voluntary and was obtained by email. Anonymity and confidentiality of the interviews were guaranteed to all participants. The interview protocol has been sent to participants before the meeting. The participant was then asked to give oral consent and to allow audio recording of the interview. We did conduct research outside our country of residence but approaching local authorities was not needed The role of regulatory science is to provide the best available scientific knowledge at a certain moment, and not the unachiev- able ideal of ‘‘perfect’’ knowledge. In line with the post-normal science proposal for addressing the robustness of science used to set policy [16], the Qualichem tool addresses more than lack of January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 2 Qualichem In Vivo because interviewees’ institutional information were not used for our project. The ethics evaluation committee of Inserm (IORG0003254, FWA00005831), the Institutional Review Board (IRB00003888) of the French Institute of medical research and Health, approved the study protocol, including the information sheet on the expert profile and the oral consent procedure (Opinion number 13-123). 2.1. The quality criteria: an original typology He only had time to give a general assessment of the study and did not use the proposed Likert scale. Due to lack of time, two of the twelve experts responded only to the questions referring to the criteria in the ‘‘Protocol’’ part of our typology (Table 1, Text S1). We used two case studies—a journal article (Tyl et al., 2002) [28] and a 4,000-page report (Stump, 2009) [29]—to test if our protocol can be used within a reasonable time frame on both short and longer studies. Both studies were funded by the chemical industry, which is common in regulatory assessment of chemical risks. 2.2. Elicitation protocol We interviewed each expert respondent individually in either 2012 or 2013. To prepare respondents for the interviews, we pasted relevant text from the study below each question. This saved respondents from having to search the study for the elements needed to answer the question or from using their memory to recall the relevant information, which could lead to imprecise responses. 2.3. Choice of respondents Respondents were either chosen through an extensive search of international peer-reviewed literature for authors of articles on BPA toxicology, were experts who had participated in BPA working groups in health agencies in Europe or were specialists in BPA and/or endocrine disrupters with expertise relevant to health agencies that were recommended by the scientists involved in our project. We searched all personal and other web pages and then listed disciplinary areas using the exact wording found in these documents, without trying to create exclusive classes. As a result, some disciplinary areas on our list overlap and some encompass others. Following this process, we contacted 64 scientists by email. Thirteen agreed to participate. Four respondents were employed by safety agencies and nine were academics. Our 45 quality criteria (defined in Text S2) are assembled into thirteen different classes (Table 1) that fall into two general categories: ‘‘Protocol’’ and ‘‘Results’’. The Protocol part of the typology includes quality criteria that are relevant to the technical and methodological aspects of best available scientific knowledge and practices. The Results part includes only two criteria for technical and methodological quality: the results analysis and results check. The remaining results criteria pertain to commu- nicational quality (such as results reporting) and normative quality (such as causal interpretation, interpretation in light of the existing epistemological background, and expert judgment of the level of evidence provided by the results) (Text S1). 2.5. Definition of controversial criteria: a measure of aggregated quality It can be cumbersome and difficult to read a graphical representation of 45 criteria. To facilitate understanding and focus on the most important results, we have defined two PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 3 Qualichem In Vivo Table 1. Typology of quality criteria for in vivo studies. Table 1. Typology of quality criteria for in vivo studies. Class Quality criteria Protocol Quality Criteria 1. Substance Check of substance properties; check of storage conditions; procedure for obtaining formulations; choice of the control 2. Experimental animals Correspondence between the characteristics of tested animals and the characteristics of exposed humans; choice of test species/strain; handling of experimental animals; monitoring of experimental animals; monitoring of controls 3. Assay Sensitivity of the assay; choice of experimental unit; number of groups tested; number of control groups; robustness of regulatory guidelines; test of a single substance or mixture 4. Measured effects Parameters observed; observation time; biological level observed; precision of effects measurement 5. Tested exposure Toxicokinetic stage for measuring exposure; level of doses tested; exposure duration; number exposure levels; route of administration; precision of exposure measurement; control of confounders 6. Laboratory procedures and human factors Experimenter bias Results Quality Criteria 7. Results reporting Results reporting; graphical data representation; abstract vs. raw data 8. Results analysis Statistical methods used; statistical unit; treatment of data for statistics; statistical power; evaluation of errors, uncertainty, variability 9. Causal interpretation Interpretation of dose-response; biological mechanism; extrapolation from animals to humans; functional relevance of changes 10. Results interpretation: epistemological context Epistemological background 11. Results check Status of peer-review; coherence with literature 12. Results interpretation: expert judgment Results vs. raw data; assumptions 13. Variability Variability doi:10.1371/journal.pone.0087738.t001 Results vs. raw data; assumptions doi:10.1371/journal.pone.0087738.t001 Controversial criteria are those for which, on the scale from 1 to 6 (Table 2): Controversial criteria are those for which, on the scale from 1 to 6 (Table 2): Controversial criteria are those for which, on the scale from 1 to 6 (Table 2): categories of criteria: controversial and critical. The subset of criteria that we call controversial or critical is specific to each study assessed with Qualichem; they are not pre-defined as such, but are based on the outcome of the evaluation. N at least one respondent gave a score of 3 or less, or N there is a difference of at least two points between any two scores. The two categories of criteria, controversial and critical, allow us to distinguish two levels of quality: The graphical representation shows all controversial criteria. All the other criteria—i.e., those that are not controversial according to our definition—received scores of 5 or 6 and were considered to have a high aggregated quality. – aggregated quality for each criterion, using the median of the expert respondents’ scores; this is an indicator of majority (consensus) views on aspects of study quality. – level of confidence for the whole study, using a decision rule based on critical criteria (see below); this is an indicator of divergence between expert respondents, and gives important weight to the scores of critical expert respondents. The graphical representation was built using a tailored Excel file (Text S8, Text S9, Text S10). The graphic is divided into three colored areas: red (including scores and median scores ,3), orange (for scores and median scores between 3 and 4) and green (for scores and median scores .4). For each criterion, a line covers the Table 2. Scale used for expert elicitation. Answer On a scale from 1 to 6, the answer corresponds to the score Agree strongly 6 Agree moderately 5 Agree slightly 4 Disagree slightly 3 Disagree moderately 2 Disagree strongly 1 I cannot answer CA Not applicable NA doi:10.1371/journal.pone.0087738.t002 PLOS ONE \| www plosone org 4 January 2014 \| Volume 9 \| Issue 1 \| e87738 Table 2. Scale used for expert elicitation. Table 2. Scale used for expert elicitation. Table 2. Scale used for expert elicitation. Controversial criteria are those for which, on the scale from 1 to 6 (Table 2): Answer On a scale from 1 to 6, the answer corresponds to the score Agree strongly 6 Agree moderately 5 Agree slightly 4 Disagree slightly 3 Disagree moderately 2 Disagree strongly 1 I cannot answer CA Not applicable NA doi:10.1371/journal.pone.0087738.t002 PLOS ONE \| www.plosone.org 4 January 2014 \| Volume 9 \| Issue 1 \| e87738 On a scale from 1 to 6, the answer corresponds to the score On a scale from 1 to 6, the answer corresponds to the score January 2014 \| Volume 9 \| Issue 1 \| e87738 January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org Qualichem In Vivo equally important, and b) choose a maximum of 15 criteria that are the most important for the overall quality of the results. full range, from the lowest score to the highest score in the group of responding experts. The median score is represented by an ‘‘x’’ and the interquartile range is represented by a rectangle. 3.1. Two in vivo case studies that assess the effects of BPA 3.1. Two in vivo case studies that assess the effects of BPA This section presents the Qualichem results for the two case studies: Tyl et al. (2002) and Stump (2009). This section presents the Qualichem results for the two case studies: Tyl et al. (2002) and Stump (2009). N they are very heterogeneous—there is a difference of 4 or 5 points between any two respondents (the maximum possible difference between the scores of two respondents is 5); As a reminder, the graphical representations of Qualichem (Fig. 1 and 2) include only controversial criteria. The assessment of the level of confidence in a study using Qualichem is based on the subset of those controversial criteria that are considered critical. N they are very low scores—at least one respondent gave a score of 1; or The average length of an interview was two hours—about 90 minutes of that was required to fill out the Qualichem survey. We assume an additional two to four hours was required for the respondent to read the study before the meeting. However, in some cases, the time for analyzing some particular studies might be much longer, e.g., when re-analysis of original raw data is done. However, we estimate that this applies to particular situations and is not the regular case of peer-reviews. N they show low or average aggregated quality—the median of scores is #4 (the ‘‘x’’ in the red or orange area, Fig. 1 and 2). To define an overall level of confidence in a study, we established a decision rule based on the number of critical criteria. A study has: is not the regular case of peer-reviews. N a high level of confidence if less than one-third of criteria (#14/45) are critical 3.1.1. Qualichem in vivo for Tyl et al., 2002. Figure 1 represents the application of Qualichem to the Tyl et al. (2002) study—eight respondents participated (Text S8). The respondents provided justification for why they assigned a score to each criterion, and these are presented in Text S3. Our goal was to synthesize their explanations without critically commenting on them. For example, bibliographic references were not included unless the respondents themselves provided them. N Low aggregated quality: median in the red area (,3). N Low aggregated quality: median in the red area (,3). In other words, if the ‘‘x’’ in figures 1, 2 and 3, is in the red area, the aggregated quality of the criterion is low. If the ‘‘x’’ is in the orange area, the aggregated quality is average, and if it is in the green area the aggregated quality is high. We did not expect a statistical correlation between respondents’ characteristics and their responses. However, these characteristics could influence their expert judgments about study quality. For the purpose of comparison, we isolated and graphically represented affiliation or disciplinary clusters in a separate figure. We created this representation for the respondents who included ‘‘endocri- nology’’ or ‘‘endocrine toxicology’’ among their fields of compe- tence. Also, we compared the results of this cluster, i.e., criteria in the red/orange area, with the results obtained on all the respondents together. Significant, easy-to-observe differences can be interpreted as an indication of the influence of discipline/ affiliation/interests. The interquartile range shown with a rectangle on the graphical representation is another indicator of inter-expert heterogeneity. Critical criteria are a subset of controversial criteria, and are used to calculate a multi-expert aggregated level of confidence in the study. The term ‘‘level of confidence’’ has a very precise meaning in statistics; however, in this paper we use ‘‘level of confidence’’ to referring to the quality of in vivo studies. We used this wording because our experience is that this formulation is easy to understand and is common wording for experts in health agencies [15]. For the future use of Qualichem, any other disciplinary, conflicting interests or affiliation clusters can be similarly isolated and compared. Such a separation can be done easily using the internet-based version of Qualichem available at URL: http:// www.qualichem.cnrs.fr/. 2.6. Definition of critical criteria: a measure of the level of confidence in a study A higher quality study will have high scores on more criteria. Depending on the scores given by the respondents, some criteria might play a greater role than others in determining the overall quality of the study. Critical criteria are defined as those controversial criteria of which the scores meet at least one of the following conditions: 3.1. Two in vivo case studies that assess the effects of BPA N an average level of confidence if between one-third and two-thirds of criteria (15 to 30/45) are critical N a low level of confidence if more than two-thirds of criteria ($31/45) are critical This decision rule is based in the assumption that all criteria have an equal weight, which may not be valid (see discussion). Additional decision rules could be established, and testing these decision rules could be the object of further research, before the method is standardized. Of the possible 45, 35 controversial criteria were identified using Qualichem. In most cases in which a six was assigned to a criterion, it was either because the study respected regulatory guidelines or because a respondent had personal experience with relevant current practice. 2.8. Influence of experts’ affiliation and background/ expertise on the use of Qualichem q g p y g The aggregated quality of an individual criterion is assigned as follows: The aggregated quality of an individual criterion is assigned as follows: Each of the respondents was asked to fill in an ‘‘expert profile’’ (Text S5) designed to identify their discipline, their publication activity (particularly on BPA and endocrine disrupters), the nature of their knowledge of BPA (i.e., experimental and/or theoretical), whether their expertise is specialized on BPA and/or endocrine disrupters or generalized on toxicology or other areas, and their institutional affiliation and financial links with industry. N High aggregated quality: median in the green area (.4) N High aggregated quality: median in the green area (.4) N Average aggregated quality: median in the orange area (ranging from 3 to 4) January 2014 \| Volume 9 \| Issue 1 \| e87738 2.7. Relative weights of different Qualichem criteria The median score for a given criterion, as a reminder, represents its aggregated quality. Thirty of the 35 controversial criteria were of high aggregated quality (the median is in the green area). Of the five remaining criteria, three were of average We assessed the relative weight of each Qualichem criteria in determining the overall (aggregated) quality of the studies. Our respondents were given two options: a) indicate that all criteria are PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 5 Qualichem In Vivo Figure 1. Quality assessment of Tyl et al. (2002), using Qualichem with eight respondents. For the study of Tyl et al. (2002), of the 45 criteria, the figure represents only the 35 controversial criteria out of the total set of 45 criteria. The remaining 10 criteria were not controversial according to our definition; they received scores of 5 or 6 and were considered to be of high aggregated quality. The figure is divided in three colored areas: red (including scores and medians ,3), orange (for scores and medians between 3 and 4) and green (for scores and medians .4). A line covers the full range, from the lowest score to the highest score in the group of responding experts. The median of the scores is represented by an ‘‘x’’ and the interquartile range is represented by a rectangle. If the median (x) is in the red area, the aggregated quality of the criterion is low. If the median is in the orange area, the aggregated quality is average. If the median is in the green area, the aggregated quality is high. The interquartile range is an indicator of inter-expert heterogeneity. Thirty of the 35 controversial criteria were of high aggregated quality (the median is in the green area). Of the five remaining criteria, three were of average aggregated quality (the median in the orange area) and two were of low aggregated quality (the median is in the red area). doi:10.1371/journal.pone.0087738.g001 Figure 1. Quality assessment of Tyl et al. (2002), using Qualichem with eight respondents. For the study of Tyl et al. (2002), of the 45 criteria, the figure represents only the 35 controversial criteria out of the total set of 45 criteria. The remaining 10 criteria were not controversial according to our definition; they received scores of 5 or 6 and were considered to be of high aggregated quality. Committee on Food [30] and the European Chemicals Bureau (ECB) [31], who considered it a very good quality, ‘‘pivotal’’ study, and suitable to use to determine the NOAEL of BPA. January 2014 \| Volume 9 \| Issue 1 \| e87738 2.7. Relative weights of different Qualichem criteria The figure is divided in three colored areas: red (including scores and medians ,3), orange (for scores and medians between 3 and 4) and green (for scores and medians .4). A line covers the full range, from the lowest score to the highest score in the group of responding experts. The median of the scores is represented by an ‘‘x’’ and the interquartile range is represented by a rectangle. If the median (x) is in the red area, the aggregated quality of the criterion is low. If the median is in the orange area, the aggregated quality is average. If the median is in the green area, the aggregated quality is high. The interquartile range is an indicator of inter-expert heterogeneity. Thirty of the 35 controversial criteria were of high aggregated quality (the median is in the green area). Of the five remaining criteria, three were of average aggregated quality (the median in the orange area) and two were of low aggregated quality (the median is in the red area). doi:10 1371/journal pone 0087738 g001 doi:10.1371/journal.pone.0087738.g001 aggregated quality (the median is between 3 and 4 and in the orange area) and two were of low aggregated quality (the median is in the red area). Committee on Food [30] and the European Chemicals Bureau (ECB) [31], who considered it a very good quality, ‘‘pivotal’’ study, and suitable to use to determine the NOAEL of BPA. 3.1.2. Qualichem in vivo for Stump (2009). Figure 2 represents the application of Qualichem to the report by Stump (2009) (Text S9). Despite the large number of competent scientists contacted by email (64), only four respondents agreed to Twenty-seven of the controversial criteria were also identified as critical, which is more than one-third of the 45 criteria. As such, confidence in Tyl et al. (2002) can be considered average. This contradicts official evaluations of this study by the Scientific January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Qualichem In Vivo Figure 2. Quality assessment of Stump (2009), using Qualichem with four respondents. For the report of Stump (2009), of the possible 45, the figure represents only the 16 controversial criteria. All the other criteria—those that are not controversial according to our definition—received scores of 5 or 6 and were considered as having a high aggregated quality. 3.2. Assessment of study quality in safety agencies Though these two studies may have been assessed by national health agencies, we focused on comparing Qualichem with quality assessments done by European institutions. These institutions play an essential role in advising decision-making on regulatory values for exposure to BPA in European countries, and it is relatively easy to access their documents. Of the possible 45, 16 controversial criteria were identified using Qualichem. Nine of these controversial criteria were of high aggregated quality (median fell in the green area). The remaining seven were all of average aggregated quality (median fell in the orange area). 3.2.1. Tyl et al. (2002). The Tyl study was used in the Opinion of the Scientific Committee on Food [30] as a pivotal study for deriving an NOAEL for BPA. This study was thought to be of good quality because of its long observation time, use of a high number of doses, and inclusion of parameters specific to endocrine disruptors such as anogenital distance, acquisition of puberty, estrous cyclicity, sperm parameters and nipple retention in males. The effects on body weight and some organ weights seen by the authors at 50 mg/kg bw/day were considered to be relevant; therefore, the NOAEL was set at 5 mg/kg bw/day. No quality weakness was highlighted in the SCF assessment of the study. 3.2.1. Tyl et al. (2002). The Tyl study was used in the Opinion of the Scientific Committee on Food [30] as a pivotal study for deriving an NOAEL for BPA. This study was thought to be of good quality because of its long observation time, use of a high number of doses, and inclusion of parameters specific to endocrine disruptors such as anogenital distance, acquisition of puberty, estrous cyclicity, sperm parameters and nipple retention in males. The effects on body weight and some organ weights seen by the authors at 50 mg/kg bw/day were considered to be relevant; therefore, the NOAEL was set at 5 mg/kg bw/day. No quality weakness was highlighted in the SCF assessment of the study. All 16 of the controversial quality criteria were also identified as critical. Because between one-third and two-thirds of the criteria were critical, the study can be labeled as providing an average level of confidence, according to our decision rule. This characteriza- tion contradicts the evaluation made by the EFSA [32], who rejected the study. 3.1.3. Two levels of quality. 2.7. Relative weights of different Qualichem criteria Because the objective was to test the tool, it is not the number of experts for each case study that is important, but the total number of experts that participated in testing Qualichem. The total number was twelve, which is well within the typical goal range in expert elicitation studies (6 to 12 experts [27]). In the next section, we compare the criteria used by SCF, ECB or EFSA with those of the Qualichem typology to explore the reasons behind their contradictory evaluations for the two studies. The respondents provided justification for why they assigned a score to each criterion, and these are presented uncritically in Text S3. 2.7. Relative weights of different Qualichem criteria The figure is divided in three colored areas: red (including scores and medians ,3), orange (for scores and medians between 3 and 4) and green (for scores or medians .4). A line covers the full range from the lowest score to the highest score in the group of responding experts. The median of the scores is represented by an ‘‘x’’ and the interquartile range is represented with a rectangle. If the median (x) is in the red area, the aggregated quality of the criterion is low. If the median is in the orange area, the aggregated quality is average. If the median is in the green area, the aggregated quality is high. The interquartile range is an indicator of inter-expert heterogeneity. Nine of the 16 controversial criteria were of high aggregated quality (median fell in the green area). The remaining 7 criteria were all of average aggregated quality (median fell in the orange area). doi:10.1371/journal.pone.0087738.g002 Figure 2. Quality assessment of Stump (2009), using Qualichem with four respondents. For the report of Stump (2009), of the possible 45, the figure represents only the 16 controversial criteria. All the other criteria—those that are not controversial according to our definition—received scores of 5 or 6 and were considered as having a high aggregated quality. The figure is divided in three colored areas: red (including scores and medians ,3), orange (for scores and medians between 3 and 4) and green (for scores or medians .4). A line covers the full range from the lowest score to the highest score in the group of responding experts. The median of the scores is represented by an ‘‘x’’ and the interquartile range is represented with a rectangle. If the median (x) is in the red area, the aggregated quality of the criterion is low. If the median is in the orange area, the aggregated quality is average. If the median is in the green area, the aggregated quality is high. The interquartile range is an indicator of inter-expert heterogeneity. Nine of the 16 controversial criteria were of high aggregated quality (median fell in the green area). The remaining 7 criteria were all of average aggregated quality (median fell in the orange area). doi:10.1371/journal.pone.0087738.g002 confidence, which shows that minority opinions are numerous and important. participate. 3.2. Assessment of study quality in safety agencies We distinguished two levels of quality, i.e., aggregated quality and level of confidence for the whole study. They provide a way to represent both majority and minority opinions, and they should be read together. For both studies, most criteria show high aggregated quality (median in the green area), which indicates that most scores were favorable. However, both studies also receive only an average level of Just one year later, the ECB reinterpreted the raw data, leading to a change in the NOAEL. In opposition to the SCF [30], the PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 7 Qualichem In Vivo ECB excluded the effects found at 50 mg/kg bw/day by considering them not ‘‘consistent’’ [31] (p. 180). The ECB concluded that ‘‘overall, this study showed 500 mg/kg bisphenol-A causes ered the study of high quality, and referred to it as ‘‘well conducted and reported’’ (pp. 179), ‘‘comprehensive, good-quality multigeneration’’ investigation, and also referred to the use of an OECD guideline Figure 3. Relative importance of the Qualichem quality criteria to the global quality of the study. Eight of the twelve respondents agreed to select a subset of up to 15 criteria that they considered to be the most important for the quality of in vivo study results. The figure shows the combined 39 criteria chosen by these eight experts. The vertical axis represents the 39 criteria, and the horizontal axis represents the percentage of respondents that selected each criterion. doi:10.1371/journal.pone.0087738.g003 Figure 3. Relative importance of the Qualichem quality criteria to the global quality of the study. Eight of the twelve respondents agreed to select a subset of up to 15 criteria that they considered to be the most important for the quality of in vivo study results. The figure shows the combined 39 criteria chosen by these eight experts. The vertical axis represents the 39 criteria, and the horizontal axis represents the percentage of respondents that selected each criterion. doi:10.1371/journal.pone.0087738.g003 ECB excluded the effects found at 50 mg/kg bw/day by considering them not ‘‘consistent’’ [31] (p. 180). The ECB concluded that ‘‘overall, this study showed 500 mg/kg bisphenol-A causes a reduction in the number of pups per litter’’ and that ‘‘the NOAEL for both parental and reproductive toxicity is 50 mg/kg/day’’. The ECB consid- ered the study of high quality, and referred to it as ‘‘well conducted and reported’’ (pp. January 2014 \| Volume 9 \| Issue 1 \| e87738 3.2. Assessment of study quality in safety agencies Furthermore, some aspects of the protocol are not clear; for example, the definition of ‘‘errors’’ in the Biel maze [33]. Two Qualichem respondents gave maximal scores of 6, saying they felt the data reporting was exhaustive. The other two gave scores of 5, noting the statistical methods used were complex and difficult to understand. These high scores indicate that reporting was not considered to be a critical quality criterion by the Qualichem respondents. The EFSA WG did not quantify this criterion so it is difficult to compare the importance given to it. Results reporting was often criticized by EFSA [32–33], referencing incomplete data for surface righting response, ambulatory count, or random effects for litter on PND62. Furthermore, some aspects of the protocol are not clear; for example, the definition of ‘‘errors’’ in the Biel maze [33]. Two Qualichem respondents gave maximal scores of 6, saying they felt the data reporting was exhaustive. The other two gave scores of 5, noting the statistical methods used were complex and difficult to understand. These high scores indicate that reporting was not considered to be a critical quality criterion by the Qualichem respondents. The EFSA WG did not quantify this criterion so it is difficult to compare the importance given to it. – The choice of the parameters observed was also found to be incomplete by the EFSA working group (WG), whose experts identified missing aspects of behavior related to anxiety, avoidance learning, schedule-controlled behavior, sexual dimorphic behavior and impulsiveness. Nevertheless, the WG considered this criterion less important than the sensitivity of the assay, as it was considered not sufficient to invalidate the study. Qualichem scores for this criterion ranged from 1 to 6, with the median in the orange area (4). The choice and treatment of data for statistical analysis and the choice of the statistical method were heavily criticized by the EFSA experts because of the lack of statistical treatment of the time-to-escape data, the data for long-term memory effects, and of data censoring. EFSA [33] considered that ‘‘the Biel maze had not been appropriately statistically analyzed and therefore conclusions drawn from the results of these analyses cannot be relied upon’’ (pp. 4). Furthermore, pooling the results diluted the possibility of finding effects at some specific moments in time. With regard to data treatment, two of the Qualichem respondents declined to answer due to a lack of specific statistical competence. 3.2. Assessment of study quality in safety agencies The two others gave maximal scores. Three Qualichem respondents answered the question about the choice of statistical analysis. One assigned a 2, giving arguments similar to EFSA’s. The two other were uncritical, giving scores of 5 and 6. – The interpretation of results as compared to raw data was comparable between Qualichem, (scores from 1 to 6) and EFSA. Those aspects where a difference between the raw data and the authors’ interpretation was identified—namely, effects of BPA on gross motor movements; convulsions and seizures; and censored and variable data on learning and memory from the Biel maze [32–33] were declared inconclusive and the respective parts of the study were considered unusable. – The epistemological background was found to be lacking mechanistic knowledge, in particular on the interaction between BPA and estrogen receptors [32]. Qualichem produced a similar result, with the median score in the orange area (3). – The treatment of assumptions was both a critical quality criterion in Qualichem (Fig. 2) and repeatedly addressed by EFSA. Qualichem respondents were not very precise, but expressed that assumptions were not reported adequately. On the contrary, EFSA [33] was very specific (pp. 7), stating that the analysis was ‘‘based on very general hypotheses’’ about the categorical variables of the model, the effects were potentially random, and the shape of the distribution of the response variable may not have been normal. The EFSA WG considered the graphical representation of the data inadequate for some results from the Biel maze test, saying it impeded interpretation and comparison of the slopes. In contrast, all four Qualichem respondents gave maximal scores for this question. – EFSA also considered the choice of the observation time insufficient (3 minutes for the Biel maze) [32–33]. All four Qualichem respondents gave maximal scores, referencing the total time of observation in the experiment (three generation, but ignoring the time of observation for particular parameters). – EFSA also considered the choice of the observation time insufficient (3 minutes for the Biel maze) [32–33]. All four Qualichem respondents gave maximal scores, referencing the total time of observation in the experiment (three generation, but ignoring the time of observation for particular parameters). – One of the most important critiques brought by the EFSA [32– 33] reports was about variability. In the learning and memory results, the variability was considered too high for any conclusion to be drawn. 3.2. Assessment of study quality in safety agencies 179), ‘‘comprehensive, good-quality multigeneration’’ investigation, and also referred to the use of an OECD guideline (p. 214). An illustrative detail is that this reference to the OECD two-generation reproduction toxicity study guideline (probably January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Qualichem In Vivo OECD 416) is incorrect, because the study actually followed an OPPTS guideline [34], and the two guidelines are not identical. Variability also seems to have been considered very important for rejecting the whole study. One of the Qualichem respondents gave a minimal score of 1 for this criterion, while the other three assigned scores of 5 or 6. The 2006 EFSA report confirmed the NOAEL of 5 mg/kg bw/ day, which was considered adequate and in accordance with a more recent, modified OECD 416-guideline study on mice [35]. The EFSA Panel considered that this NOAEL, ‘‘identified in the SCF evaluation of 2002 is still valid’’ [36] (p. 6). As well, the value of seven other quality criteria assigned by EFSA [32] opposed our Qualichem results: 3.2.2. Stump (2009). In contrast to the Tyl study, for which detailed analysis of the strengths and weaknesses of the study is not available in regulatory documents, EFSA dedicated a whole report to the Stump study. Below, we compare quality criteria addressed by these documents with the results produced with Qualichem. – For the choice of the control, our four respondents felt that a negative control was justified, and assigned scores of 5 (1 respondent) and 6 (three respondents). In contrast, EFSA [32] considered that ‘‘an oestrogenic reference compound was not included. Therefore, sensitivity of the test parameters to oestrogenic substances has not been demonstrated’’ (pp. 19). Similarly, EFSA (2010b) considers that ‘‘as no positive control group was inserted in the Stump et al. (2010) study it is not possible to know if a positive effect could have been statistically picked up if one truly existed’’ (pp. 6). However, this continues with another statement, more in line with Qualichem responses: ‘‘however, it should be noted that no generally accepted reference compounds are available for this purpose’’ (pp. 22). The Stump (2009) study was published in 2010 in the journal Toxicological Sciences [37]. 3.2. Assessment of study quality in safety agencies For six of the 16 critical quality criteria identified with Qualichem, similar assessments were done in the EFSA reports [32–33]: – Regarding the sensitivity of the assay, the Biel maze was characterized as not having the ‘‘potential to demonstrate equivalence of BPA compared to a control’’, because EFSA’s Assessment and Methodology Unit revealed ‘‘an extreme high variability, not only in the study data for the PND62 trials, but also in the data for PND22. This variability might be due to other non-modelled or not-possible-to model aspects of the experimental design or execution of the experiment’’. However, in contrast to Qualichem, this criterion was given very high weight by the EFSA working group—decisive over all the others—and was considered sufficient for declaring: ‘‘therefore, this study should be considered as inconclusive’’ [32] (pp. 25). – Regarding the sensitivity of the assay, the Biel maze was characterized as not having the ‘‘potential to demonstrate equivalence of BPA compared to a control’’, because EFSA’s Assessment and Methodology Unit revealed ‘‘an extreme high variability, not only in the study data for the PND62 trials, but also in the data for PND22. This variability might be due to other non-modelled or not-possible-to model aspects of the experimental design or execution of the experiment’’. However, in contrast to Qualichem, this criterion was given very high weight by the EFSA working group—decisive over all the others—and was considered sufficient for declaring: ‘‘therefore, this study should be considered as inconclusive’’ [32] (pp. 25). Results reporting was often criticized by EFSA [32–33], referencing incomplete data for surface righting response, ambulatory count, or random effects for litter on PND62. Furthermore, some aspects of the protocol are not clear; for example, the definition of ‘‘errors’’ in the Biel maze [33]. Two Qualichem respondents gave maximal scores of 6, saying they felt the data reporting was exhaustive. The other two gave scores of 5, noting the statistical methods used were complex and difficult to understand. These high scores indicate that reporting was not considered to be a critical quality criterion by the Qualichem respondents. The EFSA WG did not quantify this criterion so it is difficult to compare the importance given to it. Results reporting was often criticized by EFSA [32–33], referencing incomplete data for surface righting response, ambulatory count, or random effects for litter on PND62. 3.2. Assessment of study quality in safety agencies In contrast, the expected variability in motor activity between males and females was not found. – Coherence with other studies was considered absent for popcorn seizures and therefore a sufficient reason to cast ‘‘doubt on the relevance of this observation’’ [32] (p. 28). Both the study by PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 9 Qualichem In Vivo Stump and the rest of the existing literature on neurobehav- ioral toxicity were considered too low quality to be used to assess the effects of BPA. Three of the Qualichem respondents gave maximal scores, indicating that they considered the results of the study in line with other robust published results. Two respondents did not provide justification for their scores, but a third said that the Stump study was in line with other robust studies, even if it contradicted other studies considered to be of low quality. The fourth respondent could not answer, arguing that no other comparable study, in terms of number of animals and doses employed, was available in the literature. Stump and the rest of the existing literature on neurobehav- ioral toxicity were considered too low quality to be used to assess the effects of BPA. Three of the Qualichem respondents gave maximal scores, indicating that they considered the results of the study in line with other robust published results. Two respondents did not provide justification for their scores, but a third said that the Stump study was in line with other robust studies, even if it contradicted other studies considered to be of low quality. The fourth respondent could not answer, arguing that no other comparable study, in terms of number of animals and doses employed, was available in the literature. standardized guidelines, with clear experimentation procedures to follow. Other quality criteria either depend on the choices made by experimenters or are not addressed at all. Finally, GLP [41–42] only allows traceability of laboratory procedures and limits the possibility of fraud in private laboratories [13], but it is not a standard of scientific quality. There are no quality criteria in documents produced by expert groups or in the OECD 416, OECD 426, OPPTS 870.3800, OPPTS 870.6300 or GLP guidelines that are not in Qualichem (Text S6). 3.5. Influence of respondents’ disciplinary background and publication history on their quality assessments Four respondents were employed by safety agencies and the other eight were academics. Nine were, at some time, part of official expert committees involved in the assessment of BPA at different levels and in different countries in Europe. We did not have information about participation in expert committees for two respondents. Among the 12 respondents, three had not published on BPA in a peer-reviewed journal since 2006. We were not able to access a list of publications for another three (one academic and two employees of safety agencies). Six others have published at least one article addressing BPA at different levels of detail, in a peer-reviewed journal, since 2006. Figure 4 shows the disciplinary areas of the respondents—they were allowed to select multiple options. There were no quality criteria discussed in any of the SCF, ECB or EFSA reports above that was not dealt with in our typology. 3.4. Relative importance of the different criteria for the global quality of the study A criterion of ‘‘reproducibility’’ was used by EFSA [36] to argue for rejecting studies that indicate low dose effects of BPA: ‘‘the Panel considered that low-dose effects of BPA in rodents have not been demonstrated in a robust and reproducible way, such that they could be used as pivotal studies for risk assessment’’ (p. 4). This criterion has been defined rather vaguely: ‘‘low-dose effects on specific biological endpoints have been reported in some studies, but were not replicated in others’’ (p. 43). We have tested the use of ‘‘reproducibility’’ for assessing the quality of a study including a specific question in our Qualichem protocol. However, it was very difficult or impossible for our respondents to answer that question. They considered that studies could not be identically reproduced in toxicology, because the particular conditions of a specific experiment cannot be identically reproduced in another, even if an explicit aim is to confirm the results. Furthermore, toxicologists have no incentives to repeat previous studies, given that publication criteria and research funding are based on originality. The meaning given by EFSA [36] to reproducibility and the rationale for giving this criterion significant weight in its assessment of the low dose literature therefore remains unclear. We assessed the relative importance of the different Qualichem criteria in determining the final quality of the studies. Four respondents chose option a) indicate that all criteria are equally important, and eight chose option b) select a maximum of 15 criteria that are the most important for the final quality of the results. Figure 3 shows the 39 criteria chosen by at least one of the eight respondents who chose option b, in percentage of respondents that considered each criterion important. 75% or more of the respondents considered 8 of the 45 quality criteria as high priority, 60% or more of the respondents considered 18 criteria as high priority and 50% or more of the respondents considered 31 criteria as high priority. 3.2. Assessment of study quality in safety agencies This is related to the method used for developing Qualichem criteria—it began with criteria already present in safety agency reports, and completed them based on analysis of public criticism made by other stakeholders and on feedback from our respondents. – Experimenter’s bias was potentially associated with the imprecise definition of ‘‘error’’ in the Biel maze test. According to EFSA [33], ‘‘this definition is imprecise as it could be subject to different interpretations’’ (pp. 6). None of the Qualichem respondents noted this. January 2014 \| Volume 9 \| Issue 1 \| e87738 4.1. Heterogeneity in experts’ quality assessments and working procedures in safety agencies Our results show substantial heterogeneity among scientists in how they evaluate the quality of a study using the same criteria, and also differences between Qualichem assessments and those done by previous expert committees. This heterogeneity is not accounted for in an appropriate manner in current working procedures used by expert committees in health agencies. A number of factors could explain these differences. Experts may not have time to familiarize themselves with the important details of a study, may view quality differently or may just be wrong. Pragmatic considerations related to lack of time or resources can also influence how thoroughly a study’s quality is evaluated. In expert groups, consensus-based procedures sometimes favor strong personalities who take the lead in collective discussions— important minority opinions can be lost in the process of reaching a common position. Individuals can also be reluctant to express critical opinions in a group context, in particular when those opinions disagree with the group majority and/or president—a phenomenon called ‘‘the spiral of silence’’ [43]. When individual views are not heard or expressed, the overall quality of an expert group’s work can be compromised. A minority opinion in an expert committee is not necessarily a minority opinion in science, but could result from the criteria used for selecting experts, which could favour certain disciplines and competences over others. Furthermore, communicating the result of a quality assessment as a consensus masks certain quality problems, and can cause decision-makers to view a study’s results as being stronger than they really are. The institutional context of certain safety agencies, despite recent efforts, favors consensus and remains resistant to minority opinions. We know of two examples within our personal contacts of situations at EFSA in which one expert wanted to express a minority opinion but was discouraged from doing so. Qualichem avoids these problems by providing two indicators of quality: a majority-based indicator for each criterion (aggregated quality) and a multi-expert, aggregated level of confidence in a study. This allows each individual in an expert group to communicate his/her critical position, even if this position is minority. 3.3. Are Qualichem criteria already addressed in existing guidelines and in REACH? Comparison between Qualichem and other reporting and/or quality assessment frameworks. Reporting and/or quality assessment framework REACH registration, on- line version OECD 416 guideline OECD 426 guideline OPPTS 870.3800 guideline OPPTS 870.6300 guideline GLP Expert committees in safety agencies (SCF, ECB, EFSA) ARRIVE guideline Number of criteria addressed in each framework, out of the 45 Q li h it i 22/45 20/45 25/45 16/45 17/45 18/45 21/45 21/45 Qualichem (Figure 5, Text S10) for only two of these academic scientists (they assessed Tyl et al. [2002]) shows that the number of controversial criteria (30 vs 35) and critical criteria (25 vs 26) for these two respondents combined is similar to those given by the eight respondents together. However, the number of quality criteria that fall in the orange or red areas is much higher for these two respondents than for all respondents together: 16 vs 3 in the orange area, 9 vs 2 in the red area, and only 5 vs 30 in the green area. This indicates lower levels of aggregated quality for these criteria, compared to the eight respondents together. Qualichem (Figure 5, Text S10) for only two of these academic scientists (they assessed Tyl et al. [2002]) shows that the number of controversial criteria (30 vs 35) and critical criteria (25 vs 26) for these two respondents combined is similar to those given by the eight respondents together. However, the number of quality criteria that fall in the orange or red areas is much higher for these two respondents than for all respondents together: 16 vs 3 in the orange area, 9 vs 2 in the red area, and only 5 vs 30 in the green area. This indicates lower levels of aggregated quality for these criteria, compared to the eight respondents together. January 2014 \| Volume 9 \| Issue 1 \| e87738 3.3. Are Qualichem criteria already addressed in existing guidelines and in REACH? Tyl et al. (2002) meets the OPPTS 870.3800 standardized guideline on reproductive toxicity [34], corresponding to OECD 416 [38]. Stump (2009) claims to comply with the guidelines OECD 426 [39] and OPPTS 870.6300 [40] on developmental neurotoxicity. For certain Qualichem quality criteria, these standardized guidelines indicate best practices. For others, the guidelines vary. They may be flexible and leave choice of method to the discretion of the experimenters, address methodology briefly without giving precise indications, ask experimenters to report ‘‘what’’ they do but not prescribe ‘‘how’’ it should be done, or not address methodology at all. Interestingly, three of the four respondents who work in safety agencies gave scores under 5 for the criterion ‘‘Robustness of regulatory guidelines’’. The fourth was the only respondent who gave maximal scores to all quality criteria for the study assessed. This person reiterated that following a guideline is in itself a guarantee of quality, and noted that he knew the work of the people who conducted the study and trusted them. One academic scientist made a similar statement, saying that following a guideline is a sufficient guarantee of scientific quality—this person gave only one score of less than 5, for ‘‘general state of scientific knowledge’’. The number of quality criteria assigned scores under 5 was similar for the safety agency and academic respondents, indicating similar levels of criticism. In Text S6, we identify the Qualichem quality criteria that are addressed (with varying levels of precision) in the four standardized guidelines relevant for the two studies. In addition, we compared Qualichem criteria with information that REACH demands of industry and with the classes the ARRIVE guideline recommend for the scientific communication of in vivo studies [9]. This comparison also aimed to check the assumption, made by some of our respondents, that respecting regulatory guidelines ensures scientific quality for an in vivo study. As Text S6 shows (summarized in table 3), this assumption is not realistic—only some quality criteria are addressed in a precise way in The three most critical respondents gave scores lower than 5 to 14, 15 and 21 quality criteria. These were also the only respondents who included ‘‘endocrinology’’ or ‘‘endocrine toxi- cology’’ among their fields of competence. Representation of January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 10 Qualichem In Vivo Table 3. 4.2. Lowering subjectivity in regulatory quality assessments The current regulatory framework in Europe (e.g., REACH and the Regulation (EC) no 1107/2009 concerning placement of plant protection products on the market) demands that industry assess the risks of the chemicals it produces. Therefore, industry studies are frequently considered by health agencies. Research has repeatedly confirmed that conflicts of interest can bias results in favor of a study’s sponsor [44], [1]. The ability of health agencies to evaluate work done by industry would benefit from rigorous January 2014 \| Volume 9 \| Issue 1 \| e87738 11 Qualichem In Vivo Figure 4. Disciplines of Qualichem respondents. The vertical axis represents the disciplinary areas chosen by the experts. The horizontal axis represents the number of experts who chose each disciplinary area to describe his/her work. doi:10.1371/journal.pone.0087738.g004 Figure 4. Disciplines of Qualichem respondents. The vertical axis represents the disciplinary areas chosen by the experts. The horizontal axis represents the number of experts who chose each disciplinary area to describe his/her work. doi:10.1371/journal.pone.0087738.g004 quality assessment, with comparable criteria to evaluate both academic and industry studies and clear evaluation methods—an improvement over relying on fuzzy ‘‘expert judgment’’. Of course, providing a score from a scale without explanatory arguments can be as subjective as any other unstructured ‘‘expert judgment’’. For this reason, the Qualichem protocol requires respondents to support their choice of score for each quality criterion with one or more arguments that are documented together with the score (Text S7). suspicion, reduce the legitimacy of official risk assessments, give an impression of lack of transparency, waste public money by necessitating multiple expert groups, and delay decision-making. In the framework of REACH, a structured assessment of the quality of the studies submitted by industry could reduce the potential influence of conflicts of interest and provide a systematic approach that could facilitate the work of health agencies. It is currently difficult to access the original industry studies. Indeed, safety agencies do not have systematic access to the complete reports of the all studies communicated by industry in their registration dossiers. To access these full reports, safety agencies must sometimes negotiate with industry and ask for them. But, there is no guarantee that industry will provide the full reports. Access to raw data for re-analysis is even more problematic. January 2014 \| Volume 9 \| Issue 1 \| e87738 4.2. Lowering subjectivity in regulatory quality assessments The figure is divided into three colored areas: red (including scores and medians ,3), orange (for scores and medians between 3 and 4) and green (for scores and medians .4). A line covers the full range from the lowest score to the highest score in the group of responding experts. The median of the scores is represented by an ‘‘x’’ and the interquartile range is represented by a rectangle. If the median (x) is in the red area, the aggregated quality of the criterion is low. If the median is in the orange area, the aggregated quality is average. If the median is in the green area, the aggregated quality is high. The interquartile range is an indicator of inter-expert heterogeneity. The number of criteria that fell in the orange or red areas is much higher for these two respondents than for all respondents together: 16 vs 3 in the orange area, 9 vs 2 in the red area, and only 5 vs 30 in the green area. This indicates lower levels of aggregated quality for these criteria, compared to the 8 respondents together. doi:10.1371/journal.pone.0087738.g005 Figure 5. Quality assessment by two endocrinologists using Qualichem to evaluate Tyl et al. (2002). For the study of Tyl et al. (2002), of the 45 criteria, the figure represents only the 30 controversial criteria for the two respondents who included ‘‘endocrinology’’ or ‘‘endocrine toxicology’’ among their fields of competence. The figure is divided into three colored areas: red (including scores and medians ,3), orange (for Figure 5. Quality assessment by two endocrinologists using Qualichem to evaluate Tyl et al. (2002). For the study of Tyl et al. (2002), of the 45 criteria, the figure represents only the 30 controversial criteria for the two respondents who included ‘‘endocrinology’’ or ‘‘endocrine toxicology’’ among their fields of competence. The figure is divided into three colored areas: red (including scores and medians ,3), orange (for scores and medians between 3 and 4) and green (for scores and medians .4). A line covers the full range from the lowest score to the highest score in the group of responding experts. The median of the scores is represented by an ‘‘x’’ and the interquartile range is represented by a rectangle. If the median (x) is in the red area, the aggregated quality of the criterion is low. If the median is in the orange area, the aggregated quality is average. 4.2. Lowering subjectivity in regulatory quality assessments Furthermore, some study reports can be quite thick, and raw data and results are not always reported in an understandable manner, which can significantly delay the process of evaluating a study. A summary of the most relevant information contained in a report is essential to efficiently shorten the time for evaluating its quality. Furthermore, Qualichem could be further improved by adding a requirement that respondents provide scientific references from the peer-reviewed literature, where available, to support their choice of scores. There are currently unstandardized practices in health agencies on referencing expert arguments [45]. However, expert judgment can be erroneous or biased if arguments are not referenced by peer-reviewed literature—a phenomenon that tends to be important in socio-politically controversial cases [46]. The advantage of Qualichem is that it represents each quality criterion, which cannot be understood solely from a narrative description such as is presented in the SCF, ECB and EFSA reports. Furthermore, Qualichem provides a common framework that applies the same quality criteria for each study assessed and allows for quick comparison between studies and between health agencies, as shown here for our two cases. Indeed, without a common background for assessing the quality of available knowledge, different expert committees can reach opposite conclusions (e.g., ‘‘risk to all or none’’) based on the same data [47]. Such situations fuel controversies, create doubt and In the on-line version of REACH registration, industry can submit their studies according to a pre-established framework that includes administrative data, data source, materials and methods (i.e., test type, test guideline, GLP compliance, test materials, test animals), administration/exposure, examinations, results and discussions, further observations and conclusions. As shown in Table 3 and Text S6, the information currently provided in the on-line version of REACH registration dossiers for the two studies assessed includes only half of the Qualichem criteria. If January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org 12 Qualichem In Vivo information on all the Qualichem criteria were available online an similar tools for epidemiologic studies for risk assessment reports Figure 5. Quality assessment by two endocrinologists using Qualichem to evaluate Tyl et al. (2002). For the study of Tyl et al. (2002), of the 45 criteria, the figure represents only the 30 controversial criteria for the two respondents who included ‘‘endocrinology’’ or ‘‘endocrine toxicology’’ among their fields of competence. January 2014 \| Volume 9 \| Issue 1 \| e87738 4.4. Relative weights of criteria in quality assessment The weight of the same criterion could differ between studies and contexts, e.g., academic publication or regulatory assessment. An approach that asks respondents to weight each criterion, in addition to their Likert score, could be developed and tested in further work. Also, the calculation of the level of confidence could depend on the number of respondents involved, as our algorithm to identify critical criteria gives important weight to each individual respondent. There are several reasons for the contradictions between our Qualichem assessment and the evaluations made by SCF, ECB and EFSA. We showed that quality criteria used by official expert bodies differ from, and represent only a subset of, those included in Qualichem. Also, previous expert groups analyzed in this paper gave different weights to certain criteria than did Qualichem respondents. Indeed, regulatory guidelines change very slowly. For example, development of the OECD 426 guideline took 12 years [48]. Their objective is not to reflect the best scientific knowledge, but to offer a science-based political compromise among OECD member states. For this reason, there is significant potential for a gap between some OECD guidelines and rapidly advancing scientific knowledge. This is currently one of the major reasons for sociopolitical controversy about endocrine disrupters. In addition, the guidelines allow flexibility about certain aspects of the experimental protocol and leave the experimenter open to interpret the current state of scientific knowledge. However, this flexibility also allows room for experimenters to be wrong or to choose a level of scientific robustness that does not reflect available knowledge. In addition, group dynamics and procedures in official consensus-based expert groups leave only limited space for individual experts to express their insights on uncertainties and for these to be incorporated in the group’s final conclusion. Qualichem keeps track of each respondent’s criticism and values individual assessments by using the decision rule that defines critical criteria and, in turn, the ultimate level of confidence in a study. The expert groups that previously evaluated the two studies were inevitably different from Qualichem respondents, in terms of range of disciplinary domains and experience with endocrine disrupters and BPA. Some respondents are likely able to react more to some quality criteria and less to others. Our interviews showed that some respondents were not able to assess all quality criteria and responded ‘‘cannot answer’’. 4.3. Quality assessment and standardized guidelines y g Currently, regulatory guidelines and the GLP standard are given important weight in assessing the quality of studies in regulatory toxicology. While two of twelve respondents of our study stated from the very beginning of the interviews that respect of regulatory guidelines is an undisputable guarantee of scientific quality, in-depth evaluation led to very different quality assess- ments for the others. A study that respects regulatory guidelines can still present quality failures that can be judged important by certain scientists (Figures 1 and 2). Furthermore, regulatory guidance does not address several criteria that scientists consider important for assessing the quality of studies (Table 3, Text S6). For other criteria, required standards are designed to ensure the quality of work at a gross level; however, they do not account for the relevant particularities of specific substances, such as param- eters for measuring neurotoxicity. Most of the criteria that are not well addressed in regulatory guidelines were considered by our Qualichem respondents as having an important weight for the final quality of the study (Figure 3 and Text S6): control of confounders; correspondence between the experimental animals and humans; interpretation of the functional relevance of behavioral, morphologic, histological, molecular or biochemical changes; analysis of errors, uncertainty and limitations; sensitivity of the assay; status of peer-review; interpretation of raw data; choice of the toxicokinetic level for measuring exposure; data treatment for statistics; graphical representation; statistical power; precision of exposure measurement; analysis of assumptions; analysis and reporting of variability. For all these reasons, the use of standardized guidelines should not replace scientific quality assessment, but should be considered in a complementary way, as we have suggested in our interview protocols. 4.2. Lowering subjectivity in regulatory quality assessments If the median is in the green area, the aggregated quality is high. The interquartile range is an indicator of inter-expert heterogeneity. The number of criteria that fell in the orange or red areas is much higher for these two respondents than for all respondents together: 16 vs 3 in the orange area, 9 vs 2 in the red area, and only 5 vs 30 in the green area. This indicates lower levels of aggregated quality for these criteria, compared to the 8 respondents together. d i 10 1371/j l 0087738 005 doi:10.1371/journal.pone.0087738.g005 information on all the Qualichem criteria were available online, an industry study could be more easily reviewed. similar tools for epidemiologic studies, for risk assessment reports produced by health agencies, and for exposure characterization. Finally, Qualichem provides a basis for precise, reproducible and transparent assessment that could replace the Klimisch scores, which are currently subject to significant subjectivity and a confusing valuation procedure [12]. Qualichem for in vivo studies is based a generic procedure for developing quality assessment instruments and can be generalized to other types of regulatory productions. Currently, we are testing Finally, Qualichem provides a basis for precise, reproducible and transparent assessment that could replace the Klimisch scores, which are currently subject to significant subjectivity and a confusing valuation procedure [12]. PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org 13 Qualichem In Vivo of the OECD 426 guideline, such as assessment of cognitive and sensory dysfunction, are not adequately sensitive while others are overly sensitive. In addition, specific endpoints like social behavior, pharmacokinetics and neurochemistry are lacking, and there is significant variability in the endpoints that are defined, like motor activity [48]. The use of standardized guidelines as a major indication of scientific quality is currently controversial [48] [13]. of the OECD 426 guideline, such as assessment of cognitive and sensory dysfunction, are not adequately sensitive while others are overly sensitive. In addition, specific endpoints like social behavior, pharmacokinetics and neurochemistry are lacking, and there is significant variability in the endpoints that are defined, like motor activity [48]. The use of standardized guidelines as a major indication of scientific quality is currently controversial [48] [13]. 4.4. Relative weights of criteria in quality assessment This assumption is further reinforced by our results (section 3.5) showing significant differences between endocrinologists and other respondents, which reiterates previous results that indicate different approaches to endocrine disrupters in toxicology versus endocrinology [49]. Furthermore, most academic studies that could be useful for decision-making do not follow OECD or GLP guidelines and therefore start off significantly disadvantaged when it comes to regulatory assessment of the study’s quality. Robust science-based decision-making requires a more balanced playing field that considers both industry and academic studies. Systematic evalu- ation of studies as they relate to the current state of scientific knowledge is needed for well-informed decision-making, and to overcome the inevitable time delays in adapting OECD guidelines. Safety agencies do this kind of evaluation, but not in a systematic, transparent and comparable way from one agency to another. Evaluation of each of the Qualichem criteria depends on the state of knowledge at the moment of the evaluation. Applying Qualichem at the same time as the expert groups did their reports (2002, 2003 or 2010) would probably have given different results. Therefore, the most important result from our study is the Qualichem method itself; the two case studies have been used primarily to test and demonstrate the method. Guidelines have a good regulatory reputation for providing scientific quality [13]. For example, the Klimisch score calculates four levels of reliability for a study: reliable without restrictions, reliable with restrictions, not reliable, not assignable. The highest score for reliability (reliable without restrictions) is received by studies that were carried out according to standardized testing guidelines. Our work is in line with the proposals of Evidence-Based Toxicology [50]. Using Qualichem, the level of confidence in a study could be established on a clear and comparable basis, by including the views of all experts involved, including minority views. Respondents could express themselves naturally without needing cumbersome procedures such as ‘‘minority opinions’’. Reporting both consensus and controversial points could facilitate discussion in expert groups, and allow for easier representation of the quality of studies for health agency employees. However, standardized guidelines only partially deserve this reputation (see Table 3, Text S6 and respondents’ criticism on the ‘‘robustness of regulatory guidelines’’ in Fig. 1 and 2). 4.2. Lowering subjectivity in regulatory quality assessments For all these reasons, the use of standardized guidelines should not replace scientific quality assessment, but should be considered in a complementary way, as we have suggested in our interview protocols. Quality assessment and standardized guidelines 1. Lexchin J, Bero LA, Djulbegovic B, Clark O (2003) Pharmaceutical industry sponsorship and research outcome and quality: systematic review. BMJ 326: 1167–1170. Acknowledgments We are grateful to the three reviewers for the very useful comments, to David Gee for his continuous flow of excellent ideas and suggestions during many episodes of common work, to Ge´rard Arnold for careful reading and very helpful remarks, to Ce´line Vaslin for help with the figures, and to Sharilynn Wardrop for stylistic and linguistic improvements. 4.4. Relative weights of criteria in quality assessment This finding is in line with previous literature that suggests that some endpoints January 2014 \| Volume 9 \| Issue 1 \| e87738 PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e87738 14 Qualichem In Vivo Qualichem In Vivo Text S9 Spreadsheet for Stump (2009). (XLS) Text S9 Spreadsheet for Stump (2009). (XLS) Text S10 Spreadsheet for Tyl et al. (2002), scores of two endocrinologists. y) Our results indicate different levels of confidence from official quality assessments of two studies. Tyl et al. (2002) has been considered of very good quality by SCF, but the result from Qualichem indicates only an average level of confidence. The study by Stump (2009) was rejected by EFSA, but the Qualichem results also indicate an average level of confidence for the Qualichem respondents. Despite average levels of confidence for both studies, one was considered of good quality whereas the other was considered inconclusive. Comparison between criteria used by official expert committees and Qualichem criteria showed that SCF, ECB and EFSA seemed to prioritize certain criteria over others. However, as weighting was neither transparent nor explicit, it is difficult to assess the relative weight of different criteria, which can give an impression of subjectivity and even bias. 8. Wandall B, Hansson SO, Rude`n C (2007) Bias in toxicology. Arch Toxicol 81: 605–617. Author Contributions Conceived and designed the experiments: LM JVDS. Performed the experiments: LM. Analyzed the data: LM JVDS. Contributed reagents/ materials/analysis tools: LM JVDS. Wrote the paper: LM JVDS. Conceived and designed the experiments: LM JVDS. Performed the experiments: LM. Analyzed the data: LM JVDS. Contributed reagents/ materials/analysis tools: LM JVDS. Wrote the paper: LM JVDS. Conclusions As a structured and transparent way of reporting study quality assessment, Qualichem has the potential to reinforce trust in safety agencies by limiting subjectivity and transparently displaying the experts’ choices and assumptions. Furthermore, it makes inter- agency comparison of quality assessments of the same studies possible, by always applying the same set of methods and quality criteria. There is currently no clear and reproducible procedure for evaluating the quality of studies in regulatory expertise. Though considering a study of chemical risks valid or rejected can have tremendous consequences on the lives of people exposed to those risks, quality assessment remains unstructured, cannot be com- pared among expert groups and agencies, and cannot be transparently communicated. Respect of standardized OECD and GLP guidelines is currently considered a token of scientific reliability, but this is becoming more controversial. 7. Vandenbroucke JP, Von Elm E, Altman DG, Gøtzsche PC, Mulrow CD, et al. (2007) Strengthening the reporting of observational studies in epidemiology (STROBE): explanation and elaboration. Ann Intern Med 147(8): W-163-W- 194. 2. Jadad AR, Moore RA, Caroll D, Jenkinson C, Reynolds JM, et al. (1996) Assessing the quality of reports of randomized clinical trials: is blinding necessary? Control Clin Trials 17: 1–12. 3. Ansari MT, Tsertsvadze A, Moher D (2009) Grading Quality of Evidence and Strength of Recommendations: A Perspective. PLoS Med 6(9): e1000151. 4. Brozek JL, Akl EA, Compalati E, Kreis J, Terraciano L, et al. (2011) Grading quality of evidence and strength of recommendations in clinical guidelines. Part 3 of 3. The GRADE approach to developing recommendations. Allergy 66: 588–595. Text S1 Typology of quality criteria. (DOC) We have developed a tool, called Qualichem in vivo, to systematically and transparently assess the quality of in vivo studies used in chemical health risk assessment. Qualichem was usable for both short (a few pages) and extensive (4,000 page) study reports. For both studies, Qualichem contradicted the quality assessments done by expert committees in safety agencies, and confirmed that standardized guidelines only partly deserve their reputation as providers of scientific quality. Text S2 Definitions of quality criteria. (DOC) 5. Hillier S, Grimmer-Somes K, Merlin T, Middleton P, Salisbury J, et al. (2011) FORM: An Australian method for formulating and grading recommendations in evidence-based clinical guidelines. BMC Med Res Methodol 11:23. 6. Schulz KF, Altman DG, Moher D, CONSORT group (2010) CONSORT 2010 statement: updated guidelines for reporting parallel group randomized trials. PLOS Med 7(3): e1000251. Text S5 Expert profile. (DOC) – there is important heterogeneity among experts in their quality assessments, which is not well accounted for in current working procedures in health agencies Text S6 Comparison between quality criteria ad- dressed in Qualichem in vivo and quality criteria addressed in other sources. (DOC) – standardized guidelines do not appropriately include important quality criteria – different criteria have different weights for the final quality of a study. Text S7 Scale for quality assessment. (DOC) Qualichem provides two indicators of quality: a majority-based indicator for each criterion (i.e., aggregated quality) and a multi- expert, aggregated level of confidence in a study that allows each individual in an expert group to communicate his/her critical position, even if this position is a minority one (i.e., the global level of confidence in a study). Text S8 Spreadsheet for Tyl et al. (2002). (XLS) Text S2 Definitions of quality criteria. (DOC) Text S3 Analysis of arguments provided with the scores for controversial criteria: Tyl et al. (2002). (DOC) Text S4 Analysis of arguments provided with the scores for controversial criteria: Stump (2009). (DOC) Our study shows four main results: Our study shows four main results: – the 12 respondents considered the Qualichem criteria as appropriate for quality assessment of in vivo studies Text S5 Expert profile. (DOC) References 5. Hillier S, Grimmer-Somes K, Merlin T, Middleton P, Salisbury J, et al. (2011) FORM: An Australian method for formulating and grading recommendations in evidence-based clinical guidelines. BMC Med Res Methodol 11:23. g 6. Schulz KF, Altman DG, Moher D, CONSORT group (2010) CONSORT 2010 statement: updated guidelines for reporting parallel group randomized trials. PLOS Med 7(3): e1000251. 2. Jadad AR, Moore RA, Caroll D, Jenkinson C, Reynolds JM, et al. (1996) Assessing the quality of reports of randomized clinical trials: is blinding necessary? 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(2010) Flawed Experimental Design Reveals the Need for Guidelines Requiring Appropriate Positive Controls in Endocrine Disruption Research. Toxicol Sci 115(2): 612–613. 46. Maxim L, Van der Sluijs J (2010) Expert explanations of honeybee losses in areas of extensive agriculture in France: GauchoH compared with other supposed causal factors. References Environ Res Lett 5(1): 014006. 27. Knol AB, Slottje P, Van der Sluijs J, Lebret E (2010) The use of expert elicitation in environmental health impact assessment: a seven step procedure. Environ Health 9: 19. ( ) 47. Beronius A, Rude´n C, Ha˚kansson H, Hanberg A (2010) Risk to all or none? A comparative analysis of controversies in the health risk assessment of Bisphenol A. Reprod Toxicol 29: 132–146. 28. Tyl RW, Myer CB, Marr MC, Thomas BF, Keimowitz AR, et al. (2002) Three- Generation Reproductive Toxicity Study of Dietary Bisphenol A in CD Sprague-Dawley Rats. Toxicol Sci 68: 121–146. p p 48. 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https://openalex.org/W2883726018	https://iiste.org/Journals/index.php/EJBM/article/download/43258/44571	English	null	The Role of Human Resources Motivation in the Development of Hotel Enterprises in the Ferizaj Region (Republic of Kosovo)	European journal of economics and business studies	2,018	cc-by	4,042	The Role of Human Resources Motivation in the Development of Hotel Enterprises in the Ferizaj Region (Republic of Kosovo) Dr. Sc. Naim ISMAJLI AAB College, Rr. ”Elez Berisha”, nr. 56, Zona Industriale Prishtinë - Fushë Kosovë 10000 Prishtinë, Republika e Kosovës MA. Arberesha BEHLULI AAB College, Rr. ”Elez Berisha”, nr. 56, Zona Industriale Prishtinë - Fushë Kosovë 10000 Prishtinë, Republika e Kosovës PhdC. Roberta BAJRAMI AAB College, Rr. ”Elez Berisha”, nr. 56, Zona Industriale Prishtinë - Fushë Kosovë 10000 Prishtinë, Republika e Kosovës European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 www.iiste.org Abstract The main purpose of this research is to describe and sift the overall level of motivation of the employees of hotel enterprises in the region of Ferizaj, in the Republic of Kosova regarding the factors affecting it as well as measuring the relationship between motivation and quality of service.The first objective of this study is to measure and describe work relationship among the employees of hotel enterprises in the Ferizaj Region, while the second objective is to measure and describe payroll and bonuses among the employees of hotel enterprises in the Ferizaj Region.Part of the study were 467 workers of the hotel enterprises of which 36% ( n=171) were women, and 63.4% (n=296) men. Analysis of empirical data is performed using the Statistical Package for the Social Sciences (SPSS) system.From the findings of this study it has been founded that the employees of hotel enterprises in Ferizaj region are above average motivated in their work. Salary is the most important factor that has an impact on motivation, followed by the work-related relationships. However, comparing level of motivation among different places, we can see that the workers in Ferizaj and Shterpce were more motivated because more motivational techniques were used by their managers, whereas employees in Shtime and Kaqanik were less motivated.The variance analysis identified significant statistical differences in terms of motivation at work.Regarding the quality of service from the statistical data, we see that the quality of service is in right proportion to motivation. Clients were satisfied with the hotel services in general but the citizens of Ferizaj and Shterpca were more satisfied whereas citizens of Shtime and Kaqanik were less satisfied. Finally, it can be asserted that the findings of this study can help the structures of hotel establishments to use efficient techniques to motivate their employees,which could lead to the increase of the quality of service provided. p y q y Keywords: Hotel enterprises in the region of Ferizaj, motivation, quality of service Literature review Every business is intended maximizing profit. For the company to achieve this, it must, among other things, have in place and develop the human resource department ( Hashani M et al. 2015). HRM deals with the human dimension in managing an organization, so organizations through man manage human resources, selecting them, developing those skills, motivating them to the highest levels of achievement and success, and ensuring the maintenance of their level of engagement in all important activities ( Ismajli N et al. 2015). Motivation is defined as the will to exert high levels of effort to achieve organizational goals, conditioned by the ability of these efforts to satisfy some individual needs, The motivating factors are: wages, career advancement, job security, work contracts, working conditions, organizational culture, level of responsibility at work (degrees), bonuses etj.( Ismajli N et al. 2015) A motivated employee positively influences the achievements and productivity of the job. However, this result is often influenced by working conditions, organization strategy, management practices or rewarding forms. Interventions, bonuses or other incentives increase our internal motives to better perform the work that is being charged (Plantinga M. 2006). Also, great importance in the literature is devoted to the necessity of staff training, to be in trend and adequate flow of technological, logistical and business development ( Hashani M et al. 2015). ) Kosovans are recognized as very optimistic and also as strong workers, but as we all need motivation to do a better job and we can say we lack motivation ( Ismajli N et al. 2013). Benefits and revenues are complex and multifaceted factors. Salary does not only serve to help people achieve their basic needs but is also important in meeting the needs of a higher level. From the results it is seen that employees are motivated, when their pay is in line with workload, have health benefits, rewards and professional growth, security for retirement, work contract, and workplace sustainability. The greater the financial reward, the less worrying employees about their financial status, increasing their impression of the value they have in the organization, complying with community pay standards, the productivity is most likely to be the result (Igalens J et al 1999). NH1: Employment relationships affect worker motivation NH2: Wages and bonuses affect employee motivation NH1: Employment relationships affect worker motivation NH2: Wages and bonuses affect employee motivation Methodology i d Primary and secondary data are used in this research. When it comes to primary data, it is about those data that we have received from the survey of workers of hotel enterprises, while secondary data included relevant literature that corresponded to research from scientific works and from various contemporary literature. p p y The population of this study was 1551 hotel enterprises (based on data from the Ministry of Trade and Industry), while the sample was selected as 30% of the total, ie 467 surveyed employees. This paper will contain descriptive research that will describe the characteristics of different phenomena and exploratory research which sets the research relationships by showing the cause between the variables. Questionnaires were created through Google Forms, distributed across e-mails and across web pa data processing was done through the Social Science Statistical Package (SPSS). p g g g ( ) By means of econometric models, the validity of independent variables (working relationships, wages and bonuses) is verified on the dependent variables (motivation of employees). Literature review Samariha suggests that the degree of motivation at work is linked to the employees' ability to interact with others, the function of personal characteristics and the characteristics of the group to which he or she belongs. (Samariha et al. 2012). Such social relationships form an important part of the "social climate" within the workplace and provide an environment within which employees can experience meaning and identity (Aydin, T, O. 2012). g y ( y ) Employees need to be assessed and admitted unconditionally. The higher the level of cooperation and support, the more motivated they will be. Studies have shown that employees who feel valued by superiors, attach particular importance to the relationship and cooperation with them, tend to meet their expectations and accept any comment or suggestion given to them. Managers who allow employees to participate in decisions that impact the work will stimulate higher levels of satisfaction, motivation, dedication and performance, specifically in higher education. Meanwhile, relations with colleagues have also been considered extremely important. They bring a desire to work and increase results (Brady P.R, 2008). y After collecting data from 467 employees of hotel enterprises in Ferizaj Region we have the following results.We initially start with some demographic data to see who the target group was. If we refer to gender in this research, then we see that 36.6% (n = 171) were females and 63.4% (n = 296) were male from the total of 467 respondents. The age of 18 to 25 was 31% (n = 145), the age of 26 to 33 was 52.2% (n = 244), the age 34 to 41 was 15.4% (n = 72), and at the age of 50 a percentage of 1.3 (n = 6). Introduction il l Until recently, enterprises in Kosovo to increase their profits were devoted to capital investments, which was the strategy of almost all businesses, without paying attention to investment in human capital, which is key to business development. According to research, the world's enterprises are particularly concerned about the factor of man from where we can imply that they use different strategies for motivating at work such as: various staff training. rewards, better working relationships, enjoyable pay, better working conditions, and other factors that can be seen as motivational incentives. This trend of human resources management through various motivational factors has begun to be applied evenly in our country. This is best demonstrated by this research by which we understand that the leading structures of enterprises attach great importance to work relations, such as wages and rewards, as the most influential factors in motivating workers So the main purpose of this study is to describe and analyze the overall level of motivation of employees of hotel enterprises in Ferizaj Region, in the Republic of Kosovo in relation to factors affecting it and to measure the relationship between motivation and quality of service. The situation resulted such that the motivation was in proportion to the quality of the service, so with the growth of the one increasing the other. The importance of this study is great because, apart from smoothing the motivation of employees, we also analyze its impact on quality. Given the natural beauties of the Republic of Kosovo, tourism is expected to develop even more, as the Municipality of Shtërpcë has a suitable ski area. Therefore, the quality of service is of great interest. g The study was built based on a hypothesis and two under the hypothesis: The study was built based on a hypothesis and two under the hypothesis: H: Motivation of employees affects the quality of hotel services H: Motivation of employees affects the quality of hotel services 36 European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 Data and analysis Relationships at work N Min Max Average Standard deviation Variance The manager supports the professional development of employees 467 1.00 5.00 3.4325 .88214 .778 The manager treats employees fairly and impartially 467 1.00 5.00 3.4411 .88760 .788 The manager constantly gives me information about the work he does 467 1.00 5.00 3.3362 .94961 .902 My manager is efficient in delegating and distributing tasks at work 467 1.00 5.00 3.3619 1.11548 1.244 My manager is available when I have questions and I need help 467 1.00 5.00 3.4069 1.19252 1.422 My manager hears and takes into account my suicide 467 1.00 5.00 3.4069 1.26923 1.611 I'm pleased with the management skills of my manager 467 1.00 5.00 3.4004 1.21870 1.485 I get praised and rewarded when my performance is good 467 1.00 5.00 3.4283 1.21538 1.477 I get the deserved respect from my colleagues 467 1.00 5.00 3.3876 1.24374 1.547 Thi th t l t d b f th i f i l d l t t t d ll This means that employees are supported by managers for their professional development, treated equally, receive information and guidance on the job they are performing, rewarded for their performance and what is most important is mutual respect among employees because such a convenient environment makes them more comfortable with their workplace, making them more motivated. p g Another important indicator that approves the aforementioned script is the degree of compliance for this set of questions, resulting in 20.2% disagreeing that work relationships are level, 26.1% volatile for the assertions of this group, so they clicked the option "I'm not sure", and finally with a percentage of 53.48 are declared to be satisfied with interpersonal reports and reports with executives, respectively with their managers. This is an enviable and over average percentage that makes you understand that the workers of the Ferizaj Region have a positive tendency to be motivated, and on that basis give good performance. Data and analysis y After collecting data from 467 employees of hotel enterprises in Ferizaj Region we have the following results.We initially start with some demographic data to see who the target group was. If we refer to gender in this research, then we see that 36.6% (n = 171) were females and 63.4% (n = 296) were male from the total of 467 respondents. The age of 18 to 25 was 31% (n = 145), the age of 26 to 33 was 52.2% (n = 244), the age 34 to 41 was 15.4% (n = 72), and at the age of 50 a percentage of 1.3 (n = 6). 37 European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 European Journal of Business and Management ste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 As for the factors through which we measured the motivation at work, we have the following result As for the factors through which we measured the motivation at work, we have the following results: Working relationships are an important category that plays a role in employee motivation and as measured through the Likert scale where the minimum response was 1 to 5. After calculating through the SPSS program we see that the average of this category was x = 3.9 and the standard deviation ds = 1.10. Table 1. Relationships at work Table 1. g g p pp ther important indicator is the degree of compliance where we see that 20.97% are not satisfied with the bonuses given to the company, 19.32% were unsure if the claims submitted corresponded to the situation Data and analysis Table 2 Wage and Benefits Table.2 Wage and Benefits N Min Max Average Standard deviation Variance I am satisfied with my current salary 467 1.00 5.00 3.5161 1.15032 1.323 I'm delighted with the benefit package 467 1.00 5.00 3.9079 1.39053 1.934 I am delighted with the recent salary increase 467 1.00 5.00 3.8758 1.38105 1.907 I am delighted with the payment structure of the company 467 1.00 5.00 3.8223 1.39608 1.949 I am satisfied with the amounts my company pays for my benefits 467 1.00 5.00 3.8158 1.44288 2.082 I am satisfied with the wage growth interval in the company 467 1.00 5.00 3.7923 1.54396 2.384 I reward for the quality of my efforts 467 1.00 5.00 3.5268 1.28746 1.658 I experience personal financial growth in this company 467 1.00 5.00 3.5846 1.23557 1.527 Performance evaluation impacts on salary increases 467 1.00 5.00 3.5803 1.24278 1.545 Here is the opportunity for advancing the career 467 1.00 5.00 3.5803 1.27853 1.635 So surveyed workers are satisfied with pay, benefits package, are satisfied with the payment structure, are satisfied with the wage growth interval, and have career promotion opportunities. Another important indicator is the degree of compliance where we see that 20.97% are not satisfied with the pay and bonuses given to the company, 19.32% were unsure if the claims submitted corresponded to the situation Another important indicator is the degree of compliance where we see that 20.97% are not satisfied with the pay and bonuses given to the company, 19.32% were unsure if the claims submitted corresponded to the situation 38 European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 in the company, and finally with the highest percentage of compliance by all groups of questions was here with European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 in the company, and finally with the highest percentage of compliance by all groups of questions was here with 59.05%. T bl 3 Li R i in the company, and finally with the highest percentage of compliance by all groups of questions was here with 59.05%. in the company, and finally with the highest percentage of compliance by all groups of questions was here with 59.05%. Table.3 Linear Regression Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Data and analysis Error Beta 1 (Constant) .336 .084 3.978 .008 WAGE .487 .029 .558 16.611 .002 REWARD .275 .059 .294 4.632 .050 RELATIONSHIP WITH WORKERS .094 .052 .100 1.830 .068 D d V i bl MOTIVATION a. Dependent Variable: MOTIVATION Linear regression was used for the analysis of results, where correlation is measured from data and anova for finding the influence of independent variables and dependent variables. The dependent variance in this paper is the motivation of human resources, while independent variables are payouts, rewards, and magnitudes at work. e y X X X X n n + + + + + + = b b b b b ... 3 3 2 2 1 1 0 y- Motivation wage – Rewards - magnitudes at work - magnitudes at work e mw reward wage m y X X X + + + + = ) ( 094 . ) ( 275 . ) ( 487 . 336 . ) ( 3 2 1 From the formula of the model we see that despite the independent variables (influencing factors), the workers are motivated .336, while with the growth of one, a motivation payment unit rises to .487. with the growth of 1 reward unit (activity) of bonuses increased motivation for .275 and from all these less influential variables have emerged from work relations. The values given in the model are expressed on the basis of activities and have positive links, so independent variables have a direct impact on the independent variables. From the table are taken non- standardized coefficients since the unit of measurement for all variables is the same References References 1. Brady P. R, (2008). Work Motivation Scale (ISBN 978-1-59357-469-7) Published by JIST Works, an imprint of JIST Publishing, pp 2 2. Brady P. R, (2008). Work Motivation Scale (ISBN 978-1-59357-469-7) Published by JIST Works, an imprint of JIST Publishing, pp 4. 2. Brady P. R, (2008). Work Motivation Scale (ISBN 978-1-59357-469-7) Published by JIST Works, an imprint of JIST Publishing, pp 4. p g pp 3. Bull, I. H. F. (2005). The Relationship between job satisfaction and organizational commitment amongst high school teachers in disadvantaged areas in the Western Cape, University of the West Cape, pp 33. 3. Bull, I. H. F. (2005). The Relationship between job satisfaction and organizational commitment amongst high school teachers in disadvantaged areas in the Western Cape, University of the West Cape, pp 33. j ( ) 5. Hashani M, Asllani A & Bajrami R. (2015). Identifikimi i nevojave për punonjës në Ndërmarrjet e Vogla dhe të Mesme në Komunën e Ferizajt. KDU 338.92:331(496.51-2). 5. Hashani M, Asllani A & Bajrami R. (2015). Identifikimi i nevojave për punonjës në Ndërmarrjet e Vogla dhe të Mesme në Komunën e Ferizajt. KDU 338.92:331(496.51-2). j ( ) 6. Igalens J & Roussel P (1999). A study of the relationships between compensation package, work motivation and job satisfaction. Journal of organizational behavior, 20. 1003 – 1025. j ( ) 6. Igalens J & Roussel P (1999). A study of the relationships between compensation package, work motivation and job satisfaction. Journal of organizational behavior, 20. 1003 – 1025. j g 7. Ismajli N, Krasniqi I & Qosja E. (2015). Faktorët Motivues që ndikojnë në punën e nëpunësit civil në Administraten Publike Lokale në Kosovë. Journal of US-China Public Administration,Vol. 12, No. 1, pp.16. j g 7. Ismajli N, Krasniqi I & Qosja E. (2015). Faktorët Motivues që ndikojnë në punën e nëpunësit civil në Administraten Publike Lokale në Kosovë. Journal of US-China Public Administration,Vol. 12, No. 1, pp.16. 8. Ismajli N, Krasniqi I, Qosja E. (2015). Zhvillimi i Ismajli N, Zekiri J, Qosja E & Krasniqi I. (2015). Rë dë i F kt ë M ti bi f t j it K t K J l f P bli 7. Ismajli N, Krasniqi I & Qosja E. (2015). Faktorët Motivues që ndikojnë në punën e nëpunësit civil në Administraten Publike Lokale në Kosovë. Journal of US-China Public Administration,Vol. 12, No. 1, pp.16. Administraten Publike Lokale në Kosovë. Conclutions and Recommendations For other researchers who want to deal with motivational factors, it is recommended to address other factors other researchers who want to deal with motivational factors, it is recommended to address other factors For other researchers who want to deal with motivational factors, it is recommended to address oth 39 European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 influencing motivation at work because it was impossible to examine all who are mentioned in the literature, which are: institution policies, competence, success, achievement, professional growth, environment and working conditions, training, career promotion etc. European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.10, No.20, 2018 influencing motivation at work because it was impossible to examine all who are mentioned in the literature, which are: institution policies, competence, success, achievement, professional growth, environment and working conditions, training, career promotion etc. influencing motivation at work because it was impossible to examine all who are mentioned in the literature, which are: institution policies, competence, success, achievement, professional growth, environment and working conditions, training, career promotion etc. p y p 12 Samariha et al (2012), Effective Factors on Creating Job Motivation among Faculty Members of W Paper Industry Groups J. Basic. Appl. Sci. Res., 2(2)1672-1677.© 2012, TextRoad Publication Conclutions and Recommendations Quantitative methods were used to give answers to questions and objectives. Part of the study were 4 hotel workers, 36% were women and 63.4% males. The first set of questions that maths at work consists of 9 statements, found that relationships were on a satisfactory level, with an average of 3.9 from a minimum of 1 to a maximum of 5. The workers were in agreement with a sufficiently high degree of compliance to support their professional development by managers, treated fairly and impartially, assessed and rewarded when performance is high, etc. In addition, it is worth pointing out that work relationships have positively influenced the motivation of employees, thus increasing the quality of service The second group had 10 statements that motivated the job. From previous statistics we see that the group's average was 3.7 and standard deviation 1.33. According to this we came to the conclusion that employees are satisfied with their salary, benefits bonuses, and many other points mentioned in the questionnaires. So the level of compliance was quite high at 59.05%, which means that the salary positively affects the motivation of the workers, thus affecting the increase of the quality of the service. So both of them are in fair proportion, with the growth of one increasing and the other and vice versa Recommendations are attributed to enterprise hotel managers because these factors that influence motivation are internal factors, and as such should be applied to employees. Although the statistical data resulted in a high level of motivation, the recommendations help to improve the situation even further. Because a motivated worker is worth treasure. Since the subject matter of the study was labor relations as well as wages and bonuses, it is recommended that work relationships improve even more at those points where they have been poorer, also, countries such as Kaqanik and Shtime need much attention to work relations. As for the salary and rewards, it is recommended that wages are even higher, because this factor was the one with the greatest weight affecting the motivation of workers. References Journal of US-China Public Administration,Vol. 12, No. 1, pp.16. 8. Ismajli N, Krasniqi I, Qosja E. (2015). Zhvillimi i Ismajli N, Zekiri J, Qosja E & Krasniqi I. (2015). Rëndësia e Faktorëve Motivues mbi performancat e punonjesit ne Komunat e Kosoves. Journal of Public Administration and Governance, ISSN 2161-7104, Vol. 5, No. 1. 9. Ismajli N, Rexha D & Qerimi F. (2013). Statusi aktual dhe sfidat e zhvillimeve të burimeve njerëzore në Kosovë. Mediterranean Journal of Social Sciences, ISSN 2039-9340, Vol 4 No 10. 10 Ismajli N, Krasniqi I, Qosja E. 2015. Zhvillimi i karrierës dhe avancimi i i burimeve njerezore -sa ndikojnë në cilësitë e performancës se qeverisjes se administrates vendore. Issues in Business Management and Economics Vol.3 (2) 11 Plantinga M, (2006), Employee Motivation and Employee Performance in Child Care. The effects of the introduction of market forces on employees in the Dutch child-care sector. ISBN 90-367-2695-6, pp 3 12 Samariha et al (2012), Effective Factors on Creating Job Motivation among Faculty Members of Wood and Paper Industry Groups J. Basic. Appl. Sci. Res., 2(2)1672-1677.© 2012, TextRoad Publication 40
https://openalex.org/W4306780387	https://www.frontiersin.org/articles/10.3389/fpsyg.2022.904019/pdf	English	null	Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan	Frontiers in psychology	2,022	cc-by	17,877	Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan OPEN ACCESS EDITED BY Abiodun Ogunyemi, Tallinn University, Estonia REVIEWED BY Sukyung Yoon, University of Wyoming, United States Vassilis George Kaburlasos, International Hellenic University, Greece CORRESPONDENCE Marlena R. Fraune mfraune@nmsu.edu Takanori Komatsu tkomat@meiji.ac.jp Katherine M. Tsui kate.tsui@tri.global †PRESENT ADDRESS Danielle K. Langlois, Department of Computer Science, Masaryk University, Brno, Czechia; Faculty of Informatics, Masaryk University, Brno, Czechia ‡These authors share senior authorship SPECIALTY SECTION This article was submitted to Human-Media Interaction, a section of the journal Frontiers in Psychology RECEIVED 25 March 2022 ACCEPTED 16 September 2022 PUBLISHED 19 October 2022 CITATION Fraune MR, Komatsu T, Preusse HR, Langlois DK, Au RHY, Ling K, Suda S, Nakamura K and Tsui KM (2022) Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan. Front. Psychol. 13:904019. doi: 10.3389/fpsyg.2022.904019 COPYRIGHT © 2022 Fraune, Komatsu, Preusse, Langlois, Au, Ling, Suda, Nakamura and Tsui. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in OPEN ACCESS EDITED BY Abiodun Ogunyemi, Tallinn University, Estonia REVIEWED BY Sukyung Yoon, University of Wyoming, United States Vassilis George Kaburlasos, International Hellenic University, Greece CORRESPONDENCE Marlena R. Fraune mfraune@nmsu.edu Takanori Komatsu tkomat@meiji.ac.jp Katherine M. Tsui kate.tsui@tri.global †PRESENT ADDRESS Danielle K. Langlois, Department of Computer Science, Masaryk University, Brno, Czechia; Faculty of Informatics, Masaryk OPEN ACCESS EDITED BY Abiodun Ogunyemi, Tallinn University, Estonia REVIEWED BY Sukyung Yoon, University of Wyoming, United States Vassilis George Kaburlasos, International Hellenic University, Greece Marlena R. Fraune1‡, Takanori Komatsu2‡, Harrison R. Preusse1, Danielle K. Langlois1†, Rachel H. Y. Au1, Katrina Ling1, Shogo Suda2, Kiko Nakamura2 and Katherine M. Tsui3‡ Marlena R. Fraune1‡, Takanori Komatsu2‡, Harrison R. Preusse1, Danielle K. Langlois1†, Rachel H. Y. Au1, Katrina Ling1, Shogo Suda2, Kiko Nakamura2 and Katherine M. Tsui3‡ 1Department of Psychology, New Mexico State University, Las Cruces, NM, United States, 2Department of Frontier Media Science, Meiji University, Tokyo, Japan, 3Toyota Research Institute, Cambridge, MA, United States †PRESENT ADDRESS Danielle K. TYPE Original Research PUBLISHED 19 October 2022 DOI 10.3389/fpsyg.2022.904019 TYPE Original Research PUBLISHED 19 October 2022 DOI 10.3389/fpsyg.2022.904019 TYPE Original Research PUBLISHED 19 October 2022 DOI 10.3389/fpsyg.2022.904019 Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan Langlois, Department of Computer Science, Masaryk University, Brno, Czechia; Faculty of Informatics, Masaryk University, Brno, Czechia Social technology can improve the quality of older adults’ social lives and mitigate negative mental and physical health outcomes associated with loneliness, but it should be designed collaboratively with this population. In this paper, we used participatory design (PD) methods to investigate how robots might be used as social facilitators for middle-aged and older adults (age 50+) in both the US and Japan. We conducted PD workshops in the US and Japan because both countries are concerned about the social isolation of these older adults due to their rapidly aging populations. We developed a novel approach to participatory design of future technologies that spends 2/3 of the PD session asking participants about their own life experiences as a foundation. This grounds the conversation in reality, creates rapport among the participants, and engages them in creative critical thinking. Then, we build upon this foundation, pose an abstract topic, and ask participants to brainstorm on the topic based on their previous discussion. In both countries, participants were eager to actively discuss design ideas for socially facilitative robots and imagine how they might improve their social lives. US participants suggested design ideas for telepresence robots, social distancing robots, and social skills artiﬁcial intelligence programs, while Japanese participants suggested ideas for pet robots, robots for sharing experiences, and easy-to-operate instructor robots. Comparing these two countries, we found that US participants saw robots as tools to help facilitate their social connections, while Japanese participants envisioned robots to function as surrogate companions for their parents and distract them from loneliness when they were unavailable. With this paper, we contribute to the literature in two main ways, presenting: (1) A novel approach to participatory design of future technologies that grounds participants in their everyday experience, and (2) Results of the study indicating how middle-aged and older adults from the US and Japan wanted technologies to improve their social lives. Although we conducted the workshops during the COVID-19 pandemic, many ﬁndings generalized to other situations related to social isolation, such as older adults living alone. social robots, human-robot interaction, experience-grounded participatory design, older adults, US, Japan, social isolation, cross-cultural study 1. Introduction communication (Pinquart and Sorensen, 2001; Chen et al., 2018 for reviews). Little research examined how robots enhance human-human social interaction. Our study adds to this literature. Notably, it took place during a widely-enforced period of social isolation due to the COVID-19 pandemic. These unique circumstances increased the relevance of social isolation’s negative eﬀects on mental health, and the importance of developing solutions to mitigate this issue. However, social isolation was recognized as a “serious public health concern” (Cacioppo and Cacioppo, 2014) prior to the pandemic, and will likely remain so even after the social isolation caused by the pandemic is no longer in eﬀect. Loneliness and social isolation lead to negative mental and physical health, particularly for independent-living older adults (Perissinotto et al., 2012; Cudjoe et al., 2020). The quality of intimate relationships is a strong predictor of the wellbeing of older adults (Lehr et al., 2009). Without quality close relationships, older adults feel lonelier, resulting in poorer physical and mental health (e.g., Fees et al., 1999). Importantly, older adults care more about the quality rather than the quantity of their social networks (Pinquart and Sorensen, 2001). However, older adults may face social isolation for various reasons, including domestic isolation, limited social contact, and social disengagement (Philip et al., 2020). 2. Related work Although many existing online communication and robotic technologies can improve the social lives of older adults (Wada et al., 2003; Banks et al., 2008; Chen et al., 2018; Pu et al., 2019), some older adults hesitate to adopt new technologies (Barnard et al., 2013). This is true even when they are unable to meet others in person because of their health, diﬃculty traveling, being far from family, or other reasons. Social technology intended to improve the quality of older adults’ social lives and mitigate negative mental and physical health outcomes associated with loneliness should be designed collaboratively with this population to both ensure that they will use it and that it will actually improve the quality of their social lives (Gustafson et al., 2016). Frontiers in Psychology Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan These authors share senior authorship SPECIALTY SECTION This article was submitted to Human-Media Interaction, a section of the journal Frontiers in Psychology RECEIVED 25 March 2022 ACCEPTED 16 September 2022 PUBLISHED 19 October 2022 CITATION Fraune MR, Komatsu T, Preusse HR, Langlois DK, Au RHY, Ling K, Suda S, Nakamura K and Tsui KM (2022) Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan. Front. Psychol. 13:904019. doi: 10.3389/fpsyg.2022.904019 p SPECIALTY SECTION This article was submitted to Human-Media Interaction, a section of the journal Frontiers in Psychology RECEIVED 25 March 2022 ACCEPTED 16 September 2022 PUBLISHED 19 October 2022 CITATION Fraune MR, Komatsu T, Preusse HR, Langlois DK, Au RHY, Ling K, Suda S, Nakamura K and Tsui KM (2022) Socially facilitative robots for older adults to alleviate social isolation: A participatory design workshop approach in the US and Japan. Front. Psychol. 13:904019. doi: 10.3389/fpsyg.2022.904019 COPYRIGHT © 2022 Fraune, Komatsu, Preusse, Langlois, Au, Ling, Suda, Nakamura and Tsui. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 01 frontiersin.org Frontiers in Psychology Fraune et al. Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 Although we conducted the workshops during the COVID-19 pandemic, many ﬁndings generalized to other situations related to social isolation, such as older adults living alone. frontiersin.org 2.2. Social isolation and COVID-19 While the eﬀects of social isolation and loneliness on older adult populations has been a public health concern for some time, societal responses to the COVID-19 pandemic brought these issues to the forefront (Gerst-Emerson and Jayawardhana, 2015; Armitage and Nellums, 2020). COVID-19, formally known as SARS-CoV-2, is a virus which originated in Wuhan, China in December 2019, before spreading globally. By July 2021, nearly 200 million people globally had been infected by COVID-19 (Hopkins, 2020). By March 2022, over 450 million people had been infected (Worldometer, 2022). Socially facilitative robots are also widely used for companionship and communication to improve the social life of older adults. In eldercare facilities, the therapeutic seal robot PARO became a common topic of conversation, connecting both older residents and their caregivers (Wada et al., 2003). Interacting with PARO also increased the density of older adults’ social networks (Wada and Shibata, 2007) and reduced depressive symptoms in older adults (Chen et al., 2018). In response to the rapid spread of the virus, governments around the world ordered their citizens to observe various degrees of physical distancing (i.e., keeping physical distance between an individual and people who do not live in the same household), from restrictions on international travel to mandatory stay-at-home orders (Giallonardo et al., 2020; González-Rodríguez and Labad, 2020; Moreland et al., 2020). Restrictions on socializing in person increased the prevalence of mental health conditions like anxiety and depression in global populations during the COVID-19 pandemic (Lytridis et al., 2020; Saladino et al., 2020; Sikali, 2020). Older adults had higher mortality from COVID-19, leading to recommendations that family members avoid contact with the older adults to “keep them safe.” Further, traditional social outlets such as places of worship were closed, which may have put older adult populations at disproportionate risk of negative mental health outcomes (Armitage and Nellums, 2020; Tyrrell and Williams, 2020). The robotic dog, AIBO, acted a social companion of residents in long-term aged care facilities and reduced loneliness of older adults (Banks et al., 2008). While online communication and social robots may help connect older adults with friends and loved ones, some older adults may hesitate to rely upon novel technologies. Many older adults expressed diﬃculty using many modern communicative technologies (Ling et al., 2022). 2.1. Social isolation and health Social isolation, deﬁned as low quantity and quality of social and emotional connection (Shankar et al., 2011), is associated with increased feelings of loneliness along with numerous negative physical and mental health outcomes (Weiss, 1973; Van Baarsen et al., 2001; Wang et al., 2003; Tomaka et al., 2006; Shankar et al., 2011). Loneliness is the negative aﬀective state experienced when an individual feels a discrepancy between the social relationships they wish to have and those they perceive they have (Heinrich and Gullone, 2006). Loneliness correlates with negative mental health, including increased social anxiety, higher risk of depression, suicidal ideation, decreased cognitive function, and poor life satisfaction (Heinrich and Gullone, 2006; Hawkley and Cacioppo, 2010). Loneliness also predicts negative physical health, like poor cardiovascular health (e.g., high blood pressure, high cholesterol), and risky health behaviors (e.g., smoking, physical inactivity) (Hawkley and Cacioppo, 2010; Shankar et al., 2011). In this paper, we present (1) a novel participatory design (PD) workshop in which we grounded participants in their actual experience and (2) results in which we (A) uncovered older adult’s challenges with their current social lives and technology use and (B) developed concepts for socially- facilitative robots to address their needs. Speciﬁcally, we conducted the PD workshops in US and Japan because both countries are concerned about the social isolation of these older adults due to the rapid aging of their populations (Magnus, 2012; Goldin, 2016; Götmark et al., 2018), and share a strong commitment to robotics and some fundamental psychological regularities; but they diﬀer in a number of other respects, like culture, traditions and social norms. Social isolation and loneliness are of particular concern among older adult populations. National data of the United States showed that 24% of adults over 65 years of age considered to be socially isolated, and 43% of adults over 60 report feeling lonely (Perissinotto et al., 2012; Cudjoe et al., 2020). The mental and physical eﬀects of social isolation and loneliness among older adults are especially pronounced. These factors correlate Previous work on social robots for older adults has often studied the beneﬁts of robots oﬀering companionship and frontiersin.org 02 Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 with more frequent doctor visits, more rapid onset of cognitive decline and Alzheimer’s disease, and risk of all-cause mortality among the older adults (Wilson et al., 2007; Gerst-Emerson and Jayawardhana, 2015; Boss et al., 2016; Beller and Wagner, 2018). 2.3. Social technology and socially facilitative robots Current and emerging technologies may help older adults maintain social connections while physically separate from others. Use of social technology, like e-mail, online video calls, and social media, correlated with better self-reported health, fewer chronic illnesses, and reduced depressive symptoms among older adults (Chopik, 2016; Lee et al., 2021), because these social technologies helped reduce loneliness (Chopik, 2016), especially during social isolation (Fraune et al., 2022). However, people view technology-mediated communication as more psychologically demanding and enjoy it less than in- person interaction (Williams, 2021). 2.2. Social isolation and COVID-19 Despite the beneﬁts of technologies on the quality of life of seniors aged 65 and older (Heinz et al., 2013), about half of this age group did not currently use the internet and believed that it did not put them at a disadvantage. Seventy-seven percent of seniors reported that they required assistance when learning to use new technology (Smith, 2014). Reﬂective of this preference for older technology, many nursing home residents preferred letters and phone calls over video chats during the COVID-19 pandemic (Fearn et al., 2021). Social technology intended for older adults should be designed collaboratively with this population, both to ensure that they are willing and able to use it, and that the technology can improve the quality of their social lives and subsequent mental and physical health. 2.1. Social isolation and health Novel robotic technology may provide a solution to challenges with technology in communication. Robots can have unique socially facilitative eﬀects on interactions between multiple users in many settings. In human-robot cooperation tasks, human-robot teams with a robot member revealing its vulnerabilities were more likely to laugh together, console teammates who made a mistake, and communicate well than teams with a robot members not revealing its vulnerabilities (Strohkorb Sebo et al., 2018). Similarly, a robot that made vulnerable statements about itself also led their human team members to converse more, distribute speaking time more equitably, and rate their group more positively overall (Traeger et al., 2020). 2.4. Cultural diferences between US and Japan As noted by Mason (2010), qualitative studies, including PD workshops, require a longer time and therefore tend to have lower sample sizes; correspondingly, the outcomes are qualitative in nature. Although replication is important in general (Amrhein et al., 2019), statistical testing is not always appropriate in the case of the small number of subjects (Gliner et al., 2002) and it is better to listen to a small number of actual users that the researchers can obtain and interview than to have no representation of these users at all. and product-like robots (Lee et al., 2015). Other studies have explored people’s acceptance of robots across cultures (Banks et al., 2008; Cacioppo and Cacioppo, 2014; Beller and Wagner, 2018), and on cultural impact on the credibility of robot speech in US and Arabic communities (Andrist et al., 2015). However, up to now, no studies have examined to conduct the participatory design workshops for exploring robot design concepts in diﬀerent two countries. In this study, we focused on two cultures: the US and Japan. These two countries are concerned about the social isolation of these older adults due to the rapid aging of their populations (Magnus, 2012; Goldin, 2016; Götmark et al., 2018), and share a strong commitment to robotics and some fundamental psychological regularities; however, they diﬀer in a number of other respects, most notably in their broad social- cultural values [i.e., collectivism vs. individualism (Harry Hui and Triandis, 1986; Triandis et al., 1988)], religious traditions (Buddhism and Shintoism in Japan, Judeo-Christian traditions in US), and public views of robots (Nitto et al., 2017). These diﬀerences may aﬀect people’s diﬀerent perceptions and diﬀerent expected roles of the robots in their daily environment. Previous work has shown that PD workshops were particularly valuable and empowering for older adults, which is especially important for this group because they are traditionally marginalized in technology development (Lee et al., 2017; Laura Ramírez Galleguillos and Co¸skun, 2020). Older adults are often stereotyped as less willing to use new technology, but these eﬀects are accounted for by their experience with technology (Ezer et al., 2009; Flandorfer, 2012). Because older adults have diﬃculty envisioning intangible concepts and future technologies, it is imperative to customize the PD method that engages older adults’ creative thinking (Lindsay et al., 2012). 2.4. Cultural diferences between US and Japan The work in this paper examines how robots can facilitate middle- aged and older adults’ social interactions with friend and family outside of the home. Robots still have not yet become ubiquitous in domestic settings, so it is unlikely that people, particularly older adults, have no or minimal experience with robots or robotics technologies. Direct discussing robots at the start of a PD session would force people to draw on second-hand sources, such as science-ﬁction books, movies, and documentaries, or purely speculating. To preempt this, in our PD approach, we dedicated 2 of the 3 rounds of discussion to people’s own life experiences, thereby both grounding the conversation in reality, creating rapport among the group, and engaging them in creative critical thinking about robots in a grounded manner. With that foundation, then we engage the group to discuss how a robot or robotic technology might be able to help address their previously discussed challenges. 2.4. Cultural diferences between US and Japan Cross-cultural studies in Human-Robot Interaction (HRI) have documented varying attitudes toward robots across the US, Japan, the Netherlands, China, Mexico, and Germany (Bartneck et al., 2005). They have explored what assumptions people across Japan, Korea, and the US make about humanoid and animal-type robots (Nomura et al., 2008) or about humanoid Frontiers in Psychology 03 frontiersin.org 10.3389/fpsyg.2022.904019 Fraune et al. neighborhood, including a city planner. In the summer and fall of 2014, Šabanovi´c et al. (2015) conducted a series of 2 PD workshops with ﬁve older adults with co-occurring major depression and chronic physical illness (3m, 2f; ages 58–71) in conjunction with interviews of 5 staﬀmembers of a large outpatient healthcare provider; the goal of the project was to understand how Socially Assistive Robots (SARs) could be designed for and used in the homes of older adults before they become institutionalized. In 2020, Georgiou et al. (2020) conducted a PD workshop with 10 stroke survivors (7m, 3f; M = 58 years, SD = 12.4) to explore how SARs could assist stroke survivors with self-managed rehabilitation. As noted by Mason (2010), qualitative studies, including PD workshops, require a longer time and therefore tend to have lower sample sizes; correspondingly, the outcomes are qualitative in nature. Although replication is important in general (Amrhein et al., 2019), statistical testing is not always appropriate in the case of the small number of subjects (Gliner et al., 2002) and it is better to listen to a small number of actual users that the researchers can obtain and interview than to have no representation of these users at all. neighborhood, including a city planner. In the summer and fall of 2014, Šabanovi´c et al. (2015) conducted a series of 2 PD workshops with ﬁve older adults with co-occurring major depression and chronic physical illness (3m, 2f; ages 58–71) in conjunction with interviews of 5 staﬀmembers of a large outpatient healthcare provider; the goal of the project was to understand how Socially Assistive Robots (SARs) could be designed for and used in the homes of older adults before they become institutionalized. In 2020, Georgiou et al. (2020) conducted a PD workshop with 10 stroke survivors (7m, 3f; M = 58 years, SD = 12.4) to explore how SARs could assist stroke survivors with self-managed rehabilitation. Frontiers in Psychology 2.5. Participatory design Participatory design (PD) methods involve users as active collaborators, alongside traditional researchers and designers, throughout the process of designing new technology and improving existing technology to ensure that their needs are met (Lee et al., 2017). Through PD, users ﬁrst self-identify their own needs and wants, then help co-design technological solutions to meet those needs. A PD approach beneﬁts both researchers and users through mutual learning. End users have the most direct understanding of how they approach technology and the environments in which they use it (Lee et al., 2017). Using PD techniques, designers can learn from users’ experiences to develop technology that takes into account these social use contexts. Meanwhile, users learn about the current capabilities and applications of state-of-the-art technology, and they gain insight into the design process itself. The PD approach has gained popularity among HRI researchers as a way to understand the perspectives of diverse users and stake holders in a variety of contexts. The Neighborhood Networks project was interested in how an urban community in Pittsburgh, PA, would think to use robotic and environmental sensing technologies in their own neighborhood (DiSalvo et al., 2008). In summer 2007, researchers engaged the local community in a series of PD activities over the course of several weeks including a scavenger hunt activity using sensing technologies, storyboarding their concepts, “open studio” prototyping, and “science fair” presentations; although 20 residents initially participated, ultimately, 12 participants presented to each other and another 25 members of their This work of course is not the ﬁrst occurrence of using PD approaches to investigate how robots can help older adults; for example, Šabanovi´c et al. (2015) researched how socially-assistive robots might ﬁt into older adults’ daily home lives by providing them directly with social interaction and Frontiers in Psychology 04 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 Morrell et al., 2000; Sum et al., 2008), and care technologies for activities of daily living (e.g., Itoh et al., 2021). We therefore have adjusted our recruitment requirement to older adults aged 50 or above. companionship. Our study adds to these ﬁndings by examining how robots can facilitate middle-aged and older adults’ social interaction with friends and family outside the home. 1 The New Mexico State University Institutional Review Board approved the US PD workshop on August 27, 2020; “Technology and Social Activities III (Focus Group), ID: 20312.” The Ethics Committee on Human Research of Meiji University approved the Japanese PD workshops (Application Number: 2019-39). 3.2. Procedure The PD workshops in the US took place on November 2, November 10, and December 4, 2020, and the workshops in Japan took place on August 29 and September 5, 2021. Due to restrictions on in-person interactions during the COVID-19 pandemic, all workshop sessions in both countries occurred via Zoom online video chat. All PD sessions were video recorded. The researchers mailed participants study supplies (post-it notes, markers) prior to the workshop. Each session lasted approximately 75 min. 3.1. Design teams We recruited in total seven participants to join the PD workshops in US, and in 12 participants in Japan; due to the global pandemic, the PD sessions were virtual (Feil-Seifer et al., 2020). In each workshop, there were two facilitators and two to three participants. Facilitators were researchers on the current study with a background in technology research and design, while participants had no background in research and design. As part of the prior interview, participants viewed a video of current commercial robots to ensure all participants in the PD had a common understanding of the capability of current robotic technology, as opposed to drawing from movies (Sundar et al., 2016). During each session, one facilitator led the discussion and another mainly took notes. The facilitator leading the workshop posed all questions and brainstorming prompts to the participants and moderated the discussion. The note-taker paraphrased participant comments and themes of the discussion on a shared screen throughout the workshop. 2.5. Participatory design The inclusion criteria for these PD workshops was that a person was: (1) 50+ years of age, (2) able to use video conferencing software; also participants were either (3a) a resident of the US and spoke English, or (3b) a resident of Japan and spoke Japanese. The average age of the US participants was 59.29 years (SD = 5.85) and 55.42 years in Japan (SD = 4.17); demographic information of all participants is displayed in Table 1. Our sample includes current older adults (age 65+) and also people who are contemplating their upcoming retirement (middle-aged people 50+). 3. Materials and methods This method was developed for PD groups in the US and then modiﬁed with respect to culture for the PD groups in Japan1. We conducted PD to (A) identify middle-aged and older adults’ challenges with current technology-mediated social interactions, and then (B) brainstorm socially-facilitative robot concepts to address their stated needs and wants. To recruit participants in the US, we posted an invitation to the study to Reddit.com and the researchers’ personal social media page; the invitation contained basic descriptions of the study and eligibility to participate. We also recruited participants from prior related studies (Fraune et al., 2022; Ling et al., 2022) who had indicate that they wanted to be recontacted for future studies. We then used snowball sampling to increase to sample size. Participants in Japan were recruited through a human resource dispatching company. The compensation was $70 USD and ¥7,000 JPY per participant in the US and Japan, respectively. 3.1.1. Participant inclusion criteria and recruitment Our work started during the global pandemic, which limited our contact with older adults in our community due to the obvious health risks. The term “older adult" is typically used to describe people age 65 years or older, which is the ideal target population of our research. Some previous work using participatory design in the HRI literature has also used older adults aged below 60 (e.g., Šabanovi´c et al., 2015) and similarly, there is evidence of wider age bands regarding research on smartphones (e.g., Gao et al., 2015), Internet use (e.g., Each workshop session consisted of three rounds, and each focused on a diﬀerent main theme. All rounds began with a 5-min brainstorming phase, then used a ‘round-robin’ discussion format, with the facilitator allowing each participant the opportunity to share an initial idea one at a time before opening the ﬂoor to a more freeform discussion. This ensured that each participant had the chance to contribute their ideas during the early stages of each round, so that the following discussion was informed by the opinions of all present. Through all rounds, the note-taker paraphrased participant comments and main ideas on Google Slides, using it as a shared ‘digital whiteboard’ to provide a common reference point for continued discussion. Round 1 consisted of an initial discussion of the types of technologies participants currently use to communicate with Frontiers in Psychology 05 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 TABLE 1 Demographic information of the participants (P#country). e og ap c o a o o e pa c pa s ( country) US Japan Participant Age Gender Workshop session Participant Age Gender Workshop session P1US 58 Man 3 P1Japan 50 Woman 1 P2US 57 Woman 1 P2Japan 56 Woman 1 P3US 51 Woman 2 P3Japan 55 Man 1 P4US 62 Man 1 P4Japan 53 Man 2 P5US 68 Woman 1 P5Japan 50 Woman 2 P6US 64 Man 2 P6Japan 58 Woman 2 P7US 54 Woman 3 P7Japan 57 Woman 3 P8Japan 63 Man 3 P9Japan 50 Man 3 P10Japan 57 Woman 4 P11Japan 54 Man 4 P12Japan 62 Man 4 Japan The red, green, and blue shading in the ﬁgures call out the top 3 concepts from each country, and arrows show how the concept evolved. The dark black arrows indicate concepts that participants created and then carried forward their idea for development. 3.1.1. Participant inclusion criteria and recruitment The light gray arrows indicate that another participant either further developed the concept or had a similar concept. others. The facilitator encouraged participants to consider both what they liked about social experiences facilitated by technology, and what aspects of their technology-mediated interaction were missing or altered when compared to in-person interactions. Round 2 centered on a review of the social challenges participants currently faced, such as keeping up with old friends, making new connections, or socializing at large gatherings. Because the workshops occurred during the COVID- 19 pandemic, the facilitator welcomed participants to share both new challenges speciﬁc to the unique social circumstances of the time and general challenges that existed before social distancing norms. 3.3. Analysis We transcribed video-recordings of each workshop session. Researchers then analyzed and coded these transcripts along common themes. Themes were derived from open and axial coding (Glaser et al., 1968); the coding scheme is available in the Supplementary materials. The same coding rubric was utilized in both the US and Japanese PD transcripts. Interrater agreement ranged from moderate to strong in US (IRRs > 0.67) and strong in Japan (IRRs > 0.75) (Miles and Huberman, 1994). Round 3 involved a robot design session. The facilitator instructed participants to brainstorm ideas for robots to help solve one of the social challenges discussed in Round 2. The facilitator further encouraged participants to focus on ideas for a robot they would personally want to own and use and would be technically feasible within the next 3 years. After the initial brainstorm and discussion, the facilitator instructed participants to pick one of the ideas discussed (either their own or another participant’s) to improve upon, or add to, in a subsequent 3-min brainstorm session. This gave participants the opportunity to participate in iterative design. Another round-robin discussion followed this second brainstorming session. When appropriate, the facilitator re-focused the discussion or posed high-level questions, such as “What problem is the robot solving?” and “What might be some challenges of that idea?” Finally, the facilitator asked participants to create a list of their ﬁve favorite robot features discussed in this round. Frontiers in Psychology 4.1. US ﬁndings There’s nine [virtual] screens there and one of them is on a phone and ... the girlfriend’s holding the phone in the car while the guy’s driving and ... just the whole kind of big thing. (P1US) 4. Results We identiﬁed some major themes to discuss. We break them into topics that parallel the discussion rounds: (1) Beneﬁts and drawbacks of existing communicative technologies, (2) Challenges maintaining existing and developing new social relationships, and (3) Ideas for designing robots to enhance their social lives. Below we do so ﬁrst for US and then Japan PD sessions. Figures 1–3 are visualizations of each US PD session, and Figures 5–8 are visualizations of each Japanese PD session; these ﬁgures provide an overview of ﬂow during the PD sessions. Frontiers in Psychology frontiersin.org 06 Fraune et al. 10.3389/fpsyg.2022.904019 FIGURE 1 The ﬁrst US PD session was held on November 2, 2020 and had 3 participants. The light arrows indicate concepts shared between participants. ”Telepresence robots” and ”distancing robots” were highly discussed. FIGURE 1 The ﬁrst US PD session was held on November 2, 2020 and had 3 participants. The light arrows indicate concepts shared between participants. ”Telepresence robots” and ”distancing robots” were highly discussed. Frontiers in Psychology frontiersin.org 4.1.1. Current technology use In the ﬁrst round of each workshop, participants discussed their current use of social technologies. Although they viewed these technologies as convenient and helping them connect with others, they also pointed out many drawbacks and problems of current technologies. More importantly, many participants considered online communication technologies lacking important aspects of in-person communication. A participant in another session compared the more structured environment of Zoom, in which a user may already know everyone on the call, with more organic interactions on Facebook which has the potential to spark new friendships: 1) Beneﬁts of current technology: Participants across all three workshops discussed various beneﬁts of technology-mediated interactions. P1US declared that a “bright spot” of social technology was that “at least I get to see my family” during COVID-19. P2US echoed this sentiment in a separate session, indicating her gratitude for the ability to stay safely connected via video calls with her 85-year-old mother in a nursing home. Several participants discussed that video chat was preferable to texting or social media posts because they allowed some degree of non-verbal communication. Friends of friends I see comment on my post, or I comment on their posts, and so you get at least a little bit of a relationship with them, and that can spin oﬀinto more of a friendship. (P4US) P3US shared her appreciation for the Discord messaging app, as it allows her to organize all her interests and clubs in one place, while giving her the choice of participating in synchronous (voice) or asynchronous (text) conversations. P1US, a teacher, also highlighted the advances educational software platforms made during the pandemic to more closely recreate a classroom experience, by adding features like a ‘raise hand’ button. Some participants discussed their perception that not all technology-mediated social interactions are necessarily equal. P1US contrasted a “pleasant” video call with family and friends with only two participants with a particularly overwhelming family video-chat experience where: 2) Drawbacks of current technology: While participants acknowledged these beneﬁts, the consensus of all seven Frontiers in Psychology frontiersin.org 07 Fraune et al. 10.3389/fpsyg.2022.904019 FIGURE 2 The second US PD session was held on November 10, 2020 and had 2 participants. FIGURE 3 The third US PD session was held on December 4, 2020 and had 2 participants. FIGURE 2 The second US PD session was held on November 10, 2020 and had 2 participants. 4.1.1. Current technology use FIGURE 2 The second US PD session was held on November 10, 2020 and had 2 participants. FIGURE 2 The second US PD session was held on November 10, 2020 and had 2 participants. FIGURE 2 The second US PD session was held on November 10, 2020 and had 2 participants. FIGURE 3 The third US PD session was held on December 4, 2020 and had 2 participants. 08 08 Frontiers in Psychology frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 participants across all three workshops, was that technology- mediated social interactions are inferior to their in-person counterparts. Criticisms of technology-mediated social interaction generally aligned with two major themes: (1) technical issues hinder the quality of time spent socializing online, and (2) social technology highlights integral missing aspects of face-to-face social interactions. P7US, a realtor, discussed the social diﬃculties she faces when showing homes to prospective buyers via video calls. She shared that a crucial skill for realtors is knowing how to discuss a home’s minor ﬂaws, such as chipped paint, in the context of its overall attractive qualities during tours. On virtual tours, however, she felt the need to focus her phone’s camera on every minor defect in a home, which poses a novel social and professional challenge that she had not yet solved. Another participant (P2US) stated that she could not imagine trying to open Christmas presents with her family over Zoom, as video calls still “feel unreal” to her. a) Technical issues: Several participants discussed technical issues with current social technology, which frustrate users over repeated interactions and ultimately limit this technology’s usefulness. A common shared concern was the diﬃculty of teaching older or less technically proﬁcient family and friends to use new platforms, with P2US sharing a particularly frustrating experience when organizing a video call with her extended family, including her mother in a nursing home. P3US summarized participants’ overall sense of the essential elements of in-person interactions that are missing from current technology: “I think... it’s probably just camaraderie that you can mimic but not really recreate when you’re not in person.” During two sessions, participants shared stories of social interactions abruptly ending due to dropped internet connections. P6US elaborated that he expects technology to work when he needs it to, without the need to restart or troubleshoot it every time. 4.1.2. Current social challenges In the second round of each workshop, participants discussed current social challenges they faced. While participants had diﬃculty maintaining existing social relationships, they spoke at greater length on the hurdles they faced when attempting to connect with new people. b) Missing aspects from face-to-face interactions: While the above-discussed technical issues may be addressed through software updates and usability testing, participants criticized technology-mediated social interactions as lacking many components of in-person communication. P6US summarized this as the sense that “I’m just talking to a screen, rather than... a person.... Even though I can see the person’s reaction, it’s still not the same reaction that you get when you’re in person.” 1) Challenges maintaining existing social relationships: Participants across two sessions discussed challenges maintaining existing social relationships. P7US shared how her general tendency to show up late to appointments with friends leads to stress as she worries about how long people will want to keep making plans with her. P3US related the challenge of keeping her parents satisﬁed with the quality and quantity of their get togethers, especially during the COVID- 19 pandemic. While she found it important to maintain a small, self-contained social network of family and friends during the pandemic, her parents did not, and subsequently struggled to understand why she was hesitant to visit with them in-person. Almost every participant shared examples of essential features of social interaction that they felt were missing from current online platforms, such as intonation, a feeling of spontaneity, and a sense of shared space. Diﬃculties determining when it was one’s turn to speak on video calls were discussed in two separate PD sessions, with P7US stating that she felt “like I’m interrupting every time.” Several participants noted the challenge of conveying emotional tone when communicating via text: A few participants also recognized technical obstacles that challenged their ability to keep in contact with people close to them: When you talk to somebody, you can read their body language. You can see their facial expression; you can hear their intonation... You know whether or not they’re like kidding around or if they’re really emphatic, and so it’s hard when I’m reading text to ... know [if they are] ... serious about what they’re typing. (P5US) [Many online social opportunities are] based on relationships that were established before all this started and so ... 4.1.1. Current technology use P4US noted that while “fuzzy” audio quality on video calls may not detract from his understanding of what someone else is saying, it adds social distraction that is absent from face-to-face interactions. Frontiers in Psychology 4.1.3. Robot design concepts While initial ideas for robots varied widely, as participants reﬁned their designs through the round robin and iterative design phases, their ﬁnal concepts aligned along three themes: (1) telepresence robots, (2) distancing robots, and (3) artiﬁcial intelligence robots; Figures 1–3 show the conversation ﬂow. Below we discuss these concepts and then the limitations that robotic technology would still have. 1) Telepresence robots: Telepresence robots were the most discussed type, with six participants across all three sessions sharing their personal concepts of how this robot might facilitate social interaction. Participants expected a useful telepresence robot to attend physical social events in place of its user. They decided that the user should control it remotely and the robot should send audiovisual information to the user, allowing a user to have some degree of presence in a remote physical location. P6US suggested that the utility of social technology depends on the type of activity a group shares, with online meetups well- suited to ‘mental’ activities (e.g., watching a movie, playing an online game), but incapable of meeting the needs of those more interested in ‘physical’ activities (e.g., hiking). P2US discussed how she might use such a robot to attend the birthday party of a family member living on the opposite side of the United States, while P1US, an avid golfer, imagined a robot that could replicate movements (e.g., swings, putts) he made at home on a physically remote course. P5US was the most enthused by the potential of telepresence robots, oﬀering several possible applications, including for attending sci-ﬁconventions, business trips, and virtual tourism. The idea of virtual tourism, in particular, interested P2US and P4US in his session, sparking a discussion on how robots might allow one to virtually see the pyramids in Egypt, or avoid post-travel COVID-19 quarantine requirements. P3US, a recent doctoral graduate, faced novel social challenges searching for a job during the pandemic. While she felt conﬁdent maintaining a professional network in the past, she currently faced the new challenge of establishing an online presence on LinkedIn and virtually connecting with peers and recruiters. Two sessions featured discussions concerning the diﬃculty participants had building trust in people they meet online, beyond the time of COVID-19. P3US highlighted that a person’s online persona might not reﬂect who they actually are, while P7US shared that she generally relies upon an introduction from an existing friend when building trust in a new person. 4.1.2. Current social challenges it somehow seems important to maintain those contacts, but it’s more diﬃcult unless you’re very deliberate about it. (P5US) Although P5US’s mother had already passed away prior to the pandemic, P5US did not believe she would have been able to keep in contact with her mother during the COVID-19 pandemic or other situations in which in-person visits were not allowed, given the diﬃculty of teaching her mother how to use new technology. Several participants discussed how losing a shared sense of space aﬀected their social and business interactions. P4US and P5US shared a passion for attending science-ﬁction conventions, but noted that the energy was missing from online alternatives, with P4US declaring virtual conventions “totally worthless.” Frontiers in Psychology frontiersin.org 09 Fraune et al. Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 Overall, all except one participant expressed a clear preference for in-person social interactions, a sentiment summarized by P4US’s judgment of meeting people online being “much more laborious and drawn out [than] if you could sit down at a party over a beer and just chat.” The only participant who preferred technology-mediated interactions in some cases, P2US, shared that socializing online helped ease her social anxiety: 2) Challenges connecting with new people: Across all three sessions, participants discussed troubles they encountered, both personality- and technology-related, when attempting to connect with new people. Two participants discussed their ongoing experiences of social anxiety, which requires them to make a conscious and sometimes exhausting eﬀort to go to a party or reach out to new people. P7US noted the challenge of picking her word choice carefully (even outside COVID-19 and online interaction) so as not to oﬀend new people; she shared an example: ‘[realtors] can’t say ‘master bedroom’ anymore. It’s now “owner’s retreat.”’ It’s much easier to sit down and type and talk to people on Facebook through the written word than I’ve ever found it to go to a party ... and having to speak to new people... It’s not something I look forward to doing, whereas on Facebook... I can read something, decide if I want to respond at all, take my time to compose what I write as a response, and I like that security of a little bit of time to compose. (P2US) The predominance of technology-mediated social interactions during the COVID-19 pandemic posed unique challenges. 4.1.2. Current social challenges Participants across all three sessions shared that their most common methods of meeting new people (particularly other older adults) were currently unavailable, with P5US concluding that it was “pretty much impossible” to meet new people during the pandemic. Several participants noted that they typically connect with new people over common interests. However, as groups moved social interactions online during the pandemic (e.g., pre- planned Zoom calls between established social groups), it became harder to make new friends. For example, P5US enjoyed meeting new people through a board game club prior to the pandemic. While she was able to socialize with existing members of her club through online meetups, she emphasized that this was not an adequate substitute, as new members were not able to attend club meetings during this time. In another session, P1US appreciated that he was able to continue to play golf with a small group of friends, but missed having spontaneous interactions with strangers on the course, such as striking up an impromptu challenge between parties on the driving range or getting to know new people at the clubhouse bar. 4.1.3. Robot design concepts Lacking this personal introduction, P8 expressed diﬃculty imagining how a person could establish trust in a potential partner met through an online dating service. Participants further developed how they envisioned telepresence robots in context of other current technologies during the iterative design phase of the study. P2US and P5US discussed the possibility of coupling a robot with a virtual reality (VR) interface, so the robot could transmit a full three-dimensional visual scene of its location, rather than just a 2D webcam feed. In another session, participants Frontiers in Psychology 10 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 considered how robot design might include augmented reality (AR) technology, with P3US and P6US drawing inspiration from hologram communication in Star Wars. P3US imagined a device that would “project [images of] people into space,” with P6US agreeing that the added third-dimension to the conversation would “make it seem more real.” FIGURE 4 P3US drew a robot concept and indicated its features. 2) Distancing robots: Four participants across two sessions developed ideas for a robot to facilitate social distancing during the time of the COVID-19 pandemic and other situations of limited physical contact. This robot would allow users to remain socially distant while engaged in activities that typically require physical proximity. P6US imagined a robot that would transport shared items, such as a shared meal or a board game, between distanced users. P3US, the only participant who opted to sketch her robot design, imagined a robot that incorporated features from both the telepresence and social-distancing applications discussed during their session (Figure 4). Inspired by R2-D2, she imagined a “smaller” robot on wheels with a “great big processor” inside its body. Her robot would be equipped with a lens to project an augmented-reality conversation partner, 3D cameras, and speaker/microphone array to allow communication with remote users. In proximity with others, the robot used its arms with “prehensile grubby ﬁngers” and an extendable tray to carry a shared board game between players. These participants concluded that the experience of travel consists of many intangible elements, beyond the visual sights, which technology could not adequately convey. In another case, P2US immediately qualiﬁed her idea of a “friend sorter” algorithm, with concerns: 3) Artiﬁcially intelligent robot: In two sessions, participants also considered concepts using artiﬁcial intelligence (AI) to improve the quality of their social interactions, but that would not necessarily require physical embodiment inside a robot. 4.1.3. Robot design concepts P2US built on her understanding of Facebook algorithms to develop an idea for a “friend sorter” agent, which would connect strangers based on shared interests. In a separate session, P1US considered an etiquette program for communicating on social media to ensure the tone and content of a person’s posts was conveyed as intended. He imagined this as functionally similar to the spell-checking function in Microsoft Word, where the program may highlight a potentially problematic phrase for the user to reconsider. P7US agreed that this concept may help her when she wants to avoid causing oﬀense in her word choice. Part of the divides in our country are from people becoming too insular and living in echo chambers, [so] a robot that... introduce[s] us to people who only think like us might make that sort of division worse. (P2US) Another participant (P7US) shared that she still considered robots and virtual assistants to belong in the domain of movies, because real-world technology does not match the promise of stories told about them. She shared her frustrations of using the text-to-speech programs on her phone, and her desire for assistants to feature more fully-developed personalities: To me, robots don’t have personality... Personality is huge... people try to make Alexa have a personality by asking stupid question[s],... and they get back funny answers, but they don’t make sense. (P7US) 4) Limitations of robots: While all participants contributed ideas for social robots, a common point of discussion across two sessions was participants’ belief that, due to the inherent limitations of technology-mediated interactions, robots would not ultimately mitigate the challenges of their social interactions. Three participants’ (P2US, P4US, and P5US) discussion on the potential of using telepresence robots for virtual tourism turned toward a discussion of the elements of traveling that they most valued, such as getting to know a new culture, and experiencing local scents and tactile sensations: P4US held a more fundamental disagreement with the concept of proposing robotics as a means of facilitating social interaction: Almost every near-term robotic...application you can think of ... is more a substitute for human social interaction... so it would act to reinforce driving people apart because it makes it more comfortable to be apart. (P4US) In certain applications and in the current (COVID-19) environment, that’s helpful. Frontiers in Psychology 4.1.3. Robot design concepts But if I think about trips I’ve taken overseas or even within the US to interesting places, the strongest memories I have ... are the people that I met... there. (P2US) P4US further shared his belief that “the only thing I could think of that would enable social interaction from a robotics point of view would be to develop ... a real-time environmental sampling [robot].” In his conception, this robot would move Frontiers in Psychology 11 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 Because of the asynchronicity of text-based communication (not like in-person conversation), one can contact the others without worrying about their current status. P5Japan, P7Japan, and P8Japan also said that the beneﬁt of such technology- mediated communication is that they can send messages at their conveniences. through a crowd in traditional social environments such as bars or convention centers to determine if an area contained traces of a virus, therefore building conﬁdence in a return to in-person socializing, especially after COVID-19. Alongside discussing the roles socially facilitative robots might ﬁll, participants shared critical attributes about if they would be willing to use a robot. P7US shared concerns over privacy when using assistants like Alexa, while P4US stated that he would not be interested in humanoid robots. P7Japan also remarked that the other beneﬁt of such communication is to save everyone time because it does not require traveling to see the person in-person manner. COVID- 19 has eventually led to widespread the use of video conference system so this made people eliminate the need to travel. In terms of less need to travel, the other participants also said that saving time to traveling to see the others makes it easier to manage the schedules of the online meetings and appointments. P3US and P6US valued the ease-of-use of any new technology: Whatever it is, make it easy to use. Make it intuitive, so that the user doesn’t really have to do anything... know how it works or... push a lot of buttons.... It just works. (P6US) Seven out of 12 participants answered that LINE, a freeware app for instant communications on electronic devices such as smartphones2, was their current preferred social technology. As for the reason, P1Japan said, “I use LINE a lot. 4.1.3. Robot design concepts Because on this app, there is a display to show whether the message receiver has read the sent message or not, and this makes me intuitively understand whether she/he has time now or not.” Therefore, the display function whether the receiver has read the sent message or not on text-based social technologies will have a signiﬁcant role in such asynchronous communication. Both participants considered ease-of-use not merely a beneﬁt, but essential for accessibility to people even older than them. P3US considered if her parents would use a complex robot, given that using an iPhone or Kindle is already a challenge for them. P6US further shared his accessibility concerns, stating that: Especially if you’re talking robots to aid [the] elderly... they’re not going to be as technologically advanced...or...as mentally capable of handling the technology. (P6US) Another comment on the beneﬁt of LINE was the integrated function of the phone call and video chat. P3Japan commented, “There are very few people who do not install LINE on their smartphone, and you can do almost everything about communicating with others. You can make a phone call if you need, and you can also have a video chat especially when you want to see the face of that person, so this can be useful to communicating not only with my family but also with friends.” Some participants preferred such video chats over text-based communications because of rich amounts of information about the person in line including non-verbal information. These two participants considered speech recognition to be the most accessible way for older adults to interact with technology. However, P3US noted her own accessibility concerns with existing speech recognition software when relating how Siri was sometimes unable to understand her father’s slurred speech after he suﬀered a stroke. 4.2. Japanese ﬁndings 2) Drawbacks of current technology: While participants felt that such current social technologies are useful tool to connect people who are apart, they also felt that there are some issues of these technologies. These issues can be categorized into following two categories, like in US workshops: technological issues and missing aspects from face-to-face interactions. Frontiers in Psychology 2 https://line.me/en/ 2 https://line.me/en/ 4.2.1. Current technology use Like the US PD workshops, the middle-aged and older adult participants discussed their current use of social technologies and the beneﬁts and drawbacks of these technologies. While they feel the convenience of communicating with people who are apart through the current social technologies, they also raised pain points that the amount of information they can acquire through these technologies was limited compared to in-person communication. It can be said that they unwillingly use such technologies when they cannot meet people in-person manner during COVID-19, and they strongly prefer in-person communication to technologically mediated ones. a) Technical issues: P6Japan mentioned, “There is often a huge time lag during video chat conversation, while there is no such lag during in-person conversation." P11Japan also said, "I still have some troubles with internet connections, like bad or lost connections." The qualities of video chat conversation depend on the internet environments of users. Further, the more and richer information that is sent and received, the longer and slower it takes to transmit such information, which disrupts smooth communication like in-person ones. 1) Beneﬁts of current technology: P6Japan said about current technology-mediated communication, “I can send a message to my colleagues anytime and anywhere at my convenience without worrying about whether they are too busy to reply to me.” Frontiers in Psychology frontiersin.org 12 Fraune et al. Fraune et al. 10.3389/fpsyg.2022.904019 (voice call only). In terms of the use of current technology other than the phone by older adults, P7Japan said, “It is diﬃcult for older adults to master current video chat systems, so they tend to use the phone." P11Japan made assumptions about the reason why the older adults hesitate to use current technologies saying, “To master the current technologies, older adults have to get used to them, but this is quite hard, and they are somewhat reluctant to do so. This eventually leads their negative or passive attitude about new technologies.” Because online communication through current social technologies was created for one-on-one interactions, the conversations on these technologies tend to be limited on a single topic even if many people were in the conversation. P10Japan said, “With Zoom, nobody can have a conversation with a large number of people at once. And this Zoom is inconvenient for having a light chat a kind of cross-talk among the colleagues. 4.2.2. Current social challenges Next, as a technology-related challenges, P7Japan said, “Because I only can see the other person’s face or upper body in Zoom, it is diﬃcult to capture a full picture of this person or their mood through the screen.” In this regard, P4Japan and P9Japan also said that it is diﬃcult to establish new connections in on-line technologies because of the lack of information about the other person. As well as US workshops, participants discussed current social challenges they faced in terms of the following two aspects: maintaining existing social relationship, and attempting to connect with new people. 1) Challenges maintaining existing social relationships: Participants discussed the diﬃculties and problems they felt in maintaining their current relationships. P11Japan said, “In my case, I can no longer have drinking parties or reunion parties I used to have every year. The adverse eﬀect of this is that I have lost the trivial information that facilitates relationships.” As the result of COVID-19 pandemic, participants had fewer opportunities to meet their colleagues and friends in-person manner, so they lost out on opportunities to learn about current situation of these peoples. P3Japan, who mentioned a similar opinion with P11Japan, remarked that he started to contact these people more frequently than before the pandemic. 1) Challenges maintaining existing social relationships: Participants discussed the diﬃculties and problems they felt in maintaining their current relationships. P11Japan said, “In my case, I can no longer have drinking parties or reunion parties I used to have every year. The adverse eﬀect of this is that I have lost the trivial information that facilitates relationships.” As the result of COVID-19 pandemic, participants had fewer opportunities to meet their colleagues and friends in-person manner, so they lost out on opportunities to learn about current situation of these peoples. P3Japan, who mentioned a similar opinion with P11Japan, remarked that he started to contact these people more frequently than before the pandemic. 4.2.1. Current technology use So the conversation on Zoom is diﬀerent from ones in real life.” Some participants also pointed out that it is almost impossible using current social technologies for users to discuss multiple topics in parallel, like at a real party. 2) Challenges connecting with new people: Participants discussed their social challenges to connect with new people in terms of the following two aspects: human-caused challenges and technology-related ones. Regarding human-caused challenges, P3Japan mentioned that he could no longer casually meet people via social networking services (SNSs), “Before the pandemic, I used to casually meet people who have similar hobbies through Twitter or the other SNSs, but now I cannot do that at all, so my social connection with others is completely text-based one.” P5Japan, who was working as a short-period temporary worker, said that she no longer has the opportunity to meet new people because the number of jobs has drastically decreased due to COVID-19. While many participants said that they passively lost their opportunities to establish new relationships, P1Japan said that she was not actively trying to establish new relationship to prevent infections of COVID-19, “Because of this pandemic, I do not think I should go into the other’s private too much, so I am not actively trying to meet new people.” b) Missing aspects from face-to-face interaction: P1Japan argued, “One of the signiﬁcant diﬀerences between face-to- face and technology-mediated conversation is the amount of information that comes out from the person in LINE [app]. When you meet someone via in-person situations, you can see and sense their facial expressions and tones of voices. I cannot feel such non-verbal or paralinguistic information with the current social technology I am using now, so I guess this is the biggest diﬀerences.” Eight out of the 12 participants reported similar issues. In addition, P9Japan said that he could not understand where the person using LINE is actually looking at him on their screen during the video chat, so he sometimes doubted whether they were concentrated on talking with him or if they were distracted and doing other things at the same time. Frontiers in Psychology 4.2.3. Robot design concepts The participants’ ﬁnal robot design concepts eventually settled on the following three themes: (1) pet robots, (2) sharing experiences, and (3) easy operation; Figures 5–8 show the conversation ﬂow. 1) Pet robots: In Japanese PD workshops, the most common robot concept was a pet robot. In all four workshops, a pet robot was discussed, and nine out of 12 participants expressed their opinions about pet robots, as seen in Figures 5–8 (red). These participants mentioned in Round 2 that they were worried about their elderly parents who live apart, and many of them wanted to place a pet robot in their parents’ house on the participants’ to provide them with a social companion when they were unavailable to visit. The most common comments in Round 2 were about how to maintain the relationships with their elder families, especially their parents, who are far away from them. Eight of 12 participants said that they felt diﬃculty in communicating with their parents, worried about their health and social situations, so they kept in touch with their parents more frequently than before COVID-19. In addition, there were several comments that their parents were too old to master the current technologies, so they usually use phone to communicate P2Japan said, “The numbers of older adults is increasing, so robots that can watch over these older adults, including pet Frontiers in Psychology 13 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 FIGURE 5 The ﬁrst PD session in Japan was held on August 29, 2021 and had 3 participants. Dark black arrows indicate concepts that were carried forward in development. Two of the top themes were heavily discussed among the participants: sharing experiences and pet-like robots. All three participants chose a feature related to sharing experiences in their “top 5”. FIGURE 6 The second PD session in Japan was also held on August 29, 2021 and had 3 participants. Similarly, the theme of pet-like robots arose; “easy operation” was another top theme, as indicated by participants in their “top 5”. FIGURE 5 The ﬁrst PD session in Japan was held on August 29, 2021 and had 3 participants. Dark black arrows indicate concepts that were carried forward in development. Two of the top themes were heavily discussed among the participants: sharing experiences and pet-like robots. All three participants chose a feature related to sharing experiences in their “top 5”. 4.2.3. Robot design concepts FIGURE 5 The ﬁrst PD session in Japan was held on August 29, 2021 and had 3 participants. Dark black arrows indicate concepts that were carried forward in development. Two of the top themes were heavily discussed among the participants: sharing experiences and pet-like robots. All three participants chose a feature related to sharing experiences in their “top 5”. FIGURE 5 The ﬁrst PD session in Japan was held on August 29, 2021 and had 3 participants. Dark black arrows indicate concepts that were carried forward in development. Two of the top themes were heavily discussed among the participants: sharing experiences and pet-like robots. All three participants chose a feature related to sharing experiences in their “top 5”. FIGURE 6 The second PD session in Japan was also held on August 29, 2021 and had 3 participants. Similarly, the theme of pet-like robots arose; “easy operation” was another top theme, as indicated by participants in their “top 5”. FIGURE 6 The second PD session in Japan was also held on August 29, 2021 and had 3 participants. Similarly, the theme of pet-like robots arose; “easy operation” was another top theme, as indicated by participants in their “top 5”. ond PD session in Japan was also held on August 29, 2021 and had 3 participants. Similarly, the theme of pet-like robots arose; “easy on” was another top theme, as indicated by participants in their “top 5”. 14 Frontiers in Psychology frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 FIGURE 7 The third PD session in Japan was held on September 5, 2021 and had 3 participants. All participants discussed the need for easy operation. All three top themes were present in the participants’ “top 5”: sharing experiences, pet-like robots, and easy operation. FIGURE 8 The fourth PD session in Japan was also held on September 5, 2021 and had 3 participants. P10 and P12 discussed pet-like robots, and P10 and P11 discussed easy operation. All three participants noted the importance of easy operation in their “top 5”. FIGURE 7 The third PD session in Japan was held on September 5, 2021 and had 3 participants. All participants discussed the need for easy operation. All three top themes were present in the participants’ “top 5”: sharing experiences, pet-like robots, and easy operation. FIGURE 7 The third PD session in Japan was held on September 5, 2021 and had 3 participants. 4.2.3. Robot design concepts All participants discussed the need for easy operation. All three top themes were present in the participants’ “top 5”: sharing experiences, pet-like robots, and easy operation. d PD session in Japan was held on September 5, 2021 and had 3 participants. All participants discussed the need for easy operation. A op themes were present in the participants’ “top 5”: sharing experiences, pet-like robots, and easy operation. FIGURE 8 The fourth PD session in Japan was also held on September 5, 2021 and had 3 participants. P10 and P12 discussed pet-like robots, and P10 and P11 discussed easy operation. All three participants noted the importance of easy operation in their “top 5”. FIGURE 8 The fourth PD session in Japan was also held on September 5, 2021 and had 3 participants. P10 and P12 discussed pet-like robots, and P10 and P11 discussed easy operation. All three participants noted the importance of easy operation in their “top 5”. 15 frontiersin.org Frontiers in Psychology Fraune et al. 10.3389/fpsyg.2022.904019 robots, can play a signiﬁcant role in near future. These robots can be also applied to industries of nursing care; I hope the introduction of these robots will change the world eventually.” P10Japan has already given the pet robot to her parents, and her mother happily reported that she is always talking to this robot on her (P10Japan) behalf. Participants expected pet robots to be useful in relieving the loneliness of parents who are apart and in communicating and dealing with problem when they occur. so I hope there will be a tool that will allow people to experience what they have actually been through in real life.” (P1Japan) so I hope there will be a tool that will allow people to experience what they have actually been through in real life.” (P1Japan) As in the US workshops, participants discussed that there are a lot of issues especially in entertainment that are not feasible on-line manner, like the inability of traveling. In the Japanese workshops, although there were no concrete discussions about how the robots can resolve these issues, half of the participants said that they felt huge challenges of sharing experiences. As discussions on pet robots progressed, participants discussed not only the roles of the pet robots but also their required abilities or capabilities. 4.2.3. Robot design concepts Speciﬁcally, six participants mentioned the texture or appearance of the pet robots because all of these participants agreed that the pet robots commercially available today has a mechanical impression so they are looking for robots that are more like living things. P2Japan said, “I think it would be great if the robot can feel the body temperature of a distant person, for example, this robot can tell the measured temperature by its color.” People are comforted by the presence and body heat of living things, like pets, so participants discussed that a more biological robot could be realized by incorporating such body warmth mechanism into a pet robot. 3) Easy-to-operate instructor robot: Participants reported some issues keeping up with rapidly evolving technologies. Many participants require some advice from the others who are good at current technologies when they face new devices or applications. However, they were concerned that they do not always have access to someone to assist them. P11Japan said, “When we have problems about current technologies, we usually ask friends or colleagues who are good at such technologies, but we do not always have such people around, and these diﬃculties derived from new devices or application are advancing day by day. It is diﬃcult to keep up with all of these issues, so it would be very reassuring for the older adults (like us) if there were a robot with latest updated technological capabilities, and this robot beside us give instructions how to use these technologies.” (P11Japan) In addition, participants indicated that a signiﬁcant element of the pet robots is to make the person invested in the robot by spending the time and eﬀort on it. Although it is believed that robots are developed to reduce human’s workloads by taking on their tasks, some participants said that the users would feel more attracted to their pet robots if they felt attached to and needed to care for these robots, like a Tamagotchi (Cheok and Zhang, 2019). 4.2.3. Robot design concepts P4Japan said, “Pet animals are generally troublesome in that the owners should spend a lot of time and eﬀort on them; on the other hand, such eﬀorts make the owners feel more attached to them and more soothed on them.” The time and eﬀort required to take care of a robot would be a signiﬁcant factor in realizing a long-term interaction between the users and robots as well as between the owners and pet animals. He hoped that the robot will solve the situation in which there is no one to help them technically. They preferred to have such robot present with them rather than paper instruction manuals. As for the operation of such robots, the participants want a method of operation that is simple as a land-line phone or home appliances. 4) Limitation of robot: Throughout the PD workshops in Japan, although the participants discussed the design of the robots to solve their current social challenges, there were some limitations in these PD sessions. One was less concrete ideas about the robots to solve their challenges. For example, in the third round, the participants discussed how to improve sharing their experiences with people who are apart. In these discussions, they agreed that current online communication tools provide less information than in-person meetings, so they wanted new technology that can make them share more of their experiences with each other. However, while there were some remarks about speciﬁc experiences they would like to share, such as traveling or eating, they could not discuss or propose concrete and speciﬁc solutions to resolve their such challenges. P3Japan made the following remarks, “I want an exciting robot that could engage same tasks or events together, or an exciting tool that make everyone enjoyable time together, but I cannot describe or present it because it is quite diﬃcult to shape this idea concretely...” 2) Sharing experiences: In two of four Japanese workshops, participants discussed how to improve sharing experiences with people who are apart. Due to the pandemic of COVID-19 mandates and social pressure to keep others safe, it became diﬃcult to meet people in-person, and the conversations and drinking party using video conference or chat system have become popular. However, some participants felt that the quality of experiences is lower when communicating through such technologies compared to meeting in-person manner. 5. Discussion To understand middle-aged and older adults’ diﬃculty with current social technologies and develop ideas for designing socially facilitative robots to address their needs, we conducted PD workshops: three sessions in the US with seven middle- aged and older adult participants, and four session in Japan with twelve participants. Overall, participants were eager to discuss how robots might facilitate social interactions, while still harboring some reservations based on their negative experiences with current social technology and concerns about ease of use. Although we conducted the workshops during the COVID-19 pandemic, many ﬁndings generalized to other situations related to social isolation, such as older adults living alone. In this section, we present the major themes of our PD workshops while highlighting areas of focus for future PD and user- centered design intended to promote middle-aged and older adults’ use and long-term acceptance of socially facilitative robot technologies. 4.2.3. Robot design concepts P1Japan said, “I like to travel a lot, and I used to have a lot of travel friends before the pandemic. But now I cannot see them very often, so we use a kind of bulletin board system on the internet to share the information about travel. Currently, there are only text and some pictures on this bulletin board, Frontiers in Psychology frontiersin.org 16 Fraune et al. Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 In addition, some participants felt frustrated that their families and friends could not use the current social technologies to connect with them online, even as they succeeded in adopting such new technologies. P10Japan said, “People around my age and older are very timid about new technology... I tried to explain to them that it is easy to use online tools like Zoom, but they just said to me ‘no no no no’ and ‘impossible!!’ without touching these tools...” Since the participants in these PD workshops actually have certain levels of literacy of such technologies, they were frustrated by the diﬀerences in literacy levels among their families and friends who are apart. technical issues did not merely aggravate participants, but were a main cause for concern as our participants considered the diﬃculties of connecting with users, such as their parents, who may be even older or less technically proﬁcient than them. It can be a burden to people of our participants’ generations to have to teach their parents both how to technically use the social technology (what buttons to press when) but also address the communication etiquette of that social technology, particularly for the Japanese participants. While participants related several social challenges they faced when keeping up with existing friends, our middle- aged and older adult participants emphasized the challenge of building new connections with others. These challenges were particularly salient during times of social distancing due to the COVID-19 pandemic, as traditional means of socializing familiar to older adults were limited, but would also extend to situations in which older adults are conﬁned to their homes, hospitals, or elder care facilities. 5.2. Concepts for socially facilitative robots Participants expressed general excitement for the potential of robots to facilitate higher quality social interactions between people. As shown in the conversational ﬂow diagrams in Figures 1–3, 5–8 representing the PD sessions in the US and Japan respectively, our method was then successful at encouraging participants to develop and build upon a wide range of novel concepts: from robots dependent on physical embodiment in a shared space, to new applications of artiﬁcial intelligence, to representing social companionship through “body warmth.” In the US, the top three robotic concepts were telepresence robots, distancing robots, and AI to extend capability; in Japan, they were pet robots, sharing experiences, and easy operation to solve presence problems. Although these concepts may seems distinct, we ﬁnd complimentary themes in places. Frontiers in Psychology 5.1. Challenges with social technology Participants’ concepts of AI programs were targeted at improving the quality of existing social interactions. These applications might help address ongoing social issues such as avoiding potentially oﬀensive language or oﬀering reminders of scheduled times to meet with friends. This could help people in online situations regardless of age, and possibly provide diﬀerent suggestions across diﬀerent contexts to help people write more professional emails for work or more friendly emails for reaching out to new acquaintances. A “distancing robot” could allow people to safely socialize in the same shared space. Participants in the US PD sessions thought a distancing robot could enable the development of new social connections between people even when physical contact was limited. For instance, during a global pandemic, group meetings were considered unsafe. Nonetheless, with a distancing robot, individuals could share the same physical space while maintain social distancing, by having the robots delivering items between people. With such a robot, club meetings of people with shared interests could continue in a physically shared space, rather than merely online, potentially enabling that group to facilitate new connections. Although the overall sample size of our PD sessions is small, it is likely that distancing robots would have continued to be a prevalent theme, as robotics researchers had also begun prototyping this concept (Sathyamoorthy et al., 2020, 2021; Somaldo et al., 2020). Also, the idea of an “instructor robot” came from the PD sessions in Japan. Participants expressed a concern for making interactions with robots as easy as possible, particularly for their own older parents. If their parents are alone and need assistance operating a social technology, they envisioned that the instructor robot would teach and guide their parents on how to use it. This functionality speaks to lowering the technology savviness barrier and making it more accessible, which is another way to connect people socially. While the middle-aged and older adults in this study were interested in the potential of socially facilitative robots, some skepticism remained. Similar to their discussion on perceived limitations of current social technology, participants also imagined limitations of the robots they brainstormed during this session. Their concerns emphasize the need for robot designers to involve middle-aged and older adults at every stage of the design and development process, to ensure that their needs are being met and that this population are willing to use new technology. 5.1. Challenges with social technology US and Japanese participants alike recognized some beneﬁts of technology-mediated social interactions; however, they generally agreed that these mediums imperfectly replicated key aspects of in-person interactions. Both social and technical limitations of current social technology frustrate them, with all participants presenting ways in which technology-mediated interactions are inferior to traditional means of socializing. A lack of critical social cues, such as intonation and a sense of being together with others in a shared space, frustrated them over repeated interactions. Conversational cues that exist during in- person interactions, such as leaning in to indicate that you would to speak and timing of when to respond to a person, ultimately break down into cross-talk and/or stilted conversations. These social distractions detract from the quality of their interaction. In the ﬁrst theme, we note that there are opportunities for robots to both socially connect people in the capacity of a tool (i.e., telepresence, artiﬁcial intelligence enhanced social skills), and also to use its own presence for a person to interact directly with (i.e., pet robot, instructor robot). The telepresence robots conceived by participants in the US PD groups directly addressed their concerns with both maintaining current relationships and forming new connections while geographically removed from other people; positive discussions around these robots conﬁrm ﬁndings of older adults’ interest in the GiraﬀPlus telepresence robot in a PD workshop conducted in prior research (Van Baarsen et al., 2001). A review of mobile telepresence robot research prior to and during the global pandemic also indicates that the theme of telepresence would Technical issues such as dropped calls, audio lagging or breaking up, video freezing, and the lack of a turn-taking mechanisms in video calls compounded their frustration. These Frontiers in Psychology frontiersin.org 17 Fraune et al. 10.3389/fpsyg.2022.904019 likely have scaled beyond the limited number of participants in our PD sessions (Isabet et al., 2021). that current two-dimensional video calls lack the spatial sense and full-body communication integral to meaningful social interactions. Participants in both countries noted that, not only is it diﬃcult to meet new people when socially isolated, it can be diﬃcult to trust who they are and have enough information to start a conversation. Artiﬁcial intelligence (AI) is already used in suggesting likely friends based on a person’s social network, but can extend to be the entity to make that introduction and recommend topics of common interest. 5.1. Challenges with social technology If perceived as an agent, an instructor robot can also be the target of social companionship intended or otherwise. Japanese participants very much wanted pet robots to keep their parents from feeling lonely; all 4 Japanese PD groups discussed functionality they desired, so we believe that this concept would maintain its popularity if we had hosted additional PD sessions in Japan. Despite there being a plethora of academic research on robotic pets and commercially available ones (Eachus, 2001; Usui, 2011; Katsuno and White, 2022), none of the US PD groups discussed pet robots; however, in our previous work, middle-aged and older adults did show interest in pet robots in the one-on-one interviews (Ling et al., 2022). The Japanese participants had diﬃculty in expressing with the easy to use instructor robot and how to better share their experiences. However, they were very clear in articulating concrete capabilities a pet robot should have, including “body warmth” which can comfort their parents (Walsh, 2009; Zilcha- Mano et al., 2012). While not all brainstormed concepts may be immediately feasible, these PD workshops provide insights for designers based on insights into middle-aged and older adults’ expectations and reservations around the capabilities of social robots. Our participants emphasized the importance of perceived ease-of-use and reliability when making decisions in new technology use. This poses a unique problem for social technology, as users must consider not only their own technical proﬁciency, but the capabilities of those they plan to connect with. Older adults may prefer natural language voice interfaces over other input modalities, as this leverages their understanding of conversations. Finally, to avoid undue anxiety, designers must clearly communicate that the purpose of socially-facilitative robots. In the US, the goal is not to replace, but rather aid, human social interactions, while in Japan, the goal is to for robots to be a stand-in companion in the absence of people. In the second theme, participants in both the US and Japan noted that the experience of sharing was lacking. In the case of the US, participants wanted to “be there” meaning to travel, whereas the Japanese participants wanted a better way to share out their experiences with others. The US participants’ interest in the possibility of combining robotics with emerging technologies like VR and AR present a novel ﬁnding from this work on an additional area of interest to this population. These technologies could address users’ repeated observations Frontiers in Psychology 6. Conclusion and future directions While this study occurred during a time of pronounced social isolation due to the COVID-19 pandemic, many of the challenges discussed relate to social issues participants experienced prior to the pandemic, and which will be ongoing issues for older adults in the future. Many issues were not caused, but rather exacerbated, by novel social distancing measures. For example, P7US expressed trust issues with the concept of online dating prior to the pandemic, but these issues became more salient as the pandemic restricted other potential options for meeting new romantic partners. Further, many of the challenges discussed here will remain pertinent after social distancing requirements are lifted, as older adults may live far apart from their loved ones and social interactions continue to move online. In the future, we recommend that online PD sessions should have minimally 3 or ideally 4 participants; we believe this will reduce the perceived burden of participants feeling pressure to perform, like too much is riding on how their responses. We posit that a having 2 participants, as we did for US PD sessions 2 and 3, makes them feel culpable for 50% of the content, whereas with 4 people, their individual responses are only 25%. 3 or 4 participants helps build a wider variety of ideas, while still maintaining enough time for sharing out without having to signiﬁcantly extend the duration the PD session. Recent research has emphasized the importance of directly involving end users of technology as collaborators in the design process (Chen et al., 2018). It is crucial that older adults are involved in the design of robots intended to meet their particular needs (Chopik, 2016). Our research adds to the literature by (1) tailoring a PD approach to create a novel grounded approach for people to reﬂect on their own life experiences prior to giving them an abstract topic to discuss, such as robots, and (2) examining the social needs of middle-aged and older adults and how socially facilitative robots could be applied to address their self-identiﬁed needs. Our ﬁndings underscored the importance of adopting a user-centered approach for socially facilitative robot design for older adults. Further, the current workshop consisted of a relatively open brainstorm session of imagined robot functions and features which is useful at the beginning of a project. 5.3. Limitations The online nature of this study presented a notable limitation to the current research. The participant sample all had access to the Internet and exhibited some degree of technical Frontiers in Psychology 18 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 competency, as they were able to participate in a group online video call. This prevented us from studying populations of older adults who lack either perceived technical competency or access to the internet altogether. A future in-person workshop may better accommodate this population and produce new insights. conducted a study over the ﬁrst 3 months of the pandemic in the US and in which preventative and protective behaviors people engaged, including wearing a mask, washing hands, avoiding crowds and eating at restaurants, postponing or canceling social gatherings, and avoiding hosting guests in their homes. They found that as people learned over time about the risks, they adopted more of these preventative and protective behaviors. The 3 US participatory design groups were held in November and December 2020, approximately 6 months after the start of the pandemic. At that time, people were cautioned against holiday season travel and large gatherings (Abidi and Gramlich, 2020). The 4 participatory design groups in Japan were held in August and September 2021. Kim and Crimmins noted the likely possibility that people could not engage in these protective and preventative behaviors for the long term. Globally, people had been dealing with COVID-19 for well over a year and acclimated to the “new normal” (Jamaludin et al., 2020). Participants did not discuss or build upon each others’ ideas as much as we expected. We found that US participants spoke more than the Japanese participants, which could be attributed to a number of factors including participants’ lack of conﬁdence, insuﬃcient rapport among PD groups, or unintended change in facilitation style when culturally adapted for use in Japan. Another factor impacting participation in discussion might the be platform of choice used to host these online participatory design sessions and people’s familiarity with using the platform and associated social etiquette (e.g., mute when not speaking, unmute to speak, raise your hand). We used Zoom. All US participants used Zoom (n = 5) or equivalent video calling apps (n = 2), which are typically used on a computer, laptop, or larger tablet. Only 4 of the 12 Japanese participants called out Zoom, and majority (n = 8) used LINE on their smartphones. 6. Conclusion and future directions Researchers should conduct future PD workshops at diﬀerent stages of the design process, such as when drafting more speciﬁc robot designs or testing physical prototypes. There may be ethical concerns about socially facilitative robots to reduce social isolation for older adults in the US and Japan (e.g., privacy). We have mentioned these concerns as participants brought them up throughout the sessions, but researchers should conduct future work to examine what concerns may arise as the technologies are implemented. 5.3. Limitations LINE is a mobile app that has an integrated video call feature and handles other forms of social content/connection, whereas Zoom’s primary functionality is video calls on computers or tablet devices with groups of people, such as with our PD sessions. In addition, participants’ educational backgrounds and health conditions, and the economic situation, may have aﬀected their opinions in PD sessions so future studies should examine other populations. Frontiers in Psychology References Abidi, S., and Gramlich, J. (2020). As CDC warned against holiday travel, 57% of Americans say they changed Thanksgiving plans due to COVID-19. 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Data availability statement Finally, our ﬁndings should be considered with respect to people’s evolving perceptions, attitudes, and behaviors over time during the COVID-19 pandemic. Kim and Crimmins (2020) The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Frontiers in Psychology 19 frontiersin.org Fraune et al. 10.3389/fpsyg.2022.904019 10.3389/fpsyg.2022.904019 Acknowledgments The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fpsyg.2022.904019/full#supplementary-material Thank you to Aldo Chavez-Gonzalez and Danielle Benitez for coding the US data. Ethics statement Conﬂict of interest The studies involving human participants were reviewed and approved by Institutional Review Board of New Mexico State University; Ethics Committee on Human Research of Meiji University. The patients/participants provided their written informed consent to participate in this study. Author KT receives a nominal ﬁnancial award for publishing academic papers. The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding This work was supported and funded by the Toyota Research Institute. Publisher’s note HP and RA were the primary authors for the participatory design groups run in the United States. TK is the primary author for those run in Japan. SS created the conversational ﬂow diagrams. All authors contributed to the analysis and/or writing of this paper. All authors contributed to the article and approved the submitted version. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. 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https://openalex.org/W3151264539	https://zenodo.org/record/4024477/files/wallace2020exploring.pdf	English	null	Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks	Lecture notes in computer science	2,021	cc-by	5,702	ABSTRACT the output of the model. Building an understanding of these effects could aid when deciding between different approaches in generation of dance motion and other applications. It is common to explore multiple sampling approaches when gen- erating output from generative deep neural networks for creative applications. Choosing suitable sampling parameters can make or break the realism and perceived creative merit of the output. This is particularly important when attempting to simulate variations of expressive human movement. The process of selecting the correct sampling parameters is often task-specific and under-reported in many publications, which can make the reproducibility of the re- sults challenging. We explore some of the most common sampling techniques in the context of generating human body movement, specifically dance movement, and attempt to shine a light on their advantages and limitations. This work presents a Mixture Den- sity Recurrent Neural Network trained on a dataset of improvised dance motion capture data from which it is possible to generate novel movement sequences. Systematically examining the different sampling strategies allows us to further the understanding of how the sampling parameters affect motion generation, which provides evidence for utility in creative applications. Expressive movement is an intrinsic part of human life. Hand gestures during speech, body language as well as dance have the ability to efficiently convey an emotional state. Based solely on simple movement patterns such as gait or arm movement it is possible to classify characteristics like gender, personality and mood [15, 18, 20]. As such, motion analysis and generation have use cases in fields such as human-robot interaction, human activity recognition and artificial agent design [12]. While movement generation may involve all of the movement types mentioned above and more, this paper presents a system for generating dance movement. Such a system may be used as an artistic tool for choreographers, or to generate dance movement in video games or animation. In the former, the system must be able to generate novel and varied outputs, while for animation purposes realism may be a more important property than novelty. For any predictive system, a loss function would typically include some metric indicating the distance from the model output to some ground truth. This approach produces good results when generat- ing basic human motion such as gait [1]. KEYWORDS Mixture Density Networks, Movement Generation, Generative Net- works, Creative Prediction Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks Benedikte Wallace benediwa@ifi.uio.no RITMO, University of Oslo Oslo, Norway Charles P. Martin Australian National University Canberra, Australia Jim Tøressen RITMO, University of Oslo Oslo, Norway Kristian Nymoen RITMO, University of Oslo Oslo, Norway Figure 1: Dance improvisation Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks Benedikte Wallace benediwa@ifi.uio.no RITMO, University of Oslo Oslo, Norway Charles P. Martin Australian National University Canberra, Australia Jim Tøressen RITMO, University of Oslo Oslo, Norway Kristian Nymoen RITMO, University of Oslo Oslo, Norway Figure 1: Dance improvisation Benedikte Wallace benediwa@ifi.uio.no RITMO, University of Oslo Oslo, Norway Benedikte Wallace benediwa@ifi.uio.no RITMO, University of Oslo Oslo, Norway Jim Tøressen RITMO, University of Oslo Oslo, Norway Benedikte Wallace benediwa@ifi.uio.no RITMO, University of Oslo Oslo, Norway Jim Tøressen RITMO, University of Oslo Oslo, Norway Kristian Nymoen RITMO, University of Oslo Oslo, Norway Jim Tøressen RITMO, University of Oslo Oslo, Norway Jim Tøressen RITMO, University of Oslo Oslo, Norway Figure 1: Dance improvisation ABSTRACT When generating dance movements, or indeed any creative data, there is no single correct answer or ground truth to which the output can be compared as we may consider several predictions equally valid. Thus, rather than attempting to predict a single truth a good model for generating creative data should be able to generate a variety of likely outputs. A common way to approach this problem is to experiment with sampling from the probability distribution learned by the model 1 INTRODUCTION In examining the output of a model which generates creative data the results are often evaluated according to whether or not they are typical examples of their type, be it a piece of music perhaps belonging to a specific genre [21], or a painting in a certain style [23], or in our case a realistic sequence of movement. Realism is naturally a quality that has some degree of importance when gener- ating any type of data, but perhaps especially so for movement; any deviation from what is physically possible for a human to achieve is instantly recognisable. Another important aspect to consider is whether the model is able to produce novel output, different from the examples used in training. In this work, we look at creativity in movement generation, specifically dance movement. We investigate the effect of different sampling strategies for movement generated by a mixture density recurrent neural network. MDRNNs are becoming well-established tools in the generation of creative data. They have previously been applied to musical sketches in two dimensions as part of a smart- phone app [14], to sketches [8], and handwriting [7]. MDRNNs have also previously been applied to motion capture data [5, 17], which show promising results. While variation is a central aspect of generating creative data, there has been little focus on the sam- pling strategies and how they affect variation in the generated data. We aim to systematically explore the effect of applying the three sampling strategies outlined above: adjusting temperature, priming, and isolating mixture components. The following sections will de- scribe the model and motion capture recordings. The final sections present the results of sampling from the trained MDRNN. The code used to sample from the trained model has been made available in the interest of reproducibility. In order to determine the novelty or realism of a generated mo- tion sequence, it is useful to define a notion of similarity. However, the seemingly simple concept of movement similarity becomes quite complicated. In many cases, it may be sufficient to consider motions as similar if they only differ with respect to global rota- tion or spatial and temporal scaling [16], but for the purpose of comparing dance, such a definition may be unsatisfactory. Con- sider, for example, one dance sequence where the left arm is raised, and another the right arm. 1 INTRODUCTION In this paper, we will outline several common sampling strategies for Mixture Density Recurrent Neural Networks (MDRNNs). We will show that the choice of sampling strategy significantly affects Wallace et al. Wallace et al. Figure 2: Marker setup worn by the dancers during motion capture data collection with more or less “randomness”, often referred to as stochastic sampling, until the output is found to be satisfactory [6–8, 14]. This is usually performed by including a temperature parameter which is used to reweight the probability distribution. Reweighting the learned distribution allows the model to generate less likely predic- tions some of the time. If the model consistently outputs the most likely prediction, the output can become repetitive. Conversely, a flat probability distribution where all outcomes are equally likely would be equivalent to sampling at random, invariant to the learned distribution. Allowing for occasional unexpected predictions by implementing stochastic sampling, will in many cases improve the realism of the output and elicit more interesting predictions thereby improving the perceived creative capacity of the model. Another interesting approach to vary the output of a trained model is to prime the model on different unseen movement sequences. Priming consists of running an unseen sequence through the model before generating a prediction for the next time step. [7] demonstrated interesting results using this strategy, and generated handwriting examples in various styles by priming a model on examples by dif- ferent subjects. With our priming experiments, we aim to examine to what extent the results obtained for handwriting are transferable to full-body motion. Figure 2: Marker setup worn by the dancers during motion capture data collection such our network must be able to retain the nuances in our dance recordings as real-valued time-series. While other creative data sources such as music, text and images can be simplified into a dis- crete representation, it is clear that doing so would severely reduce the expressiveness of dance data. A third type of sampling strategy is to allow sampling only from part of the trained model. For example mixture density networks, learn mixture distributions consisting of several components. These may be constrained by only allowing sampling from a certain mix- ture component. The idea has been explored by [6] in the context of world models [9], finding evidence to support the theory that each component will model different stochastic events. 1 INTRODUCTION The semantic similarity between the two would not be reflected in metrics like the euclidean distance between global positions or between joint angles in consecutive frames. Further exploration of movement similarity is outside the scope of this article. Instead of comparing generated motion se- quences to the motion capture recordings, we will use the terms novelty and realism in reference to the conceptual class of artefacts that this data belongs to, namely dance, and evaluate them by visual inspection. 2 QUANTIFYING MOVEMENT Movement of a human body may be represented as real-valued mul- tidimensional time-series data unfolding in space and time. High- precision recordings of movement may be recorded with marker- based optical motion capture technology, where each marker put on the body provides a 3D position vector over time. As mentioned, dance is a particularly complex variant of human movement, with subtle features which can communicate important information. As 3 Our dataset contains 54 one minute motion capture recordings of improvised dance performed by three female dancers. Their average age is 23 and each has more than 10 years experience within modern, jazz and ballet. Each dancer performs three one Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks Figure 3: The 43 reflective markers are translated to 22 points Figure 4: Simplified mixture distribution example. The MDRNN presented in this work learns the mean (𝜇), stan- dard deviation (𝜎) and weight (𝜋) of 4 components, each hav- ing 66 dimensions, one for each of the 22 3D points. Figure 4: Simplified mixture distribution example. The MDRNN presented in this work learns the mean (𝜇), stan- dard deviation (𝜎) and weight (𝜋) of 4 components, each hav- ing 66 dimensions, one for each of the 22 3D points. Figure 3: The 43 reflective markers are translated to 22 points representing a future possible movement. By combining a recurrent neural network (RNN) with an MDN to form an MDRNN we can make real-valued predictions based on a sequence of inputs. Figure 5 shows the model architecture of the MDRNN used in this work. The RNN consists of three layers of LSTM cells [10], known to be effective in modelling temporal sequences such as music, text and speech. The three LSTM layers contain 1024, 512 and 256 hidden units respectively. The outputs of the third LSTM layer are in turn connected to an MDN. The LSTM layers learn to estimate the mean, standard deviation and weight of the 4 Gaussian distributions. minute improvisations to six different musical stimuli. The dataset was recorded using a Qualisys optical motion capture system with 12 Oqus 300/400 series cameras which capture 43 reflective markers worn by the dancers. Figure 2 shows the front and back of a dancer wearing the motion capture suit with markers. Figure 3 shows how the 43 marker positions were reduced to a 22 point skeleton representation using the MoCap Toolbox 1.5 [3]. Small gaps in the data were spline-filled using Qualisys Track Manager 2019.3 and a 2nd degree Butterworth filter with a .03Hz cutoff was applied to remove any marker jitter. 3 Recordings in our dataset have been normalized so that the root marker (a weighted average of markers 41, 42, 6 and 7 in Figure 3) is centred at the origin where x, y, and z position is 0. Body segment lengths are averaged across the three dancers ensuring that the data is invariant to global position and individual body dimensions. The data was captured at 240Hz, and downsampled to 30Hz before model training to reduce the size of each example. The resulting 54 data tensors consist of 1800 frames (60 seconds at 30Hz) with 3-dimensional positions for each of the 22 points. To optimize an MDN, we minimize the negative log-likelihood of sampling true values from the predicted GMM for each example. A probability density function (PDF) is used to obtain this likelihood value. In our case, the GMM consists of 𝐾= 4 𝑛-variate Gaussian distributions. For simplicity in the PDF, these distributions are restricted to having a diagonal covariance matrix, and thus the PDF has the form: 𝑝(𝜃;𝑥) = 𝐾 Õ 𝑘=1 𝜋𝑘N (𝜇𝑘, Σ𝑘;𝑥) (1)fi (1) where 𝜋are the mixing coefficients, 𝜇, the Gaussian distribution centres, Σ the covariance matrices and 𝑛is the 66 position values (22 points * 3 dimensions) contained in each frame. This configura- tion corresponds to 8,540,692 parameters. The loss function in our system is calculated by the keras-mdn-layer [13] Python package which makes use of Tensorflow’s probability distributions package to construct the PDF. The model is trained using the Adam opti- mizer [11] until the loss on the validation set failed to improve for 10 consecutive epochs. Two full motion capture recordings are withheld for testing while the remaining 52 examples are split into two sets, 80% are used for training and 10% for validation. Each example is sliced into overlapping sequences of 256 frames and the spatial dimensions of each of the 22 points are scaled using min-max normalisation. The input to our model thereby consists of 80 288 overlapping sequences of 256 frames. The target value of each training example is the 3D position of each point in the following frame. 1https://github.com/benediktewallace/Motion-MDRNN 5 SAMPLING FROM THE TRAINED MODELf Mixture density networks (MDNs) [2] treat the outputs of a neural network as the parameters of a Gaussian mixture model (GMM), which can be sampled to generate real-valued predictions. Figure 4 shows a simplified mixture distribution with 4 components. A GMM can be derived using the mean, weight and standard devi- ation of each component. The number of components needed to accurately represent the data is not known and is treated as a hy- perparameter for our model. For the study outlined here, we have used 4 components. We can interpret these components as each In the following sections, three different strategies for examining the creative representational capacity of the MDRNN and their effect on the model output are explored: priming, component selec- tion and temperature adjustments. The code used to sample from the trained model can be found at our working repository together with video examples.1 Figure 5: Sampling from the MDRNN. A sequence of preced- ing frames is sent through the model which outputs the pa- rameters of a mixture distribution. By sampling from this distribution the next frame is generated. Wallace et al. For our experiments, we explored a range of sigma values be- tween 0 and 1 to locate at which temperatures the effect on the output changes. Sampling with 𝜎-temperature close to or higher than 1 results in movement sequences that change rapidly between frames. The movements are “shaky” and jump in unnatural ways between time steps. We observe that this shaking effect becomes noticeable with 𝜎> 0.05. Conversely, a 𝜎-temperature closer to 0 allows for smoother motions. An example of the extremes can be seen in Figures 6a and 6b. These figures show the trajectories of hand and toe markers, with horizontal displacement indicating time evolution. Figure 6c shows a sequence wherein the temperature rises over time. 𝜎-temperatures between 10−7 and 10−4 cause small variations in the motion sequence while remaining realistic. Higher values result in sequences which contain a fair amount of noise which can also distort the form of the body, while lower values contain less overall movement. The 𝜋-temperature adjusts the probability of sampling from individual mixture components. High 𝜋-temperatures reweight the probability of sampling from each component in such a way that each component is an equally likely choice, while sufficiently low temperatures will ensure that only a single component is selected. 5.2 Isolating mixture components For these experiments, we disregard the 𝜋-temperature and instead manually select which of the 4 mixture components to sample from. This ensures that each new frame is sampled from a single compo- nent. We observed in the previous section that the entire position of the body changed as we sampled with a higher 𝜋-temperature, indicating that individual components emphasise different features. In order to examine this more closely the 𝜎-temperature is kept at a low value to make certain that we sample close to the mean of each component and each sequence is given the same starting position. Figure 8 shows the 100th frame from sequences sampled from each of the 4 components using the same starting frames and temperature parameters. These figures show how each component has predicted a different outcome, with component 1 being the For these experiments, we disregard the 𝜋-temperature and instead manually select which of the 4 mixture components to sample from. This ensures that each new frame is sampled from a single compo- nent. We observed in the previous section that the entire position of the body changed as we sampled with a higher 𝜋-temperature, indicating that individual components emphasise different features. 5 SAMPLING FROM THE TRAINED MODELf For the experiments presented here, we examine 𝜋-temperatures between 0 and 10. By selecting a 𝜋-temperature close to or higher than 1.0 we create a higher likelihood of sampling from a dif- ferent mixture component at every time step. Sampling with 𝜋- temperature close to 0, on the other hand, ensures that only the component which has the highest learned weight will be sampled from. This is equivalent to isolating this component and sampling from it at every time step. Figure 7b shows a sequence generated with a 𝜋-temperature close to 0. Figure 7c shows how the output changes as the 𝜋-temperature is increased over time. At temper- atures around 1.0 the component with the highest weight is still sampled from most frequently but, we also intermittently sample from the other 3 components according to their respective weights. Figure 7a shows the result of sampling with a high 𝜋-temperature. Here, we sample from a different component at almost every time step. Shifting between mixture components when generating mo- tion sequences causes abrupt changes in the position of the body, alluding to the notion that each component may learn a slightly different movement sequence. We examine this more closely in the upcoming section. Figure 5: Sampling from the MDRNN. A sequence of preced- ing frames is sent through the model which outputs the pa- rameters of a mixture distribution. By sampling from this distribution the next frame is generated. Figure 5: Sampling from the MDRNN. A sequence of preced- ing frames is sent through the model which outputs the pa- rameters of a mixture distribution. By sampling from this distribution the next frame is generated. 5.1 Adjusting temperature In order to examine this more closely the 𝜎-temperature is kept at a low value to make certain that we sample close to the mean of each component and each sequence is given the same starting position. Figure 8 shows the 100th frame from sequences sampled from each of the 4 components using the same starting frames and temperature parameters. These figures show how each component has predicted a different outcome, with component 1 being the When sampling from our trained model we can choose to alter the value of two temperature parameters, the 𝜋-temperature and the 𝜎-temperature. The 𝜎-temperature is used to scale the covariance matrix of each mixture component’s multivariate normal distribu- tion. Adjusting the 𝜎-temperature affects the width of each mixture component. A high 𝜎-temperature allows for samples further from the learned mean of each mixture component to be selected. Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks (a) Movement generated with 𝜎=1.0. Movements are noisy, positions of points jerk between frames. (b) Movement generated with 𝜎= 10−9 Movements are smooth and realistic. (c) Movement generated with 𝜎-temperature rising over time. Figure 6: Sequences generated using different 𝜎-temperatures. most dissimilar to the other components. Sequences sampled from are placed at unnatural angles. From these experiments, it se Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks (a) Movement generated with 𝜎=1.0. Movements are noisy, positions of points jerk between frames. (b) Movement generated with 𝜎= 10−9 Movements are smooth and realistic. (a) Movement generated with 𝜎=1.0. Movements are noisy, positions of points jerk between frames. (a) Movement generated with 𝜎=1.0. Movements are noisy, positions of points jerk between frames. (a) Movement generated with 𝜎=1.0. Movements are noisy, positions of points jerk between frames. (b) Movement generated with 𝜎= 10−9 Movements are smooth and realistic. (b) Movement generated with 𝜎= 10−9 Movements are smooth and realistic. (c) Movement generated with 𝜎-temperature rising over time.f (c) Movement generated with 𝜎-temperature rising over time. Figure 6: Sequences generated using different 𝜎-temperatures. are placed at unnatural angles. From these experiments, it seems that each component learns a slightly different movement pattern. Thereby, selecting to sample from a single component when gener- ating a movement sequence ensures that certain learned patterns appear in the predicted movement. are placed at unnatural angles. 5.1 Adjusting temperature From these experiments, it seems that each component learns a slightly different movement pattern. Thereby, selecting to sample from a single component when gener- ating a movement sequence ensures that certain learned patterns appear in the predicted movement. most dissimilar to the other components. Sequences sampled from component 1 contain almost no movement at all, while component 2 and 3 produces examples which show the highest amount of realism. The third component consistently produces the most real- istic output and is the component with the highest learned 𝜋-value. Thereby making it the component which is most likely sampled from when no adjustments are made using the 𝜋-temperature pa- rameter. Sampling from component 1 and 4 shows little overall movement and a fair amount of distortion in the body, as joints Wallace et al. (a) Movement generated with 𝜋=10. Positions of the body jerk between frames as the we alternate between mixture components. (a) Movement generated with 𝜋=10. Positions of the body jerk between frames as the we alternate between mixture components. (a) Movement generated with 𝜋=10. Positions of the body jerk between frames as the we alternate between (a) Movement generated with 𝜋=10. Positions of the body jerk between frames as the we alternate between mixture components. (b) Movement generated with 𝜋= 10−9 Movements are smooth and realistic. (c) Movement generated with 𝜋-temperature rising over time. (b) Movement generated with 𝜋= 10−9 Movements are smooth and realistic. (b) Movement generated with 𝜋= 10−9 Movements are smooth and realistic. (b) Movement generated with 𝜋= 10−9 Movements are smooth and realistic. (c) Movement generated with 𝜋-temperature rising over time. (c) Movement generated with 𝜋-temperature rising over time. 5.3 Primed sampling When generating motion with priming, a movement sequence which has not been used in training is given as input to the model. The next frame is then generated and the process is repeated. The model always predicts the next frame for a previously unseen real sequence, as opposed to non-primed sampling, wherein the models’ previous predictions become part of the sequence used to generate the following frame. We explore the effect of priming on two per- formances by different individuals using the examples that were withheld during training. The first example, hereafter referred to as primer A, was performed to rhythmical musical stimuli with a strong beat presence. The second example, primer B, was performed to slow, non-rhythmic musical stimuli. As such, the two priming examples have differing characteristics. Primer A contains more Being able to generate motion in a certain style is useful both in the context of animation and as a creative tool for choreography. Pre- vious work using MDRNNs to generate handwriting [7] show that it is possible to produce examples which display the characteristics of a particular writer when the model is primed on an example. [4] have recently shown how the characteristics of a participant’s individual style of movement are sufficiently unique to allow for classification using machine learning and as such, it is intriguing to investigate whether priming a model on an example may allow the model to generate movements in the style of a specific participant or performance. Exploring the Effect of Sampling Strategy on Movement Generation with Generative Neural Networks (a) 1st component (b) 2nd component (c) 3rd component (d) 4th component what is feasible for the human form breaks the perceived realism of the generated movement. Sampling from a single mixture compo- nent for a given sequence and altering the amount of deviation we allow away from the mean using the 𝜎-temperature instead can give varied and realistic results. Slight adjustments of 𝜎-temperature allows for some interesting variations to occur. This could have use- ful applications as a user-controlled parameter, both in a creative tool and for animation. When examining the output generated by each mixture component in isolation it is apparent that the compo- nents which achieve the highest learned weight also produce the most realistic movement sequences. 7 CONCLUSION We have presented results from exploring several common sam- pling techniques used when generating output from MDRNNs and how these techniques affect generated dance movement sequences. Our findings show that the priming technique allows the output to be shaped by an unseen example, indicating that the model could be used to generate movement sequences in a certain style. Further, the results show that changing the learned probability distribution by including temperature parameters has the potential to greatly affect the output. However, the range of viable parameter values is small, as any unnatural movement is easily detected by an ob- server. As such, temperature adjustment may be less suited for human motion than for other creative data. Alternating between mixture components when generating a motion sequence results in unnatural shifts in body positions between consecutive frames. Still, the components with lower learned weight may also contain inter- esting movement sequences. Therefore, we believe this approach warrants further exploration and analysis. movement overall and a higher speed of movement, while primer B consists of slower movements. For these experiments 𝜋and 𝜎- temperatures are both set to values close to zero ensuring that we sample from close to the mean of the component with the highest weight, component 3. Figure 9a shows a series of frames from primer A. Figure 9b shows the corresponding movement sequence generated using primer A. As can be seen in the figure, the generated movement to a large degree follows the movement of the priming sequence. Movement aspects such as the speed of motion and the overall amount of movement are similar to the priming sequence as well. Similar results were also found for primer B. Figure 8: 100th frame from sequences generated by differ- ent mixture components. Keeping all other parameters the same, each component generates a different movement se- quence. Figure 8: 100th frame from sequences generated by differ- ent mixture components. Keeping all other parameters the same, each component generates a different movement se- quence. 5.3 Primed sampling It is possible that training on a larger dataset would cause the components which now show a lot of distorted body poses to learn more realistic and useful features. This would then be an interesting parameter in a creative tool in order to create variations on a theme. Examining the output from each component also shows us the capabilities of this particular model to produce several possible futures with different degrees of variation and realism. (b) 2nd component (a) 1st component (c) 3rd component (d) 4th component (c) 3rd component From our experiments with priming, we found that the output of the model was able to continue the motion of unseen examples with some variation. This indicates that the style of movement is indeed continued when sampling with priming. Priming the model on unseen data could be useful when using the model as a creative tool to assist choreographers and animators, as it allows for priming the model with their own data and achieve predictions in a desirable style closer to their own. By combining priming and 𝜎-temperature one could produce several variations on a theme. (c) 3rd component (d) 4th component ACKNOWLEDGMENTS [10] Sepp Hochreiter and Jürgen Schmidhuber. 1997. Long Short-term Memory. Neural computation 9 (12 1997), 1735–80. https://doi.org/10.1162/neco.1997.9.8.1735 This work was partially supported by the Research Council of [11] Diederik P Kingma and Jimmy Ba. 2014. Adam: A method for stochastic opti- mization. arXiv preprint arXiv:1412.6980 (2014). This work was partially supported by the Research Council of Norway through its Centres of Excellence scheme, project number 262762. p p [12] Jina Lee and Stacy Marsella. 2006. Nonverbal Behavior Generator for Embodied Conversational Agents. In Proceedings of the 6th International Conference on Intelligent Virtual Agents (Marina Del Rey, CA) (IVA’06). Springer-Verlag, Berlin, Heidelberg, 243–255. https://doi.org/10.1007/11821830_20 6 DISCUSSION In the above results, we have explored how the output of the trained MDRNN can be affected using different sampling strategies. There are, of course, countless parameter settings and combinations to examine. Here, we have focused on outlining the extremes as well as the threshold points wherein the parameters effect changes. In future work, we will perform a study wherein trained dancers evaluate the generated movement sequences [22] using rating schemes [19] designed to evaluate the representational and cre- ative capacity of the model. This work is also part of an ongoing research effort to examine how generative deep learning can be used to capture salient features of human movement, and especially dance movement, using full-body motion capture data. Dance and music are intrinsically intertwined, and as such, we will be includ- ing the music stimuli in the training data in order to produce a sound-to-movement system. When sampling from only the component with the highest weight, we ensure that the model outputs smooth and realistic motion sequences. While stochastic sampling of mixture compo- nents is possible, our results indicate that this approach, at least for movement, does not necessarily improve the output. 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https://openalex.org/W4285589804	https://openaccess.sgul.ac.uk/id/eprint/114793/6/Musculoskeletal%20Care%20-%202022%20-%20McIlroy%20-%20The%20experiences%20and%20acceptability%20of%20a%20novel%20multimodal%20programme%20for%20the.pdf	English	null	The experiences and acceptability of a novel multimodal programme for the management of fibromyalgia: A qualitative service evaluation	Musculoskeletal care	2,022	cc-by	8,419	S E R V I C E E V A L U A T I O N S E R V I C E E V A L U A T I O N Received: 30 June 2022 - Accepted: 2 July 2022 DOI: 10.1002/msc.1672 wileyonlinelibrary.com/journal/msc - 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, pro- vided the original work is properly cited. © 2022 The Authors. Musculoskeletal Care published by John Wiley & Sons Ltd. Suzanne McIlroy1,2 \| Bethany Vaughan3 \| Heather Crowe3 \| Lindsay Bearne3,4 Suzanne McIlroy1,2 \| Bethany Vaughan3 \| Heather Crowe3 \| Lindsay Bearne3,4 1Physiotherapy Department, King's College Hospital NHS Foundation Trust, London, UK 2Health Psychology Section, Psychology Department, King's College London, London, UK 3School of Population Health and Environmental Sciences, King's College London, London, UK 4Population Health Research Institute, St George's University of London, London, UK Correspondence Suzanne McIlroy, Physiotherapy Department, King’s College Hospital NHS Foundation Trust, London, UK. Email: Suzanne.mcilroy@nhs.net 1Physiotherapy Department, King's College Hospital NHS Foundation Trust, London, UK 2Health Psychology Section, Psychology Department, King's College London, London, UK 3School of Population Health and Environmental Sciences, King's College London, London, UK 4Population Health Research Institute, St George's University of London, London, UK Correspondence Suzanne McIlroy, Physiotherapy Department, King’s College Hospital NHS Foundation Trust, London, UK. Email: Suzanne.mcilroy@nhs.net Abstract Introduction: Fibromyalgia guidelines recommend multi‐modal, non‐pharmacolo- gical interventions but there is limited evidence on the optimal programme. The Fibromyalgia Active Management and Exercise programme (FAME) aimed to improve function and quality of life. It consisted of 12 sessions delivered by a multidisciplinary team and incorporated education, exercise, Cognitive Behavioural Therapy and mindfulness approaches. S E R V I C E E V A L U A T I O N 1 \| INTRODUCTION treating clinician (e.g. physiotherapists, rheumatologists, and pain team clinicians). Fibromyalgia is a chronic disorder characterised by widespread persistent pain with a broad spectrum of symptoms including fatigue, irritable bowel syndrome, difficulty concentrating and low mood (Arnold et al., 2019). These symptoms reduce quality of life by impacting physical and psychological functioning, personal relation- ships and ability to undertake activities of daily living (Bennett et al., 2007; Skaer, 2014) resulting in high personal, healthcare and societal burden (Boonen, 2004; Soni et al., 2020). For a programme to be successfully implemented it must be acceptable to both the participants and the healthcare practitioners (HCP) delivering the programme. Acceptability is defined as a “Multi‐ faceted construct that reflects the extent to which people delivering or receiving a health care intervention consider it to be appropriate, based on anticipated experiential cognitive or emotional responses to the intervention” (Sekhon et al., 2017). Previous studies have measured acceptability from a quantitative perspective for example, drop‐out rates (Bernardy et al., 2018; Häuser et al., 2011) and, whilst important, does not provide a deep understanding of the important issues and possible solutions. The Theoretical Framework of Acceptability (TFA), considers acceptability to consist of seven con- structs (Affective Attitude; Burden; Ethicality; Intervention Coher- ence; Opportunity Costs; Perceived Effectiveness; Self‐efficacy) (Sekhon et al., 2017) that may be assessed quantitatively and quali- tatively to gain a comprehensive measure of acceptability. Robustly evaluating acceptability of FAME, from the perspective of people who receive and deliver it is an essential initial component before the scalability and sustainability is considered (Klaic et al., 2022). Fibromyalgia affects approximately 3%–7% of the general pop- ulation (Collin et al., 2017; Jones et al., 2015). More women than men are affected (female:male ratio 3:1) (Queiroz, 2013). The patho- physiology of fibromyalgia is poorly understood. It is proposed that symptoms are caused by disordered pain regulation with associated neuroendocrine changes in the central and peripheral nervous sys- tem. Physical and emotional trauma, particularly in childhood or adolescence, stressful life events and psychosocial conditions (e.g. depression, lower socioeconomic status and education levels) may be predisposing factors (Ghavidel‐Parsa & Bidari, 2021). Management of fibromyalgia aims to reduce symptoms and in- crease function (Macfarlane et al., 2016). 1 \| INTRODUCTION Multi‐modal, non‐ pharmacological interventions are recommended (Macfarlane et al., 2016; Pearson et al., 2021; UK, 2021) due to the diverse symptoms of fibromyalgia and limitations of single treatment modal- ities (Bernardy et al., 2010; Bidonde et al., 2019; Wang et al., 2018). A recent systematic review including 13 studies suggests that multi- disciplinary interventions that incorporate education, exercise and psychological approaches to support self‐management may be effec- tive (Llàdser et al., 2021). Yet there is paucity of evidence and lack of consensus on optimal content, format and mode of delivery of in- terventions (Bidonde et al., 2019; Geraghty et al., 2021). Additionally, physiotherapists and other healthcare workers report frustration and helplessness when managing people with fibromyalgia (Briones‐Voz- mediano et al., 2013). This leads to role ambiguity, lack of confidence and reluctance to treat people with fibromyalgia (Roitenberg & Shoshana, 2021). New models of care are required (Doebl et al., 2021). The study aimed to explore patients and healthcare pro- fessionals' experiences of the FAME programme, drawing on the theoretical framework of acceptability and identify areas of pro- gramme refinement. 2 \| METHODS The study is reported in accordance with the Consolidated criterion for reporting qualitative research checklist (COREQ) (Tong et al., 2007). 3.1 \| Study design This service evaluation included qualitative interviews and focus groups with attendees and healthcare professionals (HCP) delivering the FAME programme at one NHS foundation Trust in the United Kingdom. 3 \| ETHICAL APPROVAL Ethical approval was not required but the protocol registered by King's College Hospital service evaluation and audit committee. In 2019 The Fibromyalgia Active Management and Exercise programme (FAME) was developed at one NHS foundation Trust in London. FAME was a 12‐week group outpatient programme (12 people/group) that aimed to improve function and quality of life by facilitating self‐management and physical activity in people with fi- bromyalgia. It was designed and refined by a multi‐disciplinary clin- ical team. The format and duration was adapted from a successfully running 12‐week programme for people with persistent low back pain with the content adapted following a literature review (Busch et al., 2013; Karjalainen et al., 1999; Lee et al., 2014; Macfarlane et al., 2016). It consisted of weekly two‐hour interactive sessions delivered by multidisciplinary healthcare professionals. Each session focussed on a different topic such as exercise, stress management (e.g. mindfulness), and was underpinned by behaviour change ap- proaches such as Cognitive Behavioural Therapy (supplement file 1). Eligible patients (supplement file 2) were enroled onto FAME by their Abstract This qualitative service evaluation explored the experience and acceptability of FAME from the perspective of the patients' and healthcare practitioners' (HCP). Methods: All patients and HCP involved in the first FAME programme were invited to attend either one audio‐recorded focus group or an individual semi‐structured interview. Topic guides were developed a priori. Data were transcribed verbatim and analysed thematically. Results: Thirteen participants (six HCP (three physiotherapists, two nurses, one psychologist)) and seven patients (mean age 46 (7.5) years, all female,) were enroled. FAME was acceptable to HCP participants but not to all patient participants. Where patient participants understood and anticipated the aims of FAME, the programme was found to be acceptable. Whereas, patient participants who did not fully un- derstand the aims of the programme reported lower acceptability. Three themes were generated: expectations and preparation for FAME, the value of social support, and FAME as a learning opportunity. The themes could be explained by five constructs of the Theoretical Framework of Acceptability. Conclusion: FAME was acceptable to HCPs but not to all patient participants. Pa- tient and HCP participants valued social support and regarded this as central to their learning. Further adaptation of FAME is required to optimise acceptability. acceptability, exercise, fibromyalgia, physiotherapy, qualitative, self‐management This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, pro- vided the original work is properly cited. Musculoskeletal Care. 2022;1–11. 2 - MCILROY ET AL. 2 5.1 \| Findings Thirteen patients and seven HCPS were invited to take part in this study between March and July 2019. Seven patient participants (all female, age range between 32 and 56 years) and six HCP participants (four female) attended one of two 60‐min focus groups (one patient; one HCP) or a 30‐min semi‐structured interview (two telephone in- terviews with patients, one face‐to‐face interview with an HCP). Table 1 contains the participant demographics. 4.2 \| Research team and reflexivity The research team comprised a female Consultant Physiotherapist involved in the development of FAME (SMc), and two female student MSc Pre‐registration physiotherapy students (BV, HC). None of the researchers were involved with the clinical management of any interviewee or the delivery of the FAME programme. 5.2 \| Themes Overall patient participants reported mixed views about the FAME programme. Most patient participants appreciated some aspects of FAME, particularly the opportunity to meet other people with fibro- myalgia but revealed that, at times, the FAME programme did not make sense to them or align with their expectations of treatment. This affected their experience of the programme, willingness to engage with the programme content and satisfaction with the programme. 5 \| DATA ANALYSIS Interviews and focus groups followed a topic guide developed a priori, by the authors, and informed by previous literature (Bearne et al., 2017; Galea Holmes et al., 2017) and the Theoretical Frame- work of Acceptability (Sekhon et al., 2017) (supplementary file 3). The topic guides were reviewed and refined in consultation with a Psy- chologist and piloted in a focus group of student healthcare pro- fessionals. No changes to the guide were made. This data was not included in the final analysis. An inductive thematic analysis approach was conducted (Braun & Clarke, 2006). This six‐stage approach comprised of (i) familiarisation with text; (ii) coding within the computer package NVIVO 11 (QSR International Ltd., Southport, UK); (iii) categorisation of codes and (iv) building of themes and subthemes; (v) defining and naming of themes; (vi) identification of extracts and quotes to illustrate themes. BV and HC were responsible for the primary coding. The initial themes were developed in consultation with SMc and reviewed with the research team to discuss how they related to the aims and theoretical framework of acceptability (SMc, LB). A summary of the main findings were emailed to a sub‐sample of the participants (n = 3) to review, check for resonance and add to the interpretation of the findings. Participants were asked open‐ended questions about their expectations, experiences, and suggestions for refinements for the FAME programme. Core questions were included in all focus groups and the opportunity to explore relevant but unanticipated areas and reflections were provided. The focus group facilitator or interviewer used probes to encourage participants to express their views. The focus group facilitator ensured all participants had the opportunity to add their views to the discussion by inviting com- ments from each attendee, if needed. Field notes were made during each focus group by an observer and after each focus group and interview by the facilitator/interviewer to provide contextual information. participate in this qualitative service evaluation. They were provided with written information about the purpose of the evaluation and invited to attend either one of two focus groups (1 patient, 1 HCP group) at the NHS Hospital Foundation Trust. All recordings were audio‐recorded, transcribed verbatim by an external transcription agency (patient focus group) or by the re- searchers, anonymised, and checked for accuracy against the original recordings by the researchers. Data generation ceased when all participants willing to participate had been included. If patients or HCPs were unable to attend a focus group, they were offered an individual semi‐structured telephone interview (face‐to‐face or via telephone dependent upon participant prefer- ence) with one researcher. 4.1 \| Participants and recruitment All attendees at the first FAME group cohort and HCPs that contributed to the delivery of the programme were invited to MCILROY ET AL. 3 4 - MCILROY ET AL. Duration of Symptoms (Years) Number of sessions attended Health Care Professional participants Health Care Professional participants Participant Code Sex Ethnicity Profession Experience working in the profession (Years) Experience working with patients with persistent pain (Years) HCP1 Male White British Nurse 30 25 HCP2 Male White British Psychologist 10 10 HCP3 Female White British Physiotherapist 5.5 3.5 HCP4 Female White British Physiotherapist 9.5 9.5 HCP5 Female White British Physiotherapist 6 8 HCP6 Female White British Nurse 20 17 Abbreviation: HCP, Health care professional. Abbreviation: HCP, Health care professional. 4.3 \| Data generation Participants completed a sociodemographic questionnaire that included age, gender, ethnicity (self‐reported and categorised based on UK census categories (Office for National Statistics, 2021), duration of symptoms (patient participants only) and/or profession and duration of experience working with patients with persistent pain (HCP participants only)). All focus groups were conducted by one of two lead facilitators (SMc or HC) and attended by two other researchers, one acted as a co‐facilitator and the other as an observer who made field notes. All interviews were conducted by one of two researchers (BV, HC). The participants were aware of the research teams backgrounds. All HCP participants reported that FAME was acceptable to them, although they perceived that further discussion with patient participants prior to referral to the programme, alongside minor changes to the programme, could optimise its acceptability and effectiveness for some patient participants. Three themes were identified (expectations and preparation for FAME, the value of social support, and FAME as a learning opportunity, Figure 1). T A B L E 1 Participant sociodemographic and clinical characteristics Patient participants Participant code Age (Years) Ethnicity Duration of Symptoms (Years) Number of sessions attended P1 52 Other mixed 9 9 P2 45 White British Unknown 12 P3 47 Black British ‐ Caribbean Unknown 8 P4 48 Other: Ecuadorian 9 10 P5 44 White British 18 10 P6 56 Black British ‐ Caribbean 14 7 P7 32 Black British ‐ African 8 6 Health Care Professional participants Participant Code Sex Ethnicity Profession Experience working in the profession (Years) Experience working with patients with persistent pain (Years) HCP1 Male White British Nurse 30 25 HCP2 Male White British Psychologist 10 10 HCP3 Female White British Physiotherapist 5.5 3.5 HCP4 Female White British Physiotherapist 9.5 9.5 HCP5 Female White British Physiotherapist 6 8 HCP6 Female White British Nurse 20 17 Abbreviation: HCP, Health care professional. 4 - MCILROY ET AL. 4 - group setting because then it interferes with other people – HCP3 All HCP participants reflected on the importance of preparing potential attendees for FAME to ensure they were able to benefit from FAME. HCPs suggested that additional pre‐programme psy- chological assessment may be helpful to identify those potential attendees that were most likely to respond to the programme however, they reported that they may not have the resources to do this. It was also proposed that written information about FAME or a taster session followed by an active ‘opt‐in’ requirement may help potential attendees to understand the aims, format and content, and to ‘buy‐in’ to FAME. HCP and patient participants agreed that lack of written information hindered the coherence of FAME and patient participants felt that they didn't know what to expect and so were not able to fully engage or prepare for sessions. you kind of need to get them sort of geared up to what the class is going to offer, because I find if you, you kind of look down a list and see they’ve got fibromyalgia, … and you say oh we’ve got this class do you want to go to it? Without giving them any background or sort of prepping them for it, then they aren’t ready to take on the change or accept change. –HCP5 you kind of need to get them sort of geared up to what the class is going to offer, because I find if you, you kind of look down a list and see they’ve got fibromyalgia, … and you say oh we’ve got this class do you want to go to it? Without giving them any background or sort of prepping them for it, then they aren’t ready to take on the change or accept change. –HCP5 F I G U R E 1 The inter‐related themes of: expectations and preparation for FAME, the value of social support, and FAME as a learning opportunity I don’t actually think that like ‐P3 HCP participants unanimously expressed attendees at FAME should be informed about the programme aims and content prior to starting the programme so that they were prepared to engage with new perspectives and treatment options. They considered that this discussion and shared treatment decision making was appropriate regardless of duration since diagnosis. HCP participants perceived that if FAME attendees were not willing to engage with the new perspectives offered by the programme or ready to change their management approach, then FAME may not be suitable for, or acceptable to them. HCP participants were concerned that attendees who were not willing to engage with the programme content influ- enced the group interactions and potentially undermined the delivery of the programme and how it was received by others. 5.4 \| Theme 2: The value of social support So, if someone’s newly diagnosed, new to all of this, … [it] will be of benefit and will educate and help those people. People that have had it ten/twenty years plus have already covered everything ‐P5 FAME offered participants the opportunity to receive support from other people with lived experience of their condition. Both patient and HCP participants recognised the value of peer support. However, joining a group of people with the same condition was a new expe- rience for most patient participants, and they valued the opportunity to share their experiences of fibromyalgia. Patient participants re- ported that they felt understood by peers and gained a sense of belonging. Patient participants reported that their experience of peer support affected their attitude towards FAME. This influenced their attendance at FAME sessions as they valued the emotional support from their peers. These established friendships continued after the FAME programme finished as part of an ongoing independent peer support network. F I G U R E 1 The inter‐related themes of: expectations and preparation for FAME, the value of social support, and FAME as a learning opportunity group setting because then it interferes with other people – HCP3 5.3 \| Theme 1 expectations and preparation for FAME I got what I expected and that was to be in a community around people that understood me and what I was going through and we could share our pain and suffering and not feel like we were misunderstood‐ P1 Patient participants' expectations of FAME varied and were influ- enced by their perception of the aims and content of the programme. Some patient participants had little idea of what to expect of the programme or their expectations did not align with their experiences of the programme. These participants explained that FAME did not make sense to them and did not meet their needs or answer their questions about their condition. Some patient participants suggested that their referral to programme was not a shared treatment deci- sion, and they did not regard FAME as an acceptable treatment for them. I just really wanted to see, for me, what is it that they can offer in regards to relief. Not necessarily just tablets. What I could get out of it in regards to exercise groups and you know like therapy groups –P6 The need to align FAME attendees understanding of their con- dition, their needs and treatment expectations with the FAME pro- gramme content was recognised by all patient participants. However, patient participants had mixed views whether people with newly diagnosed fibromyalgia would benefit more than those with long- standing fibromyalgia. Some patient participants recommended that the programme was suitable for people regardless of the duration of their condition fibromyalgia whereas one patient participant recom- mended that those newly diagnosed would benefit more. I generally was sent here because we was hoping I’d get some answers to some things that was going on with my health but unfortunately I didn’t get that ‐P3 In contrast, some patient participants anticipated the format and content of the programme and welcomed the opportunity to share their experiences with other patient participants. In these patient participants FAME met their needs and expectations by offering in- formation, support, and complementary management techniques You know, you’ve got to work out… what your target group is. You can’t [have] newbies and oldbies in the same group and expect them to achieve the same thing ‐P5 You know, you’ve got to work out… what your target group is. You can’t [have] newbies and oldbies in the same group and expect them to achieve the same thing ‐P5 MCILROY ET AL. 5.3 \| Theme 1 expectations and preparation for FAME 5 5.5 5.5 FAME was recognised as a learning opportunity for most partici- pants. Patient participants learnt about their condition, the spectrum of symptoms and new ways of managing these. HCPs participants reported that working within a new multi- disciplinary team meant that they developed their understanding of Fibromyalgia, new ways of communicating concepts to attendees and about wider services available for people with fibromyalgia. For HCPs participants, working with this patient group provided other areas of learning, for example, managing challenging group dynamics and being able to address preconceptions about living with the condition. I think I more come because I’d made friends and it can be very lonely having fibro – P5 However, some patient participants reported that their peer support network was threatened by inconsistent attendance of some attendees and that this affected their sense of a shared community. Whilst social support from the FAME group was highly valued by patient participants, some patient participants identified that the programme did not completely remove the need for individual treatments to help them manage specific symptoms. they need to … be open to trying that more kind of psy- chological approach and treatment and kind of learn strategies for managing things, otherwise you just get those constant barriers in the way which is just not helpful in a 6 - MCILROY ET AL. HCP's also valued peer support from the multi‐professional group of FAME facilitators. This increased their confidence to deliver the FAME programme and manage challenging situations that developed. some HCPs did not get to know their individual needs and sometimes completed sessions in a perfunctory manner. sometimes it would be somebody completely different who may or may not have prior knowledge of the group as well and I think that’s the difficulty in delivering this kind of programme. I think one of the key things is that there has to be a consistent staff group ‐HCP6 I find just having the support from the MDT [multidisci- plinary team] … it was quite nice to have a bit of a debrief, either via email with everyone or after the class. –HCP5 Both HCP and patient participants felt that some attendees negatively influenced the group sessions and overpowered other group members. HCP participants found this difficult to moderate and address the perceived ‘unrelentingly’ challenges about the session content. Some patient participants reported that this conflict marred their experience of FAME and reduced their engagement with, and the acceptability of, the programme. One patient participant considered the HCPs to be overwhelmed by the FAME attendees who challenged the FAME content and management approach. Attendee selection, expectation setting and agreeing clear group rules were proposed by the HCP participants to support positive group dynamics and learning. there were some weeks when I felt overpowered by particular people moaning and that wasn’t productive‐ P6 Both groups of participants found the core components of FAME coherent (education, exercise, and mindfulness) but the value they attributed to each component varied. They recommended including an expert patient in future iterations of the programme to further enhance the opportunity for learning and the effectiveness of FAME. Inviting family members to attend FAME was viewed positively by patient participants. They reported that this allowed family members to gain an insight into what it is like to live with fibromy- algia and learn how they can provide support at home. Patient par- ticipants perceived that this increased the effectiveness of FAME. Conversely, some HCP participants considered family members attending the FAME sessions burdensome because they asked questions about themselves and their own health conditions. Some HCP participants reported that this was distracting and added complexity to facilitating the group discussions. To address this issue, one HCP identified that guidance on family member attendance and reiteration of the group's rules at each session may resolve this issue. I think there’s that old thing isn’t it, walk a mile in my shoes and unless you live it… as we’ve said we’re learning it from each other as well, so where are you going to get that in- formation from? It’s from the people that live it.‐ P3 Some of the patient participants appreciated learning from the HCPs and valued their professional advice and ability to tailor the content to their individual needs. However, other patient participants reported that some staff were not able to tailor information in the group setting. This affected their attitude towards and perceived effectiveness of FAME. Patient participants views on the support provided by the HCPs varied. Some patient participants reported HCPs worked with/ alongside them to understand and address any problems they had and that this increased their self‐efficacy to self‐manage. One patient participant reported that FAME taught everyone to exercise in the best way that suits you and [that you] were comfortable whilst doing the exercises. So that was really helpful – P7 in the best way that suits you and [that you] were comfortable whilst doing the exercises. So that was really helpful – P7 Universally, patient participants found that learning about, and practising, mindfulness was useful to help manage the pain and stress associated with fibromyalgia and valued the time spent on this during the programme. HCP participants agreed that mindfulness training was a valuable part of the programme as it provided patients a self‐ management technique and was an activity that everyone was interested and participated in. One participant suggested that mindfulness provided a way of getting a sense of control also, controlling your mind… and not letting the pain take over … it kind of does help you know ‐ P7 a way of getting a sense of control also, controlling your mind… and not letting the pain take over … it kind of does help you know ‐ P7 Patient participants reported that support from peers and family and friends influenced their attendance at FAME (i.e. perceived emotional and/or instrumental (practical) support) and that the programme influenced their confidence to tackle challenging situa- tions and treatments (e.g. physical activity). It is proposed that social support influences self‐efficacy (the confidence of the HCP or patient of their capacity to perform necessary behaviours and activities) and this could affect the acceptability of FAME. Informational support from the HCPs was also highlighted by some patient participants as important, although inconsistent staffing compromised the thera- peutic relationship at times. patient participants considered that exercise was too burdensome and reported that it exacerbated their symptoms. For example, one patient participant stated that patient participants considered that exercise was too burdensome and reported that it exacerbated their symptoms. For example, one patient participant stated that not what they expected, and this influenced their experience and acceptability of the programme. The coherence (the extent an indi- vidual understands the intervention, its contents and how it works) and perceived effectiveness (anticipated and experienced extent to which the intervention is likely to/has achieved its aims) of FAME were linked. I did do it [exercise] and it nearly killed me after that. I couldn’t walk from there to there.‐P3 I did do it [exercise] and it nearly killed me after that. I couldn’t walk from there to there.‐P3 Expectations have been found to be predictors of treatment re- sponses for many conditions including heart disease (Barefoot et al., 2011), surgery (Auer et al., 2016) and low back pain (Hayden et al., 2019). People with more positive treatment expectations are more likely to benefit from treatment (Laferton et al., 2017). Partici- pants who understood and anticipated the aims and content of FAME reported that the programme was broadly acceptable and effective. This view was shared by all HCP participants but not the patient participants. Over ambitious expectations have been found to be correlated with reduced satisfaction with treatment outcomes (Mannion et al., 2009) and health outcomes (Oettingen, 2012). Therefore, the expectations about treatment response need to be in line with the aims of the FAME programme rather than ideal expec- tations or fantasies (Laferton et al., 2017). In addition, expectations regarding structural and process‐related aspects of the treatment are also likely to influence treatment coherence and acceptability (Laferton et al., 2017). This was reflected by some patient participants who expressed the desire for individual treatment approaches, were challenging FAME sessions, inconsistent staffing and lack of pro- gramme workbook. Thus, streamlining administrative processes, ensuring attendees understand the aims and content of FAME and are active participants in the treatment decision making process may optimise the programme acceptability and treatment outcomes. In contrast, other patient participants found exercise was a positive experience and found attending FAME made exercise accessible to them. They reported that they would like more exercise within FAME. thinking a little bit more about what we need as human beings, as a person in different needs‐ P4 I was thinking that I was, I would be able to sort my problem because he was beside me– P4 Some patient participants found the breadth topics and the general nature of the content did not meet their needs and wanted more individual support. Conversely, some patient participants rec- ognised that not all topics were relevant to them but were willing to engage with FAME until they could identify topics that were useful. For example, the experience of and enthusiasm for exercise varied between patient participants. Some patient participants reported that they were pushed to complete exercise, and they did not un- derstand why exercise was such a key component of FAME. These In other cases, patient participants felt HCPs did not understand their individual needs and revealed that a more individualised approach would aid the effectiveness and the acceptability of the programme. Both participant groups identified that different staff taking each session within one FAME programme negatively affected the therapeutic relationship. HCPs acknowledged that consistent staffing was important for continuity and effectiveness of the pro- gramme. Patient participants reported that changing staff meant that MCILROY ET AL. 7 6 \| DISCUSSION This is one of the first qualitative service evaluations to explore the experiences and the acceptability of FAME from the perspective of both the patients with fibromyalgia and HCP. The HCP participants broadly viewed FAME positively whereas the patients' opinions were more diverse and influenced by their expectations of FAME. The perceptions and acceptability of the programme were explained by three themes: expectations and preparation for FAME, the value of social support, and FAME as a learning opportunity. This study confirms the importance of social support for people with long term conditions (Bearne et al., 2020). People with fibro- myalgia experience low levels of social support and significantly more loneliness than people with other Rheumatic diseases (Kool & Gee- nen, 2012). Lack of validation of their condition or illness legitimacy has been proposed to be interconnected to their physical symptoms with detrimental effects upon their illness and disability (Ghavidel‐ Parsa & Bidari, 2021). In contrast, high quality social support net- works have been associated with improved psychological wellbeing and increased levels of self‐efficacy for function and symptom man- agement (Franks et al., 2004). Thus, FAME helped provide validation and support and helps explains why the role of social support was considered a crucial beneficial aspect of the programme. Our findings The Theoretical Framework of Acceptability (TFA) (Sekhon et al., 2017) can be used to understand the acceptability of the FAME programme from both patient and HCP participant perspectives. The framework comprises seven constructs to assess acceptability and we have used five of these constructs to guide our interpretation of the results of this study (Table 2). Affective attitude (how an individual feels about the intervention before or after taking part) was broadly positive for most HCP par- ticipants, who valued the aims, format and content of FAME and perceived that most patient participants benefitted from attending the programme. However, patient participants expressed mixed views. Crucially, some participants reported that the programme was MCILROY ET AL. 6.1 The FAME programme is a new multi‐model programme for people with fibromyalgia. With some refinement, it provides a feasible and acceptable group‐based intervention that potentially improves self‐ management and health outcomes of people with fibromyalgia. Possible refinements to the programme include preparing participants for FAME to ensure their expectations align with the programmes aims and including an introduction session with active ‘opt‐in’ element; including an expert patient to facilitate learning opportu- nities; offering complementary individualised treatment components; setting and emphasising clear ground rules for expected behaviour within the group; supplementary HCP training on facilitating groups; ensuring continuity of staffing throughout the programme; and re- sources, such as an workbook, to facilitate understanding and engagement with FAME. In addition, a programme review group, with patients with a range of disease duration, is recommended. It was beyond the scope of this qualitative service evaluation to analyse costings of FAME. Future service evaluations should consider this and explore the duration of the 12‐week programme. Learning about mindfulness, but not the educational or exercise components, was universally valued by patient participants. Mind- fulness has been demonstrated to be acceptable and beneficial in terms of pain, awareness, sleep, attention and wellbeing, in people with persistent pain and the evidence for fibromyalgia is promising (Haugmark et al., 2019). However, some participants commented on the high burden (the amount of effort that was required to partici- pate) of some programme components (e.g. the perceived effort to exercise) and other patient participants reported they were already very active and this diminished the acceptability of the programme. The education components were viewed by some patient par- ticipants as too generalised and a more individualised approach required. Individualised treatment approaches have been found to be valued in other persistent pain conditions (Lin et al., 2020; Wilson et al., 2016) and could be considered as an adjunct to FAME to optimise perceived effectiveness and acceptability. A recent sys- tematic review found the best multi‐disciplinary treatments for fi- bromyalgia contained education, physical activity, pharmacology, cognitive behavioural therapy, and exercise programs with stretches 6 \| DISCUSSION T A B L E 2 Interpreting the experiences of attending FAME using the Theoretical Framework of acceptability Construct Theme Explanation Affective attitude Expectations and preparation for FAME Anticipated affective attitude: expectations of and how an individual feels about taking part ahead of FAME The value of social support; FAME as a learning opportunity Experienced affective attitude: how an individual feels about the programme, after participating in FAME Burden The value of social support; FAME as a learning opportunity Experienced burden: the amount of effort that was required to participate for example, the exercise component or the difficulty managing challenging group dynamics Intervention coherence Expectation and preparation for FAME; FAME as a learning opportunity The extent to which an individual understands the FAME programme, its contents and how it works that is, its aims and the contents of the programme Perceived effectiveness Expectation and preparation for FAME; The value of social support; FAME as a learning opportunity Anticipated and experienced extent to which FAME is likely to/has achieved its aims Self‐efficacy Expectation and preparation for FAME; The value of social support; FAME as a learning opportunity The patient or the HCP confidence of their capacity to perform the behaviours required by FAME Abbreviation: FAME, Fibromyalgia active management and exercise programme. The patient or the HCP confidence of their capacity to perform the behaviours required by FAME and aerobic exercise (Llàdser et al., 2021). Whilst this reinforces the theme expectations and preparation for FAME, refinement of FAME may be necessary to make the delivery of components feasible and more acceptable. are concordant with a qualitative study on peer support in women with fibromyalgia (Sallinen et al., 2011) who found that peer‐support provided an impetus for reconstruction of identity, acceptance and coping with fibromyalgia. Whilst education (informational support and learning) can reas- sure patients, legitimise symptoms and support self‐management (Rooks et al., 2007), learning from peers was seen as more credible by some of our patient participants than learning from HCP. This is corroborated by a qualitative study including 11 fibromyalgia pa- tients found that sharing information with peers improved the speed at which patients could access useful information about the condition by explaining things in an understandable and relatable way (van Uden‐Kraan et al., 2008) potentially explaining why learning from peers was such a valuable part of FAME to participants. 8 \| CONCLUSION Barefoot, J. C., Brummett, B. H., Williams, R. B., Siegler, I. C., Helms, M. J., Boyle, S. H., Clapp‐Channing, N. E., & Mark, D. B. (2011). Recovery expectations and long‐term prognosis of patients with coronary heart disease. Archives of Internal Medicine, 171(10), 929–935. https://doi.org/10.1001/archinternmed.2011.41 FAME is a new multi‐modal management programme for people with fibromyalgia. It was broadly acceptable for HCPs and some patient participants. Expectations and preparation for FAME, social support and the opportunity to learn about fibromyalgia and new treatment approaches all influenced the acceptability of FAME. Adaptation, including working collaboratively with stakeholders, is required to optimise acceptability for people with fibromyalgia. Bearne, L. M., Bieles, J., Georgopoulou, S., Andrews, J., Tully, A., Stolarchuk‐ Prowting, K., Williamson, T., Suarez, B. S., Nel, L., D’Cruz, D., & Lempp, H. (2020). Fatigue in adults with primary antiphospholipid syndrome: Findings from a mixed‐methods study. Lupus, 29(8), 924–933. https:// doi.org/10.1177/0961203320928421 Bearne, L. M., Manning, V. L., Choy, E., Scott, D. L., & Hurley, M. V. (2017). Participants' experiences of an Education, self‐management and upper extremity eXercise Training for people with Rheumatoid Arthritis programme (EXTRA). Physiotherapy, 103(4), 430–438. https://doi.org/10.1016/j.physio.2016.12.002 Robustly evaluating acceptability of FAME from the perspective of people who receive and deliver it is an essential initial component before the scalability and sustainability is evaluated. Bennett, R. M., Jones, J., Turk, D. C., Russell, I. J., & Matallana, L. (2007). An internet survey of 2,596 people with fibromyalgia. BMC Musculoskel- etal Disorders, 8(1), 27–31. https://doi.org/10.1186/1471‐2474‐8‐27 CONFLICT OF INTEREST The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Bidonde, J., Busch, A. J., Schachter, C. L., Webber, S. C., Musselman, K. E., Overend, T. J., Góes, S. M., Dal Bello‐Haas, V., & Boden, C. (2019). Mixed exercise training for adults with fibromyalgia. Cochrane Database of Systematic Reviews, 5(5), CD013340. https://doi.org/10. 1002/14651858.CD013340 DATA AVAILABILITY STATEMENT The datasets generated during and/or analysed during the current study are not publicly available. If required please contact the cor- responding author. There are some limitations. While all FAME attendees were invited to participate; the views of patients who did not attend all FAME sessions or those who declined to attend FAME were not captured. The patient participants interviewed had all had fibromy- algia for a long time and we were unable to include people with more recently diagnosed Fibromyalgia. Neither FAME nor the topic guide were coproduced with patients. Had FAME been designed in part- nership with people with fibromyalgia, acceptability may have been enhanced from the outset. Furthermore, developing the topic guide with patients may have led to different avenues of enquiry and ensured we asked about subjects pertinent to them. ACKNOWLEDGEMENTS The authors wish to thank all those involved in the development and delivery of FAME in the physiotherapy department at King's College Hospital in particular: Jessica Moss, Francesca Howes, Clare Jarrett, Alice Margetts, Caroline Condon, Emmett Scanlan and Nicky Wilson. We would also like to thank all the participants of this study. No funding was received for this project. Bernardy, K., Füber, N., Köllner, V., & Häuser, W. (2010). Efficacy of cognitive‐behavioral therapies in fibromyalgia syndrome ‐ A sys- tematic review and metaanalysis of randomized controlled trials. Journal of Rheumatology, 37(10), 1991–2005. https://doi.org/10. 3899/jrheum.100104 Bernardy, K., Klose, P., Welsch, P., & Häuser, W. (2018). Efficacy, acceptability and safety of cognitive behavioural therapies in fibro- myalgia syndrome–A systematic review and meta‐analysis of ran- domized controlled trials. European Journal of Pain, 22(2), 242–260. https://doi.org/10.1002/ejp.1121 AUTHOR CONTRIBUTION Suzanne McIlroy and Lindsay Bearne conceptualised the study, Suzanne McIlroy, Bethany Vaughan and Heather Crowe undertook the focus groups and interviews. All authors were involved in ana- lysing the data and developing the themes. The manuscript was drafted by Suzanne McIlroy. All authors contributed to and reviewed the final manuscript. Boonen, A. (2004). Large differences in cost of illness and wellbeing be- tween patients with fibromyalgia, chronic low back pain, or anky- losing spondylitis. Annals of the Rheumatic Diseases, 64(3), 396–402. https://doi.org/10.1136/ard.2003.019711 Braun, V., & Clarke, V. (2006). Using thematic analysis in psychology. Qualitative Research in Psychology, 3(2), 77–101. https://doi.org/10. 1191/1478088706qp063oa Briones‐Vozmediano, E., Vives‐Cases, C., Ronda‐Pérez, E., & Gil‐González, D. (2013). Patients' and professionals' views on managing fibromy- algia. 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The experiences and acceptability of a novel multimodal programme for the management of fibromyalgia: A qualitative service evaluation. Musculoskeletal Care, 1–11. https://doi.org/10.1002/msc.1672 How to cite this article: McIlroy, S., Vaughan, B., Crowe, H., & Bearne, L. (2022). The experiences and acceptability of a novel multimodal programme for the management of fibromyalgia: A qualitative service evaluation. Musculoskeletal Care, 1–11. https://doi.org/10.1002/msc.1672 Wang, C., Schmid, C. H., Fielding, R. A., Harvey, W. F., Reid, K. F., Price, L. L., Driban, J. B., Kalish, R., Rones, R., & McAlindon, T. (2018). Effect of tai chi versus aerobic exercise for fibromyalgia: Comparative
https://openalex.org/W2057414709	https://europepmc.org/articles/pmc3157756?pdf=render	English	null	Prevalence of Congenital Malaria in Minna, North Central Nigeria	Journal of tropical medicine	2,012	cc-by	3,471	Innocent Chukwuemeka James Omalu,1 Charles Mgbemena,2 Amaka Mgbemena,1 Victoria Ayanwale,1 Israel Kayode Olayemi,1 Adeniran Lateef,3 and Victoria I. Chukwuemeka1 1Department of Biological Sciences, Federal University of Technology, Minna, Nigeria 2Dentistry Department, Niger State General Hospital, Minna 900002, Nigeria 3Department of Biochemistry/Physiology, University of Abuja, FCT, Nigeria Correspondence should be addressed to Innocent Chukwuemeka James Omalu, omalu icj@hotmail.com Received 14 April 2011; Revised 24 June 2011; Accepted 28 June 2011 Academic Editor: Demba Sarr Copyright © 2012 Innocent Chukwuemeka James Omalu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The study was designed to determine the true prevalence of congenital, cord, and placental malaria in General Hospital Minna, North Central Nigeria. Peripheral blood smears of near-term pregnant women, as well as the placental, cord, and peripheral blood smears of their newborn babies, were examined for malaria parasites, using the Giemsa staining technique. Out of 152 pregnant women screened, 21 (13.82%) of them were infected with malaria parasites. Of the 152 new born babies, 4 (2.63%) showed positive peripheral parasitaemia. Placental parasitaemia was 7/152 (4.61%), while cord blood parasitaemia was 9/152 (5.92%). There were strong associations between peripheral and cord malaria parasitaemia and congenital malaria (P < 0.05). Plasmodium falciparum occurred in all, and none had mixed infection. The average birth weights of the babies delivered of nonmalarious pregnant women were higher than those delivered by malarious pregnant women, though not signiﬁcant (P > 0.05). Malaria parasitaemia occurred more frequently in primigravidae than multigravidae. Hindawi Publishing Corporation Journal of Tropical Medicine Volume 2012, Article ID 274142, 5 pages doi:10.1155/2012/274142 Hindawi Publishing Corporation Journal of Tropical Medicine Volume 2012, Article ID 274142, 5 pages doi:10.1155/2012/274142 1. Introduction Reduced lymphoproliferative response sustained by ele- vated levels of serum cortisol, loss of cell-mediated immunity in the mother, the presence of placenta, a new organ in the primigravidae, allows the parasite to bypass the existing host immunity, or allows placenta-speciﬁc phenotypes of P. falciparum to multiply; pregnant women display a bias to- wards type-2 cytokines and are therefore susceptible to diseases requiring type-1 responses for protection like TB, malaria, and P. falciparum has the unique ability of cytoad- hesion; chondroitin sulfate A and hyaluronic acid are the adhesion molecules for parasite attachment to placental cells [4]. Congenital malaria was ﬁrst described in 1876 [1]. It can be acquired by transmission of parasites from mother to child during pregnancy or perinatally during labour [2]. Congenital malaria has been documented for many years, but it was previously thought to be uncommon especially in indigenous populations; more recent studies, however, suggest that incidence has increased, and values between 0.30 to 33.00% have been observed from both endemic and nonendemic areas [3]. Malaria and pregnancy are generally believed to be mutu- ally aggravating conditions. The pathological changes due to malaria and the physiological changes associated with preg- nancy have a synergistic eﬀect on the course of each other [4]. In pregnancies complicated by malaria, both fetal growth retardation and preterm birth contribute to low birth weight [6]. Malaria control still remains the foremost public health challenge in Africa, and world malaria report, which indi- cated that Nigeria accounts for a quarter of all malaria cases in the 45 malaria endemic countries in Africa, clearly showed the enormity of the socioeconomic and health burdens of the disease in the country, even in the face of dearth of Pregnancy exacerbates malaria through a nonspeciﬁc hormone-dependant depression of the immune system; pro- tective antiplasmodial activity is suppressed at pregnancy [5]. The following hypotheses to explain the altered immu- nity associated with pregnancy were oﬀered. 2 Journal of Tropical Medicine 2 Table 1: Prevalence of Plasmodium falciparum in blood samples of diﬀerent groups of women and tissues at General Hospital Minna, North Central Nigeria. Table 1: Prevalence of Plasmodium falciparum in blood samples of diﬀerent groups of women and tissues at General Hospital Minna, North Central Nigeria. 2. Materials and Methods 2.1. Study Site and Population. The study was carried out at General Hospital Minna, North Central Nigeria. Minna, the capital of Niger State, Nigeria, is located within longitude 6◦33′E and latitude 9◦37′N, covering a land area of 88 km2 with a population of 1.2 million. Minna has a tropical climate with mean annual temperature, relative humidity, and rainfall of 30.20◦, 61.00%, and 1334.00 cm, respectively. The climate presents two distinct seasons: a rainy season (April–October) and dry season (November–March). Minna is an endemic area for malaria. The ages, types of birth, and weights of newborn babies were recorded. 2.4. Parasitological Examination. Thick and thin blood ﬁlms were stained with 10% Giemsa and read for malaria parasites by two trained microscopists following standard quality con- trol procedure. Parasitaemia was expressed as the number of asexual forms of P. falciparum per microlitre; a result was considered negative after a reading of 1000 leucocytes in the microscope (×1000). Parasitaemia was graded as low (1– 999/µL), moderate (1000–9999/µL), and high (>10000/µL). Transplacental passage of Plasmodium falciparum was con- ﬁrmed by detection of malaria parasite within 7 days of birth. The study was conducted from October 2010 to Febru- ary 2011. The subjects were near-term (close to delivery) pregnant women who were delivered of their babies at the hospital. Sample sizes were determined from the number of pregnant women that attended antenatal care during the period of study. The nonpregnant women served as control to help compare malaria prevalence in pregnancy only. The pregnant women did not take any chemoprophylaxis and did not use mosquito treated nets for protection. The HIV status of the subjects were not determined. 2.5. Statistical Analysis. All data were analysed using SPSS version 10.1 for windows. Descriptive statistics were com- puted for all relevant data. Chi square analysis was used to compare proportions within and among groups, for statisti- cal signiﬁcance. 2.2. Ethical Considerations. All work was performed accord- ing to the guidelines for human experimentations in clinical research stated by the Federal Ministry of Health of Nigeria. This study was approved by the ethical committee of General Hospital Minna, Nigeria. All women gave oral informed consent. 1. Introduction Source of blood Overall Primigravidae Multigravidae No examd No positive Infect rate (%) No examd No positive Infect rate (%) No examd No positive Infect rate (%) Near-term Pregnant women 152 21 13.82 76 12 15.79 76 9 11.84 Nonpregn women 100 13 13.00 — — — — — — Neonate 152 4 2.63 — 2 — — 2 — Placenta 152 7 4.61 76 3 3.95 76 4 5.26 Cord 152 9 5.92 76 4 5.26 76 5 6.58 Total 708 54 7.63 226 21 9.29 228 20 8.77 separation of placenta, and peripheral blood of neonates was collected by heel pricking using a sterile blood lancet on clean glass slides. Labelled ethylene-diamine tetraacetic acid (EDTA) bottles were used in collecting blood samples for parasitological examinations. Blood collections were made possible with the assistance of midwives on duty during child delivery. Blood samples were also collected from 100 nonpregnant women. information on the epidemiology of the disease. This, thus, informed this study on the prevalence of congenital malaria in Minna, an often neglected but germane aspect of the epidemiology of malaria in endemic communities [7]. 3. Results Examination of the 152 placentae gave a prevalence of 4.63% (7) for malaria parasite, out of which 4 placentae were for the 4 babies with positive peripheral parasitaemia and known showed any clinical symptom. The only malaria parasite encountered in this study was the Plasmodium falciparum. The prevalence of cord blood parasitaemia was 5.92% (9/152), 5 of which came from multigravidae (6.58%) and 4 from primigravidae (5.26%). The average parasite density was 1,415 p/uL for pregnant women which was signiﬁcantly (P < 0.05) higher than the 411 p/uL recorded for nonpregnant women. Average parasite density for newborns was 128 p/uL, slightly lower than that for the cords which was 196 p/uL; however, both were signiﬁcantly lower than parasite density recorded in the placentae (mean = 689 p/µL). Parasite density was signiﬁcantly (P < 0.05) higher in ne- onates from primigravid (1281/uL) mothers than multi- gravids (134/uL) (Table 2). Parasite stages encountered were dominated by trophozoites (64.50%), distantly followed by gametocytes (26.80%) in pregnant women; the same trend was observed for nonpregnant women, placenta, cord, and neonates (Table 3). The prevalence of malaria in nonpregnant controls in this study was 13%, although this was lower than the pregnant subjects; the diﬀerence was not signiﬁcant. The inclusion of nonpregnant women in the study allows comparison of malaria prevalence in pregnancy with nonpregnant female subjects. Parasite densities for pregnant women were com- paratively higher than that of nonpregnant women. This is consistent with the ﬁndings of Agomo et al. [8], in a study done in Lagos, Nigeria. Although none of the pregnant women screened took malaria chemoprophylaxis during pregnancy, nor used any form of mosquito nets, the parasite density ﬁgures were low. However, one may conclude that this may have to do with the period of the study, October to February, that is, early to mid-dry season. A season that coincides with decreasing densities of female Anopheles species and therefore reduced inoculation with parasites. The 4 parasitized newborns all had birth weights above 3 kg. On the average, however, the birth weights of babies from parasitized mothers were lower than those from non- parasitized mothers, albeit, the diﬀerence was not signiﬁcant (P > 0.05). Also, newborns from primigravid mothers weighed averagely less than those from multigravid mothers; again the diﬀerence was not signiﬁcant (Table 4). Low parasites densities were also recorded for the placen- tae, cords, and peripheral blood of newborns. 3. Results Table 2: Average parasite densities (Parasites/uL) of blood of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. Parasite densities Pregnant women Nonpregnant women Newborn Placenta Cord PG MG Parasites/uL 1415 411 128 689 196 1281 134 PG: Neonate with primigravid mothers, MG: Neonate with multigravid mothers. Table 3: Erythrocytic stages of Plasmodium falciparum in blood samples of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. Table 3: Erythrocytic stages of Plasmodium falciparum in blood samples of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. S f bl d N i i TR GM SCH TR & SCH TR & GM TR & GM & SCH Total Table 3: Erythrocytic stages of Plasmodium falciparum in blood samples of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. Source of blood No positive TR GM SCH TR & SCH TR & GM TR & GM & SCH Total No % No % No % No % No % No % Pregnant women 21 60 64.50 25 26.80 — — — — 8 8.60 — — 93 Nonpreg women 13 15 46.80 12 37.50 — — — — 5 15.60 — — 32 Newborn 4 5 45.50 5 45.50 — — — — 1 9.00 — — 11 Placenta 7 20 62.50 10 31.20 — — — — 2 6.30 — — 32 Cord 9 12 36.30 10 30.30 — — — — 3 9.00 — — 33 Total 54 112 62 19 201 TR: trophozoites, GM: gametocytes, SCH: schizonts. women in this study was 14%; though consistent with the reported Nigerian situation, the relatively low percentage could be due to the fact that the study was carried out during the dry season, a period of low mosquito density and, per- haps, low level malaria transmission rates. This observation supports the position that in areas of malaria endemicity, pregnancy is associated with increased susceptibility to malaria, arising from pregnancy-induced altered immunity [6], Immunosuppression from raised serum cortisol, loss of cell-mediated immunity, eﬀects of a new organ, the placenta, and loss of type-1 cytokine responses [4]. Of the 152 newborn babies screened, 4 (2.63%) had positive peripheral parasitaemia. 3. Results Each placentae of the 4 parasitaemia neonates in this study also had positive parasitaemia. This agrees with the position of Chedraui et al. [6], that placental infection is a prerequisite for, but does not predict, congenital malaria. A strong correlation between placental and congenital para- sitaemia has been shown [12]. 3. Results The prevalence of malaria infection in blood smears of near- term pregnant mothers, their newborns, cords, placentae, and nonpregnant women was shown in Table 1. Out of a total of 152 near-term pregnant women screened, 21 (13.82%) had parasite in their peripheral blood. Among 100 nonpregnant women screened, 13 (13.00%) had peripheral malaria parasite. Infection rate was higher in pregnant women but not signiﬁcant (P > 0.05) using the chi-square. The inclusion of nonpregnant women helped to compare the prevalence of malaria. Also, there was no signiﬁcant diﬀerence in infection rates between the primigravidae (15.79%) and the multigravidae (11.84%), (P > 0.05). 2.3. Sample Collections. Blood samples were obtained from the peripheral blood of 152 near-term pregnant women using sterile syringes. After delivery, blood was collected from the placentae by placental biopsies. The placentae were incised between the maternal and foetal surface and a small quantity of blood pipette out from the intervillous with a sterile syringe. Cord blood was collected immediately after 3 Journal of Tropical Medicine Journal of Tropical Medicine Table 2: Average parasite densities (Parasites/uL) of blood of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. Parasite densities Pregnant women Nonpregnant women Newborn Placenta Cord PG MG Parasites/uL 1415 411 128 689 196 1281 134 PG: Neonate with primigravid mothers, MG: Neonate with multigravid mothers. Table 3: Erythrocytic stages of Plasmodium falciparum in blood samples of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. Source of blood No positive TR GM SCH TR & SCH TR & GM TR & GM & SCH Total No % No % No % No % No % No % Pregnant women 21 60 64.50 25 26.80 — — — — 8 8.60 — — 93 Nonpreg women 13 15 46.80 12 37.50 — — — — 5 15.60 — — 32 Newborn 4 5 45.50 5 45.50 — — — — 1 9.00 — — 11 Placenta 7 20 62.50 10 31.20 — — — — 2 6.30 — — 32 Cord 9 12 36.30 10 30.30 — — — — 3 9.00 — — 33 Total 54 112 62 19 201 TR: trophozoites, GM: gametocytes, SCH: schizonts. Table 2: Average parasite densities (Parasites/uL) of blood of diﬀerent groups of women and tissues at General Hospital Minna, North central Nigeria. References [1] S. Romand, P. Bouree, J. Gelez, B. Bader-Meunier, F. Bisaro, and J. P. Dommergues, “Congenital malaria. Infected twins born to an asymptomatic mother,” Presse Medicale, vol. 23, no. 17, pp. 797–800, 1994. [2] D. A. Opara, “Congenital malaria in newborn twins,” Ghana Medical Journal, vol. 44, no. 2, pp. 76–78, 2010. Infection with Plasmodium falciparum was the only one encountered in this study. This agrees with a similar study done in Libreville, Gabon by Bouyou-Akotet et al. [16], wherein only Plasmodium falciparum was encountered. It also agrees with widely accepted view that Plasmodium falciparum is the predominant species in Nigeria [16]. The predominant forms of this parasite seen were the tropho- zoites (ring stages). [3] S. A. Sotimehin, T. I. Runsewe-Abiodun, O. T. Oladapo, O. F. Njokanma, and D. M. Olanrewaju, “Possible risk factors for congenital malaria at a tertiary care hospital in Sagamu, Ogun State, South-West Nigeria,” Journal of Tropical Pediatrics, vol. 54, no. 5, pp. 313–320, 2008. [4] B. S. Kakkilaya, “Malaria and pregnancy,” 2009, http://www .malariasite.com [5] O. O. Okwa, “The status of malaria among pregnant women: a study in Lagos, Nigeria,” African Journal of Reproductive Health, vol. 7, no. 3, pp. 77–83, 2003. g g The only stillbirth encountered in this study resulted from foetal distress. The mother of the baby was negative for malaria parasites (nonmalarious). [6] P. A. Chedraui, J. Daily, B. Wylie, P. F. Weller, S. M. Ramin, and V. Barss, “Overview of malaria in pregnancy,” 2009, http://www.uptodate.com. On the average, the birth weights of babies from parasitaemia mothers were lower than those of babies from nonparasitaemia mothers, but not signiﬁcantly so. This dis- agrees with the widely held view that babies with parasitized placentae often have low birth weight. It also contradicts the ﬁndings of Adebami et al. [17], who stated that babies of mothers with parasitized placentae have mean birth weight signiﬁcantly lower than babies of mothers with nonpara- sitized placentae. The generally low parasite densities found in this study may explain the minimal eﬀect on birth weight, as observed by Opara [2] who recorded parasitized neonate twins with normal birth weights. Primigravidae are more susceptible to malaria infection than multigravidae in endemic areas [18]. The ﬁndings of this study supports this position, albeit, the diﬀerence in prevalence was not signiﬁcant. [7] World Health Organisation, “World Malaria Report,” WHO, Geneva, Switzerland, pp. 99-101, 2008. [8] C. O. Agomo, W. A. References Oyibo, R. I. Anorlu, and P. U. Agomo, “Prevalence of malaria in pregnant women in Lagos, South- West Nigeria,” Korean Journal of Parasitology, vol. 47, no. 2, pp. 179–183, 2009. [9] O. O. Okwa, “The status of malaria among pregnant women: a study in Lagos, Nigeria,” African Journal of Reproductive Health, vol. 7, no. 3, pp. 77–83, 2003. [10] M. B. Kagu, M. B. Kawuwa, and G. B. Gadzama, “Anaemia in pregnancy: a cross-sectional study of pregnant women in a Sahelian tertiary hospital in Northeastern Nigeria,” Journal of Obstetrics and Gynaecology, vol. 27, no. 7, pp. 676–679, 2007. [11] C. J. Uneke, F. E. Iyare, P. Oke, and D. D. Duhlinska, “Assessment of malaria in pregnancy using rapid diagnostic tests and its association with HIV infection and hematologic parameters in South-Eastern Nigeria,” Haematologica, vol. 93, no. 1, pp. 143–144, 2008. Congenital malaria was previously thought to be rare, especially, in areas of malaria endemicity. Though this study appears not to support this view, an increasing prevalence of congenital malaria among newborns in other areas of malaria endemicity has been observed. [12] O. A. Oduwole, G. C. Ejezie, and M. Meremekwu, “Congenital Malaria,” American Journal of Tropical Medicine and Hygiene, vol. 84, pp. 386–389, 2011. [13] C. Falade, O. Mokuolu, H. Okafor et al., “Epidemiology of congenital malaria in Nigeria: a multi-centre study,” Tropical Medicine and International Health, vol. 12, no. 11, pp. 1279– 1287, 2007. Conﬂict of Interests The authors declare that there is no conﬂict of interests. [14] P. O. Obiajunwa, J. A. Owa, and O. O. Adeodu, “Prevalence of congenital malaria in Ile-Ife, Nigeria,” Journal of Tropical Pediatrics, vol. 51, no. 4, pp. 219–222, 2005. 4. Discussion Many researchers have reported high prevalence of malaria in pregnancy in diﬀerent parts of Nigeria, ranging from 19.70% to 72.00% [8–11]. The prevalence of malaria in pregnant Journal of Tropical Medicine 4 Table 4: Mean (±SD) birthweights (kg) of neonate of pregnant women at General Hospital Minna, North Central Nigeria. Pregnant women Primigravidae Multigravidae Male Female Male Female Positive pregnant women 3.03 ± 0.5 2.83 ± 0.44 3.18 ± 0.31 2.83 ± 0.45 Negative pregnant women 3.01 ± 0.52 2.89 ± 0.25 3.31 ± 0.14 2.94 ± 0.14 facilities of General Hospital Minna and the Department of Biological Sciences, Federal University of Technology, Minna. However, an increasing trend in prevalence of congenital malaria has been reported recently. In a multicentre study done at Ibadan a prevalence of 5.10% was reported in University College Hospital [13]. A prevalence of 46.70% was reported in a study of 120 newborn babies at Ile-Ife, Southwestern Nigeria [14]. Also a prevalence of 13.00% was reported among 546 in-born neonates at Calabar Teaching Hospital [15]. These ﬁndings represent a new trend since parasitaemia in peripheral blood of newborns was considered rare in highly endemic areas. Acknowledgments The authors gratefully acknowledge the assistance of the technologists and midwives and for the use of laboratory [15] A. D. Ekanem, M. U. Anah, and J. J. Udo, “The prevalence of congenital malaria among neonates with suspected sepsis in 5 Journal of Tropical Medicine 5 Calabar, Nigeria,” Tropical Doctor, vol. 38, no. 2, pp. 73–76, 2008. [16] M. K. Bouyou-Akotet, D. E. Ionete-Collard, M. Mabika- Manfoumbi et al., “Prevalence of Plasmodium falciparum infection in pregnant women in Gabon,” Malaria Journal, vol. 2, no. 1, article 1, p. 18, 2003. [17] O. J. Adebami, J. A. Owa, G. A. Oyedeji, O. A. Oyelami, and G. O. Omoniyi-Esan, “Associations between placental and cord blood malaria infection and fetal malnutrition in an area of malaria holoendemicity,” American Journal of Tropical Medicine and Hygiene, vol. 77, no. 2, pp. 209–213, 2007. [18] O. A. Idowu, C. F. Maﬁana, and S. Dapo, “Malaria among pregnant women in Abeokuta, Nigeria,” Tanzania Health Research Bulletin, vol. 8, no. 1, pp. 28–31, 2006.
https://openalex.org/W2747647717	https://link.springer.com/content/pdf/10.1007%2Fs00415-017-8596-7.pdf	English	null	What proportion of AQP4-IgG-negative NMO spectrum disorder patients are MOG-IgG positive? A cross sectional study of 132 patients	Journal of neurology	2,017	cc-by	3,794	J Neurol (2017) 264:2088–2094 DOI 10.1007/s00415-017-8596-7 ORIGINAL COMMUNICATION What proportion of AQP4‑IgG‑negative NMO spectrum disorder patients are MOG‑IgG positive? A cross sectional study of 132 patients Hamid shahd.hamid@thewaltoncentre.nhs.uk Daniel Whittam daniel.whittam@thewaltoncentre.nhs.uk Kerry Mutch kerry.mutch@thewaltoncentre.nhs.uk Samantha Linaker Samantha.Linaker@thewaltoncentre.nhs.uk Tom Solomon tsolomon@liverpool.ac.uk Kumar Das kumar.das@thewaltoncentre.nhs.uk Maneesh Bhojak maneesh.bhojak@thewaltoncentre.nhs.uk 1 The Walton Centre NHS Foundation Trust, Liverpool L9 7LJ, UK 2 Institute of Infectious Disease and Global Health, University of Liverpool, The Walton Centre NHS Foundation Trust, Liverpool, UK * Anu Jacob anu.jacob@thewaltoncentre.nhs.uk Shahd H. M. Hamid shahd.hamid@thewaltoncentre.nhs.uk Daniel Whittam daniel.whittam@thewaltoncentre.nhs.uk Kerry Mutch kerry.mutch@thewaltoncentre.nhs.uk Samantha Linaker Samantha.Linaker@thewaltoncentre.nhs.uk Tom Solomon tsolomon@liverpool.ac.uk Kumar Das kumar.das@thewaltoncentre.nhs.uk Maneesh Bhojak maneesh.bhojak@thewaltoncentre.nhs.uk 1 The Walton Centre NHS Foundation Trust, Liverpool L9 7LJ, UK 2 Institute of Infectious Disease and Global Health, University of Liverpool, The Walton Centre NHS Foundation Trust, Liverpool, UK Introduction 73–90% of neuromyelitis optica spectrum disorder (NMOSD) patients diagnosed according to the 2015 Interna- tional panel on NMO diagnosis have aquaporin-4 antibodies (AQP4-IgG) [1, 2]. It is presumed that at least a proportion of the remaining 10–27% of patients, classified as seron- egative NMOSD have another disease specific antibody. Antibodies to myelin oligodendrocyte glycoprotein (MOG- IgG) have been increasingly reported in a variety of CNS neuroinflammatory conditions including patients with phe- notypes typical for NMOSD [3]. We aimed to determine the prevalence of MOG-IgG in AQP4-IgG-negative NMOSD. Maneesh Bhojak maneesh.bhojak@thewaltoncentre.nhs.uk 1 The Walton Centre NHS Foundation Trust, Liverpool L9 7LJ, UK What proportion of AQP4‑IgG‑negative NMO spectrum disorder patients are MOG‑IgG positive? A cross sectional study of 132 patients Shahd H. M. Hamid1,2 · Daniel Whittam1 · Kerry Mutch1 · Samantha Linaker1 · Tom Solomon2 · Kumar Das1 · Maneesh Bhojak1 · Anu Jacob1 Received: 11 April 2017 / Revised: 10 August 2017 / Accepted: 11 August 2017 / Published online: 24 August 2017 © The Author(s) 2017. This article is an open access publication Abstract Antibodies to myelin oligodendrocyte glyco- protein (MOG-IgG) have been described in patients with neuromyelitis optica spectrum disorders (NMOSD) without aquaporin-4 antibodies (AQP4-IgG). We aimed to identify the proportion of AQP4-IgG-negative NMOSD patients who are seropositive for MOG-IgG. In a cross sectional study, we reviewed all patients seen in the National NMO clinic over the last 4 years (after the availability of MOG-IgG testing), including clinical information, MRI, and antibody tests. 261 unique patients were identified. 132 cases satisfied the 2015 NMOSD diagnostic criteria. Of these, 96 (73%) were AQP4- IgG positive and 36 (27%) were AQP4-IgG negative. These 36 patients were tested for MOG-IgG and 15/36 (42%) tested positive. 20% (25/125) of the patients who did not satisfy NMOSD criteria had MOG-IgG. Approximately half of seronegative NMOSD is MOG-Ig seropositive and one in five of non-NMOSD/non-MS demyelination is MOG-IgG positive. Since MOG-associated demyelinating disease is likely different from AQP4-IgG disease in terms of underly- ing disease mechanisms, relapse risk and possibly treatment, testing for MOG-IgG in patients with AQP4-IgG-negative NMOSD and other non-MS demyelination may have signifi- cant implications to management and clinical trials. Keywords Neuromyelitis optica · Aquaporin-4 antibodies · Myelin oligodendrocytes glycoprotein * Anu Jacob anu.jacob@thewaltoncentre.nhs.uk Shahd H. M. Hamid shahd.hamid@thewaltoncentre.nhs.uk Daniel Whittam daniel.whittam@thewaltoncentre.nhs.uk Kerry Mutch kerry.mutch@thewaltoncentre.nhs.uk Samantha Linaker Samantha.Linaker@thewaltoncentre.nhs.uk Tom Solomon tsolomon@liverpool.ac.uk Kumar Das kumar.das@thewaltoncentre.nhs.uk Maneesh Bhojak maneesh.bhojak@thewaltoncentre.nhs.uk 1 The Walton Centre NHS Foundation Trust, Liverpool L9 7LJ, UK 2 Institute of Infectious Disease and Global Health, University of Liverpool, The Walton Centre NHS Foundation Trust, Liverpool, UK * Anu Jacob anu.jacob@thewaltoncentre.nhs.uk Shahd H. M. Hamid shahd.hamid@thewaltoncentre.nhs.uk Daniel Whittam daniel.whittam@thewaltoncentre.nhs.uk Kerry Mutch kerry.mutch@thewaltoncentre.nhs.uk Samantha Linaker Samantha.Linaker@thewaltoncentre.nhs.uk Tom Solomon tsolomon@liverpool.ac.uk Kumar Das kumar.das@thewaltoncentre.nhs.uk Maneesh Bhojak maneesh.bhojak@thewaltoncentre.nhs.uk 1 The Walton Centre NHS Foundation Trust, Liverpool L9 7LJ, UK 2 Institute of Infectious Disease and Global Health, University of Liverpool, The Walton Centre NHS Foundation Trust, Liverpool, UK * Anu Jacob anu.jacob@thewaltoncentre.nhs.uk Shahd H. M. Results We also assessed how many of the MOG-IgG patients with NMOSD phenotype had a relapsing course. Thir- teen patients (86%) had a relapsing course. However, a relapsing course was the reason for referral to the clinic in the first place (n = 13/13). The median duration of ill- ness for the relapsing patients was 4.7 years (2–16 years). The median inter-attack interval was 1 year (0.16–17) and median EDSS in the relapsing MOG group at last follow-up was 3 (0–9, Table 1). All relapsing patient are on immunosuppressants (Table 1). 261 unique patients with non-MS/atypical CNS inflamma- tory conditions attended the clinic and were assessed for NMOSD. All patients were tested for AQP4-IgG. 132 cases satisfied the 2015 NMOSD diagnostic criteria. Of these, 96 (73%) were AQP4-IgG positive and 36 (27%) AQP4-IgG negative. These 36 patients, were tested for MOG-IgG and 15/36 (42%) tested positive. This would account for 11% (15/132) of the total cohort of NMOSD patients (Fig. 1; Table 1). All MOG-IgG-negative patients were Caucasians with a median age of onset of 18 years (8–44 years) and median disease duration of 4.7 years (2–16 years). The pre- dominant clinical phenotype of the demyelinating event was ON (60%), TM (21%), brain (12%), and brainstem (4%). We also assessed the proportion of patients with optic neuritis and long myelitis who fulfill Wingerchuk 2006 criteria [6] that are MOG-IgG positive, as this is a clinical question often posed. Of the whole cohort of 261 patients, 75 patients had long myelitis and optic neuritis. Of these 49 were AQP4-IgG positive (66%) and 10 were MOG-IgG positive (13%, or 38% of AQP4-IgG-negative patients) and 16 remained seronegative (21%). Serial testing where done in 14/15 patients (13 relapsing); MOG-IgG was detected in all. Treatment with steroid or immunosuppression does not seem to have an effect on MOG-IgG serostatus in this cohort of predominantly relapsing patients (Table 2). While we tested all AQP4-IgG-negative patients for MOG-IgG (n = 36), only a proportion (33%) of AQP4- IgG-positive patients (n = 32) were tested (as double positives are exceptionally rare) (Fig. 1). None were defi- nitely positive. However, one patient was ‘low positive/ possibly negative. This patient with one episode of long myelitis also had antinuclear antibodies (1/80 titre with 1 3 Fig. 1 Classification of non- MS/atypical demyelination based on 2015 NMOSD criteria, AQP4-IgG, and MOG-IgG testing. Methods 2 Institute of Infectious Disease and Global Health, University of Liverpool, The Walton Centre NHS Foundation Trust, Liverpool, UK The Walton Centre Neurosciences NHS Trust in Liverpool, United Kingdom, is a tertiary neurology hospital that hosts Vol:.(12345678 1 3 l:.(1234567 3 J Neurol (2017) 264:2088–2094 2089 one of the two national multidisciplinary specialist clinics for patients with NMOSD and non-MS demyelinating disor- ders as part of the UK NMOSD service. We systematically reviewed all patients seen in this clinic over the last 4 years (after the availability of MOG-IgG testing), including clini- cal information, MRI, and antibody tests. Both AQP4-IgG and MOG-IgG were tested using a validated live cell-based assay with high specificity (John Radcliffe Hospital, Oxford, UK) [4, 5]. This study was approved by Research Ethics Service, NRES Committee London—Hampstead, Ref. no. 15/LO/1433. one of the two national multidisciplinary specialist clinics for patients with NMOSD and non-MS demyelinating disor- ders as part of the UK NMOSD service. We systematically reviewed all patients seen in this clinic over the last 4 years (after the availability of MOG-IgG testing), including clini- cal information, MRI, and antibody tests. Both AQP4-IgG and MOG-IgG were tested using a validated live cell-based assay with high specificity (John Radcliffe Hospital, Oxford, UK) [4, 5]. This study was approved by Research Ethics Service, NRES Committee London—Hampstead, Ref. no. 15/LO/1433. homogenous pattern (nuclear antigens all negative) and was ‘low positive’ for anti-glycine antibodies too. The significance of the MOG-IgG in the context of these addi- tional antibodies is uncertain and may reflect a heightened humoral autoimmune response rather than truly patho- genic dual positivity. This patient has not been included in the MOG cohort in this paper. We also tested the majority of patients with a demyeli- nating syndrome referred to the service who did not fulfill the NMOSD criteria (125/129, 97%). Twenty-five (20%) were positive for MOG-IgG. Details of these cases will be the subject of an upcoming separate research paper and are not discussed further here. Results NMOSD neuromyelitis optica spectrum disorder, AQP4 IgG Antibody to aquaporin 4, MOG-IgG antibody to myelin oligodendrocyte glycoprotein, OSD optico-spinal demyelina- tion with normal brain MRI 261 Non-MS /Atypical demyelination 132 NMOSD 96 AQP4-IgG+ve (73%) 129 Other CNS demyelination Optico-spinal demyelination (OSD) Isolated or relapsing transverse myelitis (TM) Optic neuritis (ON) Brainstem demyelination Cerebral demyelination Other combinations 36 AQP4-IgG-ve (27%) 15 MOG-IgG+ve (42% ) 33% Tested for MOG-IgG (n=32) 0 MOG-IgG+ve 100 % tested for MOG-IgG 97% tested for MOG-IgG (n= 125) 25 MOG-IgG+ve (20%) Fig. 1 Classification of non- MS/atypical demyelination based on 2015 NMOSD criteria, AQP4-IgG, and MOG-IgG testing. NMOSD neuromyelitis optica spectrum disorder, AQP4 IgG Antibody to aquaporin 4, MOG-IgG antibody to myelin oligodendrocyte glycoprotein, OSD optico-spinal demyelina- tion with normal brain MRI 261 Non-MS /Atypical demyelination 132 NMOSD 96 AQP4-IgG+ve (73%) 129 Other CNS demyelination Optico-spinal demyelination (OSD) Isolated or relapsing transverse myelitis (TM) Optic neuritis (ON) Brainstem demyelination Cerebral demyelination Other combinations 36 AQP4-IgG-ve (27%) 15 MOG-IgG+ve (42% ) 33% Tested for MOG-IgG (n=32) 0 MOG-IgG+ve 100 % tested for MOG-IgG 97% tested for MOG-IgG (n= 125) 25 MOG-IgG+ve (20%) Fig. 1 Classification of non- MS/atypical demyelination based on 2015 NMOSD criteria, AQP4-IgG, and MOG-IgG testing. NMOSD neuromyelitis optica spectrum disorder, AQP4 IgG Antibody to aquaporin 4, MOG-IgG antibody to myelin oligodendrocyte glycoprotein, OSD optico-spinal demyelina- tion with normal brain MRI 261 Non-MS /Atypical demyelination 129 Other CNS demyelination Optico-spinal demyelination (OSD) Isolated or relapsing transverse myelitis (TM) Optic neuritis (ON) Brainstem demyelination Cerebral demyelination Other combinations 132 NMOSD 33% Tested for MOG-IgG (n=32) 100 % tested for MOG-IgG 97% tested for MOG-IgG (n= 125) 15 MOG-IgG+ve (42% ) 25 MOG-IgG+ve (20%) 0 MOG-IgG+ve g (42% ) (42% ) 3 J Neurol (2017) 264:2088–2094 2090 2090 J Neurol (2017) 264:2088 209 1 3 Table 1 Demographic, clinical, and radiological characteristics of the 15 NMOSD patients with MOG-IgG Patient no. Age Sex Age at onset Disease duration (years) Course Total no. of events Clinical phe- notype (no. Discussion ab e ( ) Patient no. Age Sex Age at onset Disease duration (years) Course Total no. of events Clinical phe- notype (no. of attacks) First inter- attack interval Spinal MRI Baseline brain MRI CSF oligo- clonal bands EDSS Current treatment 10 28 M 19 8.2 R 4 ON (3) TM (1) 6 years LETM Normal Unknown 4 Mycophenolate 11 44 M 13 31 R 5 ON (3) TM (2) 17 years LETM Normal Negative 3.5 Azathioprine 12 39 F 36 3.1 R 2 Brain stem (1) ON (2) 2.2 years Normal Lesion on pons Negative (161) 3 Mycophenolate and oral predni- solone 13 42 M 38 3.6 R 2 TM (1) Brain stem (1) 2 months LETM Peri ependymal pons lesion Unknown 6 Azathioprine and oral predniso- lone 14 28 M 26 2 Single event 1 ON + LETM Simultaneously LETM Normal Positive 1.5 Mycophenolate 15 45 M 40 5 Single event 1 ON + LETM Simultaneously LETM Normal Negative 2 None F f l M l R l i ON ti iti TM t liti LETM l it di ll t i t liti d IVIG i t i l b li In a cohort of well-characterised NMOSD patients (n = 132), 73% were AQP4-IgG and 11% were MOG-IgG seropositive and 16% remained seronegative. MOG-IgG disease accounts for 42% of the AQP4 IgG-negative seron- egative cohort. MOG-IgG was present in 38% of patients with long myelitis and optic neuritis who do not have AQP4 IgG. 86% (13/15) of our patients who satisfy criteria for NMOSD who are MOG-IgG-positive patients have relaps- ing disease, similar to a recent study [7] who reported that 80% of their MOG-IgG-positive cohort (n = 50) fol- lowed a relapsing course. However, a relapsing course was the reason for referral to the clinic in the first place (n = 13/13) making this a biased sample. Long-term fol- low-ups of a cohort of MOG-IgG-positive patients after the very first event is required to obtain the true risk of relapse. Importantly, 20% of patients with non-MS/atypical demyelination who do not satisfy criteria for NMOSD tested positive for MOG-IgG (Fig. 1). Double positive cases (both AQP4-IgG and MOG-IgG) are rare [8–10] with none of the tested patients were definite positives. Since we have tested only 52% (68/132) of the total NMOSD cohort for MOG-IgG, this requires further clarification in future studies. Results of attacks) First inter- attack interval Spinal MRI Baseline brain MRI CSF oligo- clonal bands EDSS Current treatment 1 31 F 18 13.4 R 13 ON (13) TM (1) 3 years LETM Normal Negative 4 Subcutaneous IGs (immuno- globulins) and oral predniso- lone 2 55 M 44 11 R 7 ON (2) TM (1) brain- stem (1) brain syndrome (5) 7 years Short mid tho- racic lesion Brain stem, cortical and subcortical extensive demy Positive 3.5 Steroid & mycophenolate 3 31 F 15 16.4 R 2 ON (1) TM (1) 4 years LETM Normal Negative 9 Azathioprine and oral predniso- lone 4 21 M 18 2.5 R 5 Brain stem (1) Brain syndrome (1) TM (1) ON (5) 2 months Multiple short lesions on thoracic cord Large area of high T2 signal in the poste- rior brainstem both sides of mid brain Negative 1.5 Azathioprine switched to rituximab 5 22 M 17 4.7 R >7 ON (>7) and TM (2) 2 months LETM Normal Unknown 3 Tocilizumab, IVIG six weekly and oral prednisolone 6 30 F 28 2 R 2 ON (1) TM (1) 1 year LETM Cerebral ring enhanc- ing lesion supracallosal subcortical Negative 0 Mycophenolate 7 23 F 8 14.4 R 3 ON (2) TM (2) Brain syn- drome (1) 3 years LETM Multiple non- specific white matter lesions Negative 6 Azathioprine and oral predniso- lone 8 24 F 17 6.9 R 2 ON (1) TM (1) Brain syndrome (1) 3 months LETM Brainstem, left cerebral peduncle, and few non- specific white matter lesions Negative 1 Azathioprine and oral predniso- lone 9 14 F 10 4 R 3 Brain syndrome (1) ON (3) TM (1) 3 month LETM Bilateral hemispheric white matter changes Negative 2.5 Rituximab and mycophenolate J Neurol (2017) 264:2088–2094 2091 Discussion Discussion In conclusion, our study provides the best possible answers at the current time on several questions on the frequency of MOG-IgG patients: NMOSD who are AQP4- IgG negative and MOG-IgG positive (42%), NMO (as per Wingerchuk 2006) with optic neuritis and long myeli- tis who are AQP4-IgG negative but MOG-IgG (13%). We also found that MOG-IgG is found in 20% of non- NMOSD/non-MS demyelination. It is also estimated that at least 11% of all NMOSD (as per 2015 criteria) is MOG- IgG positive. Our study has important practical implications. First, the definite diagnosis of MOG-IgG-associated disease offers patients and physicians a better diagnostic label than seron- egative NMOSD. Second, as nearly one in every two of seronegative NMOSD, and 1/5 of atypical non-MS demy- elination is MOG-Ig positive, testing for these cohorts will be of high yield and worthwhile, compared to testing every demyelination (which in most Caucasian predominant popu- lations is likely to be MS) with attendant costs and risk of false-positive results. Third, it is likely that the long-term disease course and therefore treatment strategies of AQP4- IgG and MOG-IgG is different. If this is the case, MOG-IgG status, should be part of inclusion/exclusion criteria or a var- iable for stratification in clinical trials. The latter issue may have importance for currently recruiting trials that include seronegative NMOSD. 1 3 3 J Neurol (2017) 264:2088–2094 2092 Table 2 MOG-IgG testing in relation to disease course and immunosuppressive treatment. NA: not available Patient no. Discussion Date of onset Date of first relapse Last relapse Date of start on steroid Date of start on maintenance immunosuppressive treat- ment First-positive MOG-IgG test Subsequent MOG test year Titre Comments 1 Jan 02 May 05 Jul 05 Jan 08 2009 2011 2013, 2014 both positive NA Data not clear if was on steroid in first or last relapse, but was on immunosuppressant when tested positive for MOG-IgG 2 2004 2011 2015 2014 2014 2014 2015, 2016, 2017 all positive 300 Patient was not on steroid in first or last relapses, but was on immunosuppressant when tested positive for MOG-IgG after diagnosis and remained positive 3 Jan 99 Apr 03 May 03 Unknown 2003 Apr 14 Jul 14 positive NA Data not clear if was on steroid in first or last relapse, but was on immunosuppressant when tested positive for MOG-IgG subsequently 4 Sep 14 Nov 14 May 17 Nov 14 Dec 14 2014 2015 positive 300 Patient was not on steroid in first relapse, but was on steroid and immunosup- pressant in last relapse and when MOG-IgG tested and remained positive 2016 positive 400 5 Sep 10 Oct 10 Jul 13 At onset 2011 2012 2014, 2015, 2016 all positive NA Patient was on reducing dose of steroid in first relapse, and on immunosuppressant and steroid in last relapse and when MOG-IgG was tested and remained positive 6 Aug 13 Sep 14 Sep 14 Sep 14 May 15 Sep 14 2016, 2017 both positive NA Patient was not on steroid in first relapse, was on steroid when tested for MOG-IgG initially and in 2016 but off steroid in 2017 and remained positive 7 2001 2004 2010 At onset 2010 2013 2014, 2016 both positive NA Patient was not on steroid in first or last relapse, she was on immunosuppressant when tested for MOG-IgG subse- quently. 2001 J Neurol (2017) 264:2088–2094 2093 2 (continued) no. Discussion Date of onset Date of first relapse Last relapse Date of start on steroid Date of start on maintenance immunosuppressive treat- ment First-positive MOG-IgG test Subsequent MOG test year Titre Comments Jul 08 Nov 08 Nov 08 At onset Nov 08 Apr 11 May 11 positive NA Data unavailable if patient was on steroid in first relapse, she was on immunosuppres- sant when tested positive for MOG-IgG Apr 12 Jul 12 Aug 15 At onset 2012 2012 2015, 2016 positive NA Patient was on steroid in first relapse and when tested posi- tive for MOG-IgG. She was also positive when was on steroid and immunosuppres- sant in subsequent relapses. Mar 07 Jul 13 Dec 15 At onset Jul-14 Apr 14 2016 positive NA Patient was not on steroid in first relapse, or first MOG- IgG test. He was on immu- nosuppressant in last relapse and when remained positive in subsequent testing 1984 2001 Mar 13 At onset 2013 2015 No further tests NA No available data whether patient was on steroid in first or last relapse, but he was on immunosuppressant when tested positive for MOG-IgG. May 12 Aug 14 Aug 14 At onset May 15 May 15 2016 positive NA Patient was not on steroid in first relapse, but was on steroid when tested posi- tive for MOG-IgG and was on immunosuppressant on subsequent positive test Oct 12 Jan 13 Jan 13 At onset Aug 13 Jul 13 2014 negative 2015 positive NA Patient was on steroid in first relapse, however, immuno- suppressant was initiated after MOG-IgG returned positive in 2013, later test one year apart was negative in 2014, and subsequent test in 2015 was positive while still on immunosuppressant Mar 14 At onset Apr 14 Apr 14 2015, 2016, 2017 all positive NA Only one event but patient chose to go on treatment Jun 12 At onset Not on immunosuppressant Jun 12 2015 positive NA Not on immunosuppression 2094 J Neurol (2017) 264:2088–2094 3. Ramanathan S, Dale RC, Brilot F (2016) Anti-MOG antibody: the history, clinical phenotype, and pathogenicity of a serum bio- marker for demyelination. Autoimmun Rev 15(4):307–324 Author contributions Dr. SHMH collected and compiled the data and wrote the manuscript. KM, MB, KD, SL collected data. Prof. TS critically reviewed the manuscript. DW collected data and reviewed the manuscript. Dr. AJ collected the data and critically reviewed the manuscript. Simple statistical analysis done by authors. Ethical standards This study meets UK ethical standards. Ethical standards This study meets UK ethical standards. Ethical standards This study meets UK ethical standards. 7. Jarius S, Ruprecht K, Kleiter I, Borisow N, Asgari N, Pitarokoili K et al (2016) MOG-IgG in NMO and related disorders: a multi- center study of 50 patients. Part 1: frequency, syndrome specific- ity, influence of disease activity, long-term course, association with AQP4-IgG, and origin. J Neuroinflamm 13(1):279 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea- tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. l 8. Jarius S, Ruprecht K, Kleiter I, Borisow N, Asgari N, Pitarokoili K et al (2016) MOG-IgG in NMO and related disorders: a multi- center study of 50 patients. Part 2: epidemiology, clinical presen- tation, radiological and laboratory features, treatment responses, and long-term outcome. J Neuroinflamm 13(1):280 l 9. Hoftberger R, Sepulveda M, Armangue T, Blanco Y, Rostasy K, Cobo Calvo A et al (2015) Antibodies to MOG and AQP4 in adults with neuromyelitis optica and suspected limited forms of the disease. Mult Scler 21(7):866–874 Discussion 4. Waters PJ, McKeon A, Leite MI, Rajasekharan S, Lennon VA, Vil- lalobos A et al (2012) Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays. Neurology 78(9):665–671 (discussion 9) Funding The study is not industry sponsored. Funding The study is not industry sponsored. Compliance with ethical standards 5. Waters P, Woodhall M, O’Connor KC, Reindl M, Lang B, Sato DK et al (2015) MOG cell-based assay detects non-MS patients with inflammatory neurologic disease. Neurol Neuroimmunol Neuroinflamm 2(3):e89 Conflicts of interest All authors declare no conflict of interest. Conflicts of interest All authors declare no conflict of interest. Conflicts of interest All authors declare no conflict of interest. l 6. Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Wein- shenker BG (2006) Revised diagnostic criteria for neuromyelitis optica. Neurology 66(10):1485–1489 References 1. Hamid SH, Elsone L, Mutch K, Solomon T, Jacob A (2017) The impact of 2015 neuromyelitis optica spectrum disorders criteria on diagnostic rates. Mult Scler 23(2):228–233 10. Sato DK, Callegaro D, Lana-Peixoto MA, Waters PJ, de Haidar Jorge FM, Takahashi T et al (2014) Distinction between MOG antibody-positive and AQP4 antibody-positive NMO spectrum disorders. Neurology 82(6):474–481 2. Hyun JW, Jeong IH, Joung A, Kim SH, Kim HJ (2016) Evaluation of the 2015 diagnostic criteria for neuromyelitis optica spectrum disorder. Neurology 86(19):1772–1779 2. Hyun JW, Jeong IH, Joung A, Kim SH, Kim HJ (2016) Evaluation of the 2015 diagnostic criteria for neuromyelitis optica spectrum disorder. Neurology 86(19):1772–1779 1 3 1 3 3
https://openalex.org/W1558713119	https://uscholar.univie.ac.at/detail/o:446241.pdf	English	null	African soft power in China	African East-Asian affairs	2,015	cc-by	7,616	AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Issue 2 June 2015 African soft power in China African soft power in China by Adams Bodomo* Department of African Studies, and Global African Diaspora Studies (GADS) Research Platform, University of Vienna Vienna, Austria by Adams Bodomo* Department of African Studies, and Global African Diaspora Studies (GADS) Research Platform, University of Vienna Vienna, Austria © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Abstract This paper comprises an account of outstanding performance by some Africans living in China in the practice of their professions, in their studies, in public service, and in their general interaction with the Chinese people and the Chinese state. The paper argues that these activities, recognised as outstanding by the Chinese and widely reported in the Chinese and international media, constitute the building blocks of African soft power in China. The consequences of this argumentation include the fact that if the governments of Africa and China want to promote people- to-people relations in the third decade of 21st Century Africa-China relations under the aegis of the Forum for Africa-China Cooperation (FOCAC), more of such activities by diaspora Africans in China and diaspora Chinese in Africa must be recognised, promoted, rewarded, and institutionalised within the formal FOCAC framework. *Adams Bodomo is Professor of African Studies (holding the Chair of African Languages and Literatures) at the University of Vienna, Austria, where he directs a new research centre called the Global African Diaspora Studies (GADS) Research Platform. 76 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS AFRICAN EAST-ASIAN AFFAIRS © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Adams Bodomo “African soft power in China“ Soft power These positive socio-economic and socio-cultural contributions by Africans in China may be seen as the basis of a nascent African soft power in China. Soft power is a notion that has become very important in the second decade of the 21st Century Africa-China relations. There are many definitions and conceptualisations of soft power (Nye 1990a&b, 2004; Bodomo 2009; King 2013). For instance, Joseph Nye, the scholar who first used the term, described soft power or co-optive power in the following terms: “The second aspect of power – which occurs when one country gets other countries to want what its wants – might be called co-optive or soft power in contrast with the hard or command power of ordering others to do what it wants”. (Nye 1990b:166). Introduction 77 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS communities on either side of the partnership have a major role to play in developing stronger Africa-China cooperation (for example Bodomo 2010, 2012; Bodomo and Ma 2010, 2012; Bodomo and Silva 2012; Sautman and Yan 2014). I continue to argue for this second position in this paper, showing that despite challenges posed by some members of the African Diaspora and some members of the Chinese Diaspora, these Africans in China and Chinese in Africa are playing positive socio-economic, socio-political, and socio-cultural roles, even if sometimes unconsciously, towards strengthening Africa-China cooperation. Introduction After more than a decade (2000 to 2013) of intense co-operation between Africa and China within the framework of the Forum on Africa-China Cooperation (FOCAC), it is time to take stock of these relations and find ways to develop them further in the second and subsequent decades. In this paper, I shall briefly examine the role of Diaspora Africans in China and Diaspora Chinese in Africa with the aim of finding out if these diaspora groups can contribute to the development of Africa-China relations. There are now more than two million Chinese in Africa and half a million Africans in China; and many academic studies including journal articles, books and theses have already been produced on Africans in China (examples include: Bodomo 2009, 2010, 2012, 2013a and b, 2014; Bredeloup 2012; Bork et al. 2011; Castillo 2014; Cissé 2013; Hall et al. 2014; Han 2013; Haugen 2011, 2012, 2013; Lan 2014; Li et al. 2009, 2012; Lyons et al. 2008, 2012, 2013; McLaughlin 2014; Pieke 2012) and on Chinese in Africa (for example: Park 2008; Mohan and Lampert 2013; Sautman and Yan 2014). These numbers are the basis of nascent diasporan communities on either side of the partnership; a Diasporan community being defined as a transnational or deterritorialised group of people organising themselves into networks or relating to themselves and to their host communities based on some common socio-cultural features and institutions like language, food, and clothing; and who have links, whether permanent or tenuous, back to their historical homelands. This diasporic presence on either side of the partnership is not without debate. What challenges do these Diasporas pose for their hosts and for general Africa-China relations? Do they play any positive roles and how can we further harness these positive roles to strengthen Africa-China cooperation? On the one hand there are media reports (for example CNN, BBC and SCMP) and academic analyses (for example Zhou, 2011; and Mathews and Yang 2012 for Africans in China; Mohan and Lampert 2013 for Chinese in Angola, Ghana and Nigeria; and Sautman and Yan 2014 for Africans in Zambia) that show that these communities indulge in activities that create negative perceptions, tensions and divisions and thus threaten the strengthening of relations between the two parties. There are, however, those that believe that diaspora © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. It goes further to state: “Co-optive power is the ability of a country to structure a situation so that other countries develop preferences or define their interests in ways consistent with its own. This power tends to arise from such resources as cultural and ideological attraction as well as rules and institutions of international regimes”. (Nye, 1990b:168). In this study I conceptualise soft power a bit differently as being the gamut of positive socio-political and socio-cultural influences a polity and its citizens have on another polity and its citizens without the threat of “gun-boat” diplomacy or even © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 78 AFRICAN EAST-ASIAN AFFAIRS Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS outright violence. This definition captures the original core idea of soft power, as a mostly, though not always, an asymmetrical system in which polities use alternative means other than traditional balance of power politics – as regulated mainly by military superiority – to positively influence other polities. It however puts the emphasis on ordinary African citizens consciously or unconsciously acting on behalf of their polities to promote positive views of their countries and continent. How and in what ways have Africans in China employed their positive contributions to Chinese society as a means to increase African soft power, to attain socio-cultural positive views and outlook of Africa in China? Based on extensive fieldwork in China, using a mixed mode of qualitative and quantitative socio-cultural research methods, I draw on the theoretical notions of soft power and of Diaspora communities acting as bridges interconnecting host and source communities to illustrate this notion of positive Diaspora contributions to Africa-China co-operation. Section 2 sketches the challenges involved; sections 3 and 4 outline some contributions, with the latter focusing empirically on the contributions of two prominent Africans in China; section 5 makes some recommendations for FOCAC support of people-to-people relations; while section 6 summarises the points raised and draws some conclusions to buttress the argument made in the paper. Africa-China relations: the diaspora challenge On July 16, 2009, The South China Morning Post (SCMP), Hong Kong's main English-language newspaper, had on its front page a bold headline and a picture of Africans in Guangzhou demonstrating against police brutality on members of the African community1. This was one of the very first times, if not the first, that many people in China and worldwide came to the realisation that there now reside sizeable groups of Africans in Guangzhou and other cities in China such as Hong Kong, Macau, Yiwu, Shanghai and, of course, Beijing. Since then many more negative news reports (but also positive ones) have appeared in Chinese and International news media such as China Daily, CNN, BBC, CCTV, and The New York Times. Most of these reports centre on what has come to be known as the three immigration © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 79 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS illegalities (san fei in Chinese): illegal entry, illegal stay, and illegal engagement in employment. Alongside these media reports are academic writings (for example Zhou 2011) which, based on what are called “perception studies” or even “attitude studies”, document instances of negative attitudes and opinions that Chinese have about their African hosts in cities like Guangzhou. With respect to Chinese in Africa, the two most prominent instances of negative media reports come from Chinese in Zambia, with regards to the ill-treatment of workers by Chinese bosses2 and Chinese in Ghana, with regards to illegal immigration, illegal stay, and more prominently, illegal employment activities in the mining sector as reported in several international news media3. Besides these prominent cases on Chinese in Africa, there are numerous other reports about Diaspora Chinese indulging in other negative activities such as selling fake medicines, poaching, and undercutting prices to outcompete local businessmen. If one were to stay with these scenarios of negative reports and their academic analyses, one would find that our target Diaspora communities serve as great challenges and indeed a huge distraction for building more harmonious relations between Africa and China. However, this picture of negativity is far from complete, as we see in the next section of the paper. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Africa-China cooperation: the diaspora contributions Diaspora communities, given the definition as presented above and by most prominent scholars of global Diaspora studies throughout the world (for example Vertovec 1997), if presented with the right conditions, have often served as vital links between source and host communities. In several works (Bodomo 2010, 2012; Bodomo and Ma, 2012; Bodomo and Silva,2012), I have developed a theoretical framework showing that Africans in China serve as link points between Africa and China. Africans in Africa and Africans arriving newly in China learn about the Chinese in large part through Africans already in China. Chinese, for their part, learn a lot about Africa, whether negatively or positively, through their interactions with Africans in China. In the same vein, Africans in Africa learn a great deal about China and the Chinese by interacting with Chinese in Africa, and Chinese in Africa 80 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS AFRICAN EAST-ASIAN AFFAIRS teach their fellow Chinese in China a great deal about Africa and indeed serve as some of the first points of contact for newly arriving Chinese in Africa. In short, Diaspora Africans in China and Diaspora Chinese in Africa function as bridges interconnecting Africa and China in many respects. We may outline three main perspectives to illustrate this bridge theory of migrant-indigene relations: socio- economic, socio-political, and socio-cultural. Socio-economic contributions Worldwide, the global African Diaspora sent back to Africa 52 billion dollars in 2010, according to the World Bank, far more than the 43 billion dollars of Overseas Development Assistance (ODA) to Africa in that year. Indeed the African Union (AU) recognises and defines the global African diaspora in terms of the contributions it expects it to make to African development (Edozie, 2012). Though the focus of this paper is on African soft power in China, it needs to be mentioned for comparative purposes that Chinese in Africa are also performing similar functions as their African counterparts. Much of the Chinese presence in Africa in terms of trade is undertaken by private Chinese businessmen resident in Africa, and many scholars have reported on small scale Chinese enterprises dotted all over the 50-odd African countries- be it in Lesotho, Gabon, Nigeria or Senegal. In terms of investment, many private companies are being started up throughout Africa by individual Chinese resident in Africa. Chinese restaurants are dotted throughout Africa; these private businesses complement the government businesses led by the top 10 Chinese investment companies in Africa including Sinopec, China National Petroleum, State Grid Corporation, Industrial and Commercial Bank of China, China Railway Construction, Sicofor (Sino Congo Foret), CITIC-CRCC, China International Fund (CIF), China State Construction Engineering Corporation Limited (CSCEC), and Federated Steel (Bodomo, 2013). Socio-economic contributions Socio-economically, Diaspora Africans and Diaspora Chinese serve as prominent actors in cementing economic relations, frameworks, and platforms between Africa and China. They serve as traders, investors, and remitters. Many of the Africans in China are traders. They know more than most people what their people back home in Africa need, so they select and source out the most appropriate goods from Chinese factories for the African market; some of them have graduated into investors, starting up their own shipping and even manufacturing companies, sometimes in conjunction with Chinese businessmen to manufacture goods including textiles, household utensils, and farm equipment for the African market and worldwide. Africans remit a substantial amount of money back home to their families in Africa. Says the Chairman of the Guinea Association in Guangzhou: “Hi Professor, the amount sent home is often a function of many factors: how much the person is getting here on average, how the person's family is organised back home, etc. In general, an adult [male] Guinean in GZ [Guangzhou] has a wife (or wives) and kids back home in Conakry, the capital (or another big town), and his parents and other relatives (his and his wife’s/wives’ relatives) staying in the villages. It is normal for all these people to be supported by the adult living in GZ. Moreover, it is the ambition of each Guinean in the diaspora to build a house (where he can return) and start a business (shop or plantation, etc.) while still abroad. So, a guestimate for average remittances between 25000Rmb and 150000 Rmb per year is in place”. If all the half a million Africans plying their business in China were doing this, © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 81 81 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS Africans in China would be sending anywhere between five and 50 billion yuan a year or more – and this doesn't even include the value of all the merchandise that they buy from China and send back to Africa for sale, which even if not countered as remittance, still constitutes a good contribution from Diaspora Africans in terms of trade. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Socio-political contributions In performing these economic activities, Diaspora Africans and Diaspora Chinese also invariably perform socio-political functions. A number of African diplomatic personnel have told me at informal meetings and gatherings how essential some prominent members of the African communities in China are in helping them perform their official consular duties away from Beijing in places like Guangzhou, Yiwu, and Hong Kong. For instance, the office of the Chairman of the Nigerian © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 82 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS AFRICAN EAST-ASIAN AFFAIRS Association in Guangzhou was a de facto consulate for many Nigerians and even other Africans during much of the period of my field research in Guangzhou (2008 to present). African community leaders have met there to strategize and resolve immigration problems involving their fellow Africans. Many visiting political and business leaders have sought out established members of the African communities in Hong Kong, Macau, Guangzhou, and Shanghai for support to perform their duties. Some African community members have served as consultative and advisory committee members at the ruling Community Party meetings in Yiwu and other municipalities. Chinese community members in Africa, while generally shying away from overt dealings with the political establishment in their country of residence and even with their own embassy and government back in China, are mediating political contacts between their members and African governments, especially in times of crises. The situation needs to be investigated more closely in each country. For instance, in the case of Ghana, when a group of alleged illegal miners were arrested and faced deportation, community leaders emerged from their ranks to mediate with the Ghanaian government ministries involved and also to galvanise their members into a pressure group demanding that their Embassy and their government back in China intervene to resolve the problem in their favour. Some members of the Chinese Diaspora continue to develop socio-political links with Africa even when they are back in China. A case in point is a one-time Honorary Consul of Ghana in Hong Kong (the late Jonas Wu) who lobbied to be appointed to the position on returning to Hong Kong from an extended stay in Ghana. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Socio-cultural contributions Socio-culturally, Africans in China and Chinese in Africa serve as cultural ambassadors, whether consciously or unconsciously, to showcase and promote their cultures in the societies in which they live. Africans in China and Chinese in Africa are learning each other’s languages and ways of communicating. Africans in China are becoming more and more proficient in Chinese and even in the various local dialects of the cities in which they live, such as Cantonese in Guangzhou, Hong © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 83 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Kong, and Macau. In a survey of more than 700 Africans throughout China (Bodomo 2012) more than 50 per cent considered that they spoke Chinese at least only ‘a little’, with more than 10 per cent being confident that they spoke it very well. We need more of such sociolinguistic surveys to ascertain the levels of language proficiencies in these Diasporas. Two interesting linguistic phenomena that I noticed in Guangzhou are that Chinese who trade and interact with Africans in various ways are beginning to speak English with quite audible African accents and African ways of structuring their vocabularies and communication patterns. A second salient way, described at length in Bodomo (2012) is the phenomenon of calculator communication in which Africans and Chinese who do not share a specific lingua franca like English and Chinese communicate together with the help of a calculator. Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS AFRICAN EAST-ASIAN AFFAIRS Issue 2 June 2015 Kong, and Macau. In a survey of more than 700 Africans throughout China (Bodomo 2012) more than 50 per cent considered that they spoke Chinese at least only ‘a little’, with more than 10 per cent being confident that they spoke it very well. We need more of such sociolinguistic surveys to ascertain the levels of language proficiencies in these Diasporas. Two interesting linguistic phenomena that I noticed in Guangzhou are that Chinese who trade and interact with Africans in various ways are beginning to speak English with quite audible African accents and African ways of structuring their vocabularies and communication patterns. Case studies Many of the over 500,000 Africans in China have lived exemplary lives, some have even excelled in various ways to the extent that they have been recognised by the communities in which they live as deserving of awards. In this case study, I focus on two African males. In terms of methodology towards collecting and collating this data, I searched many internet fora and selected these two as Africans that have been broadly acknowledged by their communities. Then I contacted them and they kindly agreed to have me administer questionnaires to both of them; I followed up with a face-to-face interview with one of them on the side-lines of a conference in Paris in May 2014, and did email chatting with the other one to complement the information I collected from the questionnaire4. I shall in the following sub-sections summarise the profile of each of these two African soft power brokers in China, as one may call them. Socio-cultural contributions A second salient way, described at length in Bodomo (2012) is the phenomenon of calculator communication in which Africans and Chinese who do not share a specific lingua franca like English and Chinese communicate together with the help of a calculator. Beyond matters of language, Africans in China are creating cross-cultural spaces like ethnic restaurants, barbershops, football clubs, and small quasi-religious groups for worship and social networking, and are organising cultural programmes to showcase African culture like music, drumming, and dancing. Chinese in Africa are also beginning to learn the official languages of the countries in which they live, like English, French, Portuguese, and Arabic, along with prominent local lingua franca like Swahili, Hausa, Zulu, Amharic, Yoruba, Akan, Bambara, and Wolof. What is needed are comprehensive sociolinguistic surveys among members of the Chinese communities to get more specific linguistic profiles and repertoires to gauge the level of acculturation going on among Chinese communities in Africa, just as done in Bodomo (2012) for Africans in China. Beyond linguistic issues, Chinese in Africa are instrumental in getting Africans to have a taste of Chinese culture through their business activities in matters of cuisine and health, among others. Chinese restaurants are now dotted throughout the cities and towns in all the 50-odd countries of Africa. The African middle class is beginning to consume Chinese medicinal products, such as herbal tea, and awareness about Chinese culture is likely to increase because of the increasing presence of Chinese in Africa and also because of the spread of Chinese media like CCTV and China Daily. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 84 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS AFRICAN EAST-ASIAN AFFAIRS © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Dominique Saatenang: an African Bruce Lee Dominique Saatenang is a Cameroonian by nationality and was in his late 30s at the time of my interview. He says that the Chinese call him the African Eagle, the Europeans call him the Black Chinese while the Africans call him the African Bruce Lee. Equipped with an MBA degree, he considers himself as primarily a businessman and a Wushu master, having trained at the Shaolin Wushu Temple in China5. He is quite multi-lingual, mentioning French as his native language and other languages spoken as Chinese, English, and Bamileke. Dominique has lived for eight years in China at Defeng, Zhengzhou (Shaolin Temple), and in Beijing (at the Beijing Sports University). In terms of goodwill and charity, activities that have attracted attention to him are his excellent performance in sports at the Shaolin Temple and the Beijing Sports University. He lists a number of prizes and achievements to his credit, having won many exceptional medals in Wushu practice, including the 2006 Double Vice-World Champion award in Wushu organised in China. He mentions that in 2009, he won a UNESCO medal for his exceptional activities in Wushu; and that in 2011, he won a World Prize for his movie “Bring Charm to China”; and that in that year he was © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 85 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS officially named Wushu ambassador of the Shaolin Temple by the Spiritual Chief of the Temple. As charity activities, he mentions that he has brought 10 Africans to study in China for free during his five years at the Shaolin Temple. Asked if he had any ideas about how good African image can be promoted worldwide, he said: “We must just ty to be the best for what we come to do in China in our chosen activities”. Summary These case studies have illustrated – by concentrating on two Africans widely recognised in Chinese society as extraordinary members of the community – that Africans are contributing to the promotion of socio-cultural relations between Africa and China. These two and many other Africans located in many places in China including Guangzhou, Hong Kong, Macau, Yiwu, Shanghai and Beijing are emerging as agents of African soft power in China. Adam Musa: an African Lei Feng Adam Musa was a 29-year old PhD student in International Relations at Central China Normal University in Wuhan Hubei Province, China at the time of my interview. He is a Nigerian national and his mother tongue is Hausa, and he lists languages such as English and Mandarin Chinese as other languages that he speaks. Altogether, he has spent six years in China and outside Nigeria. Adam has excelled in many charity events, including blood donation for three years at the time of the interview, visiting houses for the elderly to help them, visiting orphanages, and volunteering as a teacher for migrant workers’ schools since 2010. He mentions that he has also been involved in many environmental protection exercises code-named “Keep Wuhan Clean”. Adam is a highly decorated community activist. To the question: “Have you won any prizes or been recognised in any way by the community or local authorities?”, he provides the following impressive list: 2011 Central China Normal University Excellent student Award 2011 Hubei province University Students Excellent Award 2011 Hubei province University Students Excellent Award 2012 Hubei Province Excellent Social Work Award 2012 Hubei Province Excellent Social Work Award 2012 China Youth Volunteer Excellent Award 2013 Central China Normal University Excellent Talent Award 2014 International Cultural Exchange Friendship Award Indeed, Adam’s work has been widely recognised and talked about in the community, and he has been featured in many Chinese media such as the China © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 86 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS Adams Bodomo “African soft power in China“ © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Adams Bodomo “African soft power in China“ Discussion: an alternative hypothesis? In this section, we address a few other issues that go to contextualise the case studies involving these two African individuals and then pose the question whether we are indeed dealing with African soft power or there could be an alternative hypothesis at work that claims that this is rather a manifestation of Chinese soft power7. The first issue is how the skills of the two individuals are related to conditions in China. Their skills including martial arts, volunteering, care for one's neighbours and proficiency at several languages all relate to the Chinese socio-cultural conditions, and are much appreciated by the Chinese as people. A second issue is what cultural framework the decorated individuals are seen to be excelling in. Even though they lived in China at the time of the study, I believe that they excel in both African and Chinese cultural frameworks, and even beyond. One of them got a UNESCO prize - these are individuals who have demonstrated universal values of charity and selflessness! It cannot therefore be the case that these individuals are influenced only by the Chinese cultural framework to do good. They are in China, so they have to © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 87 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS Issue 2 June 2015 operate in this framework, but it is not only the Chinese who are appreciating them, Africans are also appreciating them - one of them brought 10 Africans to study in China; these individuals surely appreciate that gesture. A third issue also has to be raised as to what specific benefits Africa is getting for the excellence these Africans are demonstrating in China. This must be seen, not in terms of monetary gains, but in terms of enhancing Africa’s image in China. Africa's image is very often gutted in China by the frequent tensions and clashes between Africans in China and mostly law-enforcement authorities and these specific positive contributions by the Africans would go a long way to changing the mentality of some Chinese citizens about Africa. Fourthly, it must be mentioned that these individuals are not just demonstrating exclusively Chinese cultural values or exclusively African cultural values. Rather, they possess some important cross-cultural nuances or even universal values: hard work, generosity (in terms of giving out their money and time), a caring nature, valour, and so on! Discussion: an alternative hypothesis? Finally, the question arises as to why we have limited ourselves to only two individuals. It is because these are case studies; the author is embarking on a series of articles on soft power and cannot document a throng of African individuals in one paper, even if their ethnographic profiles were readily available. In sum then, many of the issues raised in this section may lead to an alternative hypothesis that postulates that these individuals, rather than being conduits of African soft power in China, are indeed demonstrating that they have been imbued with Chinese soft power; in other words, that it is Chinese soft power, rather than African soft power, that is at play here because the Chinese are somehow only appreciating these Africans as a way of infusing Chinese soft power in them, rather than having a positive view of Africa because of the activities of these individuals. Several issues may be at play here that do not in any way negate the hypothesis of African soft power as argued throughout the paper, and must therefore be teased out. First, it is plausible that the Chinese admire these individuals for learning Chinese culture and speaking the Chinese language very well - but so do they do with regards to other Africans who have learnt Chinese culture and speak the Chinese language © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 88 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS AFRICAN EAST-ASIAN AFFAIRS fluently. It is not everybody who so integrates themselves into Chinese society who are awarded such high prizes. These individuals are bringing something else to the table, and that extra something is the basis of the African soft power as argued here. The two individuals (and many more that could be uncovered through further research) are excelling in terms of their volunteerism, kindness, generosity, valour at Wushu, and intelligence and the Chinese see many of these as attributes they brought from Africa and thus have a positive view of these Africans. This is clearly in line with aspects of our earlier definition of soft power as "...the positive...socio-cultural influences a polity and its citizens have on another polity and its citizens....". Discussion: an alternative hypothesis? If we found Chinese in Africa of the same stature as these Africans in China, there would be no doubt that they would be seen as spreading Chinese soft power, so there is no reason to doubt that these Africans are spreading African soft power in China. Second, it may be the nature of soft power such that when it is deployed in one direction, there could be a reciprocal effect; that is, soft power effects of entity A unto entity B may engender soft power effects of entity B unto entity A, though not always. Soft power can be symmetrical and bidirectional, unlike hard power, which is necessarily asymmetrical and unidirectional. Seen this way, even if the Chinese are preoccupied with trying to infuse Chinese soft power into these individuals, they are in turn getting infused with African soft power, as they cannot help but admire these individual Africans and the attributes they may have brought from Africa. So, the hypothesis of an African soft power, rather than being negated, is indeed strengthened. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Looking ahead: recommendations for a people-to-people approach We have demonstrated in the above analysis that Diaspora African communities in China and Diaspora Chinese communities in Africa act as vital economic, political, and cultural links – bridges – between Africa and China, and especially that a good number of Africans in China are engaging in activities that go to form the building blocks of an emergent African soft power in China. This soft power building process may be occurring among Chinese in Africa and awaits a closer investigation. It therefore goes without saying that if we are looking for ways to strengthen Africa- © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 89 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR Issue 2 June 2015 China co-operation, we must involve Diaspora Africans and Diaspora Chinese. I recommend that while the first decade of the co-operation was more of a government -to-government approach, which was necessary to create a solid base, the second decade should be dedicated to strengthening a people-to-people approach to Africa- China cooperation. A people-to-people approach involves creating avenues for different groups of people from each side of the partnership, like professional associations, academic groups, business groups, youth and student movements, artists, and sportsmen to interact more often. Diaspora community members can play a vital role in facilitating contact between all these groups mentioned, since they are indeed already doing this on their own and are sitting with a lot of experience about contacts at all the levels. The 5th edition of the Forum on Africa-China Cooperation (FOCAC2012 in Beijing) did touch briefly on the idea of people-to-people relations, presumably based on the exhortations some of us scholars have already been making through our writings, but it did not actually stress it enough, and said very little about what we could do to achieve it. I recommend that the 6th edition (FOCAC2015 in South Africa) should dedicate a whole working session and theme on the role of the two Diasporas involved in the partnership to find out how we can enhance their formal participation in strengthening Africa-China cooperation if they so wish. Two ways in which we can enhance their participation is, first, by streamlining immigration rules on both sides of the partnership. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Looking ahead: recommendations for a people-to-people approach There must be clearer paths to permanent residency and citizenship for Africans in China and Chinese in Africa. Police and other security forces on both sides of the partnership must respect the rights of Diaspora members, including treating even those on the wrong side of the law, such as African visa over-stayers in Guangzhou or Chinese illegal miners in Ghana, humanely. A second way is to create structures and incentives to get Diaspora Africans and Diaspora Chinese keenly interested in strengthening Africa-China cooperation. Funds can be set aside for competitive bidding by various Diaspora Community groups to be used in promoting cultural activities that go towards enhancing the intermingling of Africans and Chinese. It is now time for governments on both sides 90 Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS Adams Bodomo “African soft power in China“ AFRICAN EAST-ASIAN AFFAIRS of the partnerships to start promoting Diaspora cultural festivals, Diaspora business meetings, academic conferences on the African and Chinese Diasporas, and Diaspora Sports festivals. There should also be reward and recognition systems to highlight excellence in service and leadership among Diaspora community members on both sides of the partnership. Summary and conclusions As Africa and China move closer to each other for mutual benefits, Diaspora communities will grow on each side of the partnership. Media and academic reports and analyses are documenting both negative and positive activities involving members of these Diaspora communities, suggesting that the communities that are the subject of this study can indeed be double-edged swords, in that their activities can pose both challenges and opportunities to the development of Africa-China relations. I have argued here that, after balancing things out, the Diaspora account is indeed in the positive digits since the communities in question play far more positive than negative roles in strengthening Africa-China co-operation. Many members of the African communities in China are indeed engaging in activities that may go to promote the good image of Africa in China, and may indeed be building the bricks for an African soft power in China. This may well be the case for Chinese in Africa, and this needs further investigation. If the recommendations outlined above are implemented, Diaspora Africans in China and Diaspora Chinese in Africa can serve as agents of soft power that promote cultural diplomacy, and as useful links or bridges to the further development of Africa-China cooperation. © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. Endnotes 1. The SCMP article was titled: “Africans protest in Guangzhou after Nigerian feared killed fleeing visa check” SCMP, July 16, 2009: http:// www.scmp.com/article/686919/africans-protest-guangzhou-after-nigerian- feared-killed-fleeing-visa-check (accessed: August 19, 2014). © Centre for Chinese Studies, Stellenbosch University All Rights Reserved. 91 Issue 2 June 2015 AFRICAN EAST-ASIAN AFFAIRS THE CHINA MONITOR AFRICAN EAST-ASIAN AFFAIRS 2. Lusaka News. 9 October 2010. Ill-treatment of workers, disgraceful – ZFE [Online]. Available: http://www.lusakatimes.com/2010/10/19/illtreatment- workers-disgraceful-zfe/ (2014, August 19). 3. BBC News. June 6, 2013. Ghana arrests Chinese for ‘illegal mining’ [Online]. Available: http://www.bbc.co.uk/news/world-asia-china- 22793659 (2014, August 19). 4. The criteria for inclusion were quite stringent: for anyone to be on my list they had to have won a recognition in the society in which they were operating. This is a case study and if the research is pushed further other individuals, including females, would show up. 4. The criteria for inclusion were quite stringent: for anyone to be on my list they had to have won a recognition in the society in which they were operating. This is a case study and if the research is pushed further other individuals, including females, would show up. 5. A video clip of his activities as a Wushu practitioner can be viewed at this youtube link: https://www.youtube.com/watch?v=oXGYsN-VLdY 5. A video clip of his activities as a Wushu practitioner can be viewed at this youtube link: https://www.youtube.com/watch?v=oXGYsN-VLdY 6. China Daily has one of several reports on Adam Musa, the latest being an article titled: “Lei Feng’s African brother” (Lei Feng being ‘China’s icon of selflessness’ at this weblink: http://usa.chinadaily.com.cn/epaper/2013-08/06/ content_16874364.htm, China Daily Aug 6, 2013 (accessed on August 19, 2014) 7. I thank an anonymous reviewer for suggesting this alternative hypothesis. Adams Bodomo “African soft power in China“ Bibliography Bodomo, A. 2014. The African traveller and the Chinese customs official: Ethnic Minority Profiling in Border Check Points in Hong Kong and China? Journal of African American Studies. New York: Springer Science and Business Media. Bodomo, A. 2013. The Globalization of Investment: Chinese Companies in Africa, Modern Ghana [Online]. Available: http://www.modernghana.com/ news/444094/1/the-globalization-of-investment-chinese-companies-.html Bodomo, A. 2013. The Globalization of Investment: Chinese Companies in Africa, Modern Ghana [Online]. 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https://openalex.org/W2901789930	https://europepmc.org/articles/pmc6349567?pdf=render	English	null	Diagnosis of Mucopolysaccharidosis Based on History and Clinical Features: Evidence from the Bajio Region of Mexico	Curēus	2,018	cc-by	4,770	DOI: 10.7759/cureus.3617 © Copyright 2018 Colmenares-Bonilla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Mucopolysaccharidosis (MPS) are infrequent deposit diseases; generally, the diagnosis is delayed until symptoms appear. Age or presentation is related to the severity of the disease. A substantial number of patients are misdiagnosed since they describe nonspecific initial symptoms and signs in common. The aim of this study is to describe the common characteristics of patients with mucopolysaccharidosis already diagnosed, treated in hospitals of the Guanajuato Health System, with a special focus on early manifestations in order to review early clinical suspect manifestations. Open Access Original Article Open Access Original Article Open Access Original Article Methods A multicenter, descriptive, observational study was conducted to evaluate the cases of mucopolysaccharidosis treated and diagnosed. The study was carried out in the Pediatric departments of five big important hospitals of Bajio Mexico region in the period from February to August 2016. Results Eighteen patients were identified, 13 men and five women, with an average age of 8.6 years. The most frequent mucopolysaccharidosis was type IV A (Morquio) in seven patients, followed by type I (Hurler) in four patients, three patients for type III (San Filippo), two patients for type II (Hunter), and two patients for type VI (Maroteaux-Lamie). The commonest clinical manifestations at diagnosis were dimorphism, triangular dorsal hump, skeletal alterations (genu valgus, short stature, and flat feet), and a limited range of movement in the major joints. Non-skeletal manifestations, such as an umbilical/inguinal hernia and hepato-splenomegaly, were very frequent. In a majority of patients with mucopolysaccharidosis, the radiological data of the disease were found: they were most severe in type IV and type VI, mild in type I and II, and none in MPS III. A diagnosis was made in all patients by a clinical and radiological evaluation and confirmed by an enzymatic study. Received 09/26/2018 Review began 10/05/2018 Review ended 11/17/2018 Published 11/20/2018 How to cite this article Colmenares-Bonilla D, Colin-Gonzalez C, Gonzalez-Segoviano A, et al. (November 20, 2018) Diagnosis of Mucopolysaccharidosis Based on History and Clinical Features: Evidence from the Bajio Region of Mexico. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 Diagnosis of Mucopolysaccharidosis Based on History and Clinical Features: Evidence from the Bajio Region of Mexico Douglas Colmenares-Bonilla Dr. , Christian Colin-Gonzalez , Alejandra Gonzalez-Segoviano , Enrique Esquivel Garcia , Ma Martha Vela-Huerta , Fanny Guadalupe Lopez-Gomez 1 2 3 2 4 5 1. Pediatric Orthopaedics, Hospital Regional De Alta Especialidad Del Bajio, Leon, Guanajuato, MEX 2. Ortopedia Y Traumatologia, Hospital General De Leon, Leon, MEX 3. Ortopedia Pediatrica, Hospital De Especialidades Pediatricas De Leon, Leon, MEX 4. Neonatology Department, Hospital General De Leon, Leon, MEX 5. Pediatrics, Hospital General De San Luis De La Paz, San Luis de La Paz, MEX  Corresponding author: Douglas Colmenares-Bonilla Dr., douglas_cb@yahoo.com Disclosures can be found in Additional Information at the end of the article Received 09/26/2018 Review began 10/05/2018 Review ended 11/17/2018 Published 11/20/2018 © Copyright 2018 Colmenares-Bonilla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. the need to strengthen collaboration patterns between different specialities play an important role in the early diagnosis and treatment of these conditions. the need to strengthen collaboration patterns between different specialities play an important role in the early diagnosis and treatment of these conditions. Categories: Family/General Practice, Pediatrics, Miscellaneous Categories: Family/General Practice, Pediatrics, Miscellaneous Keywords: mucopolysaccharidosis, mexico, diagnosis, short stature, dwarfism, orphan disease, lysosomal disease Conclusions In all rare diseases, a suspicion diagnosis is based on subtle characteristics that manifest themselves in a few different organs and systems may be mild. Suspicion by the physician and How to cite this article Colmenares-Bonilla D, Colin-Gonzalez C, Gonzalez-Segoviano A, et al. (November 20, 2018) Diagnosis of Mucopolysaccharidosis Based on History and Clinical Features: Evidence from the Bajio Region of Mexico. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 How to cite this article Colmenares-Bonilla D, Colin-Gonzalez C, Gonzalez-Segoviano A, et al. (November 20, 2018) Diagnosis of Mucopolysaccharidosis Based on History and Clinical Features: Evidence from the Bajio Region of Mexico. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 Introduction The term mucopolysaccharidosis (MPS) groups a series of rare conditions, characterized by defects in the catabolism of glycosaminoglycans (GAGs) [1], which causes the progressive accumulation of these substances in cellular lysosomes on different tissues [2], resulting in pathologic progressive changes in multiple systems that will affect the quality of life in the short or medium term [3-5]. Generally, manifestations of these diseases will be related to the type of GAGs accumulating in cellular lysosomes. Accumulation is as follows: dermatan and heparan sulfate in the Hurler (MPS I) and Hunter (MPS II) syndromes; heparan sulfate in Sanfilippo syndrome (MPS III); keratan and chondroitin sulfate in Morquio syndrome (MPS IV A) [6]; and dermatan sulfate in Maroteaux-Lamy syndrome (MPS VI) [7-8]. Currently, there are seven described types of MPS, caused by the deficiency of 10 hydrolases [9]; these are mentioned in Table 1. 2 of 12 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 TYPE NAME STORED GLYCOSAMINOGLYCAN ENZYME I Hurler Heparan sulfate. Dermatan sulfate. α-L-iduronidase Hurler-Scheie Scheie II Hunter. Heparan sulfate. Dermatan sulfate. Iduronate-2-sulfatase III San Filippo A Heparan sulfate. Heparan N-sulfatase San Filippo B α-N-acetyl glucosaminidase San Filippo C α-glucosaminide acetyltransferase San Filippo D N-acetylglucosamine 6- sulfatase IV Morquio A Keratan sulfate + Chondroitin-6-sulfate. Keratan sulfate. N-acetylgalactosamine-6- Sulfatase Morquio B β-D-galactosidase VI Maroteaux- Lamie. Dermatan sulfate. N-acetylgalactosamine-4- sulfatase VII Sly. Heparan sulfate + Dermatan sulfate + Chondroitin-4, -6 sulfate. β-glucuronidase IX - Hyaluronan Hyaluronidase TABLE 1: Current classification of mucopolysaccharidosis, name, stored glycosaminoglycan, and enzymatic deficiency Name, stored glycosaminoglycan and enzymatic deficiency. Diagnosis can represent a challenge, and an agreement between clinical and radiological findings is required. At birth, patients phenotypically have a normal appearance. The onset of the development of symptoms varies depending on the severity of the disease [10]; those severely affected show signs and symptoms in the first year of life while the attenuated forms do so in childhood or adolescence [11]. The age of first clinical signs in patients with a severe phenotype can be evidenced from birth, diagnostic suspicion, and referral to the specialist as early as four months; however, in attenuated patients, this suspicion may be delayed even after 10 or 12 years [12]. Diagnosis confirmed by an enzymatic study varies between 20 and 522 months of age, in some reports, with an average of 62 months [13-14]. Among the clinical findings found in MPS are general manifestations, such as short stature, infiltrated facies, thick eyebrows, as well as orthopedic manifestations (joint hypermobility or joint stiffness without inflammation, skeletal deformities, and multiple dysostosis), cardiac 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 3 of 12 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 manifestations (murmur, myocardiopathy, mitral-aortic valve disease, cardiomegaly), and respiratory manifestations (hypertrophy of tonsils, adenoids, frequent respiratory infections). In other patients, there are umbilical and/or inguinal hernias, global retardation of psychomotor development, and the progressive loss of motor and intellectual skills they had already acquired in all but attenuated phenotypes and MPS IV and VI [3-4,6,10-11,13,15- 16] (Figure 1). manifestations (murmur, myocardiopathy, mitral-aortic valve disease, cardiomegaly), and respiratory manifestations (hypertrophy of tonsils, adenoids, frequent respiratory infections). In other patients, there are umbilical and/or inguinal hernias, global retardation of psychomotor development, and the progressive loss of motor and intellectual skills they had already acquired in all but attenuated phenotypes and MPS IV and VI [3-4,6,10-11,13,15- 16] (Figure 1). FIGURE 1: Radiographic features in an MPS IV A male patient a) Central beaking of the first lumbar vertebrae and posterior vertebral scalloping. b) Severe acetabular dysplasia, coxa plana, broad neck of femur. c) Flattened ribs, hepatosplenomegaly. d) Genu valgus with proximal and lateral tibial dysplasia, widening of the physeal line. MPS: mucopolysaccharidosis FIGURE 1: Radiographic features in an MPS IV A male patient a) Central beaking of the first lumbar vertebrae and posterior vertebral scalloping. b) Severe acetabular dysplasia, coxa plana, broad neck of femur. c) Flattened ribs, hepatosplenomegaly. d) Genu valgus with proximal and lateral tibial dysplasia, widening of the physeal line. MPS: mucopolysaccharidosis Each type of glycosaminoglycan has major storage organs: heparan sulfate produces predominantly neurological symptoms, as in MPS I, II, and III; keratan sulfate produces corneal opacities and skeletal alterations without the neurological involvement observed in MPS IV A and VI; and dermatan sulfate produces cardiomyopathy and valvular heart disease in MPS I, II, and VI [5-6,10,13,17]. 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 4 of 12 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 MPS: mucopolysaccharidosis To confirm the diagnosis in a patient with suspected MPS, a dry peripheral blood spot test is taken on filter paper, determining the activity of the deficient enzyme. If the result is positive, it must be confirmed with the measurement of the enzyme's serum activity or mutational molecular analysis. Often, a mutational analysis is not routine and is only used for genetic counseling or research purposes [22]. Early diagnosis is important for prompt, specific treatment with enzyme replacement therapy (ERT), which is currently available for types I, II, IV and VI. Early start of treatment helps reduce the accumulation of intracellular GAGs and the deceleration of progressive multiorgan worsening, improving lung and heart function and final growth rate [1,23-25]. MPS are grouped into rare or orphan diseases. The main differential diagnosis is skeletal dysplasias [12,26]. The incidence varies upon the population studied and the type of MPS described; for example, for MPS IV A, the incidence ranges from 1:640000 live births in Australia to 1:76000 in Northern Ireland [13]. In Mexico, the diagnosis is confirmed around 4.5 years, usually by the geneticist or orthopedic surgeon [27]. Scarpa and collaborators reported in 2011, a European incidence from 1:140000 to 1:156000 live newborns for the MPS II [15]. Regard MPS VI, in 2007, an incidence from 1:238095 to 1:300000 live newborns were reported [14]. There is scarce literature addressing the common characteristics of these diseases and giving the clinician strong tools for a diagnostic suspicion. Diffusion will improve early diagnosis or refine diagnosis in patients with similar diseases. The aim of the present report is to describe the clinical-radiographic features of patients with mucopolysaccharidosis in hospitals of the health system of Guanajuato. It is recommended to carry out a clinical and radiographic evaluation of different regions of the skeleton for patient assessment. These radiographs are cranial in the lateral view, complete lateral spine, anteroposterior pelvis, and extremities, particularly knees, and hands and wrists in anteroposterior views [10,18-20]. Some radiological findings expected in MPS include odontoid hypoplasia, atlantoaxial dislocation, J-shaped sella turcica, flattening of the ribs, Pectus carinatum, small iliacus bone, acetabular dysplasia, flattened capital femoral epiphysis, coxa plana, platyspondyly, central beaking of the first lumbar vertebrae and posterior vertebral scalloping, short ulna, and carpal and metacarpal bone dysplasia [2,4,10-11,13,15,18,20-21], as shown in Figure 2. p y p [ , , , , , , ], g FIGURE 2: Clinical common characteristics in MPS patients a) Semiflexion contracture in elbows, wrists, hips, knees, and ankles. b) Contracture flexion in shoulders, incapacity to raise hands above the head, umbilical hernia. c) and d) Maximum active range of hand mobility with loss of grip force and hand skills. (This picture shows an MPS VI male patient). FIGURE 2: Clinical common characteristics in MPS patients a) Semiflexion contracture in elbows, wrists, hips, knees, and ankles. b) Contracture flexion in shoulders, incapacity to raise hands above the head, umbilical hernia. c) and d) Maximum active range of hand mobility with loss of grip force and hand skills. (This picture shows an MPS VI male patient). MPS: mucopolysaccharidosis 5 of 12 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 Results A total of 18 patients are reported on, Morquio A syndrome (MPS IV-A) predominates in seven patients (38%), followed by Hurler syndrome (MPS I) (22%) in four, San Filippo syndrome (MPS III) (16.6%) in three, and Hunter syndrome (MPS II) and Maroteaux-Lamie syndrome (MPS VI) in two cases each (11%, 11%, respectively). Materials And Methods In this retrospective study, all patients with a clinical diagnosis of mucopolysaccharidosis, who had confirmation of the enzymatic activity in the plasma, were included in the study, regardless of whether they were undergoing enzymatic replacement therapy (ERT) or not, in the hospitals of the Guanajuato Health System, including the Bajío High Specialty Regional Hospital, León General Hospital, Leon Pediatric Specialties Hospital, and General of San Hospital Luis de la Paz, in a period from January to December 2016. Those patients who were lost to follow-up or incomplete records were eliminated. By direct clinical examination and data from the files, somatometry (weight, height, and body mass index) was obtained, as well as MPS features (corneal opacity, hernias, respiratory infections, joint movement, and orthopedic manifestations) since the first visit (before the diagnosis) and data that would lead to a suspicion of diagnosis. Simple radiographs were used: pelvis posteroanterior, skull lateral, both knees posterior- anterior, hand anteroposterior, and lateral spine. We collected the main findings on electronic data sheets and an analysis was carried out by computer software. Results were aggregated on Excel spreadsheets (Microsoft Corporation, Redmond, WA, US), formatted prior to analysis, and described with measures of central dispersion. 6 of 12 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 Half of the patients had a family history of this type of diseases, and six of the patients (33%) had consanguineous parents. Patient MPS H CO D DD M TD HS LL DH GV FF 1 IV-A ● ● ● ● ● ● ● ● ● ● 2 IV-A ● ● ● ● ● ● ● ● ● ● 3 IV-A ● ● ● ● ● ● ● ● 4 IV-A ● ● ● ● ● ● ● ● ● 5 II ● ● ● 6 III ● ● ● ● 7 III ● ● ● ● ● 8 IV-A ● ● ● ● ● ● ● ● ● ● 9 IV-A ● ● ● ● ● ● ● ● ● ● ● 10 VI ● ● ● ● ● ● ● ● ● ● 11 VI ● ● ● ● ● ● ● ● ● 12 IV-A ● ● ● ● ● ● ● ● ● 13 I ● ● ● ● ● ● ● ● ● ● 14 II ● ● ● ● ● ● ● 15 III ● ● ● ● ● ● ● ● 16 I ● ● ● ● ● ● ● ● ● ● 17 I ● ● ● ● ● ● ● ● ● ● ● 18 I ● ● ● ● ● ● ● ● ● ● TABLE 2: Physical characteristics of present study MPS population MPS: type of mucopolysaccharidosis. H: umbilical or inguinal hernia. CO: corneal opacity. D: dimorphism. DD: dental diastasis. M: macroglossia. TD: thoracic deformity. HS: hepatosplenomegaly. LL: ligamentous laxity. DH: dorsal hump. GV: genu valgus. FF: flat feet TABLE 2: Physical characteristics of present study MPS population MPS: type of mucopolysaccharidosis. H: umbilical or inguinal hernia. CO: corneal opacity. D: dimorphism. DD: dental diastasis. M: macroglossia. TD: thoracic deformity. HS: hepatosplenomegaly. LL: ligamentous laxity. DH: dorsal hump. GV: genu valgus. FF: flat feet MPS: type of mucopolysaccharidosis. H: umbilical or inguinal hernia. CO: corneal opacity. D: dimorphism. DD: dental diastasis. M: macroglossia. TD: thoracic deformity. HS: hepatosplenomegaly. LL: ligamentous laxity. DH: dorsal hump. GV: genu valgus. FF: flat feet The time between the first observed manifestation and accurate diagnosis ranged between one month and six years (with an average of 2.7 years). The average age at diagnosis was 5.6 years (range from one month to 15 years). All patients but one had a history of frequent respiratory infections (more than six events per year). Two of the patients died of complications thereof. Features at diagnosis The first clinical manifestation related to the disease occurred at 3.1 years on average (range: birth to nine years). Earliest data were on intrauterine pneumonia in a patient with MPS I and the latest data were on knee valgus in a nine-year-old patient with MPS VI. The most commonly encountered alteration was genu valgus in 14 patients (77%) patients, of which four were severe (all MPS IV A), two moderate (all MPS VI), and the rest were mild. Other first manifestations were hernia (umbilical or inguinal) in five patients. Hernia together with infiltrated facies was the first finding in two patients and in three patients, hernia was the only first suspicion data. In three patients, the first manifestation was deformity in the thoracic cage (kyphosis or scoliosis). The rest of the patients showed limitations in joint mobility (rigidity), flat feet, and short stature, as shown in Table 2. 7 of 12 All patients have dimorphism (infiltration) in variable ranges and relative macroglossia. Except for two patients (who died during the study) and one with MPS II, all of them showed a triangular dorsal hump. Other physical characteristics were hepatosplenomegaly in 15 patients, flat feet in 15 patients (88.3%), and genu valgus in 14 patients (ranging from mild to severe angulation). Hernia (inguinal or umbilical) was observed in 12 patients (66%). Fifty-five percent showed corneal opacity, 88.88% dental diastasis, and 100% relative macroglosia. Radiographic studies showed 15 patients (83.33%) shared at least two or more characteristics, such as odontoid hypoplasia, atlantoaxial subluxation, J-shaped sella, widening of anterior ribs, pectus carinatum, flared ilium laterally with inferior constriction (wineglass shape), acetabular dysplasia, poorly formed femoral epiphyses, widened femoral necks, coxa valga, platispondilia, hypoplastic vertebra at the thoracolumbar junction, anterior inferior vertebral body beaking, short ulna, conical (bullet-shaped) proximal bases of metacarpals. All patients with MPS III (n = 3) did not show any radiological alteration. All patients with MPS IV A have undergone orthopedic surgical procedures at the time of the study. Physical features 8 of 12 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 Discussion Currently, seven types of MPS are known, caused by the deficiency of 10 hydrolases [8-9,22]. Within our study, only five types were presented, with a higher frequency of MPS IV A because the small sample size is not representative of the population, nor as an average to calculate the incidence. For example, in Western Australia, the general incidence of MPS is calculated as one in 29,000 live births, whereas the incidence of MPS III is the highest (one in 58000), followed by MPS I and, less frequently, MPS IV A (one in 640000) [28]; however, in Switzerland and Japan, the general incidence is calculated as 1.56 per 100000 live births; The first place in incidence corresponds to MPS. II (55%), followed by I, III, and IV A (12%, 24%, and 24%) [29]. A Pakistani report states that MPS I dominates in more than 80% of patients, followed by MPS IV A and III. The different data suggest that the marked difference in incidence depends on many factors, among which are consanguinity, cultural characteristics, and the underdiagnosis of these conditions [2,11-12]. The age of onset in more than 50% of patients with the severe form is 2.2 years, although the enzymatic diagnosis is usually delayed at least three years, which leads to a mean age for diagnosis of 4.9 years [11]. In the present report, the first manifestation was presented at 3.15 years with a diagnosis delay of 2.45 years average and age at enzymatic diagnosis was 5.6 years. On average for the Mexican MPS IV A population, the diagnosis is at 4.5 years in severe cases, like the present report, where the average age is five years [27]. Among the most common historical antecedents in these patients were: frequent respiratory infections, affected relatives, and consanguinity, as well as outstanding clinical characteristics, such as dental diastasis, macroglossia, hepatosplenomegaly, and infiltrated facies, in the same way as described by other publications. [2,6,17,21-22]. Orthopedic manifestations may be the first to be described by parents or guardians. In the present report, 11 of the 18 showed alterations of this kind, with genu valgus as the most commonly referred to; this agreed with the literature [3,18-19]. About 83.33% of the patients studied showed radiological characteristics like those reported in the literature [18,26]. About 83.33% of the patients studied showed radiological characteristics like those reported in the literature [18,26]. 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 Each type of Mucopolysaccharidosis has predominant, specific deposition organs. In case of MPS III, enzyme deficiency in the production of heparan sulfate has repercussions with the development of neurological symptoms, without skeletal muscle involvement as observed in the present series, where three patients with this disease did not present radiological data [12,16]. The severity of clinical characteristics seems to determine early or late diagnosis [30]. In the present report, the diagnosis was suspected on clinical and radiological findings, followed by filter paper screening and a confirmatory exam in the enzymatic test, according to the guidelines of international literature [8,22]. Discussion DOI 10.7759/cureus.3617 9 of 12 Disclosures Human subjects: Consent was obtained by all participants in this study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Conclusions Commonest clinical manifestations for diagnostic suspicion were dimorphism, skeletal alterations, triangular dorsal hump, genu valgus, restriction to mayor joint movement, and pes planus. Other frequent manifestations were short stature and umbilical or inguinal hernia. In the majority of the patients with mucopolysaccharidosis, radiological data of the disease were found, except for MPS III. The diagnosis was made by clinical and radiological evaluation and confirmed by an enzymatic study. In all rare diseases, the diagnosis of suspicion is based on subtle characteristics that manifest themselves in different organs and systems. Suspicion by the physician and the need to strengthen collaboration patterns between different specialties play an important role in the early diagnosis and treatment of these conditions. Greater familiarity with these diseases is necessary in first-contact physicians in order to improve the detection and decrease the complications of disease progression. It is a study whose main limitation is the small size of the sample, which only represents patients diagnosed in a specific geographical area of the center of a country. 2018 Colmenares-Bonilla et al. Cureus 10(11): e3617. DOI 10.7759/cureus.3617 care. 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https://openalex.org/W3118148042	https://pub.uni-bielefeld.de/download/2949970/2955542	English	null	Intraindividual Variability and Temporal Stability of Mid-Sleep on Free and Workdays	Journal of biological rhythms	2,020	cc-by	13,219	Intraindividual Variability and Temporal Stability of Mid-Sleep on Free and Workdays *Department of Psychology, University of Warwick, Coventry, UK, †Faculty of Brain Sciences, University College London, London, UK, ‡Estonian Genome Center, University of Tartu, Tartu, Estonia, §Department of Psychology, Bielefeld University, Bielefeld, Germany, \|\|Division of Health Sciences, Warwick Medical School, University of Warwick, Coventry, UK, ¶Institute of Psychology, University of Tartu, Tartu, Estonia, and #The Estonian Academy of Sciences, Tallinn, Estonia Abstract People differ in their sleep timings that are often referred to as a chro- notype and can be operationalized as mid-sleep (midpoint between sleep onset and wake-up). The aims of the present studies were to examine intraindividual variability and longer-term temporal stability of mid-sleep on free and work- days, while also considering the effect of age. We used data from a 2-week expe- rience sampling study of British university students (Study 1) and from a panel study of Estonian adults who filled in the Munich Chronotype Questionnaire twice up to 5 years apart (Study 2). Results of Study 1 showed that roughly 50% of the variance in daily mid-sleep scores across the 14-day period was attributed to intraindividual variability as indicated by the intraclass correlation coeffi- cient. However, when the effect of free versus workdays was considered, the intraindividual variability in daily mid-sleep across 2 weeks was 0.71 the size of the interindividual variability. In Study 2, mid-sleep on free and workdays showed good levels of temporal stability—the retest correlations of mid-sleep on free and workdays were 0.66 and 0.58 when measured twice over a period of 0-1 to 5 years. The retest stability of mid-sleep scores on both free and workdays sharply increased from young adulthood and reached their peak when partici- pants were in late 40 to early 50 years of age, indicating that age influences the stability of mid-sleep. Future long-term longitudinal studies are necessary to explore how age-related life circumstances and other possible factors may influ- ence the intraindividual variability and temporal stability of mid-sleep. Keywords chronotype, mid-sleep, intraindividual variability, temporal stability, ques- tionnaire validation preference or natural inclination to go to and get out of bed (Horne and Östberg, 1976). The two are strongly related, yet distinct, constructs (Zavada preference or natural inclination to go to and get out of bed (Horne and Östberg, 1976). 842 JBRXXX10.1177/0748730420974842Journal of Biological RhythmsLenneis et al. / Intraindividual Variability and Stability of 842 JBRXXX10.1177/0748730420974842Journal of Biological RhythmsLenneis et al. / Intraindividual Variability and Stability of https://doi.org/10.1177/0748730420974842 JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 36 No. 2, April 2021 169–184 DOI: 10.1177/0748730420974842 © 2020 The Author(s) Article reuse guidelines: sagepub.com/journals-permissions 1. To whom all correspondence should be addressed: Anita Lenneis, Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK; e-mail: a.lenneis@warwick.ac.uk; and Anu Realo, Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK; e-mail: a.realo@warwick.ac.uk Intraindividual Variability and Temporal Stability of Mid-Sleep on Free and Workdays The two are strongly related, yet distinct, constructs (Zavada Timing of sleep is an indicator of chronotype (Roenneberg et al., 2003) and is not identical with morningness-eveningness, which refers to one’s 1. To whom all correspondence should be addressed: Anita Lenneis, Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK; e-mail: a.lenneis@warwick.ac.uk; and Anu Realo, Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK; e-mail: a.realo@warwick.ac.uk 169 JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 170 et al., 2005). Chronotype, on one hand, refers to a time of day, namely, when an individual’s endog- enous clock synchronizes (entrains) to the 24-h day (Roenneberg et al., 2004) and is often opera- tionalized as mid-sleep, that is, midpoint between sleep onset and wake-up (Terman et al., 2001). Morningness-eveningness, on the other hand, is a diurnal preference for what time to go to and get out of bed, including at what time during the day an individual is most alert and can perform best (Horne and Östberg, 1976). People with an early or morning chronotype tend to wake up and to go to bed relatively early whereas those with a late or evening chronotype tend to sleep longer in the morning and to go to bed later. Chronotype has a strong biological and genetic basis with roughly half of the variance being attributed to heritability (see for example Hur, 2007; Koskenvuo et al., 2007; Barclay et al., 2010), whereas the remaining variance can be accredited to environmental and social factors such as the exposure to natural sunlight (Roenneberg et al., 2007), artificial light (Vetter et al., 2011), social and professional demands (Leonhard and Randler, 2009; Abbott et al., 2017), and personality (Lipnevich et al., 2017). morningness-eveningness may be different across generations. g Most of the studies that have looked into the tempo- ral stability of chronotype and morningness-evening- ness have done so over relatively short periods of time in the course of validating and establishing test-retest reliabilities of chronotype and morningness-evening- ness questionnaires. Supplemental Table S1.1 gives an overview of the previously conducted studies investi- gating test-retest correlations in sleep-time-based assessments of chronotype and preferential morning- ness-eveningness questionnaires over both shorter and longer periods of time. Intraindividual Variability and Temporal Stability of Mid-Sleep on Free and Workdays Generally speaking, the test-retest reliabilities of both types of questionnaires have been found to be quite high (i.e., range between r = 0.76 and 0.97) when the same participants were tested 1 to 4 months apart from each other. However, the test-retest reliabilities seem to decrease with lon- ger time intervals. A longitudinal study by Urner et al. (2009), for instance, used actigraphy-based measures of mid-sleep on free days (MSF; chronotype) and mid- sleep on workdays (MSW) in a sample of 17- to 19-year- old students and found that the mid-sleep scores on free and workdays were correlated at r = 0.55 and −0.58 (p < 0.05) across two measurements 5 years apart, respectively. Also, when measuring the stability of diurnal type in a small subsample of the Older Finnish Twin Cohort, the findings of Koskenvuo and colleagues (2007) showed that there was a significant shift toward being a more morning person with increasing age across a 6-year period. The Aims of the Present Studies To the best of our knowledge, none of the existing studies has examined the daily intraindividual vari- ability in mid-sleep (i.e., a marker for chronotype) over a consecutive period of time in a natural setting. The handful of test-retest studies reporting on the temporal stability of chronotype thus far indicate that chronotype is remarkably stable across shorter time scales ranging from 2 weeks up to 1 year. However, only few studies have investigated the temporal sta- bility of chronotype over longer periods of time and in different age groups. To fill these gaps in knowl- edge, we present two studies that extend previous research in several ways. y p g In Study 2, we investigated the temporal stability of chronotype over a period of 0-1 to 5 years and across different parts of the life span. As we reviewed above, both cross-sectional and a few longer-term longitudi- nal studies have shown that chronotype can—and mostly does—change across the life span. However, due to a limited age range of their samples, the existing studies have not been able to address the question of whether there is any systematic variation in the lon- ger-term stability of chronotype across the life span. By using a sample of adults with an age range from 18 to 87 years who completed the MCTQ (Roenneberg et al., 2003) twice up to 5 years apart, we will explore the degree of rank-order stability of mid-sleep on both free and workdays across different stages of adulthood. y In Study 1, we examined the daily intraindividual variability in chronotype during a consecutive 14-day period among a sample of undergraduate students from the United Kingdom. More specifi- cally, we were interested in the daily variability of the midpoint of sleep (i.e., midpoint between sleep onset and wake-up) which significantly predicts an individual’s dim light melatonin onset (DLMO) which is the gold standard for a circadian phase marker (Terman et al., 2001; Pandi-Perumal et al., 2007; Kantermann et al., 2015). Similar to previous studies (Urner et al., 2009; Zwart et al., 2018), we were not only interested in people’s MSF, which is often used to assess chronotype (Roenneberg et al., 2019), but also in people’s timing of MSW. The Aims of the Present Studies In other words, we aimed to find out how large the intraindi- vidual variability in daily mid-sleep scores is during the period of 14 days and to what extent the daily variability of mid-sleep is influenced by free and workdays. As mid-sleep is composed of sleep onset and wake-up time, we were also interested in the intraindividual variability of these two sleep vari- ables and how they relate to mid-sleep. y g The pseudodata and scripts for Study 1 as well as the pseudodata and scripts for Study 2 can be down- loaded at https://osf.io/e7q6y/?view_only=9a48bec d4430488584f70eddb57cad82. For access to the real data, please apply at https://genomics.ut.ee/en/bio- bank.ee/data-access. Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 171 to report on their typical behavior over a certain period of time (e.g., the MCTQ asks about sleep behavior over the past 4 weeks) often fail to ade- quately characterize intraindividual variations over time and therefore, produce more biased estimates than momentary or daily assessments (Shiffman et al., 2008). Because studies have shown mixed agreement between objectively and subjectively assessed esti- mates of sleep (Lockley et al., 1999; Girschik et al., 2012), it is important to assess both types of sleep mea- surement. Thus, another aim of Study 1 was to vali- date the recall-based mid-sleep estimates on free and workdays as obtained with the MCTQ and the actig- raphy-derived measures of mid-sleep against the daily mid-sleep assessments averaged over 2 weeks. however, there are many studies that have investi- gated the inter- and intraindividual variability of cir- cadian rhythms in non- human animals. These studies have consistently shown that the interindividual variability of circadian markers is greater than the intraindividual variability (see for example Sharma, 1996; Refinetti and Piccione, 2005; Romeijn and Van Someren, 2011; Wassmer and Refinetti, 2019). Intraindividual Variability and Temporal Stability of Chronotype and Morningness-Eveningness g g y p The intraindividual or within-person variability in chronotype, that is how an individual’s chronotype or mid-sleep fluctuates from one day to another (Mroczek et al., 2003), has rarely been examined. Such studies need intensive repeated measurement designs and hence are harder to conduct. A 14-day experience sampling study, for instance, that investigated night- to-night variability in various sleep behaviors and measures found that both participants with and with- out chronic insomnia had substantial night-to-night variations in quantitative and qualitative sleep diary measures as well as in actigraphy-based measures of sleep (Buysse et al., 2009). A related study found that adolescents with attention deficit hyperactivity disor- der (ADHD) had greater intraindividual variability in their bedtimes and wake-up times than adoles- cents without ADHD (Langberg et al., 2019). These studies suggest that there might also be daily fluctua- tions in mid-sleep time points. When investigating biological markers of circadian rhythms during three 24-h sessions in the laboratory, Selmaoui and Touitou (2003) reported that participants had little daily intra- individual variability of cortisol circadian rhythm parameters and daily intraindividual variability in melatonin parameters. Differently from humans, Despite sleep being an area of fast-developing interest due to its important role in people’s physical and mental health (e.g., Espie and Morin, 2012), the temporal stability of chronotype and morningness- eveningness across time has received far less atten- tion. What is known from cross-sectional studies is that chronotype and morningness-eveningness vary with age: People are typically morning-oriented in childhood, become later chronotypes during adoles- cence with chronotype and morningness-evening- ness peaking in lateness around the age of 18 to 20 years, and then gradually become earlier chrono- types again with increasing age, even more so from the age of 50 years (see Adan et al., 2012, for a review). However, cross-sectional studies do not allow us to examine intraindividual variability or change over time as any findings in cross-sectional studies may be due to cohort or period effects rather than age- related changes per se (Realo and Dobewall, 2011; Jacob and Ganguli, 2016). A study by Broms and col- leagues (2014), for instance, examined morningness- eveningness across generations in the 1980s and 2000s and showed that there were less morning peo- ple and more evening people in the 2000s compared with the 1980s, indicating that the distribution of Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 17 Materials MCTQ p During each stage of data collection, participants visited the laboratory twice, usually in groups of four to six. During the first introductory session, partici- pants gave their informed consent and filled in an online questionnaire, which included sociodemo- graphic questions as well as validated questionnaires about sleep quality, chronotype (i.e., the MCTQ), per- sonality, diet, and affect. They then downloaded an application on their smartphones for the experience sampling study. To link the online questionnaire with the experience sampling data, the participants were given a randomly created unique identification code. At the end of the introductory session, participants received £5. The next morning, the experience sam- pling study started for the 2-week period. If partici- pants had questions, they were advised to email the experimenters. After the 2-week period, participants came back to the laboratory for a debrief session. They were asked to fill in a short feedback question- naire, return the actigraphs, and collect a remaining honorarium of up to £35 (depending on the number of surveys that they had filled in; one survey was equivalent to approximately £0.63). MCTQ. Participants were asked to complete the English version of the MCTQ (Roenneberg et al., 2003) during the first introductory meeting before the 14-day experience sampling study began. The MCTQ consists of 17 items that ask about typical sleep behavior over the past 4 weeks separately for workdays and free days to take into account our modern lifestyle which often leads to a clash between biological and social clocks (Roenneberg et al., 2019). The MCTQ was designed to assess chronotype as biological phase of entrainment rather than prefer- ences (Roenneberg et al., 2003; Roenneberg, 2015) and it measures chronotype as MSF after correcting for accumulated sleep debt over the week. p In our study, however, we did not correct MSF for sleep debt on workdays because MSF corrected for sleep debt on workdays is not defined as a daily score as it takes into account the sleep duration on free and workdays. It is also problematic to correct MSF for sleep debt on workdays when assessing the validity of the MCTQ scores as the ratio of free and workdays can differ considerably across participants while the correction uses 2 free days and 5 workdays (Kühnle, 2006). Method Participants. A total of 129 undergraduate students from a University in the United Kingdom signed up to take part in the study. We recruited them through the Student Mental Health and Resilience in Transition’s (SMaRT) online questionnaire where respondents indicated whether they were interested in participating in an experience sampling study. We also advertised the study on SONA—a system used across the University for advertising and booking into research studies. Of the 129 partici- pants, 13 were not able to participate in the experi- ence sampling study since they could not download y p We also examined the correspondence between the average daily estimates of mid-sleep both on free and workdays over the period of 2 weeks and (a) the recall-based estimates of mid-sleep as obtained with the Munich Chronotype Questionnaire (MCTQ; Roenneberg et al., 2003) as well as (b) actigraphy- derived estimates of mid-sleep. Research suggests that global retrospective measures which ask participants 172 JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 about the previous day’s physical activity, diet, social media usage, and sleep but also about when partici- pants woke up on the day of the survey. In addition, participants were randomly prompted to fill in a shorter momentary survey five times a day between 0800 h and 2200 h from Monday to Friday and between 1000 h and 2200 h Saturday and Sunday. At each prompt, participants had 20 min to fill in the survey and were advised to complete it as soon as they had received the prompt. During the momen- tary surveys, participants were asked about their cur- rent mood, well-being, what they were doing, and their social media usage. In the present study, only the data from the open survey will be analyzed. the application on their phones that we used for the study. One participant dropped out after the first day of the study. We excluded four participants from the analyses since they did not provide enough informa- tion on their sleep to calculate average sleep scores. p g p As a result, the final sample consisted of 111 University undergraduates, 71 (63.96%) identified as female, 40 (36.04%) as male. Their mean age was 19.71 (SD = 1.58) years, ranging from 18 to 32 years. Of those, 104 (93.69%) had actigraphy data available. Method The dataset has been used in other studies (Das- Friebel et al., 2020) but it has not been used for the present purpose. Procedure. Participants took part in a 2-week experi- ence sampling study between October 2017 and March 2018. Due to a limited number of actigraphs, only a maximum of 25 participants could partake in the study at a time. Therefore, the data collection took place in five consecutive stages during the above- mentioned period. p y y Participants were also asked to wear an actigraph for the course of the study. Participants’ sleep was recorded with actigraphy the same night following the introduction session whereas the experience sam- pling study started the next day. We will use the daily actigraphy data for validation purposes. Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 17 173 Finally, we calculated average daily mid-sleep scores for free (MSFES) and work (MSWES) days across the 2-week study period. The free and workdays were set differently for each participant, depending on their answers to the question if the day on which they completed the daily measurement of sleep was a regular work-/university day for them or not. The average number of measurements for computing MSFES was 4.61 (SD = 2.14) and 7.45 (SD = 2.25) for MSWES. The correlation between the MSFES and MSWES was r = 0.87, p < 0.001. The average daily mid-sleep score on free days (MSFES; M = 5.26, SD = 1.48) was significantly higher than on workdays (MSWES; M = 4.78, SD = 1.23), t(107) = 7.34, p < 0.001. also computed a midpoint of sleep on workdays (MSWMCTQ) in the same way as the MSFMCTQ. Lower scores of both MSFMCTQ and MSWMCTQ indicate a greater tendency to an early chronotype whereas higher scores indicate a disposition toward a later chronotype (Roenneberg et al., 2003). The MSFMCTQ and MSWMCTQ scores were correlated at r = 0.84 (p < 0.001). The MSFMCTQ (M = 5.69; SD = 1.57) was significantly higher than MSWMCTQ (M = 4.49; SD = 1.24), t(110) = 14.91, p < 0.001, indicating that people sleep later on free days than on workdays. Measurement of mid-sleep in the experience sampling study. Since the MCTQ assesses mid-sleep retro- spectively over the period of past 4 weeks, we had to adjust for this in the experience sampling study. More specifically, on each day of the experiment, partici- pants were asked to answer the following questions: (a) “At what time did you get into bed last night?” (b) “At what time did you switch off the lights to fall asleep last night?” [getting ready to fall asleep]; (c) “How long did it take for you to fall asleep last night?” [time it takes to fall asleep]; (d) “At what time did you wake up this morning?” [wake-up time]; (e) “Did you use an alarm clock to wake up this morn- ing?” (yes/no); and (f) “Is today a regular working/ university day for you?” (yes/no). We used the last question to differentiate between MSW and MSF. To calculate the daily mid-sleep scores, we first had to calculate sleep onset for each day: Actigraphy-derived measurements of mid-sleep. Next, we calculated the sleep duration for each day: sleep duration wake up time sleep onset - = − . Materials MCTQ Furthermore, in a study by Kantermann and Burgess (2017), MSF correlated at r = 0.71 (p < 0.001) with the DLMO, which is currently seen as the gold standard for a circadian phase marker, whereas MSF corrected for sleep debt accumulated on workdays correlated at r = 0.68 (p < 0.001) with the DLMO, indicating that both measures assess chronotype or circadian rhythm equally well. We therefore refer to MSF as chronotype (MSFMCTQ) which we calculated as the midpoint between sleep onset (the time some- one falls asleep) and wake-up time on free days. We The 14-day experience sampling study was con- ducted with Ilumivu’s mobile ecological momentary assessment app (mEMA; https://ilumivu.com/) which was compatible with both major mobile oper- ating systems (i.e., Android OS and iOS). Participants were told that each day of the study they would receive two types of surveys—open and momentary surveys. The open survey launched every day at 0800 h and was left open for the next 24 h so that partici- pants could complete the survey any time during the day. They received a reminder to fill it in at the start of the time window. It mainly consisted of questions Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 17 We used ActiGraph wGT3X-BT devices manufactured by ActiGraph to get objective estimates of mid-sleep. The actigraph recorded information about partici- pants’ movements and activity using a three-axis accelerometer. Participants were not able to indi- cate on their actigraphs at what time they tried to fall asleep and gotten out of bed. Therefore, we used the information extracted from the sleep diaries (i.e., the daily open surveys of the experience sampling study) as anchoring points. We calculated the daily mid-sleep scores the same way as in the experience sampling study and then calculated an average score for MSFACT and MSWACT. The two scores correlated at r = 0.77, p < 0.001, with each other. The actigra- phy-derived average daily mid-sleep score on free days (MSFACT; M = 5.65, SD = 1.51) was significantly higher than on workdays (MSWACT; M = 4.86, SD = 1.18), t(100) = 8.44, p < 0.001. sleep onset getting ready to fall asleep time it takes = + to fall asleep. sleep onset getting ready to fall asleep time it takes = + to fall asleep. sleep onset getting ready to fall asleep time it takes = + to fall asleep. time it takes + to fall asleep. Next, we calculated the sleep duration for each day: Results We found significant effects for the two main effects, workday today, F(1, 101.55) = 68.10, p < 0.001, and workday yesterday, F(1, 101.87) = 8.62, p = 0.004. Daily mid-sleep scores were later on free days for both factors, that is, par- ticipants’ mid-sleep scores were later when they went to bed and got out of bed on a free day. Furthermore, the effect of workday today on daily mid-sleep scores was more pronounced than for workday yesterday, meaning that participants’ mid- sleep scores were mostly influenced by whether the day they woke up was a free or workday. The inter- action between both factors did not reach signifi- cance, F(1, 972.21) = 2.46, p = 0.117. variance can be explained by between-participant effects. However, the ICC is only well-defined for random-intercept-only models and thus we will not be using it in the following model. Our first model did not allow for the possibility to examine systematic differences in daily mid-sleep scores between free and workdays. More specifi- cally, one could imagine that people’s mid-sleep dif- fers systematically between days they have to work versus days they do not have to work when they get up (e.g., compare Friday vs. Saturday in a standard European workweek)—we call this factor workday today (with two levels, workday vs. free day). In addition, another factor that might affect mid-sleep is whether the previous day was a free day versus workday (e.g., compare Saturday vs. Sunday in a standard European workweek)—we call this factor workday yesterday (with two levels, workday vs. free day). Finally, we examined the intraindividual variance of daily mid-sleep scores in the second model when considering the effects of free versus workdays. When taking the above elaborated effect of free ver- sus workday into account, the intraindividual vari- ability (1.09; SD = 1.04) in daily mid-sleep scores was 0.71 the size of the interindividual variability (1.53; SD = 1.24). This indicates that when the effects of free versus workday are controlled for, the daily mid-sleep scores differ less within than between participants. We coded each day in our data on these two vari- ables based on participants’ self-reports and esti- mated a second mixed model on the daily mid-sleep scores with fixed effects for factors workday today and workday yesterday, as well as for their interaction. Results The daily mid-sleep scores were calculated using the following formula: Intraindividual Variability of Mid-sleep Over the Period of 14 Days. The main focus of Study 1 was to examine the amount of intraindividual variability in self- reported daily mid-sleep scores across a 2-week period. We performed the analyses using linear mixed models employing the afex package (Sing- mann et al., 2020) in R. mid sleep sleep onset sleep duration - = + 2 . mid sleep sleep onset sleep duration - = + 2 . 2 Overall, there were 1361 complete daily measure- ments of mid-sleep across participants out of 1547 possible measurements (104 participants × 14 days and 7 participants × 13 days due to technical issues), with the overall response rate being 87.98%. Across all participants, we further excluded 21 daily mid- sleep scores due to several reasons (Please refer to Supplemental Material S2). The average number of daily measurements of mid-sleep per participant was 12.25 (SD = 2.32), ranging from 3 to 14. The average daily mid-sleep score across the 14 days across all participants was 5.07 (SD = 1.27) which corresponds roughly to 0500 h. In a first model, we solely compared within- and between-individual variability across the full 2-week period. The model which had daily mid-sleep as the dependent variable only contained by-subject ran- dom intercepts and no further fixed effects. Results showed that intraindividual variability in daily mid-sleep scores (1.41; SD = 1.19) was approxi- mately equal to the interindividual variability in daily mid-sleep scores (1.43; SD = 1.20). This can be expressed in terms of an intraclass correlation coef- ficient (ICC) according to which 50.46% of the 174 JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 174 Figure 1. Violin plot of the mixed model depicting mid-sleep including work and free days today and yesterday. Mean scores across the sample are depicted as bold points together with their 95% confidence intervals. The violin plots depict per participant aggregated data (Study 1). Figure 1. Violin plot of the mixed model depicting mid-sleep including work and free days today and yesterday. Mean scores across the sample are depicted as bold points together with their 95% confidence intervals. The violin plots depict per participant aggregated data (Study 1). The test of the fixed effects was based on the Satterthwaite approximation, model predictions are displayed in Figure 1. Discussion Wake-up time. The intercept-only model indicated that the intraindividual variability in daily wake-up time (1.86, SD = 1.36) was larger than the interin- dividual variability (1.36, SD = 1.17). The ICC indi- cated that 42.24% of variance could be explained by between-subject effects. We then came up with mod- els that included workday today and workday yesterday. Our final model revealed a main effect for workday today, F(1, 110.03) = 73.90, p < 0.001, and workday yes- terday, F(1, 103.69) = 8.68, p = 0.004. We also found a significant interaction between the two factors, F(1, 89.41) = 8.01, p = 0.006. These results indicate that participants woke up later when they both went to bed and woke up on a free day. However, when participants got up on a workday, it did not matter whether it was a free or workday the day before. The intraindividual variability of the wake-up time (1.35, SD = 1.16) was 1.05 times the size of the interindi- vidual variability (1.29, SD = 1.14). Intraindividual Variability of Mid-Sleep Over the Period of 14 Days. The intercept-only model (first model) showed that about half the variance in daily mid-sleep scores across the period of 14 days can be explained by within-person differences and the other half by between-person differences, suggesting that people’s daily mid-sleep scores fluctuate as much within-person from day to day as they do between participants. This is similar to the findings of a 14-day experience sampling study by Buysse and colleagues (2009) that also found that there was substantive intraindividual variability in various quantitative and qualitative sleep measures (e.g., bedtime and wake- up time) assessed with sleep diaries and actigraphy. However, when we included free and workdays into a mixed model, we found that the intraindivid- ual variability in daily mid-sleep was 0.71 times the size of the interindividual variability, so that daily mid-sleep scores differed less within than between participants. This might be due to the differences in the amount of free and workdays participants had in a week. It supports the findings of previous studies that have shown that the interindividual variability in circadian rhythms is greater than the intraindivid- ual variability, both in humans and animals (see, for example, Sharma, 1996; Selmaoui and Touitou, 2003; Refinetti and Piccione, 2005; Romeijn and Van Someren, 2011; Wassmer and Refinetti, 2019). Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 175 before and after midnight, we subtracted 24 from the times before midnight to have the scores centered on midnight. on the basis of the participants’ reported daily sleep times during the 2 weeks of the experience sampling study. On free days, the retrospective (MSFMCTQ) and the average daily scores of mid-sleep across 2 weeks (MSFES) were correlated at r = 0.73 whereas the cor- relation between the respective scores on workdays (i.e., MSWMCTQ and MSWES) was r = 0.79, both corre- lations significant at p < 0.001. The two correlations did not differ significantly from each other, z = 1.04, p = 0.300. Sleep onset. The intercept-only model showed that intraindividual variability in daily sleep onset (2.21, SD = 1.49) was higher than the interindividual variability (1.91, SD = 1.38). According to the ICC, 46.37% of the variance was due to between-partici- pant effects. Our final model that included workday today and workday yesterday showed a significant main effect for workday today, F(1, 99.35) = 25.98, p < 0.001. There was no significant effect of workday yesterday, F(1, 109.03) = 2.76, p = 0.099, and the inter- action between the two factors was also not signifi- cant, F(1, 1002.07) = 0.00, p = 0.996. Thus, our results indicate that the participants fell asleep later when it was a free day on the next day. The intraindividual variability of sleep onset (1.90, SD = 1.38) was 0.90 times the size of the interindividual variability (2.12, SD = 1.46). The actigraphy-derived score for mid-sleep on free days (MSFACT) correlated at r = 0.80 with the average daily scores of mid-sleep across 2 weeks (MSFES) whereas the correlation between the respective scores on workdays (i.e., MSWACT and MSWES) was r = 0.97, both correlations significant at p < 0.001. The two correlations differed significantly from each other, z = −6.74, p < 0.001. Results For the random-effects structure, we initially started with the maximal random-effects structure justified by the design (Barr et al., 2013), which entailed by- participants random intercepts, by-participant ran- dom slopes for the two fixed effects and their interaction, as well as the correlation among the ran- dom slopes. Because this model showed a singular fit, we removed the random-slope for the interaction of the two fixed effects (this model still allowed us to retain the correlation among the remaining random- effect parameters). Intraindividual Variability of Bedtimes and Wake-up Times Over the Period of 14 Days. As mid-sleep is cal- culated as the midpoint between sleep onset and wake-up time, we were also interested to investigate the intraindividual variability in sleep onset and wake-up times. Therefore, we used the same kind of analysis as for mid-sleep. As participants fell asleep Intraindividual Variability of Bedtimes and Wake-up Times Over the Period of 14 Days. As mid-sleep is cal- culated as the midpoint between sleep onset and wake-up time, we were also interested to investigate the intraindividual variability in sleep onset and wake-up times. Therefore, we used the same kind of analysis as for mid-sleep. As participants fell asleep JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 Participants’ mid-sleep times were affected by free and workdays so that participants had different sleep routines on free days compared with workdays (i.e., that they systematically went to bed and got up later on free days than on workdays), and yet, they had similar mid-sleep scores on free days (i.e., that on all free days they went to bed and got up around the same time) and on workdays (i.e., that on all work- days they went to bed and got up around the same time) during the study period. The intraindividual variability of sleep onset was 0.90 times the size of the interindividual variability which is similar to the pro- portion of inter- and intraindividual variability in mid-sleep. However, participants’ wake-up times dif- fered more within than between participants which might be due to the fact that wake-up times on work- days are largely predetermined by social and work/ university demands. This aligns with previous results that showed that sleep onset is dependent on chrono- type on workdays whereas wake-up time is not (Roenneberg et al., 2003). Thus, the variability of mid- sleep cannot fully be explained by the variability of its composing factors. Please see Supplemental Material S3 for a more detailed discussion about the implications of the results for the MCTQ. MCTQ with the average MSF score of a 6-week long sleep log in people who exhibit a normal chronotype (i.e., the MSF score corrected for sleep debt was between 2.17 and 7.25), r = 0.86 (p < 0.001). However, the correlation was much higher within the normal chronotype spectrum than among those with either earlier and later chronotypes (MSF corrected for sleep debt below 2.17 or above 7.25); rs = 0.56 and 0.41 (ps < 0.001), respectively. Our participants were individ- uals at the end of their adolescence or early adult- hood who typically exhibit later chronotypes (Adan et al., 2012), which was also confirmed by the rela- tively late daily mid-sleep scores we found in our sample. Thus, the ecological validity of the MCTQ might be dependent on chronotype, that is, the vari- ability in MSF may be higher in earlier and later chro- notypes than in normal chronotypes. This might make it harder for earlier and later chronotypes to accurately remember their sleep times and thus harder to fill out the MCTQ. JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 The actigraphy-derived estimates of MSF and MSW were highly correlated with the average daily estimates of MSF and MSW extracted from the sleep diaries (rs = 0.80 and 0.97, respectively). This con- firms the assumption that actigraphs and sleep diary derived sleep timings show good correlations (Lockley et al., 1999), indicating that participants can estimate quite well at what time they fall asleep and wake up. The estimations seem to be better on work than on free days as participants seem to better remember the sleep times on workdays. Comparison of Retrospective, Actigraphy-Derived, and Average Daily Assessments of Mid-Sleep. The cor- relations between the retrospective MSFMCTQ and the MSWMCTQ scores with their corresponding average daily mid-sleep scores over the period of 2 weeks were strong and significant (rs = 0.73 and 0.79, respectively) and this indicates that the MCTQ (Roen- neberg et al., 2003) is a relatively accurate measure to assess participants’ sleeping patterns. Participants might already think of their average bedtimes when filling out the questionnaire since they are asked to report on their typical sleep behavior over the past 4 weeks. Our findings, however, do not support the results of Santisteban et al. (2018) who reported that participants depict their sleep times more accurately on free days than on workdays. On the contrary, our findings indicated that the correlations between the retrospective scores of mid-sleep and experience- sampling-based average daily assessments of mid- sleep were higher on workdays than on free days. Even though the difference between the two correla- tions was not significant at p < 0.05, it seems reason- able to assume that one might retrospectively assess one’s sleeping patterns during the week better than during free days. During the week, one might have a certain routine at what time to go to bed and get up. However, on free days, one might engage in a variety of different activities that are less predictable. Discussion Differently from Selmaoui and Touitou (2003), we tested participants in their natural environments and not in the laboratory which adds to the validity and generalizability of the findings as participants were able to follow their normal work/university and sleep routines during the study period. Correspondence Between the Retrospective Assessments of Mid-sleep (MCTQ) and Actigraphy-derived Mid-sleep With Average Daily Mid-sleep Scores Over the Period of 14 Days. Finally, we examined the correspondence of retrospective assessments of mid-sleep and actigra- phy-derived mid-sleep with the average daily mid- sleep scores on free and workdays over the period of 2 weeks. To this aim, we used the two recall-based mid-sleep scores on free and workdays obtained with the MCTQ before the experience sampling study (MSFMCTQ and MSWMCTQ, respectively), the actigra- phy-derived scores of mid-sleep (MSFACT and MSWACT), and the average scores of mid-sleep on free (MSFES) and workdays (MSWES) that were calculated 176 Measures Measures MCTQ. The Estonian version of the MCTQ by Roenneberg and his colleagues (2003) was used. It is a 17-item retrospective questionnaire that assesses chronotype. Similar to Study 1, the mid-sleep scores on free (MSF) and work (MSW) days were extracted from the questionnaire. For the sake of consistency with Study 1, we did not correct mid-sleep on free days (MSF) for sleep debt on workdays (MSFsc) in Study 2. However, as suggested by an anonymous reviewer, we also repeated all the analyses using MSFsc and found similar trends. The results of these analyses are reported in Supplemental Material S5, Tables S5.1 and S5.2 and Figures S5.1 to S5.4. Analyses. We used IBM SPSS Statistics 24 for statistical analyses. For each participant, we computed Asendor- pf’s (1992) coefficient for individual stability both for MSF and MSW to obtain a measure of intraindividual change in rank-order stability over time (cf. Terracci- ano et al., 2010). This score is calculated as such: The final sample consisted of 681 participants, 344 (50.51%) of them were female. Their mean age at T1 was 47.73 years (SD = 15.89), ranging from 18 to 87 years. At T1, 69 (10.12%) persons had basic education, 363 (53.30%) completed secondary education/sec- ondary vocational education, and 249 (36.56%) com- pleted higher education. i z z 12 1 2 2 1 2 = − − ( ) , p g Fifteen participants (2.20%) completed the MCTQ (Roenneberg et al., 2003) for the second time in the same year (ranging from 1 to 11 months apart), 48 (7.05%) completed the questionnaires 1 year apart, 220 (32.31%) 2 years apart, 293 (43.02%) 3 years apart, 54 (7.93%) 4 years apart, and 51 (7.49%) 5 years apart. On average, the time between two measurements was 2.70 years (SD = 1.05), ranging from 0 (40 days) to 5 years. Due to the small number of participants who filled out the MCTQ in the same year, we com- bined those with the group that completed the MCTQ 1 year apart. We performed a one-way analysis of variance (ANOVA) to test whether the groups with different retest intervals differed in terms of age. The results revealed that the five groups did not signifi- cantly differ in age either at T1, F(4, 676) = 0.13, p = 0.971, or at T2, F(4, 676) = 0.77, p = 0.545. Method Participants. The participants for Study 2 were a sub- sample of the Estonian Biobank cohort (currently more than 200,000 participants), which is a large- scale population-based sample of the Estonian adults (Leitsalu et al., 2014). A part of the Estonian Biobank cohort has been followed up longitudinally and, in this study, we use a subsample of the cohort who have completed the MCTQ (Roenneberg et al., 2003) twice. Recruitment and data collection were assisted by a unique network of data collectors, that is, Gen- eral Practitioners and other medical personnel in pri- vate practices and hospitals, but also recruitment offices at the Estonian Genome Center. Participants gave their informed consent which can be found at https://www.geenivaramu.ee/en/access-biobank. Doctors conducted a standardized health examina- tion of each participant. Participants gave blood Kühnle (2006) also reported a high ecological validity of the MCTQ when comparing MSF of the Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 177 samples and filled in and completed a Computer Assisted Personal Interview (CAPI) on health-related topics and various clinical diagnoses described in the World Health Organization (WHO) International Classification of Diseases–10th revision (ICD-10) (Leitsalu et al., 2014). N = 681) = 31.73, p < 0.001, so that there were far fewer women than expected in the group of partici- pants who completed the MCTQ 2 years apart and far more women than expected in the group who were retested 5 years later. Table 1 describes the five groups according to their age, gender, and educational attain- ment at both time points. ( ) Supplemental Figure S4.1 depicts a flowchart of how participants from the Estonian Biobank were selected. Overall, 1111 participants completed the MCTQ twice over the period of 1 to 9 years. The first time they filled it in was between 2007 and 2010, while the second time was between 2009 and 2016. However, we had to exclude participants either at the first (T1) or at the second (T2) point of measurement due to (a) an average sleep duration of shorter than 4 h, (b) taking medications that influence sleep (catego- rized with the WHO’s ATC/DDD Index), (c) doing shift work, or (d) missing data. Method We also excluded 10 participants who had completed the MCTQ for the second time more than 5 years later (i.e., five partici- pants filled in the questionnaires 6 years apart, two 7 years apart, two 8 years apart, and one 9 years apart). It is a well-known fact that stability and consistency generally decline with longer retest intervals, but we did not have enough participants to test this effect in a more systematic way. Measures We also performed a chi-square test of independence to com- pare the frequency of gender and educational level across the six groups. While the groups did not differ in terms of the highest level of educational attain- ment, χ2(8, N = 681) = 2.53, p = 0.961, the gender distribution was not equal across the groups, χ2(4, where z1 and z2 are the z-transformed scores at T1 and T2. The higher the score is, the more stable are the scores between the two measurement points. A negative score indicates that the scores are less stable. The population mean matches the correlation r12 between the two assessments. Since the coefficients of individual stability were strongly skewed to the left, we transformed the scores as proposed by Asendorpf (1992): Ti i i for i i 12 12 12 12 12 1 2 1 001 1 001 0 1 1 1 = + −     ≤ ≤ −     ln . . ln  <       for i 12 0 . We plotted the t-transformed scores for MSF and MSW with age and fitted a curve that matched the data best (polynomial curve of two degrees). We divided our participants into age groups to identify how the stability of mid-sleep changes with age. To inform our analyses, we ran a series of hierarchi cal linear regression models to test whether the We plotted the t-transformed scores for MSF and MSW with age and fitted a curve that matched the data best (polynomial curve of two degrees). We divided our participants into age groups to identify how the stability of mid-sleep changes with age. To inform our analyses, we ran a series of hierarchi cal linear regression models to test whether the JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 178 Table 1. Sociodemographics and mean scores of mid-sleep on free days and workdays across the five groups who completed the MCTQ twice either 0-1, 2, 3, 4, or 5 years apart (Study 2). Measures Year 0-1 2 3 4 5 Total n 63 220 293 54 51 681 Age at T1 (SD) 47.16 (16.81) 47.68 (15.68) 47.90 (16.25) 48.67 (16.25) 46.69 (14.89) 47.73 (15.89) Age at T2 (SD) 48.56 (16.86) 50.33 (15.71) 51.34 (16.31) 52.98 (15.00) 52.14 (14.90) 50.95 (15.96) Gender Female (%) 35 (55.56%) 90 (40.91%) 143 (48.81%) 35 (64.81%) 41 (80.39%) 344 (50.51%) Education at T1 Basic (%) 6 (9.52%) 25 (11.36%) 28 (9.56%) 6 (11.11%) 4 (7.84%) 69 (10.12%) Secondary/ vocational (%) 30 (47.62%) 114 (51.82%) 160 (54.61%) 29 (53.70%) 30 (58.82%) 363 (53.30%) Higher (%) 27 (42.86%) 81 (36.82%) 105 (35.84%) 19 (35.19%) 17 (33.33%) 249 (36.56%) Mid-sleep scores MSF at T1 (SD) 3.83 (1.12) 3.81 (1.26) 3.76 (1.18) 3.81 (1.06) 3.77 (1.16) 3.79 (1.19) MSF at T2 (SD) 3.62 (1.23) 3.82 (1.22) 3.68 (1.20) 3.76 (1.02) 3.58 (1.06) 3.71 (1.18) MSW at T1 (SD) 2.91 (0.83) 2.90 (0.85) 2.92 (0.81) 2.94 (0.77) 2.82 (0.93) 2.91 (0.83) MSW at T2 (SD) 2.80 (0.78) 2.98 (0.98) 2.92 (0.87) 2.86 (0.73) 2.80 (0.90) 2.92 (0.89) Abbreviations: MCTQ = Munich Chronotype Questionnaire; Year = the difference between the first (T1) and the second (T2) completion of the MCTQ in years; MSF = mid-sleep score on free days; MSW = mid-sleep score on workdays; secondary/vocational = secondary education and secondary vocational education. All percentages are within the specific group (year difference when filling out the questionnaires). Table 1. Sociodemographics and mean scores of mid-sleep on free days and workdays across the five groups who completed the MCTQ twice either 0-1, 2, 3, 4, or 5 years apart (Study 2). Abbreviations: MCTQ = Munich Chronotype Questionnaire; Year = the difference between the first (T1) and the second (T2) completion of the MCTQ in years; MSF = mid-sleep score on free days; MSW = mid-sleep score on workdays; secondary/vocational = secondary education and secondary vocational education. All percentages are within the specific group (year difference when filling out the questionnaires). t-transformed coefficients of individual stability in MSF and MSW were influenced by the age of par- ticipants at T1, the quadratic term of age at T1, as well as the time difference between T1 and T2. p < 0.001). The test-retest correlations for MSF and MSW for groups with different retest intervals rang- ing from 0-1 to 5 years are shown in Supplemental Figure S6.1. Measures Broadly speaking, the retest correlations of MSF and MSW were very similar across the groups with different retest intervals, and varied between 0.63 (tested 2 years apart) and 0.70 (tested 3 years apart) for MSF and between 0.51 (tested 1 year apart) to 0.65 (tested 5 years apart) for MSW, respectively. The retest stability of MSF was consistently higher (median retest correlation = 0.65) than the stability of MSW (median retest correlation = 0.54) across all five groups with different retest intervals. Results As for MSW, the individual stability coeffi- cients increase from young adulthood until mid-40s and then decrease from late 40s onward. When we fitted a quadratic model on the data, it accounted for 2.40% of the variance in MSW individual stability coefficients compared with the linear model, which only accounted for 0.27%, with an equation of y = −0.001x² + 0.085x – 0.300. In any case, the individual stability of mid-sleep both on free and workdays seems to reach its peak in middle age, namely in 40s and 50s. To further elaborate on how the rank-order stabil- ity of MSF and MSW is influenced by age, we divided participants into six age categories at T1: 18 to 25 (n = 69), 26 to 35 (n = 114), 36 to 45 (n = 134), 46 to 55 (n = 121), 56 to 65 (n = 124), and 66 to 87 (n = 119). We then calculated test-retest correlations for MSF and MSW for each group. Figure 2 depicts these test-retest correlations by age group. The rank- order stability of MSF seems to reach a plateau when participants are in late 40 to early 50 years of age (r = 0.66, p < 0.001) whereas the rank-order sta- bility of MSW also reaches its peak when partici- pants are 46 to 55 years old (r = 0.74, p < 0.001) and then decreases again in older participants. Since stability of psychological traits tends to decline with longer retest intervals (Roberts and DelVecchio, 2000; Terracciano et al., 2006) and because the retest interval varied across the participants of our study, we ran a series of hierarchical regression analyses where we predicted the individual stability coefficients (MSF and MSW in separate models) from participant’s age and the square of age at T1 (to account for both linear and non-linear relationships) when also controlling for retest interval. The results of the hierarchical multiple regression analyses for the individual stability of MSF and MSW are shown in Supplemental Material (Table S8.1 and S8.2, respec- tively). In both models, age and age square had a significant effect on the stability of mid-sleep at p < 0.001. Time difference in the retest interval was a sig- nificant predictor of the stability of MSF at p = 0.045 but not of the stability of MSW (p = 0.477). Results As can be seen from Supplemental Figure S7.1, the individual stability of MSF increases from young adulthood to early 50s and then starts to decline again from mid-50s onward. When we fitted a qua- dratic model on the data (equation of y = −0.001x² + 0.074x – 0.240), it accounted for 3.53% of the variance in MSF individual stability coefficients compared with the linear model, which only accounted for 1.63%. As for MSW, the individual stability coeffi- cients increase from young adulthood until mid-40s and then decrease from late 40s onward. When we fitted a quadratic model on the data, it accounted for 2.40% of the variance in MSW individual stability coefficients compared with the linear model, which only accounted for 0.27%, with an equation of y = −0.001x² + 0.085x – 0.300. In any case, the individual stability of mid-sleep both on free and workdays seems to reach its peak in middle age, namely in 40s and 50s. To further elaborate on how the rank-order stabil- ity of MSF and MSW is influenced by age, we divided participants into six age categories at T1: 18 to 25 (n = 69), 26 to 35 (n = 114), 36 to 45 (n = 134), 46 to 55 (n = 121), 56 to 65 (n = 124), and 66 to 87 (n = 119). We then calculated test-retest correlations for MSF and MSW for each group. Figure 2 depicts these test-retest correlations by age group. The rank- order stability of MSF seems to reach a plateau when participants are in late 40 to early 50 years of age (r = 0.66, p < 0.001) whereas the rank-order sta- bility of MSW also reaches its peak when partici- pants are 46 to 55 years old (r = 0.74, p < 0.001) and then decreases again in older participants. Results Descriptive Statistics. Across all participants, the average mid-sleep score on free days (MSF) was 3.78 (SD = 1.18) at T1 and 3.72 (SD = 1.18) at T2. The scores did not significantly differ from each other t(678) = 1.71, p = 0.087. The average mid-sleep scores on workdays (MSW) also did not differ between T1 (M = 2.91; SD = 0.83) and T2 (M = 2.92, SD = 0.89), t(678) = −0.33, p = 0.740. Table 1 gives an overview about these scores according to the year difference between filling out the questionnaires. The correlations between MSF and MSW were rs = 0.70 and 0.69 at T1 and T2, respectively (both signifi- cant at p < 0.001). Individual and Group-level Stability of Mid-sleep Across the Life Span. Finally, we were interested in finding out if and to what extent the individual stability coef- ficients for MSF and MSW depend on age. Supple- mental Figures S7.1 and S7.2 depict age at the first time of assessment (T1) on the x-axis and the Asen- dorpf’s (1992) t-transformed coefficients of individual stability of MSF and MSW on the y-axis. A t-trans- formed coefficient of individual stability of 3.8 corre- sponds to an individual stability coefficient of 1 and a t-transformed coefficient of 2.6 to a coefficient of 0.99. Test-retest Reliabilities of Mid-sleep Scores for the Groups With Different Retest Intervals. The test-retest correla- tions for MSF and MSW for the full sample were r = 0.66 and r = 0.58, respectively (both significant at Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 179 Figure 2. Test-retest correlations of MSF and MSW by age at T1 (Study 2). Abbreviations: MSF = mid-sleep on free days; MSW = mid- sleep on workdays. Figure 2. Test-retest correlations of MSF and MSW by age at T1 (Study 2). Abbreviations: MSF = mid-sleep on free days; MSW = mid- sleep on workdays. As can be seen from Supplemental Figure S7.1, the individual stability of MSF increases from young adulthood to early 50s and then starts to decline again from mid-50s onward. When we fitted a qua- dratic model on the data (equation of y = −0.001x² + 0.074x – 0.240), it accounted for 3.53% of the variance in MSF individual stability coefficients compared with the linear model, which only accounted for 1.63%. JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 In their most recent critical review of their work, Roenneberg et al. (2019) argued that chronotype should rather be seen as a state and not a trait since zeitgeber signals people are exposed to vary in strength and timing. This might indicate that the life circumstances of younger and older people may vary more than those of middle-aged participants. During adulthood, humans experience a variety of major life events which in turn might have an impact on their bedtimes. MSF seems to change the most when par- ticipants are at the age of starting something new, for example, a job (de Souza et al., 2014), living together with a partner (Hida et al., 2012), or starting a family (Leonhard and Randler, 2009). Therefore, it is not as surprising that the stability of MSF reached a plateau in the age group of 46 to 55 years of age who most likely had already experienced such life events. MSW also reached its peak of stability in the same age group (i.e., 46-55 years) but decreased again in older age groups, meaning that people’s sleep habits on workdays seem to become less stable when they reach the retirement age (Hagen et al., 2016) and when their daily routines are no longer determined by work and school hours. of psychology suggests that global retrospective measures, especially summary measures that ask participants to report on their typical behavior over several past weeks or months, are often biased because people use mental heuristics to recall infor- mation (Shiffman et al., 2008). Study 1 aimed to fill this gap in literature and contribute to a better understanding of intraindividual variability of chro- notype over a period of 2 weeks, as well as to exam- ine correspondence between recall-based estimates of chronotype (i.e., MCTQ) and actigraphy-derived estimates of mid-sleep with average real-time esti- mates of MSF and MSW. Furthermore, only few studies have investigated the temporal stability of chronotype; the majority of those studies evaluated the test-retest reliability of chronotype question- naires during relatively short periods of time while not bearing in mind how age might affect the tempo- ral stability of chronotype (see, for example, Smith et al., 1989; Kühnle, 2006; Di Milia et al., 2013). Thus, Study 2 examined the stability of mid-sleep over lon- ger periods of time while also considering the effect of age. JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 g When the daily variability in mid-sleep was exam- ined across the study period of 2 weeks (Study 1), we found that the intraindividual variability was about equal to the interindividual variability in daily mid- sleep scores (ICC = 50.46%), meaning that there was as much variability between participants’ daily mid- sleep scores as in within each participant. However, when the effect of free versus workday was consid- ered, people’s mid-sleep scores fluctuated more across than within participants. Our findings also speak for the relatively high levels of intraindividual consistency in chronotype, meaning that even though people have different mid-sleep points on work and free days, they tend to have a routine of going to bed and getting up on workdays and another routine on free days. We also found that waking up on a free day has the biggest influence on one’s mid-sleep—not surprisingly, people wake up later on free days than on workdays—but interestingly, going to bed on a free day also delays one’s mid-sleep, meaning that people go to bed and wake up the latest when both the day they go to bed and the day they wake up are free days. y Due to the nature of our study, participants filled out the questionnaire for the second time at different years apart from each other. The results of the hierar- chical regression analyses showed that age and its square were more important than the year difference when predicting the stability of MSF and that the year difference in filling out the questionnaires did not matter when predicting the stability of MSW. A possible reason for this could be that the time differ- ence between filling out the questionnaires was quite small, ranging from 0 to 5 years. Overall, both MSF and MSW showed strong test- retest correlations when participants filled out the MCTQ up to 5 years apart from each other. The retest stability of MSF was higher than the retest stability of MSW at both time points, which shows that one’s bedtimes on free days are more stable than those on workdays. Future studies should establish how lon- ger time intervals between filling out the question- naires will affect the stability of both MSF and MSW. JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 y The recall-based retrospective mid-sleep scores on free (MSF) and work (MSW) days extracted from the MCTQ (Roenneberg et al., 2003) correlated highly with the respective average mid-sleep scores from the experience sampling study (rs = 0.73-0.79). This is consistent with previous research (Kühnle, 2006; Kantermann et al., 2015; Santisteban et al., 2018) and speaks for high ecological validity of the MCTQ. It seems though that our participants were slightly more accurate in retrospectively estimating their sleep times on workdays than on free days which Discussion Previous cross-sectional and a few longitudinal studies have shown that chronotype changes across the life span (Koskenvuo et al., 2007; Adan et al., 2012; Broms et al., 2014). We wanted to find out whether there was any systematic variation in the longer-term stability of chronotype across life span. Our results indicate that the rank-order stability of mid-sleep on both free and workdays varies with age and is the highest when participants are in their late 40s to early 50s. 180 Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 18 Interestingly though, the stability of MSF reaches a plateau and levels off when participants are in their late 40s to early 50s whereas the stability of MSW decreases again when people reach the retirement age. This seem to suggest that the differences in the stability of mid-sleep across the life span are likely not solely due to biological age effects but also to social life-cycle effects (e.g., finish- ing school, finding a job, getting married, settling down, retiring) that are intertwined with the biologi- cal process of aging (Glenn, 2003). These findings are confirmed by individual-level stability analyses (see Supplemental Figures 7.1 and 7.2), which provided further evidence in support of the view that mid- sleep stability changes over the life span. Overall, both group and individual-level analyses clearly indicate that when examining the stability of chrono- type or mid-sleep, the effect of age (either biological or social in nature) strongly needs to be considered. p Study 2 contributed to important insights into the change and stability of chronotype over a longer period of time and across different stages of life span. We found relatively high retest correlations for MSF and MSW when examining the retest stability of mid- sleep during the periods of 0-1 to 5 years. The median retest correlations of MSF and MSW at T1 with T2 across different time periods were 0.65 and 0.54, respectively, which are comparable (if slightly lower) with the retest stability coefficients of the Big Five personality traits assessed in middle adulthood with a testing interval of 3 to 10 years (Hampson and Goldberg, 2006; Terracciano et al., 2010). Our esti- mates were, however, a bit lower than those reported in previous studies when participants’ chronotype was tested twice during 1 to 24 months using mostly preferential questionnaires of chronotype or morn- ingness and eveningness (see, for example, Smith et al., 1989; Greenwood, 1994; Caci et al., 2000; Griefahn et al., 2001; Kühnle, 2006; Wood et al., 2009; Di Milia et al., 2013). The lower retest stability indica- tors in our study could be due to a longer time span between the two measurements since it is known that the stability of psychological traits declines with lon- ger test-retest intervals (Roberts and DelVecchio, 2000; Terracciano et al., 2006). Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 18 181 could be explained by the fact that there are more restrictions and less flexibility in sleep times due to university-related responsibilities (e.g., classes, semi- nars) on weekdays compared with free days (cf. Paine and Gander, 2016), and thus, sleep times can be more accurately recalled. However, it should be noted that the sleeping times assessed in the MCTQ asked about the 4 weeks before the start of the experience study and therefore did not overlap with the sleeping times extracted from the experience sampling study. This means that the bedtimes extracted from the MCTQ could have also differed from the average bedtimes during the course of the experience sampling study. As we found high correlations between the average daily self-reported mid-sleep scores and the average mid-sleep scores assessed with actigraphy during the same period of 14 days, we can be quite certain that participants can estimate well at what times they fall asleep and wake up. However, the correlations might be this high because we anchored the actigraphy- derived sleep times on the sleep times extracted from the sleep diaries. Interestingly, across the whole sample, the retest stability of MSF (0.66) was greater than on workdays (0.58) over the periods of up to 5 years. During a lon- ger time interval, several life circumstances might change due to changing opportunities and constraints characteristic of different stages in life (Heckhausen et al., 2010). These could have affected one’s MSW, for example, having children and their entry into school, getting a promotion, or retiring. g g p g One of the novel aspects of Study 2 was to examine the retest stability of chronotype across different stages of life span. Even though MSF and MSW remained relatively stable over a period of up to 5 years, the retest stability varied greatly in different age groups: the retest stability coefficients both for MSF and MSW were the lowest when participants were in late teens and early 20s and the highest when participants were in their late 40s to early 50s. Even though mid-sleep fluctuates little within young adults over a period of 2 weeks (Study 1), the tempo- ral stability coefficients of mid-sleep are very low when participants are tested twice over much longer periods of time (Study 2). Lenneis et al. / INTRAINDIVIDUAL VARIABILITY AND STABILITY OF MID-SLEEP 18 As our testing interval varied from 0-1 to 5 years, we also conducted hierar- chical linear regression analyses to examine the effects of age and the year difference on the stability of mid-sleep. The results showed that age was more important than the year difference between two mea- surements in predicting the stability of MSF. The sta- bility of MSW was only affected by age and not the year difference between the measurements. General Discussion Even though chronotype has been a topic of extensive research over the past decades, most stud- ies have used preferential (e.g., The Morningness- eveningness Questionnaire; Horne and Östberg, 1976) or retrospective recall-based measures of chro- notype (e.g., MCTQ; Roenneberg et al., 2003), which typically do not examine the daily intraindividual variability in chronotype. Research in different fields Authors’ Note Adan A, Archer SN, Paz Hidalgo M, Di Milia L, Natale V, and Randler C (2012) Circadian typology: a compre- hensive review. Chronobiol Int 29:1153-1175. Study 1 was approved by the Ethics Committee of the Department of Psychology at the University of Warwick on 16 October 2017. The research using data from the Estonian Biobank (Study 2) was approved by the Research Ethics Committee of the University of Tartu (approvals: 236/M- 29, 14 May 2014; 206/T-4, 22 August 2011; 170/T-38, 28 April 2008; 166/T-21, 17 December 2007). Secondary data analysis of the Estonian Biobank data was approved by the Ethics Committee of the Department of Psychology at the University of Warwick on 19 November 2018. Asendorpf JB (1992) Beyond stability: predicting inter- individual differences in intra-individual change. Eur J Pers 6:103-117. Barclay NL, Eley TC, Buysse DJ, Archer SN, and Gregory AM (2010) Diurnal preference and sleep quality: same genes? A study of young adult twins. Chronobiol Int 27:278-296. Barr DJ, Levy R, Scheepers C, and Tily HJ (2013) Random effects structure for confirmatory hypothesis testing: keep it maximal. J Mem Lang 68:255-278. Broms U, Pitkaniemi J, Backmand H, Heikkila K, Koskenvuo M, Peltonen M, Sarna S, Vartiainen E, Kaprio J, and Partonen T (2014) Long-term consistency of diurnal-type preferences among men. Chronobiol Int 31:182-188. Limitations and Future Research Our approach was not without limitations though. First, the participants from both studies differed in age. The participants from Study 1 were university students whereas the participants from Study 2 were part of a large-scale sample of Estonian adults ranging from 18 to 87 years in age at T1. Therefore, the results of Study 1 might not be applicable to older participants whereas we did consider the effect of age in Study 2. In Study 1, we were able to show that intraindivi dual variability in mid-sleep is a lot smaller than JOURNAL OF BIOLOGICAL RHYTHMS / April 2021 182 interindividual variability when the type-of-day vari- able was controlled for. This can be partly explained by the difference in the amount of free and workdays our participants reported. Future research could use experi- ence sampling methodology of mid-sleep using a repre- sentative population over a longer period of time to generalize our findings of intraindividual stability of chronotype. It would also be interesting to explore whether early and late chronotypes show a different intraindividual variability in mid-sleep. Development Fund Strategic Award to Dieter Wolke and Nicole K. Y. Tang, the Department of Psychology of the University of Warwick, and an institutional research fund- ing (IUT2-13) from the Estonian Ministry of Education and Science to Jüri Allik. We are grateful to the Estonian Genome Center of the University of Tartu and its director, Andres Metspalu, for their help in collecting the data and for their kind permission to use the data in the current study. The assistance of Lauren Jones in running the SMaRT study is also gratefully acknowledged. y p When examining the temporal stability of mid- sleep across the life span in Study 2, we compared the stability of mid-sleep in different age groups that con- sisted of different participants. Thus, future studies need to confirm our findings by applying a longitudi- nal approach that would allow to examine the intrain- dividual change of the stability of mid-sleep in the same individuals across the life span (cf. Terracciano et al., 2010). Furthermore, long-term longitudinal stud- ies will be necessary to explore how life circumstances and other possible factors influence one’s mid-sleep. References Abbott SM, Reid KJ, and Zee PC (2017) Circadian disorders of the sleep-wake cycle. In: Kryger MH, Roth T, and Dement WC, editors. Principles and practice of sleep medi- cine. Philadelphia (PA): Elsevier, p. 414-423.e415. Notes Supplementary material is available for this article online. Supplementary material is available for this article online. ORCID iD p p In sum, our studies have given important insights on the intraindividual variability and temporal stabil- ity of mid-sleep. Using experience sampling and lon- gitudinal methodologies, we were able to complement the weaknesses of cross-sectional studies. Our results show that mid-sleep varies less within than between participants when the effect of free and workdays is controlled for and that the stability of mid-sleep of both MSF and MSW is largely dependent on age. However, future research is needed to investigate how intraindividual variability of mid-sleep is depen- dent on chronotype and how the temporal stability of mid-sleep systematically changes with age. 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https://openalex.org/W2019607185	https://www.scielo.br/j/rsp/a/9Hr54wPh5jdKK4p6bw5fRnx/?lang=pt&format=pdf	Portuguese	null	Uso racional de medicamentos antineoplásicos e ações judiciais no Estado de São Paulo	Revista de saúde pública/Revista de Saúde Pública	2,010	cc-by	5,699	Rev Saúde Pública 2010;44(4):620-8 Rev Saúde Pública 2010;44(4):620-8 Artigos Originais RESUMO OBJETIVO: Avaliar a racionalidade das ações judiciais e pedidos administrativos recebidos pela Secretaria de Estado da Saúde de São Paulo segundo evidências cientíﬁ cas de eﬁ cácia e segurança. MÉTODOS: Estudo descritivo, transversal, baseado em informações da Secretaria de Saúde sobre os medicamentos antineoplásicos solicitados por via judicial, com maior impacto ﬁ nanceiro para o Sistema Único de Saúde em 2006 e 2007. Os fármacos foram avaliados quanto às evidências clínicas de eﬁ cácia e segurança, com base na classiﬁ cação do Micromedex®, em metanálises e revisões sistemáticas. As indicações foram confrontadas com aquelas aprovadas em agências reguladoras. RESULTADOS: Os medicamentos bevacizumabe, capecitabina, cetuximabe, erlotinibe, rituximabe, imatinibe e temozolomida geraram gastos superiores a R$ 40 milhões para atender 1.220 solicitações, com custo médio de R$ 33,5 mil por paciente. Os estudos selecionados não recomendam parte das indicações dos medicamentos prescritos. Cerca de 17% dos pedidos não tinham evidência para a indicação mencionada no pleito, o que equivale a um gasto inadequado de, no mínimo, R$ 6,8 milhões. I Programa de Pós-graduação em Ciências Farmacêuticas. Universidade de Sorocaba (Uniso). Sorocaba, SP, Brasil CONCLUSÕES: Os resultados reforçam a necessidade de qualiﬁ cação técnica para tratar as demandas judiciais e exige capacitação dos proﬁ ssionais no manejo da literatura cientíﬁ ca, na seleção adequada dos fármacos e na escolha da melhor conduta terapêutica para cada condição clínica. Dessa forma será possível garantir o acesso a tecnologias eﬁ cazes e seguras, e assim aprimorar o modelo de assistência farmacêutica em oncologia. II Curso de Graduação em Farmácia. Uniso. Sorocaba, SP, Brasil III Núcleo de Assistência Farmacêutica. Escola Nacional de Saúde Pública Sérgio Arouca. Fundação Oswaldo Cruz. Rio de Janeiro, RJ, Brasil DESCRITORES: Antineoplásicos, provisão & distribuição. Decisões judiciais. Legislação de Medicamentos. Gastos em Saúde. Assistência Farmacêutica. Correspondência \| Correspondence: Luciane Cruz Lopes Programa de Pós-Graduação stricto sensu em Ciências Farmacêuticas Rod. Raposo Tavares, km 92.5 18023-000 Sorocaba, SP, Brasil E-mail: luslopes@terra.com.br Recebido: 22/4/2009 Aprovado: 20/12/2009 Artigo disponível em português e inglês em: www.scielo.br/rsp Correspondência \| Correspondence: Luciane Cruz Lopes Programa de Pós-Graduação stricto sensu em Ciências Farmacêuticas Rod. Raposo Tavares, km 92.5 18023-000 Sorocaba, SP, Brasil E-mail: luslopes@terra.com.br Recebido: 22/4/2009 Aprovado: 20/12/2009 Artigo disponível em português e inglês em: www.scielo.br/rsp Correspondência \| Correspondence: Luciane Cruz Lopes Programa de Pós-Graduação stricto sensu em Ciências Farmacêuticas Rod. Uso racional de medicamentos antineoplásicos e ações judiciais no Estado de São Paulo Luciane Cruz LopesI Silvio Barberato-FilhoI Augusto Chad CostaII Claudia Garcia Serpa Osorio- de-CastroIII a Brasil. Lei nº 8.080, de 19 de setembro de 1990. Dispõe sobre as condições para a promoção, proteção e recuperação da saúde, a organização e o funcionamento dos serviços correspondentes e dá outras providências. In: Vieira JL, editor. Código sanitário do Estado de São Paulo. Normas técnicas e legislação complementar. Bauru: Edispro; 2000. (Série Legislação). b Ministério da Saúde. Portaria nº 2.981, de 26 de novembro de 2009. Aprova o componente especializado da assistência farmacêutica. Diario Oﬁ cial Uniao. 30 nov 2009;Seção1:725. c Ministério da Saúde. Portaria nº 2.982, de 26 de novembro de 2009. Aprova as normas de execução e de ﬁ nanciamento da assistência farmacêutica na atenção básica. Diario Oﬁ cial Uniao. 30 nov. 2009;Seção1:771. d Nogueira RWL. Saúde, medicamentos, desenvolvimento social e princípios orçamentários. Jus Navigandi. 2005[citado 2009 jan 20]9(542). Disponível em: http://jus2.uol.com.br/doutrina/imprimir.asp?id=6127 RESUMO Raposo Tavares, km 92.5 18023-000 Sorocaba, SP, Brasil E-mail: luslopes@terra.com.br Recebido: 22/4/2009 Aprovado: 20/12/2009 Artigo disponível em português e inglês em: www.scielo.br/rsp 621 Rev Saúde Pública 2010;44(4):620-8 ABSTRACT OBJECTIVE: To assess the rationality of legal suits and administrative requests requiring anticancer drugs ﬁ led against and submitted to the São Paulo State Department of Health, in view of scientiﬁ c evidence on efﬁ cacy and safety. METHODS: A descriptive cross-sectional study was carried out based on information on lawsuits ﬁ led by cancer patients requiring anticancer drugs were furnished by the Department of Health. These drugs are among those having the greatest ﬁ nancial impact on the Brazilian Health System in 2006 and 2007. The drugs were assessed according to clinical evidence on efﬁ cacy and safety, based on Micromedex® categorization, on systematic reviews and meta-analyses. Indications present in the legal documentation were compared to the indications approved by regulatory agencies. RESULTS: Bevacizumab, capecitabine, cetuximab, erlotinib, rituximab, imatinib, and temozolomide accounted for expenses over R$ 40 million to meet 1220 requests and lawsuits, at an average cost of R$ 33,500 per patient. Selected studies do not recommend all the indications for the prescribed drugs. Approximately 17% of requests and lawsuits did not provide evidence for the required indication, and these amounted to inappropriate expenses of, at least, R$ 6.8 million. CONCLUSIONS: The results reinforce the need for technical expertise in dealing with legal suits and for capacity-building of health professionals in approaching the scientiﬁ c literature, in order to appropriately select drugs and to ensure the best therapeutic decision for each clinical condition, and thus guarantee access to safe and effective health technologies and, therefore, to enhance the quality of the Brazilian pharmaceutical services model in oncology. DESCRIPTORS: Antineoplastic Agents, supply & distribution. Judicial Decisions. Legislation, Drug. Health Expenditures. Pharmaceutical Services. INTRODUÇÃO das sentenças judiciais que determinam o fornecimento de medicamentos. Essas ações, impetradas contra o Estado, têm se destacado como via alternativa de acesso a medicamentos no SUS.9 O Sistema Único de Saúde (SUS) garante aos usuá- rios assistência terapêutica integral, incluindo a farmacêutica.a No entanto, à margem da interpretação de que integral inclui assistência de alta, média e baixa complexidade, diferentes compreensões do termo insistem em entender integralidade como toda e qual- quer opção terapêutica existente, disponível ou não no Sistema. Isso resulta em distorções quanto às estratégias de acesso a tecnologias, entre elas medicamentos.13 Por outro lado, o atendimento dessa demanda pela via judicial não está vinculado à reserva orçamentária, consumindo recursos consideráveis e causando diﬁ - culdades para garantir aquisição de medicamentos previstos na legislação e aqueles pactuados nas Comissões Intergestoras.b,c Muitas vezes, esses medi- camentos não são essenciais, conforme determina a Política Nacional de Medicamentos, nem há garantias quanto à sua segurança e eﬁ cácia.d No Brasil, problemas de gestão da assistência farmacêu- tica são freqüentes nas três esferas de governo. Esses problemas, aliados à constante pressão por incorpora- ções de novas tecnologias no SUS, resultam no aumento 622 Antineoplásicos e ações judiciais Lopes LC et al Em todo o País, segundo informações provenientes do Ministério da Saúde, os valores gastos com ações judi- ciais no ano de 2007 ultrapassam R$ 500 milhões nas esferas federal, estadual e municipal.e Só no Ministério da Saúde, o valor anual gasto passou de R$ 188 mil em 2003a para R$ 52 milhões em 2008.f No Paraná, entre 2002 e 2007, o valor gasto com ações judiciais aumentou de R$ 200 mil para R$ 14 milhões.g antineoplásicos solicitados por via judicial com maior impacto ﬁ nanceiro sobre o SUS. A amostra de ações foi construída com base nessa lista de medicamentos, cuja indicação é para o tratamento de diversos tipos de câncer e fazem parte da terapia antineoplásica direcionada a alvos moleculares especíﬁ cos, representando nova abordagem de tratamento.5 Além das ações judiciais, foram incluídos os pedidos administrativos – solicitação formal, ao ente estatal, de medicamentos que o paciente não conseguiu obter nas unidades de saúde do SUS – contendo os medicamentos indicados, conforme sua presença no Sistema de Controle Jurídico da SES-SP. As variáveis investigadas foram: indicação clínica, prescritor, representação jurídica e origem do pleito. e Jungmann M. Ministério da Saúde classiﬁ ca de “epidêmico” volume de ações judiciais contra o SUS. Brasília; 2007[citado 2009 set 10]. Disponível em: http://www.aids.gov.br/main.asp?View=%7BDA56F374-128A-40FB-B16F-D08A1F5DD07B%7D&Team=&params=itemID=% 7B9016699E-670E-4209-B9DD-06C65CA88037%7D%3B&UIPartUID=%7BD90F22DB-05D4-4644-A8F2-FAD4803C8898%7D f Conselho Regional de Farmácia do Estado do Rio de Janeiro. Ministério defende equilíbrio nas ações judiciais de saúde. Rio de Janeiro; 2009[citado 2009 set 10]. Disponível em: http://www.crf-rj.org.br/crf/noticia/2009/5/minist%C3%A9rio_defende_equil%C3%ADbrio_ nas_a%C3%A7%C3%B5es_judiciais_de_sa%C3%BAde.htm g Ministério da Saúde. Ações judiciais colocam em risco o Sistema Único de Saúde. Brasília; 2007[citado 2009 jan 03]. Disponível em: http:// portal.cnm.org.br/003/00301009.asp?ttCD_CHAVE=71155 h Vianna SM, Nunes A, Góes G, Silva, JR, Santos, RJM. Atenção de alta complexidade no SUS: desigualdades no acesso e no ﬁ nanciamento. Brasília: Ministério da Saúde/Instituto de Pesquisa Econômica Aplicada; 2005. [citado 2009 set 10]. Disponível em: http://getinternet.ipea.gov. br/economiadasaude/adm/arquivos/destaque/alta_complexidade.pdf INTRODUÇÃO As ações judiciais têm ocupado lugar na mídia, sobre- tudo os gastos empreendidos pelas secretarias estaduais e municipais de Saúde e pelo Ministério da Saúde na aquisição de medicamentos. O fornecimento de medi- camentos de forma indiscriminada acaba privilegiando segmentos de usuários com mais recursos ﬁ nanceiros para pagar advogados, ou mais acesso à informação, em detrimento daqueles mais necessitados.4 Nesse contexto, os gestores têm demandado informações consistentes sobre os benefícios das tecnologias e a repercussão ﬁ nanceira sobre a esfera pública, com a ﬁ nalidade de subsidiar a formulação de políticas de saúde e a efetiva tomada de decisão.3 Para estimar o gasto com a aquisição desses medica- mentos, foram calculados a quantidade comprada no período e os preços, obtidos do Banco das Atas de Registro de Preços da SES-SP. As indicações terapêuticas constantes nas ações foram confrontadas com as evidências respaldadas pela lite- ratura disponível nas bases PubMed, Lilacs, SciELO e Biblioteca Cochrane, na forma de revisões sistemáticas e metanálises. O critério utilizado para a seleção dos artigos foi avaliar pelo menos um dos sete medica- mentos estudados, para qualquer indicação terapêutica, nos quais o tipo de intervenção foi comparado com outro tratamento ou com placebo. Foram selecionadas 40 metanálises e 38 revisões sistemáticas, publicadas até julho de 2008. As conclusões desses trabalhos coin- cidem com as evidências descritas na base Thomson Micromedex®. A área oncológica apresenta grande demanda, devido ao custo elevado e à soﬁ sticação tecnológica.h No município de São Paulo, em 2005, as ações judiciais para aquisição de antineoplásicos representaram 7,2% do total de itens solicitados, gerando gastos de R$ 661 mil, equivalentes a 75% do gasto com a aquisição de medicamentos por determinação judicial.12 Assim, o objetivo do presente estudo foi avaliar a racio- nalidade das ações judiciais relativas aos medicamentos antineoplásicos, considerando as evidências cientíﬁ cas de eﬁ cácia e segurança. Além disso, foram estimados os gastos com o fornecimento desses medicamentos em casos não respaldados pela literatura, visando contribuir para o modelo de assistência farmacêutica em oncologia no Sistema Único de Saúde. Dados de aprovação dos medicamentos foram obtidos de agências reguladoras, como Agência Nacional de Vigilância Sanitária (Anvisa), Food and Drug Administration (FDA) e European Medicines Agency (Emea), permitindo investigar se a indicação clínica constante nos pleitos correspondia àquelas aprovadas. RESULTADOS colorretal em 30% (em 2006) e 40% (em 2007) das ações judiciais e pedidos administrativos recebidos pela SES-SP. Os pedidos de capecitabina para tratamento do câncer de mama diminuíram de 46,2% (2006) para 28,1% (2007), enquanto o uso off-label (indicações não- aprovadas pelas agências reguladoras) está aumentando (23,0% das solicitações em 2006 e 31,2% em 2007 tinham outras indicações, como, por exemplo, câncer pancreático, para o qual não há sequer registro em nenhuma das agências reguladoras consultadas). Foram incluídos no estudo 1.220 pedidos envolvendo os antineoplásicos: bevacizumabe, capecitabina, cetu- ximabe, erlotinibe, imatinibe, rituximabe e temozolo- mida. A Tabela 1 mostra o número de pedidos recebidos pela SES-SP nos anos de 2006 e 2007, o gasto total e o gasto médio por pedido. O gasto total da Secretaria com os sete medicamentos foi 120% maior em 2007 do que em 2006, explicado pelo aumento do número de pedidos. Para alguns casos, apesar do maior número de pedidos, o gasto médio por pedido diminuiu (capecita- bina, cetuximabe, rituximabe). Por outro lado, o gasto médio por pedido de imatinibe quase quintuplicou. Mais de 80% das ações judiciais e pedidos admi- nistrativos de cetuximabe visavam ao tratamento de câncer colorretal. Embora exista benefício deﬁ nido para o tratamento do câncer colorretal metastático com expressão de EGFR-receptor do fator de crescimento epidermal, e esta indicação tenha sido aprovada pelo FDA e pelo Emea,7 no Brasil ela não foi aprovada. A indicação de cetuximabe para o tratamento de câncer de cabeça e pescoço, células escamosas, local ou regional- mente avançado, em combinação com radioterapia é a única indicação para o produto no Brasil com evidência para uso. Porém, menos de 5% das ações judiciais e pedidos administrativos estavam relacionados a algum tipo de câncer de cabeça e pescoço. Por outro lado, o uso em indicações ainda não aprovadas pelas agências reguladoras consultadas ﬁ cou em torno de 14% em 2006 e 10% em 2007. A Tabela 2 mostra características dos pedidos adminis- trativos e judiciais. Com algumas exceções decorrentes da incorporação de medicamentos pela SES-SP, os pedidos se concentraram na via judicial. Parte das demandas judiciais decorreu de prescrições do próprio SUS, em desacordo com os protocolos vigentes. A origem dos pedidos se concentrou em nove prescritores e sete advogados; um único médico foi responsável por quase 40% das prescrições de erlotinibe e um único advogado por 70% das ações judiciais envolvendo o cetuximabe. MÉTODOS As informações das ações judiciais foram obtidas de um programa informatizado da SES-SP denominado Sistema de Controle Jurídico. Houve garantia de conﬁ dencialidade e sigilo quanto à identiﬁ cação de todos os sujeitos evidenciados pelo estudo. O trabalho foi aprovado pelo Comitê de Ética em Pesquisa da Universidade de Sorocaba, em 20/12/2007 (Documento 052/2007). Estudo descritivo, transversal, cuja unidade de análise do estudo foi o Estado de São Paulo e o objeto as ações judi- ciais para antineoplásicos. A investigação contemplou o período de janeiro de 2006 a dezembro de 2007. Foi solicitado à Secretaria Estadual de Saúde de São Paulo (SES-SP) que indicasse os sete medicamentos 623 Rev Saúde Pública 2010;44(4):620-8 Fonte: Secretaria de Estado da Saúde de São Paulo. Fonte: Secretaria de Estado da Saúde de São Paulo. RESULTADOS As prescrições de cinco médicos resultaram em gastos da ordem de R$ 7 milhões, em dois anos. Ações impe- tradas contra a SES-SP por cinco advogados, responsá- veis pela maioria delas, resultaram em gasto de R$ 16 milhões. A Tabela 3 mostra os valores individualizados dos principais prescritores e advogados envolvidos. A indicação de erlotinibe para o câncer de pâncreas, embora não aprovada pela Anvisa, já recebeu aprovação do FDA e Emea, além de existir benefício deﬁ nido para o tratamento de câncer de pâncreas localmente avançado ou metastático, como tratamento de primeira linha, em combinação com gencitabina. Cerca de 12% em 2006 e 6% em 2007 das ações judiciais e pedidos administrativos estavam relacionados a algum tipo de câncer de pâncreas. O uso do erlotinibe em indicações ainda não aprovadas pelas agências reguladoras consul- tadas não alcançou 5% em 2006 ou 2007. Cada fármaco selecionado tem sido empregado em inúmeras indicações terapêuticas, algumas sem apro- vação nas agências reguladoras consultadas. Das 16 indicações para o bevacizumabe, duas atenderam aos critérios de evidência e grau de recomendação consi- derados aceitáveis. Cerca de 30% dos pedidos estavam relacionados a outros tipos de câncer para os quais não existem evidências que sustentem sua indicação. A capecitabina foi utilizada para o tratamento de câncer Aproximadamente 86% das ações e pedidos de imati- nibe estavam relacionados ao tratamento de leucemia Aproximadamente 86% das ações e pedidos de imati- nibe estavam relacionados ao tratamento de leucemia Tabela 1. Pedidos administrativos e judiciais de medicamentos e respectivos gastos. Estado de São Paulo, 2006–2007. Medicamento Número de pedidos Gasto (R$) Gasto médio por pedido (R$) 2006 2007 2006 2007 2006 2007 Bevacizumabe 97 125 4.618.108,74 6.463.859,07 47.609,37 51.710,87 Capecitabina 14 33 136.796,79 292.231,23 9.771,20 8.855,49 Cetuximabe 28 48 3.001.670,98 4.046.478,75 107.202,54 84.301,64 Erlotinibe 73 160 1.852.682,80 4.352.155,20 25.379,22 27.200,97 Imatinibe 126 164 1.402.226,16 8.543.003,08 11.128,78 52.091,48 Rituximabe 49 170 460.168,58 1.301.591,20 9.391,20 7.656,42 Temozolomida 46 87 1.232.857,38 3.079.281,75 26.801,25 35.394,04 Total 433 787 12.704.511,43 28.078.600,28 29.340,67 35.678,02 F t S t i d E t d d S úd d Sã P l 624 Antineoplásicos e ações judiciais Lopes LC et al Tabela 2. Caracterização de pedidos (administrativos e judiciais) segundo o tipo de demanda, a origem do pleito, os principais prescritores e advogados. Estado de São Paulo, 2006–2007. DISCUSSÃO Os sete medicamentos estudados foram selecionados pela SES-SP, conforme critérios de custo e demanda em 2006 e 2007. Em outros biênios, os medicamentos provavelmente serão diferentes, pois o processo é dinâmico e as necessidades se modiﬁ cam, exigindo constantes avaliações. Em 2006, cerca de 90% das ações e pedidos de ritu- ximabe e, em 2007, 75% visavam ao tratamento de linfoma não-Hodgkin. Embora o rituximabe tenha seu uso aprovado nas três agências reguladoras para o tratamento da artrite reumatóide (moderada a severa, em combinação com metotrexato, em pacientes que apresentem resposta inadequada a outras terapias com antagonistas de fatores anti-TNF), é indicação não respaldada por evidências da literatura para a incor- poração da tecnologias (classe IIb - recomendado em alguns casos, mas não na maioria). Houve aumento das ações e pedidos para essa indicação, de 1,1% em 2006 para 17,1% em 2007; 4,7% e 8,1% dos pedidos em 2006 e 2007, respectivamente, foram para uso em indicações sem evidência clínica de eﬁ cácia e segurança. Os resultados não permitem estimar com precisão o impacto econômico dos medicamentos selecionados sobre a SES-SP, pois não foram fornecidas as infor- mações relativas ao gasto total com medicamentos e com ações judiciais no estado de São Paulo, em 2006 e 2007. As ações judiciais e os pedidos administrativos dobraram em número no período estudado. Em 2006, para cada pedido, cerca de sete ações eram impetradas contra a SES-SP, proporção mantida em 2007. Os pedidos administrativos se concentram para o trata- mento de leucemia mielóide crônica e linfoma não- Hodgkin, enquanto as ações judiciais se concentram em câncer colorretal e câncer de pulmão. Considerando as prevalências de câncer no Brasil,i recomenda-se que a SES-SP mantenha atualizada a pesquisa sobre as evidências clínicas dos tratamentos desses tipos de câncer, pois resultam em pressão administrativa e ﬁ nanceira ao Estado de São Paulo. Ações e pedidos de temozolomida (80% em 2006 e 86% em 2007) se dirigiam ao tratamento de glioblastomas e astrocitomas. A indicação de temozolomida para o tratamento de melanoma metastático foi aprovada apenas no Brasil, mesmo sem evidências clínicas que justiﬁ quem seu emprego nessa indicação. Porém, 1,6% das ações e pedidos de temozolomida em 2007 apre- sentavam essa indicação. Não há registro de pedidos dessa natureza em 2006. Os sete medicamentos selecionados comprometeram quase R$ 30 milhões do orçamento estadual em 2007. RESULTADOS Variável Bevacizumabe Erlotinibe Capecitabina Rituximabe Imatinibe Cetuximabe Temozolomida 2006 (n = 97) 2007 (n = 125) 2006 (n = 73) 2007 (n = 160) 2006 (n = 14) 2007 (n = 33) 2006 (n = 49) 2007 (n = 170) 2006 (n = 126) 2007 (n = 164) 2006 (n = 28) 2007 (n = 48) 2006 (n = 46) 2007 (n = 87) Demanda judicial 99 96,8 100 97,5 100 90.9 100 80 59,3 61,6 100 100 93,3 88,5 Demanda administrativa 1 3,2 0 2,5 0 9,1 0 20 40,7 38,4 0 0 6,7 11,5 Origem do pleito: setor privado 81,7 86, 1 52,1 52,2 64,2 75,8 18,8 22,7 44,9 45,9 92,6 75 60,9 62,1 Origem do pleito: setor público 18,3 13,9 47,9 47,8 35,8 24,2 81,2 77,3 55,1 54,1 7,4 25 39,1 37,9 Principal prescritor 23,7 Presc. 1 9,6 Presc. 1 38,4 Presc. 2 31,3 Presc. 2 14,3 Presc. 3 12,1 Presc. 1 22,4 Presc. 4 16,3 Presc. 5 13,0 Presc. 6 11,0 Presc. 6 17,9 Presc. 7 10,6 Presc. 7 13,0 Presc. 8 11,5 Presc. 9 Principal advogado 48, 4 Adv. 1 33,3 Adv. 1 39,7 Adv. 2 31,4 Adv. 2 33,3 Adv. 1 35,7 Adv. 1 60,4 Adv. 3 31,3 Adv.4 15, 7 Adv. 5 Adv. 6 12,6 Adv. 6 70,4 Adv. 1 43,8 Adv. 1 16,7 Adv. 7 9,5 Adv. 1 Adv. 7 Fonte: Secretaria de Estado da Saúde de São Paulo. Tabela 3. Gastos gerados pela prescrição de cada medicamento segundo o prescritor e ação judicial. Estado de São Paulo, 2006-2007. Bevacizumabe Capecitabina Cetuximabe Erlotinibe Imatinibe Rituximabe Temozolomida Total 2006 2007 2006 2007 2006 2007 2006 2007 2006 2007 2006 2007 2006 2007 Prescritor 1 1.094.491,77 620.530,47 35.359,97 213.118,63 343.950,69 227.879,98 326.411,64 2.861.743,18 2 711.430,19 1.362.224,57 2.073.654,77 6 182.289,40 939.730,33 1.122.019,74 8 160.271,45 212.470,44 372.741,90 9 80.135,72 354.117,40 434.253,13 Subtotal 6.864.412,72 Advogado 1 2.235.164,63 2.152.465,07 45.553,33 104.326,55 2.113.176,37 1.772.357,69 329.777,54 500.497,85 292.531,77 9.545.850,79 2 735.515,07 1.366.576,73 2.102.091,80 6 193.960,57 271.482,08 75.959,99 195.846,98 220.149,51 1.076.418,39 146.710,03 249.421,82 2.429.949,37 5 220.149,40 990.988,35 1.211.137,86 9 147.779,48 213.307,35 335.857,74 696.944,56 Subtotal 15.985.974,38 625 Rev Saúde Pública 2010;44(4):620-8 mielóide crônica (LMC). RESULTADOS Há benefício deﬁ nido para uso em LMC cromossomo Filadélﬁ a positivo, fase crônica, recentemente diagnosticado ou após falha da terapia com alfainterferona, sendo essas indicações aprovadas pela Anvisa, FDA e Emea.7 As indicações de imatinibe cujo uso não mostra evidência de eﬁ cácia e segurança aumentou de 10,4% (2006) para 14,4% (2007). i Ministério da Saúde. Secretaria de Atenção à Saúde. Instituto Nacional de Câncer. Coordenação de Prevenção e Vigilância de Câncer. Estimativas 2008: Incidência de Câncer no Brasil. Rio de Janeiro; 2007. [citado 2009 set 10]. Disponível em: http://bvsms.saude.gov.br/bvs/ publicacoes/estimativa_incidencia_cancer_2008.pdf j Castilhos WO. Impacto do câncer no SUS. São Paulo: Agência FAPESP; 2007. [citado 2008 jul 27]. Disponível em: http://www.agencia. fapesp.br/materia/8104/especiais/o-impacto-do-cancer-no-sus.htm p cto do câncer no SUS. São Paulo: Agência FAPESP; 2007. [citado 2008 jul 27]. Disponível em: http://www.agencia. 4/especiais/o-impacto-do-cancer-no-sus.htm DISCUSSÃO Especiﬁ camente para o tratamento do câncer, os custos aumentaram 450% desde 1995 e não melhoraram a sobrevida dos pacientes nem aumentaram os índices de cura da doença.j Esses achados mostram que parte das solicitações não tem respaldo cientíﬁ co que fundamente o uso eﬁ caz e, sobretudo, seguro para o paciente. Os resultados mostram que as ações judiciais envol- vendo esses medicamentos correspondem a poucos prescritores e advogados. Essa constatação é suﬁ ciente para justificar uma auditoria das prescrições, dos prescritores e dos advogados, de modo a investigar suas relações, diretas e indiretas, com a indústria farmacêutica. A Tabela 4 mostra as indicações clínicas dos sete fármacos selecionados com nível de evidência (A e B) e grau de recomendação (classes I e IIa) e suas respec- tivas aprovações pela Anvisa, FDA e Emea. Algumas indicações regulamentadas por outras agências regu- ladoras não foram aprovadas no Brasil; entretanto, existem ações judiciais e pedidos administrativos para tais usos. Vários trabalhos destacam as inﬂ uências que o pres- critor sofre para decidir a opção terapêutica: concepções sobre o processo saúde–doença; qualidade da formação técnica, condições socioculturais e econômicas da população que atende; disponibilidade de medica- mentos no serviço em que atua; acesso a fontes de informações, assédio da indústria farmacêutica, entre outros.1,2,10 A análise dos percentuais de ações judiciais e pedidos administrativos para indicações sem evidência clínica mostra gasto inadequado de R$ 6.870.926,83. Bevacizumabe (59,5%) e rituximabe (31%) foram os medicamentos com maior percentual de solicitações sem evidência nos anos estudados (Tabela 5). 626 Antineoplásicos e ações judiciais Lopes LC et al Tabela 4. Indicações clínicas com nível de evidência (A e B) e grau de recomendação (classes I e IIa) e suas respectivas aprovações pelas agências reguladoras. Estado de São Paulo, 2006–2007. DISCUSSÃO Fármaco/Indicação Aprovação Anvisa FDA Emea Bevacizumabe Câncer colorretal metastático, usado em combinação com terapia baseada em 5-ﬂ uorouracila como tratamento de primeira ou segunda linha Sim Sim Sim Câncer de pulmão não-pequenas células, não-ressecável, localmente avançado, recorrente ou doença de células não-escamosas metastática, tratamento de primeira linha, em combinação com paclitaxel e carboplatina NC Sim Sim Capecitabina Em combinação com docetaxel está indicado para o tratamento de pacientes com câncer de mama localmente avançado ou metastático Sim Sim Sim Câncer de mama metastático, como monoterapia, quando resistentes a regimes contendo paclitaxel/antraciclina ou resistentes ao paclitaxel e não um candidato para tratamento posterior com antraciclina Sim Sim Sim Câncer do cólon, adjuvante terapêutico, Dukes C, quando o tratamento com uma ﬂ uoropirimidina sozinha é preferido NC Sim Sim Câncer esofagogástrico, avançado ou metastático, em combinação com agentes quimioterápicos NC NC NC Câncer gástrico, tratamento de primeira-linha para doença avançada ou metastática, em combinação com epirrubicina e oxaliplatina ou cisplatina NC NC NC Cetuximabe Câncer colorretal metastático, com expressão do EGFR, como monoterapia em pacientes que não responderam ao tratamento com irinotecano e oxaliplatina NC Sim Sim Câncer colorretal metastático, com expressão do EGFR, em associação com irinotecano, em pacientes refratários ao irinotecano isolado NC Sim Sim Câncer de cabeça e pescoço, células escamosas local ou regionalmente avançado, em combinação com radioterapia Sim Sim Sim Erlotinibe Câncer de pulmão, não-pequenas células, localmente avançado ou metastático (após falha da quimioterapia prévia) Sim Sim Sim Carcinoma do pâncreas, localmente avançado, não-ressecável, ou metastático, primeira linha de tratamento em combinação com gencitabina NC Sim Sim Imatinibe Leucemia mielóide crônica, cromossomo ﬁ ladélﬁ a positivo, fase crônica, após falha da terapia com alfainterferona Sim Sim Sim Leucemia mielóide crônica, cromossomo ﬁ ladélﬁ a positivo, fase crônica, recentemente diagnosticado Sim Sim Sim Leucemia linfóide aguda, cromossomo ﬁ ladélﬁ a positivo, recentemente diagnosticado, como parte da quimioterapia combinada NC NC Sim Rituximabe Linfoma não-Hodgkin, difuso, células-B grandes, CD20-positivo, em combinação para a primeira linha de tratamento Sim Sim Sim Linfoma não-Hodgkin, recorrente ou refratário, baixo grau ou folicular, CD20-positivo, células-B Sim Sim Sim Leucemia linfóide crônica, em combinação com tratamento de primeira linha NC NC NC Temozolomida Glioblastoma multiforme do cérebro recentemente diagnosticado concomitantemente com Erlotinibe Imatinibe Temozolomida 627 Rev Saúde Pública 2010;44(4):620-8 Tabela 5. Gasto estadual com ações judiciais e pedidos administrativos sem evidência clínica. Estado de São Paulo, 2006– 2007. Fármaco Gastos (R$) % de pedidos sem evidência Gastos com pedidos sem evidência (R$) 2006 2007 2006 2007 2006 2007 Bevacizumabe 4.618.108,74 6.463.859,07 28,1 31,4 1.297.688,56 2.029.651,75 Capecitabina 136.796,79 292.231,23 17,4 19,2 23.802,64 56.108,40 Cetuximabe 3.001.670,98 4.046.478,75 13,9 10,1 417.232,27 408.694,35 Erlotinibe 1.852.682,80 4.352.155,20 2,8 4,4 51.875,12 191.494,83 Imatinibe 1.402.226,16 8.543.003,08 10,4 14,2 145.831,52 1.213.106,44 Rituximabe 460.168,58 1.301.591,20 5,8 25,2 26.689,78 328.000,98 Temozolomida 1.232.857,38 3.079.281,75 20,0 14,1 246.571,48 434.178,73 Total 12.704.511,43 28.078.600,28 - - 2.209.691,36 4.661.235,47 Fonte: Secretaria de Estado da Saúde de São Paulo pela SES-SP quanto aos protocolos de uso. Ademais, solicitações provenientes dos hospitais públicos e/ou conveniados precisam ser sistematicamente auditadas pela SES-SP, pois o fornecimento de medicamentos deve estar vinculado às normas do SUS. Além disso, várias entidades brasileiras de defesa de usuários são ﬁ nanciadas por laboratórios farmacêuticos, visando incluir seus medicamentos no âmbito do SUS. Uma entidade que representa pacientes com linfoma e leucemia, com sede em São Paulo, recebeu R$ 1,5 milhão de oito multinacionais em 2007.k O uso clínico de um medicamento em indicações diferentes daquelas cuja literatura mostra evidências de eﬁ cácia e segurança não é recomendado. Entre as conseqüências, pode acarretar o emprego de um tratamento de eficácia duvidosa, com importantes efeitos adversos, e onerar o SUS. Além disso, para registrar uma nova indicação nas agências reguladoras, o laboratório produtor precisa comprovar, por meio de ensaios clínicos bem conduzidos, a eﬁ cácia e a segu- rança do medicamento para determinada enfermidade. O fornecimento do medicamento para indicações não aprovadas, por imposição da via judicial, equivale ao ﬁ nanciamento, pelo SUS, de pesquisas cuja responsa- bilidade é do laboratório inovador. O ﬁ nanciamento de pesquisa cientíﬁ ca pela indústria farmacêutica é muito comum em todas as áreas da Medicina. Essa é uma aliança necessária e valiosa. Entretanto, estudos financiados pela indústria têm maior probabilidade de relatar resultados favoráveis a seus produtos do que os independentes. Friedberg et al6 (1999) encontraram resultados desfavoráveis em 5% dos estudos ﬁ nanciados pelos laboratórios produ- tores de medicamentos utilizados em oncologia. Já em estudos independentes, essa taxa subiu para 38%. A avaliação crítica da literatura cientíﬁ ca determina a qualidade, a força da evidência e seus limites. k Colucci C, Westin E. Indústria farmacêutica ﬁ nancia ONGs. Folha de S Paulo, 2008 maio 15;Cotidiano[citado 2008 maio 28]. Disponível em: www.folha.uol.com.br/fsp/cotidian/ff1805200801/htm l Santos L. Saúde: conceito e atribuições do Sistema Único de Saúde. Jus Navigandi. 2005[citado 2009 jan 21]9(21). Disponível em: http://jus2.uol.com.br/doutrina/imprimir.asp?id=7378. AGRADECIMENTO Justiﬁ cativa de uso dos medicamentos e informações clínicas sobre o real estado de saúde do paciente são À Secretaria Estadual de Saúde de São Paulo pelo fornecimento dos dados. Temozolomida Dessa forma, com base em informações cientíﬁ cas de boa qualidade, podem ser formuladas políticas públicas que contribuam para a racionalização do uso de tecno- logias médicas. A comprovação no presente estudo de que cerca de 17% dos pedidos não tinham evidência para aquela indicação informada no processo equivale a um gasto inadequado de, no mínimo, R$ 6,8 milhões no período estudado. A limitação do presente estudo quanto ao diagnóstico impreciso referido no pedido – e não o Código Internacional de Doenças – subdimensiona o percentual de indicações clínicas solicitadas sem evidência, pois a força de recomendação da utilização é especíﬁ ca para cada condição. Portanto, o gasto da SES-SP com o fornecimento de medicamentos sem o devido respaldo cientíﬁ co pode ser muito superior ao valor aqui estimado. A incorporação tecnológica no campo da saúde deve avaliar corretamente sua eﬁ cácia e segurança, bem como seus efeitos sobre os gastos públicos com os serviços de saúde. Deve ser pautada pelo necessário, oportuno, razoável, conveniente e essencial para garantir a saúde coletiva e individual, e não pela sua existência no mercado.l Mais de 50% das ações judiciais e cerca de 40% dos pedidos administrativos eram provenientes do setor privado. Dessa forma, recomenda-se que as solici- tações provenientes desse setor sejam monitoradas Políticas sanitárias bem planejadas resultam na eﬁ ci- ência das ações de promoção e prevenção à saúde, 628 Antineoplásicos e ações judiciais Lopes LC et al indispensáveis para a avaliação do pleito e para a decisão judicial de fornecimento pelo SUS. O estabe- lecimento de protocolos clínicos para o uso de imuno- biológicos, que apresentam indicações restritas e riscos elevados, pode racionalizar o uso destes medicamentos, contribuindo para o modelo de assistência farmacêutica em oncologia no SUS. traduzindo-se em medicina preventiva. Em contraste, na medicina curativa, os medicamentos assumem impor- tância exagerada na assistência à saúde.8,11 Além disso, considerando a eqüidade na distribuição dos recursos, que constitui um dos dilemas mais complexos enfren- tados pelo SUS, é clara a necessidade de qualiﬁ cação técnica para tratar as demandas judiciais. Isso exige capacitação dos proﬁ ssionais para a seleção adequada dos fármacos e para a escolha da melhor conduta tera- pêutica em determinada condição clínica.m Pesquisa ﬁ nanciada pelo Ministério da Saúde/Secretaria de Estado da Saúde de São Paulo/Conselho Nacional de Desenvolvimento Cientíﬁ co e Tecnológico/Fundação de Amparo à Pesquisa do Estado de São Paulo (edital PPSUS 2006/2007, Processo Nº 2006/61512-5) Pesquisa ﬁ nanciada pelo Ministério da Saúde/Secretaria de Estado da Saúde de São Paulo/Conselho Nacional de Desenvolvimento Cientíﬁ co e Tecnológico/Fundação de Amparo à Pesquisa do Estado de São Paulo (edital PPSUS 2006/2007, Processo Nº 2006/61512-5). Costa AC foi apoiado pela Universidade de Sorocaba (Bolsa de iniciação cientíﬁ ca). Trabalho apresentado no Congresso Latinoamericano de Farmacologia, Coquimbo, Chile, em 2008; no I Simpósio de Pesquisas sobre Uso Racional de Medicamentos, Sorocaba, SP, em 2008; no I Congresso Brasileiro de Medicina Baseada em Evidências e Direito à Saúde, Brasília, DF, em 2009; e no 12th World Congress on Public Health, Istambul, Turquia, em 2009. Os autores declaram não haver conﬂ itos de interesses. m Vieira FS, Lopes LC, Barberato-Filho S. Relatório Final Oﬁ cina. Gestão da assistência farmacêutica e ações judiciais: estratégias que favorecem o uso racional e o acesso a medicamentos. I Simpósio de Pesquisas sobre Uso Racional de Medicamentos. Sorocaba: Universidade de Sorocaba; 2008[citado 2009 set 10]. Disponível em: http://www.cebes.org.br/media/File/direito%20sanitrio/relatorio_ﬁ nal_oﬁ cina_uniso_ dez_2008.PDF Costa AC foi apoiado pela Universidade de Sorocaba (Bolsa de iniciação cientíﬁ ca). Trabalho apresentado no Congresso Latinoamericano de Farmacologia, Coquimbo, Chile, em 2008; no I Simpósio de Pesquisas sobre Uso Racional de Medicamentos, Sorocaba, SP, em 2008; no I Congresso Brasileiro de Medicina Baseada em Evidências e Direito à Saúde, Brasília, DF, em 2009; e no 12th World Congress on Public Health, Istambul, Turquia, em 2009. Os autores declaram não haver conﬂ itos de interesses. REFERÊNCIAS 7. Klasco RK, editor. Drugdex System. Thomson Micromedex. Greenwood Village: Thomson; 2008. 1. Arnau JP, Laporte JR. Promoção do uso racional dos medicamentos e preparação de guias farmacêuticos. In: Laporte JR, Tognoni G, editores. Epidemiologia do medicamento: princípios gerais. São Paulo: Hucitec; 1989. p.57-74. 8. Lefévre F. O medicamento como mercadoria simbólica. São Paulo: Cortez; 1991. 9. Messeder AM, Osorio-de-Castro CGS, Luiza VL. Mandados judiciais como ferramenta para garantia do acesso a medicamentos no setor público: a experiência do Estado do Rio de Janeiro, Brasil. Cad Saude Publica. 2005;21(2):525-34. DOI:10.1590/ S0102-311X2005000200019 2. Barros JAC. Propaganda de medicamentos: Atentado à saúde? São Paulo: Hucitec/Sobravime; 1995. 2. Barros JAC. Propaganda de medicamentos: Atentado à saúde? São Paulo: Hucitec/Sobravime; 1995. 3. Ministério da Saúde. Secretaria de Ciência, Tecnologia e Insumos Estratégicos. Departamento de Ciência e Tecnologia. Avaliação de Tecnologias em saúde: institucionalização das ações no Ministério da Saúde. Rev Saude Publica. 2006;40(4):743-7. DOI:10.1590/ S0034-89102006000500029 10. Santos V, Nitrini SMO. Indicadores do uso de medicamentos prescritos e de assistência ao paciente de serviços de saúde. Rev Saude Publica. 2004;38(6):819- 26. DOI:10.1590/S0034-89102004000600010 4. Conselho Nacional dos Secretários Municipais de Saúde. A saúde no banco dos réus. Rev Conasems. 2004;(8):25-31. 4. Conselho Nacional dos Secretários Municipais de Saúde. A saúde no banco dos réus. Rev Conasems. 2004;(8):25-31. 11. Scott DK, Ferner RE. The strategy of desire and rational prescribing. Br J Clin Pharmacol. 1994;37(3):217-9. 12. Vieira FS, Zucchi P. Distorções causadas pelas ações judiciais à política de medicamentos no Brasil. Rev Saude Publica. 2007;41(2):214-22. DOI:10.1590/ S0034-89102007000200007 12. Vieira FS, Zucchi P. Distorções causadas pelas ações judiciais à política de medicamentos no Brasil. Rev Saude Publica. 2007;41(2):214-22. DOI:10.1590/ S0034-89102007000200007 5. Del Debbio CB, Tonon LM, Secoli SR. Terapia com anticorpos monoclonais: uma revisão de literatura. Rev Gaucha Enferm. 2007;28(1):133-42. 6. Friedberg M, Saffran B, Stinson TJ, Nelson W, Bennett CL. Evaluation of conﬂ ict of interest in economic analyses of new drugs used in oncology. JAMA. 1999;282(15):1453-7. DOI:10.1001/ jama.282.15.1453 13. Vieira FS. Qualiﬁ cação dos serviços farmacêuticos no Brasil: aspectos inconclusos da agenda do Sistema Único de Saúde. Rev Panam Salud Publica. 2008;24(2):91-100. DOI:10.1590/S1020- 49892008000800003 m Vieira FS, Lopes LC, Barberato-Filho S. Relatório Final Oﬁ cina. Gestão da assistência farmacêutica e ações judiciais: estratégias que favorecem o uso racional e o acesso a medicamentos. I Simpósio de Pesquisas sobre Uso Racional de Medicamentos. Sorocaba: Universidade de Sorocaba; 2008[citado 2009 set 10]. Disponível em: http://www.cebes.org.br/media/File/direito%20sanitrio/relatorio_ﬁ nal_oﬁ cina_uniso_ dez_2008.PDF
W4365514179.txt	https://www.urovest.ru/jour/article/download/677/453	en		Pathological findings in semen analysis from COVID-19 survivors and the impact of various SARS-COV-2 vaccines on spermatogenesis	Vestnik urologii	2,023	cc-by	5,573	ОБЗОРЫ ЛИТЕРАТУРЫ Х.С. Ибишев, Я.О. Прокоп ПАТОЛОГИЧЕСКИЕ НАХОДКИ В СПЕРМОГРАММЕ У ПАЦИЕНТОВ, ПЕРЕНЁСШИХ COVID-19, И ВЛИЯНИЕ РАЗЛИЧНЫХ ВАКЦИН ОТ SARS COV-2 НА СПЕРМАТОГЕНЕЗ УДК 616.69-008.8: 616.98:615.37 https://doi.org/10.21886/2308-6424-2023-11-1-125-133 Патологические находки в спермограмме у пациентов, перенёсших COVID-19, и влияние различных вакцин от SARS-COV-2 на сперматогенез © Халид С. Ибишев, Ян О. Прокоп Ростовский государственный медицинский университет [Ростов-на-Дону, Россия] Аннотация Репродуктивное здоровье мужчин зависит от множества факторов, к которым в том числе относится и наличие или отсутствие инфекционных заболеваний в репродуктивной системе организма. В последние три года во всём мире, в том числе и в России, растёт количество людей, заболевших новой вирусной инфекцией (COVID-19), вызванной коронавирусом SARS-CoV-2, который вызывает дисфункцию и оказывает негативное действие на многие органы и системы организма, в том числе и репродуктивные органы, что отражается в аномальных показателях спермограммы. Несмотря на высокую эффективность и небольшое количество побочных эффектов, обнаруженных в клинических испытаниях, только 56% людей в США и 49% в России сообщили о желании получить вакцину. Одной из причин недоверия к вакцинам является потенциальное негативное влияние на фертильность. Обзор публикаций посвящён изучению влияния SARS-CoV-2 и вакцинации на репродуктивное здоровье мужчин. Поиск проведён с использованием баз данных Medline, PubMed, EMBASE. Ключевые слова: репродуктивное здоровье; инфертильность; коронавирусная инфекция; COVID-19; SARS-CoV-2; спермограмма; вакцинация Финансирование. Исследование не имело спонсорской поддержки. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Этическое заявление. Исследование выполнено в рамках диссертационной работы, одобренной локальным независимым этическим комитетом ФГБОУ ВО РостГМУ Минздрава России (Протокол № 1/22 от 13 января 2022 года). Вклад авторов. Х.С. Ибишев — концепция исследования, критический обзор, научное редактирование; научное руководство; Я.О. Прокоп — обзор литературы, систематизация данных; написание текста рукописи.  Корреспондирующий автор: Халид Сулейманович Ибишев; e-mail: ibishev22@mail.ru Поступила в редакцию: 02.10.2022. Принята к публикации: 10.01.2023. Опубликована: 26.03.2023. Для цитирования: Ибишев Х.С., Прокоп Я.О. Патологические находки в спермограмме у пациентов, перенёсших COVID-19, и влияние различных вакцин от SARS-COV-2 на сперматогенез. Вестник урологии. 2023;11(1):125-133. DOI: 10.21886/2308-6424-2023-11-1-125-133. Pathological findings in semen analysis from COVID-19 survivors and the impact of various SARS-COV-2 vaccines on spermatogenesis © Khalid S. Ibishev, Jan O. Prokop Rostov State Medical University [Rostov-on-Don, Russian Federation] Abstract Male reproductive health depends on many factors, including whether infectious diseases occur in the reproductive system. Such changes may be reflected in the semen analysis. In the past three years, the number of individuals who fell ill with COVID-19 caused by SARS-CoV-2 has been growing worldwide, including in Russia. This infection causes dysfunction and a negative effect on many organs and systems, including reproductive organs, which is reflected in abnormal semen parameters. Despite the high efficacy and small number of side effects found in clinical trials, only 56% of the population in the US and 49% in the Russian Federation reported wanting the vaccine. One of the reasons for distrust of vaccines is the potential negative impact on fertility. A literature review is devoted to the study of the effect of SARS-CoV-2 and vaccination on male reproductive health. The search was carried out using Medline, PubMed, and EMBASE databases. Keywords: reproductive health; infertility; COVID-19; SARS-CoV-2; semen analysis; vaccination Вестник урологии Vestnik Urologii 2023;11(1):125-133 ISSN 2308–6424 UROVEST.RU 125 K.S. Ibishev, J.O. Prokop PATHOLOGICAL FINDINGS IN SEMEN ANALYSIS FROM COVID-19 SURVIVORS AND THE IMPACT OF VARIOUS SARS-COV-2 VACCINES ON SPERMATOGENESIS REVIEW ARTICLES Financing. The study was not sponsored. Conflict of interests. The authors declare no conflict of interest. Ethical statement. The study was performed as part of a thesis approved by the Ethics Committee of the Rostov State Medical University (Protocol No. 1/22 signed January 13, 2022). Authors' contribution: Kh.S. Ibishev — study concept, critical review, scientific editing; supervision; Ya.O. Prokop — literature review, data acquisition; drafting the manuscript.  Corresponding author: Khalid S. Ibishev; e-mail: ibishev22@mail.ru Received: 10/02/2022. Accepted: 01/10/2023. Published: 03/26/2023. For citation: Ibishev Kh.S., Prokop Ya.O. Pathological findings in semen analysis from COVID-19 survivors and the impact of various SARSCOV-2 vaccines on spermatogenesis. Urology Herald. 2023;11(1):125-133. (In Russ.). DOI: 10.21886/2308-6424-2023-11-1-125-133. Введение С момента первоначальной вспышки в декабре 2019 года новая коронавирусная болезнь (COVID-19) быстро распространилась и превратилась в глобальную чрезвычайную ситуацию в области здравоохранения, затрагивающую многие аспекты повседневной жизни людей. Всемирная организация здравоохранения (ВОЗ) объявила, что на сегодняшний день COVID-19 подтверждён более чем у 489 миллионов человек во всем мире. Коронавирус, вызвавший эту эпидемию, названный ВОЗ коронавирусом тяжёлого острого респираторного синдрома 2 (SARS-CoV-2), на 79% совпадает с последовательностью человеческого коронавируса SARS-CoV, вызвавшего пандемию атипичной пневмонии в 2002 году [1 – 3]. Аналогично, по сравнению с SARS-CoV, SARS-CoV-2 также использует ангиотензин, превращающий фермент II (ACE2) в качестве рецептора для проникновения в клетки-хозяева [2 – 4]. Анализ профилей экспрессии одноклеточной РНК человека выявил экспрессию ACE2 в сперматогониях, клетках Leydig и Sertoli, а также в эпителиальных клетках предстательной железы, а также вызывать нарушения в гипоталамогипофизарной цепи, что вместе приводит к нарушениям репродуктивного здоровья у пациентов, перенёсших COVID-19 [5, 6]. Неоднократно публиковались исследования о том, что РНК SARS-CoV-2 не была обнаружена в образцах спермы, перенёсших острую инфекцию выздоравливающих или выздоровевших пациентов [7 – 9]. Вместе с тем, существует множество работ, доказывающих наличие изменений в спермограмме у пациентов, перенёсших COVID-19. Наиболее актуальные из них на момент публикации будут рассмотрены в данной статье. Механизм возникновения данных нарушений является дискутабельным вопросом. Однако уже на данном этапе понятно, что данные патологические изменения приводят к снижению репродуктивного потенциала мужского населения. 126 ISSN 2308–6424 UROVEST.RU Цель исследования: провести анализ литературы, изучающей влияние SARS-CоV-2, и наиболее доступных вакцин от COVID-19 на репродуктивный потенциал мужчин. Материалы и методы Электронный поиск литературы был проведён с использованием базы данных Medline, PubMed, EMBASE для того, чтобы выделить соответствующие исследования, проведённые в 2020 – 2021 годах, которые имеют отношение к анализу влияния SARSCоV-2 на репродуктивное здоровье мужчин. В настоящий обзор вошли 12 источников. Отбор литературы осуществляли по критериям практической значимости, а также импакт-фактору журнала, в котором была опубликована статья. Ключевыми словами, по которым проходил поиск статей, являлись «COVID-19», «SARS COV-2», «semen», «vaccine». Из выборки были исключены литературные обзоры других статей и мнения других авторов о проведённых исследованиях. Результаты Механизм воздействия SARS-CoV-2 на репродуктивные органы и патологические находки в них. Патогенность коронавируса зависит от его проникновения через связывание его вирусного шиповидного белка (S) с рецептором ангиотензин превращающего фермента 2 (АПФ2) и от праймирования S-белка трансмембранной сериновой протеазой II типа (TMPRSS2) [2 – 4, 10 – 13]. Рецептор АПФ 2 — регулятор артериального давления через ренин-ангиотензинальдостероновую систему, который обеспечивает проникновение вируса в клетки человека и его патогенность. АПФ 2 обнаруживается во множестве органов, включая яички и их придатки, простату, почки и кишечник [14]. TMPRSS2 активирует шиповидный белок на внешней оболочке вируса и облегчает проникновение в клетку. Экспрессия гена TMPRSS2 стимулируется только через Вестник урологии Vestnik Urologii 2023;11(1):125-133 ОБЗОРЫ ЛИТЕРАТУРЫ Х.С. Ибишев, Я.О. Прокоп ПАТОЛОГИЧЕСКИЕ НАХОДКИ В СПЕРМОГРАММЕ У ПАЦИЕНТОВ, ПЕРЕНЁСШИХ COVID-19, И ВЛИЯНИЕ РАЗЛИЧНЫХ ВАКЦИН ОТ SARS COV-2 НА СПЕРМАТОГЕНЕЗ рецептор андрогена и увеличивается при воздействии андрогенов [15 – 18]. В последнее время сообщения о частоте патологии яичек при COVID-19 становятся всё более убедительными. Посмертные исследования яичек человека позволяют предположить, что инфекции SARS-CoV-2 приводят к воспалению яичек, отёку и дегенерации сперматогенных клеток в связи с инфильтрацией CD3 + и CD68 + иммунными клетками по аналогии с SARS [19]. Данные изменения можно заподозрить при клиническом наблюдении, у пациентов отмечается снижение гемодинамики тестикул и снижение уровня тестостерона сыворотки [20], также отмечается, что из-за снижения уровня тестостерона снижается иммунный ответ у пациентов, что может отрицательно сказаться на тяжести COVID-19 [21 – 23]. В исследовании, опубликованном в сентябре 2020 года в журнале Европейской Ассоциации Урологов, Dr. Mig Yang с соавторами по результатам патологоанатомического исследования обнаружил гибель клеток Leydig и Sertoli у больных, скончавшихся от COVID-19 [24]. Наше исследование, проведённое на базе РостГМУ, в котором участвовали пациенты с идиопатическим бесплодием, только подтверждает находку доктора Mig Yangи его коллег [25]. Патология эякулята во время и после COVID-19. Тема влияния COVID-19 на репродуктивное здоровье мужчин имеет огромное значение. Множество исследователей обращают внимание на нарушения, возникающие у пациентов перенёсших СOVID-19. Так, в краткосрочном исследовании, проведённом в США доктором G. Erbay et al., приняли участие 69 мужчин, перенёсших COVID-19, из которых 26 мужчин — с лёгким течением заболевания, 43 мужчины — со среднетяжёлым течением заболевания [26]. Из исследования были исключены пациенты моложе 20 и старше 45 лет, пациенты с приёмом гормональных препаратов в анамнезе, которые могут повлиять на сперматогенез, пациенты с олигозооспермией или азооспермией, пациенты с операциями на яичках в анамнезе (неопущение яичка, варикоцеле, водянка яичка, перекрут яичка, опухоль яичка), те, у кого в анамнезе была лучевая терапия органов малого таза, те, кто перенёс меВестник урологии Vestnik Urologii 2023;11(1):125-133 тоды вспомогательной репродукции, и те, кто получил медикаментозную терапию для увеличения общего количества сперматозоидов и подвижности сперматозоидов. В исследовании не было обнаружено статистически значимой разницы между группами, с точки зрения демографических данных и медикаментозного лечения COVID-19. В качестве лечения применялись фавипиравир, гидроксихлорохин, эноксапарин натрия, парацетамол при лихорадке и болевом синдроме, антибактериальная терапия и терапия глюкокортикостероидами не применялись. Лихорадка присутствовала у 18 из 26 пациентов в группе с лёгкими симптомами и у 31 из 43 — в группе с умеренными симптомами. Эндотрахеальная интубация не потребовалась ни одному пациенту. В группе с лёгкой симптоматикой прогрессивная и общая подвижность и жизнеспособность сперматозоидов снизились по сравнению с периодом до болезни, в то время как в группе с умеренной симптоматикой заболевание отрицательно повлияло на все параметры сперматозоидов. Пациенты в группе с лёгкими симптомами были дополнительно разделены на две подгруппы с лихорадкой и без неё, было изучено влияние лихорадки на параметры спермы. Обнаружено, что в обеих подгруппах не было отрицательного влияния на параметры спермы из-за лихорадки. Можно сказать, что основоположником наблюдения за изменениями в фертильном статусе мужчин, возникающими после перенесённого COVID-19, является группа N. Holtmann [27]. В 2020 году ими было проведено исследование, в котором участвовали 34 пациента: 18 мужчин перенесли COVID-19, 2 — находились в острой фазе заболевания, 14 человек составили контрольную группу. Исследуемая группа не получала противовирусного лечения и терапии кортикостероидами. РНК SARSCoV2 не была обнаружена с помощью ПЦР в сперме, в том числе в образцах спермы двух пациентов в острой стадии COVID-19. У пациентов со среднетяжёлым течение заболевания отмечались выраженная астенозооспермия и олигоспермия. Обращает на себя внимание тот факт, что у пациентов с лихорадкой подобные изменения имели более выраженный характер, а у 9 из 10 больных отмечалась лейкоспермия. ISSN 2308–6424 UROVEST.RU 127 K.S. Ibishev, J.O. Prokop PATHOLOGICAL FINDINGS IN SEMEN ANALYSIS FROM COVID-19 SURVIVORS AND THE IMPACT OF VARIOUS SARS-COV-2 VACCINES ON SPERMATOGENESIS Со временем сообщений о нарушении в спермограмме стали появляться чаще. J. C. Best вместе с коллегами в июле 2021 года опубликовали статью о своём исследовании, в котором приняло участие бóльшее количество пациентов, чем представлено в работе N. Holtmann [28]. В исследовании приняли участие тридцать мужчин с диагнозом «Острая инфекция SARS-CoV-2», заболевание было подтверждено с использованием ПЦР тестирования в реальном времени из образцов мазка из глотки. Образцы спермы были собраны у каждого человека с использованием рассылаемых по почте наборов. Суммарно в исследовании участвовали 60 мужчин, из которых 30 имели подтверждённую ПЦР новую Коронавирусную инфекцию, 30 были здоровыми пациентами, средний возраст исследуемых составил 40 лет. Средняя продолжительность между положительным тестом на SAR-CoV-2 и сбором спермы составила 37 дней. У пациентов с COVID-19 средняя концентрация сперматозоидов была на уровне 11,5 млн/мл, а общее количество — 12,5 млн, что значительно ниже по сравнению с группой не имевших COVID-19, где средняя концентрация была на уровне 21,5 млн/мл, а среднее общее количество — на уровне 59,2 млн. Насколько долго могут сохраняться изменения в спермограмме после COVID-19? Другие исследователи также находили изменения в спермограмме после перенесённого COVID-19 и попытались узнать, насколько продолжительными могут быть эти изменения. В исследовании T. H. Guo et al., проведённом в 2021 году, был задействован 41 пациент, выздоровевший после COVID-19, и 50 пациентов контрольной группы [29]. Из 41 исследуемого было выявлено 4 пациента с олигозооспермией, 10 — с тератозооспермией, 18 — с астенозооспермией. По сравнению с контрольной группой концентрация сперматозоидов, подвижность и прогрессивная подвижность были значительно снижены у пациентов при первом отборе проб, который был проведён в среднем через 56 дней после выписки из больницы. При сравнении же характеристик сперматозоидов через некоторое время после выздоровления от COVID-19 общее количество сперматозоидов концентрация сперматозоидов и количество подвижных 128 ISSN 2308–6424 UROVEST.RU REVIEW ARTICLES и прогрессивно подвижных сперматозоидов у пациентов были значительно увеличены при втором отборе проб, который проводился в среднем через 84 дня после выписки из больницы. В исследование коллег из Ирана, проведённое в Masih Daneshvari Hospital (Teheran, Iran), включало в себя 84 пациента и 105 человек контрольной группы [30]. Из 84 человек 37 получали терапию глюкокортикостероидами и 58 — противовирусную терапию. Среди 84 пациентов с положительным результатом на COVID-19 у 1,2% (1/84) было «лёгкое течение», у 27,4% (23/84) — «средней тяжести», у 32,1% (27/84) — «тяжёлое течение», у 39,3% (33/84) — «критическое состояние». Группа, в которую входили пациенты с COVID-19, имела значительно более низкие уровни объёма спермы, прогрессивной подвижности, морфологии сперматозоидов, концентрации сперматозоидов и количества сперматозоидов по сравнению с контрольной группой. Наблюдалось увеличение количества сперматозоидов по сравнению с исходным уровнем в период от 10 до 60 дней в группе COVID-19, в то время как прогрессивная подвижность увеличивалась по сравнению с исходным уровнем в период от 30 до 60 дней. COVID-19 приводит к иммунологическому бесплодию? В исследовании G. Donders et al., проведённом в Бельгии, среди 120 мужчин выявили патологические параметры в спермограмме [31]. Четверть (25,4%; 30/118) всех обследованных мужчин после заражения SARS-CoV-2 имели олигозооспермию, 44,1% (52/118) — астенозооспермию, две трети (67,0%; 79/118) — тератозооспермию. После заражения SARS-CoV-2 морфология сперматозоидов была нарушена больше, чем подвижность, а концентрация сперматозоидов была затронута меньше всего. Только 24,6% (29/118) участников имели полностью нормальные параметры сперматозоидов (концентрация, подвижность и морфология). Около трети (38/118) имели астенозооспермию (n = 10) или тератозооспермию (n = 28), 25,4% (30/118) имели два аномальных параметра спермы (9 олигозооспермия и тератозооспермия; 21 астенозооспермия и тератозооспермия), 17,8% (21/118) имели три (олигозооспермия, астенозооспермия и тератозооспермия). У 106 участников было достаточно подвижных сперматозоидов для выполнения Вестник урологии Vestnik Urologii 2023;11(1):125-133 ОБЗОРЫ ЛИТЕРАТУРЫ Х.С. Ибишев, Я.О. Прокоп ПАТОЛОГИЧЕСКИЕ НАХОДКИ В СПЕРМОГРАММЕ У ПАЦИЕНТОВ, ПЕРЕНЁСШИХ COVID-19, И ВЛИЯНИЕ РАЗЛИЧНЫХ ВАКЦИН ОТ SARS COV-2 НА СПЕРМАТОГЕНЕЗ прямого теста MAR как на IgA, так и на IgG. В 61% образцов спермы (65/106) были обнаружены IgA-антитела, преимущественно локализованные в хвостиках сперматозоидов. У одного участника процент IgA антител сперматозоидов был выше 40%, что указывает на иммунологическое бесплодие (1/106; 0,9%), тогда как у 14 участников процент IgA антител сперматозоидов составлял от 10 до 40% (13,2%, снижение рождаемости). IgG ASA выявлялись реже, чем IgA ASA после COVID-19 (34/106; 32,1%). У двух субъектов более 90% подвижных сперматозоидов содержали IgG-антитела, которые были связаны со всеми сперматозоидами. Может ли COVID-19 повлиять на результаты ЭКО? Изменения в сермограмме и изменения иммунологического статуса после перенесённого COVID-19 отрицательно сказываются на возможность зачатия у мужчин. Некоторым из них возможно придётся прибегнуть к применению вспомогательных репродуктивных технологий. Исследователи из КНР в Tongji Hospital (Wuhan, PRC) во главе с доктором М. Wang провели исследование, направленное на оценку влияния инфекции SARS-CoV-2 на параметры спермы и изучение влияния инфекции на результаты экстракорпорального оплодотворения (ЭКО) [32]. В исследовании участвовали 148 здоровых мужчин, не имевших в анамнезе новой короновирусной инфекции и показавших отрицательные тесты на наличие IgG\IgM к SARS-CoV-2, и 50 перенёсших COVID-19. Из этих 50 пациентов 6 (6/50; 12,0%) перенесли COVID-19 в лёгкой форме, в основном отмечая лихорадку, кашель, головную боль, мышечную боль и утомляемость, однако рентгенологических изменений в лёгких у них не было. Один пациент сообщил об аносмии. Семь участников (7/50; 14,0%) были классифицированы как имеющие умеренную инфекцию с типичными проявлениями КТ органов грудной клетки. У остальных 37 (37/50; 74,0%) пациентов инфекция была бессимптомной. Эти пациенты были диагностированы в период с января по март 2020 года. От первого диагноза инфекции SARS-CoV-2 до лечения ЭКО прошло не менее четырёх месяцев. Среди 50 мужчин с инфекцией SARSCoV-2 в анамнезе 26 ранее подвергались одному анализу спермы до заражения. После инфицирования у мужчин оказалось Вестник урологии Vestnik Urologii 2023;11(1):125-133 меньше нормальных сперматозоидов с точки зрения их морфологии, хотя все значения всё ещё находились в пределах нормального референтного диапазона. Анализ данных ЭКО показал, что инфекция SARS-CoV-2 у мужчин не может значительно ухудшить исходы ЭКО с точки зрения клинических исходов, но в то же время в инфицированной группе наблюдалось снижение скорости образования бластоцист и количества доступных бластоцист, что свидетельствует о потенциальном негативном влиянии на эмбриональное развитие. Влияние вакцинации от COVID-19 на сперматогенез. За период пандемии было разработано несколько высокоэффективных вакцин, которые успешно применяются для профилактики COVID-19. Любая вакцинация имеет побочные реакции. Введение вакцин против COVID-19, как и любых других вакцин, может вызывать побочные эффекты, преимущественно в диапазоне от лёгких до умеренно выраженных, которые самостоятельно разрешаются в течение нескольких дней. К типичным побочным эффектам относятся боль в месте инъекции, лихорадка, утомляемость, головные боли, боли в мышцах, озноб и диарея. К наиболее опасным осложнениям вакцин против COVID-19 исследователи относят тромбозы, перикардиты, эндокардиты и, как следствие, возникающие на их фоне инфаркты миокарда [33 – 35], также отмечено возникновение перитонитов после ведения некоторых вакцин от SARS-CoV-2 [36]. С самого начал существовало несколько довольно устойчивых мифов об их патологическом влиянии на различные системы организма. Данные мифы снижают доверие к вакцинации, что, безусловно, подвергает популяцию опасности, но самое печальное в том, что уровень доверия к вакцинации от COVID-19 низок и среди медицинского персонала [37 – 40]. Одной из причин недоверия к вакцинам является мнение общественности о потенциальном негативном влиянии на фертильность [41]. В настоящее время для профилактики COVID-19 активно используются несколько вакцин. По состоянию на 19 августа 2021 в число вакцин, одобренных Всемирной организацией здравоохранения для экстренного применения, вошли препараты Pfizer / BioNTech, Moderna, AstraZeneca, ISSN 2308–6424 UROVEST.RU 129 K.S. Ibishev, J.O. Prokop PATHOLOGICAL FINDINGS IN SEMEN ANALYSIS FROM COVID-19 SURVIVORS AND THE IMPACT OF VARIOUS SARS-COV-2 VACCINES ON SPERMATOGENESIS Johnson & Johnson, Sinopharm и Sinovac. Отталкиваясь от возможного негативного влияния на репродуктивное здоровье самого вируса SARS-COV-2, были проведены несколько исследований направленные на то, чтобы изучить влияние вакцинации от COVID-19 на репродуктивное здоровье мужчин. В исследовании D. C. Gonzalez et al. были отобраны 45 мужчин, средний возраст которых составлял 28 лет [42]. Мужчины прошли предварительный скрининг, чтобы убедиться, что у них нет проблем с фертильностью. Те, у кого были симптомы COVID-19 или положительный результат теста в течение 90 дней, были исключены. Участники предоставили образец спермы после 2 – 7 дней воздержания до получения первой дозы вакцины и примерно через 70 дней после второй. Из 45 мужчин 21 (46,7%) получили BNT162b2 (вакцина Pfizer-BioNTech), а 24 (53,3%) получили мРНК-127 (Moderna). Восемь из 45 мужчин имели олигоспермию до введения вакцины (средняя концентрация 8,5 млн/мл [МКИ, 5.1-12]). Из этих 8 у 7 мужчин концентрация сперматозоидов увеличилась до нормозооспермии при последующем наблюдении, а у 1 мужчины осталась олигоспермия. Ни один мужчина не имел азооспермии после вакцинации. Россия, являясь лидером в разработке вакцины от COVID-19, также провела исследование влияние препарата Гам-Ковид-Вак, хорошо известного под названием Спутник V: были проанализированы средние показатели эякулята в 3 независимых группах, сформированных среди пациентов, обследованных в лаборатории генетики нарушений репродукции ФГБНУ «МГНЦ» в 2021 году: группа сравнения — 759 непривитых мужчин, группа из 73 пациентов, вакцинированных препаратом «Гам-Ковид-Вак» [43]. Срок между вакцинацией двумя компонентами препарата и сдачей эякулята не превышал 75 дней, и группа из 58 пациентов, прошедших вакцинацию более чем за 75 дней до сдачи анализа. При сравнении показателей спермограмм одних и тех же пациентов до и после прохождения процедуры вакцинации не было выявлено статистически значимых различий. Было обнаружено незначительное снижение прогрессивной подвижности сперматозоидов (с 19,0 ± 0,4 до 13,8 ± 1,1%) и снижение количества морфологически нормаль- 130 ISSN 2308–6424 UROVEST.RU REVIEW ARTICLES ных форм сперматозоидов (с 4,53 ± 0,12 до 3,55 ± 0,31%) в группе прошедших вакцинацию в срок менее 75 дней до сдачи анализа. В группе прошедших вакцинацию в срок более 75 дней до сдачи анализа показатели не отличались от таковых в группе не привитых. В отличие от американских и российских аналогов 2 китайские вакцины на данный момент, используемые в КНР, являются инактивированными вариантами SARSCoV2. Sinovac-CoronaVac представляет собой инактивированную цельновирусную вакцину с адъювантом гидроксида алюминия. Sinopharm BBIBP-CorV (вакцина Sinopharm COVID-19) — ещё одна широкодоступная инактивированная цельновирусная вакцина, которая также показала высокую эффективность (78,1%) и мало побочных эффектов в клинических исследованиях. Китайские коллеги, основываясь на полученных ранее данных о патологическом влиянии инфицирования SARS-CoV-2 на сперматогенез, провели когортное исследование в котором участвовало 260 мужчин из которых 105 были привиты инактивированными вакцинами из них 70 SinovacCoronaVac и 35 Sinopharm COVID-19 [44]. В результате исследования не удалось выявить никаких существенных различий с точки зрения объёма, концентрации сперматозоидов, количества сперматозоидов, прогрессивной подвижности, общей подвижности и общего количества подвижных сперматозоидов (е) между вакцинированной группой и контрольной группой. Точно так же не было обнаружено статистических различий в качестве эмбрионов после ЭКО между этими двумя группами. Обсуждение В последнее время сообщения о частоте патологии яичек при COVID-19 становятся все более убедительными. Посмертные и прижизненные исследования тканей яичек позволяют предположить, что инфекция SARS-CoV-2 приводит к иммунным нарушениям в тканях тестикул [13, 14]. Подобные нарушения находят отражение в анализах спермограммы. Анализ параметров спермы является одним из самых основных и важных способов оценки мужской фертильности. Снижение параметров спермы тесно связано с мужским бесплодием. В настоящее время опуВестник урологии Vestnik Urologii 2023;11(1):125-133 ОБЗОРЫ ЛИТЕРАТУРЫ Х.С. Ибишев, Я.О. Прокоп ПАТОЛОГИЧЕСКИЕ НАХОДКИ В СПЕРМОГРАММЕ У ПАЦИЕНТОВ, ПЕРЕНЁСШИХ COVID-19, И ВЛИЯНИЕ РАЗЛИЧНЫХ ВАКЦИН ОТ SARS COV-2 НА СПЕРМАТОГЕНЕЗ бликовано несколько исследований, оценивающих параметры спермы при COVID-19, большинство из них представлены в данном обзоре. И, хотя вирус SARS-CoV-2 не обнаруживается в сперме, у пациентов, перенёсших COVID-19, отмечается олигозооспермия, астенозооспермию, тератозооспермия. Примерно в 20% случаев имеет место комбинация данных нарушений. У пациентов, перенёсших COVID-19, отмечается рост титра антиспермальных антител, что может говорить об иммунологическом аспекте нарушений фертильности, данные факты подтверждаются гистологическими находками. Несмотря на то, что снижение параметров качества спермы было наибольшим у мужчин, прошедших тестирование вскоре после выздоровления от COVID-19, изменения сохранялись длительный срок. Поскольку цикл сперматогенеза у человека в среднем составляет примерно 72 дня, эти данные свидетельствуют о том, что последствия COVID-19 могут сохраняться в течение одного цикла сперматогенеза. Об этом стоит предупреждать пациентов, планирующих беременность. Внушает надежду тот факт, что перенесённая коронавирусная инфекция не влияет на результаты ЭКО и пациентам, перенёсшим COVID-19 и имеющим проблемы с зачатием, можно рекомендовать ЭКО как один из методов вспомогательных репродуктивных технологий, но стоить помнить, что в исследовании, проведённом в г. Ухань (Китай), отмечена возможность потенциального негативного влиянии перенесённого COVID-19 на эмбриональное развитие, но данный факт требует более детального наблюдения и исследования. На данный момент существующие вакцины показали высокую эффективность и достаточную безопасность и обоснованно рекомендуются как одно из средств защиты от COVID-19. В отношения влияния изученных вакцин на фертильность не выявлено статистически значимых изменений репродуктивного потенциала у мужчин, привитых существующими вакцинами. Выводы 1. Доказанны патологические изменения в репродуктивных органах у пациентов перенесших COVID-19 2. Заболевание, вызываемое вирусом SARS-CoV-2, приводит к нарушениям в спермограмме, которые могут быть обнаружены у пациентов, недавно перенёсших COVID-19. 3. Патологические изменения у пациентов, перенёсших COVID-19, могут сохраняться на протяжении длительного срока до 72 дней, что в среднем составляет 1 цикл сперматогенеза. 4. Механизм возникающих нарушений может иметь иммунологический характер из-за роста уровня антиспермальных антител у пациентов, перенёсших Новую Коронавирусную инфекцию. 5. Существующие вакцины, защищающие от тяжёлых форм COVID-19, достаточно эффективны в этом отношении, относительно безопасны и не влияют на репродуктивное здоровье вакцинированных мужчин. Список литературы \| References 1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W; China Novel Coronavirus Investigating and Research Team. A Novel Coronavirus from Patients with Pneumonia in China, 2019. 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Прокоп ПАТОЛОГИЧЕСКИЕ НАХОДКИ В СПЕРМОГРАММЕ У ПАЦИЕНТОВ, ПЕРЕНЁСШИХ COVID-19, И ВЛИЯНИЕ РАЗЛИЧНЫХ ВАКЦИН ОТ SARS COV-2 НА СПЕРМАТОГЕНЕЗ ОБЗОРЫ ЛИТЕРАТУРЫ Reproduction. 2021;161(3):319-331. DOI: 10.1530/REP-20-0382 31. Donders GGG, Bosmans E, Reumers J, Donders F, Jonckheere J, Salembier G, Stern N, Jacquemyn Y, Ombelet W, Depuydt CE. Sperm quality and absence of SARS-CoV-2 RNA in semen after COVID-19 infection: a prospective, observational study and validation of the SpermCOVID test. Fertil Steril. 2022;117(2):287-296. DOI: 10.1016/j.fertnstert.2021.10.022 32. Wang M, Hu J, Huang B, Yang Q, Liu S, Li Z, Yang L, Xi Q, Zhu L, Jin L. Investigating the impact of SARS-CoV-2 infection on basic semen parameters and in vitro fertilization/ intracytoplasmic sperm injection outcomes: a retrospective cohort study. Reprod Biol Endocrinol. 2022;20(1):46. DOI: 10.1186/s12958-022-00918-1 33. Carli G, Nichele I, Ruggeri M, Barra S, Tosetto A. Deep vein thrombosis (DVT) occurring shortly after the second dose of mRNA SARS-CoV-2 vaccine. Intern Emerg Med. 2021;16(3):803-804. DOI: 10.1007/s11739-021-02685-0 34. Jeet Kaur R, Dutta S, Charan J, Bhardwaj P, Tandon A, Yadav D, Islam S, Haque M. Cardiovascular Adverse Events Reported from COVID-19 Vaccines: A Study Based on WHO Database. Int J Gen Med. 2021;14:3909-3927. DOI: 10.2147/IJGM.S324349 35. Hippisley-Cox J, Patone M, Mei XW, Saatci D, Dixon S, Khunti K, Zaccardi F, Watkinson P, Shankar-Hari M, Doidge J, Harrison DA, Griffin SJ, Sheikh A, Coupland CAC. Risk of thrombocytopenia and thromboembolism after covid-19 vaccination and SARS-CoV-2 positive testing: self-controlled case series study. BMJ. 2021;374:n1931. DOI: 10.1136/bmj.n1931 36. Elimam A, Al-Dhahir S. Pfizer COVID-19 vaccine-induced peritonitis. Clin Med (Lond). 2022;22(Suppl 4):53. DOI: 10.7861/clinmed.22-4-s53 37. Barbari A. COVID-19 Vaccine Concerns: Fact or Fiction? Exp Clin Transplant. 2021;19(7):627-634. DOI: 10.6002/ect.2021.0056 38. Kricorian K, Civen R, Equils O. COVID-19 vaccine hesitancy: misinformation and perceptions of vaccine safety. Hum Vaccin Immunother. 2022;18(1):1950504. DOI: 10.1080/21645515.2021.1950504 39. Prabani KIP, Weerasekara I, Damayanthi HDWT. COVID-19 vaccine acceptance and hesitancy among patients with cancer: a systematic review and meta-analysis. Public Health. 2022;212:66-75. DOI: 10.1016/j.puhe.2022.09.001 40. Navin MC, Oberleitner LM, Lucia VC, Ozdych M, Afonso N, Kennedy RH, Keil H, Wu L, Mathew TA. COVID-19 Vaccine Hesitancy Among Healthcare Personnel Who Generally Accept Vaccines. J Community Health. 2022;47(3):519-529. DOI: 10.1007/s10900-022-01080-w 41. Schaler L, Wingfield M. COVID-19 vaccine - can it affect fertility? Ir J Med Sci. 2022;191(5):2185-2187. DOI: 10.1007/s11845-021-02807-9 42. Gonzalez DC, Nassau DE, Khodamoradi K, Ibrahim E, Blachman-Braun R, Ory J, Ramasamy R. Sperm Parameters Before and After COVID-19 mRNA Vaccination. JAMA. 2021;326(3):273-274. DOI: 10.1001/jama.2021.9976 43. Сорокина Т.М., Брагина Е.Е., Сорокина Е.А., Курило Л.Ф., Штаут М.И., Седова А.О., Черных В.Б. Оценка и сравнительный анализ спермиологических показателей у мужчин до и после вакцинации препаратом «Спутник V» (Гам-КОВИД-Вак). Андрология и генитальная хирургия. 2021;22(4):45-53. Sorokina T.M., Bragina E.E., Sorokina E.A., Kurilo L.F., Shtaut M.I., Sedova A.O., Chernykh V.B. Evaluation and comparative analysis of sperm characteristics in men before and after vaccination with Sputnik V (Gam-COVID-Vac). Andrology and Genital Surgery. 2021;22(4):45-53. (In Russian). DOI: 10.17650/1726-9784-2021-22-4-45-53 44. Xia W, Zhao J, Hu Y, Fang L, Wu S. Investigate the effect of COVID-19 inactivated vaccine on sperm parameters and embryo quality in in vitro fertilization. Andrologia. 2022;54(6):e14483. DOI: 10.1111/and.14483 Сведения об авторах Information about the authors Халид Сулейманович Ибишев — доктор медицинских наук, профессор; профессор кафедры урологии и репродуктивного здоровья человека (с курсом детской урологии-андрологии) ФГБОУ ВО РостГМУ Минздрава России г. Ростов-на-Дону, Россия http://orcid.org/0000-0002-2954-842X ibishev22@mail.ru Ян Олегович Прокоп — аспирант кафедры урологии и репродуктивного здоровья человека (с курсом детской урологии-андрологии) ФГБОУ ВО РостГМУ Минздрава России, г. Ростов-на-Дону, Россия http://orcid.org/0000-0001-5340-9406 unprocop@mail.ru Khalid S. Ibishev — M.D., Dr.Sc. (Med), Full Prof.; Prof., Dept. of Urology, Pediatric Urology and Reproductive Health, Rostov State Medical University Rostov-on-Don, Russian Federation http://orcid.org/0000-0002-2954-842X ibishev22@mail.ru Вестник урологии Vestnik Urologii 2023;11(1):125-133 Yan O. Prokop — M.D, Urologist; Postgrad. student; Dept. of Urology, Pediatric Urology and Reproductive Health, Rostov State Medical University Rostov-on-Don, Russian Federation http://orcid.org/0000-0001-5340-9406 unprocop@mail.ru ISSN 2308–6424 UROVEST.RU 133
https://openalex.org/W198798444	https://europepmc.org/articles/pmc4471243?pdf=render	English	null	Critical care in a maternity hospital: a retrospective observational study	Critical care	2,015	cc-by	275,066	Introduction To assess cerebral hemodynamics in an experimental sepsis model. Methods The study was a prospective, observational pilot study conducted in our hospital. Consecutive adult patients with severe sepsis, on a mechanical ventilator with an IL-6 blood concentration ≥100 pg/ml in the acute phase, defi ned as being up to the 28th day of illness in the ICU, were entered in this study between June 2011 and December 2012. Subjects were divided into those who were treated with steroids (steroid treatment group) and those who were not (no- steroids group) during the target period, because steroids strongly aff ect IL-6 blood levels. Methods Nineteen juvenile female Hungahib pigs were subjected into control group (n = 9) or septic group (n = 10). Under general anesthesia in animals of the sepsis group, Escherichia coli culture (2.5 × 105/ml; strain: ATCC 25922) was intravenously administrated in a continuously increasing manner as follows: 2 ml in the fi rst 30 minutes, then 4 ml in 30 minutes and afterwards 16 ml/hour for 2 hours (so a total of 9.5 × 106 E. coli within 3 hours). In the control group the anesthesia was maintained for 8 hours, infusion was administered as a similar volume of isotonic saline solution and no other intervention was made. Hemodynamic monitoring of all animals was performed by PiCCo monitoring system. The middle cerebral artery of the pigs was insonated through the transorbital window and cerebral blood fl ow velocity (MCAV) and pulsatility index was registered. Results The subjects were fi ve adult patients in the acute phase of severe sepsis on a mechanical ventilator. Gastrointestinal motility was measured for a total of 62,399 minutes: 31,544 minutes in three subjects in the no-steroids group and 30,855 minutes in two subjects in the steroid treatment group. In the no-steroids group, the bowel sound counts were negatively correlated with IL-6 blood concentration (r = –0.76, P <0.01), suggesting that gastrointestinal motility was suppressed as IL-6 blood concentration increased. However, in the steroid treatment group, gastrointestinal motility showed no correlation with IL-6 blood concentration (r = –0.25, P = 0.27). The IL-6 blood concentration appears to have decreased with steroid treatment irrespective of changes in the state of sepsis, whereas bowel sound counts with the monitoring system refl ected the changes in the state of sepsis, resulting in no correlation. MEETING ABSTRACTS MEETING ABSTRACTS P1 but long-term observation and objective evaluation of gastrointestinal motility are diffi cult. We developed a non-invasive monitoring system capable of quantifying and visualizing gastrointestinal motility in real time. In the study gastrointestinal motility was performed in patients with severe sepsis using this developed bowel sound analysis system, and the correlation between bowel sounds and changes over time in blood concentrations of IL-6, which is associated with sepsis severity, was evaluated. P1 Cerebral autoregulation testing in a porcine model of intravenously administrated E. coli induced fulminant sepsis L Molnar1, M Berhes1, L Papp1, N Nemeth2, B Fulesdi1 1Deoec Aneszteziologia, Debrecen, Hungary; 2University of Debrecen, Hungary Critical Care 2015, 19(Suppl 1):P1 (doi: 10.1186/cc14081) P1 Cerebral autoregulation testing in a porcine model of intravenously administrated E. coli induced fulminant sepsis L Molnar1, M Berhes1, L Papp1, N Nemeth2, B Fulesdi1 1Deoec Aneszteziologia, Debrecen, Hungary; 2University of Debrecen, Hungary Critical Care 2015, 19(Suppl 1):P1 (doi: 10.1186/cc14081) Introduction To assess cerebral hemodynamics in an experimental sepsis model. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Introduction To assess cerebral hemodynamics in an experimental sepsis model. Results In the septic group, as expected, all animals developed fulminant sepsis and died within 3 to 7 hours two animals in 3 to 4 hours, and three in 6 to 7 hours). In the septic animals the heart rate rose and mean arterial pressure dropped, their ratio increased signifi cantly compared with both the base values (at the 6th hour: P <0.001) and the control group (P = 0.004). The control animals showed stable condition over the 8-hour anesthesia. MCAV signifi cantly decreased during the development of sepsis (from 23.6 ± 6.6 cm/s to 16.0 ± 3.9 cm/s, P <0.01) and pulsatility indices increased (from 0.68 ± 0.22 to 1.37 ± 0.58, P <0.01), indicating vasoconstriction of the resistance vessels. A signifi cant relationship was fund between percent change of the MAP and the pulsatility index in septic animals (R2 = 0.32) referring to maintained cerebral autoregulation. Conclusion The new real-time bowel sound analysis system provides a useful method of continuously, quantitatively, and non-invasively evaluating gastrointestinal motility in severe patients. Furthermore, this analysis may predict disease severity in septic patients. Conclusion Cerebral autoregulation is preserved in the pig model of experimentally induced fulminant sepsis. P3 Usefulness of sepsis screening tools and education in recognising the burden of sepsis on hospital wards EJ Galtrey, C Moss, H Cahill Guy’s and St Thomas’ Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P3 (doi: 10.1186/cc14083) P2 New real-time bowel sound analysis may predict disease severity in septic patients J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) 35th International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 17-20 March 2015 Publication charges for this supplement were funded by ISICEM. p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 1University of Cambridge, UK; 2Ipswich Hospital, Ipswich, UK Critical Care 2015, 19(Suppl 1):P5 (doi: 10.1186/cc14085) 1University of Cambridge, UK; 2Ipswich Hospital, Ipswich, UK Critical Care 2015, 19(Suppl 1):P5 (doi: 10.1186/cc14085) fi Methods All referrals to our critical care response team with a diagnosis of sepsis over a 3-month period (September to November 2014) were investigated to determine how many had an EPR sepsis alert comprising a prompt for blood cultures, serum lactate measurement, fl uid challenge if hypotensive, and antibiotics within 1 hour. Introduction Severe sepsis results in ~36,800 UK deaths each year [1]. Prior studies demonstrate the benefi t of early recognition and treatment of sepsis in reducing mortality [2]. The Sepsis Six [1] bundle aims to optimise the fi rst hour of sepsis management. We assessed the proportion of emergency department (ED) patients with severe sepsis receiving the Sepsis Six bundle and whether this was improved by a combination of staff education and use of Sepsis Six management stickers in patient notes. l g yp Results Only 25/174 (14%) patients with a diagnosis of sepsis had an EPR sepsis alert. There was no signifi cant diff erence between acute and nonacute ward areas in their likelihood of using the screening tool or alert, in contrast to previous audits of the alerted population which showed that acute areas such as A&E and medical acute admission wards had higher utilisation and bundle completion rates. Methods A closed loop audit was completed in the ED at Ipswich Hospital, UK. Each cycle was 14 days with interventions made in a 4-week period between the two cycles. The interventions consisted of: Sepsis Six management stickers and posters placed in the ED; two training sessions for all ED nurses on sepsis recognition and management; a teaching session for all middle-grade doctors; and a trolley in the ED with equipment required for the Sepsis Six. The notes of all patients with lactate ≥2 mmol/l were retrospectively reviewed. Those with ≥2 systemic infl ammatory response syndrome criteria and a documented suspicion of infection were deemed to have severe sepsis. The times at which these patients had each of the Sepsis Six completed were recorded, as were the fi nal diagnosis and 7/28 day mortality. P5 tool and electronic order set (EPR alert) alongside an education programme to improve delivery of the SSC bundle. Previous audits showed only 43% full bundle compliance in those that were alerted, and this raised concerns regarding the burden of unalerted sepsis. We sought to estimate the number of unalerted sepsis episodes to assess the effi cacy of our screening tool and improve early recognition. p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 Results In Cycle 1, 31/106 patients met the criteria for severe sepsis, compared with 36/120 in Cycle 2. The delivery of the Sepsis Six interventions was highly variable. In Cycle 1 lactate levels and i.v. access had the highest 60-minute completion rates (90.3%, 83.9% respectively). Blood cultures and i.v. fl uid resuscitation were completed for 61.3% and 64.5% of patients within 60 minutes. Only 38.7% of septic patients were given i.v. antibiotics within 60 minutes. In total, 58.9% of patients received antibiotics in accordance with trust guidelines. High fl ow oxygen, catheters and fl uid balance charts had the lowest 60-minute completion rates (35.5%, 6.5%, 6.5% respectively). In Cycle 2, post intervention, there was no signifi cant change in the percentage of patients receiving the Sepsis Six bundle. Methods A closed loop audit was completed in the ED at Ipswich Hospital, UK. Each cycle was 14 days with interventions made in a 4-week period between the two cycles. The interventions consisted of: Sepsis Six management stickers and posters placed in the ED; two training sessions for all ED nurses on sepsis recognition and management; a teaching session for all middle-grade doctors; and a trolley in the ED with equipment required for the Sepsis Six. The notes of all patients with lactate ≥2 mmol/l were retrospectively reviewed. Those with ≥2 systemic infl ammatory response syndrome criteria and a documented suspicion of infection were deemed to have severe sepsis. The times at which these patients had each of the Sepsis Six completed were recorded, as were the fi nal diagnosis and 7/28 day mortality. Conclusion Despite these interventions, most patients still do not receive the full recommended treatment bundle. These fi ndings have prompted a point prevalence audit at ward level, which will examine all patients’ notes for the preceding 24 hours to ascertain if sepsis is truly unrecognised or whether it is simply that our current tool is not a helpful adjunct to care. With national guidelines expected within the year, we will redesign and re-launch our screening tools and education programme to improve awareness and management of this common medical emergency. Continuous versus intermittent temperature measurement in the detection of fever in critically ill patients p g p Conclusion The low rates of Sepsis Six completion require improvement to meet the targets set out by the College of Emergency Medicine. Our results suggest that simple interventions are ineff ective in increasing Sepsis Six completion and thus lend support to the case for integrated interventions such as electronic recording and alert systems. References Introduction An elevated body temperature is one of the four criteria in diagnosing systemic infl ammatory response syndrome (SIRS), and is often used at the bedside to trigger diagnostic investigations for infection. Standard intermittent temperature measurement may, however, delay the detection of an elevated temperature or miss this altogether. The aim of the study is to compare intermittent non- invasive versus continuous invasive body temperature measurement as a tool to detect an elevated body temperature. P4 P4 Continuous versus intermittent temperature measurement in the detection of fever in critically ill patients A Heyneman, V Bosschem, N Mauws, D Van Der Jeught, E Hoste, J Decruyenaere, J De Waele Ghent University Hospital, Ghent, Belgium Critical Care 2015, 19(Suppl 1):P4 (doi: 10.1186/cc14084) New real-time bowel sound analysis may predict disease severity in septic patients J G t 1 K M t d 1 N H ii1 T M i hi1 M Y i 1 D H d 1 p p J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) Introduction Sepsis is defi ned as the presence of infection with systemic signs of infection, and severe sepsis as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion [1]. Since the Surviving Sepsis Campaign (SSC) in 2002, the Health Service Ombudsman for England published recommendations for improving recognition and treatment of sepsis [2], the Royal College of Physicians issued a toolkit for the management of sepsis in the acute medical unit [3], and NHS England released a patient safety alert to support prompt recognition and treatment of sepsis [4]. In 2012 our Trust introduced a sepsis screening Introduction Healthy bowel function is an important factor when judging the advisability of early enteral nutrition in critically ill patients, © 2015 BioMed Central Ltd © 2015 BioMed Central Ltd © 2015 BioMed Central Ltd S2 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 interventions References 1. Daniels et al. Emerg Med J. 2011;28:507-12. 1. Daniels et al. Emerg Med J. 2011;28:507-12. 1. Daniels et al. Emerg Med J. 2011;28:507-12. 2. Kumar et al. Crit Care Med. 2006;34:1589-96. 2. Kumar et al. Crit Care Med. 2006;34:1589-96. References 1. Dellinger R, et al. Crit Care Med. 2013;41:580-637. 2. http://bit.ly/1nzV3Kp. 3. http://bit.ly/12Y3KHq. 4. http://bit.ly/10PYi8G. 1. Dellinger R, et al. Crit Care Med. 2013;41:580-637. 2. http://bit.ly/1nzV3Kp. 3. http://bit.ly/12Y3KHq. 4. http://bit.ly/10PYi8G. ,i g y y Results In Cycle 1, 31/106 patients met the criteria for severe sepsis, compared with 36/120 in Cycle 2. The delivery of the Sepsis Six interventions was highly variable. In Cycle 1 lactate levels and i.v. access had the highest 60-minute completion rates (90.3%, 83.9% respectively). Blood cultures and i.v. fl uid resuscitation were completed for 61.3% and 64.5% of patients within 60 minutes. Only 38.7% of septic patients were given i.v. antibiotics within 60 minutes. In total, 58.9% of patients received antibiotics in accordance with trust guidelines. High fl ow oxygen, catheters and fl uid balance charts had the lowest 60-minute completion rates (35.5%, 6.5%, 6.5% respectively). In Cycle 2, post intervention, there was no signifi cant change in the percentage of patients receiving the Sepsis Six bundle. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P5 Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia y q p C Joya-Montosa, MD Delgado-Amaya, E Trujillo-García, E Curiel-Balsera Hospital Regional de Málaga, Spain C Joya-Montosa, MD Delgado-Amaya, E Trujillo-García, E Curiel-Balsera Hospital Regional de Málaga, Spain p g g p ritical Care 2015, 19(Suppl 1):P9 (doi: 10.1186/cc14089) Introduction The aim of the study is to evaluate the calibration and discrimination of two specifi c risk scores for community-acquired pneumonia (CAP) in patients with this illness who required ICU admission. Results Severe sepsis criteria were fulfi lled in 31% (n = 91) of the patients. These were older (median age: 79 years vs. 71 years) and experienced more symptoms (mean: 2.2, SD 0.9 vs. mean: 1.4, SD 0.7) than patients without severe sepsis, while there was no diff erence in C-reactive protein levels (OR per 50 mg/l: 1.07, 95% CI: 0.96 to 1.20). Among individual symptoms, altered mental status (OR: 4.4, 95% CI: 2.2 to 9.0), dyspnea (OR: 3.5, 95% CI: 2.1 to 5.9), and muscle weakness (OR: 2.2, 95% CI: 1.0 to 4.4) were signifi cantly related to severe sepsis. Adjusting for age and sex, altered mental status (adj. OR: 4.1, 95% CI: 2.0 to 8.4) and dyspnea (adj. OR: 3.1, 05% CI: 1.8 to 5.3) remained signifi cant. Conclusion General symptoms, especially altered mental status and dyspnea, appear to be more common in severe sepsis than in milder infections. These symptoms might be utilized as a diagnostic aid for severe sepsis in the clinical setting, complementing vital signs and laboratory tests. Methods A retrospective descriptive study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical and epidemiological variables and APACHE II, SAPS III, CURB-65 and Pneumonia Severity Index (PSI) that were recorded in the fi rst 24 hours. We used the Student t test to compare means and the chi-square test for univariate analysis. The standardized mortality ratio (SMR) and Hosmer–Lemershow test were calculated to analyze the calibration and ROC curve analysis for discrimination of diff erent scores. Results We analyzed 111 patients aged 57.5 ± 17.7 years, with 63.1% (70) males. The APACHE II score at admission was 19.8 ± 17.7 and SAPS III was 60.6 ± 16.7. ICU mortality was 29.7% (33). There was association between the four scores and mortality. P9 weakness, gastrointestinal symptoms, localized pain, altered mental status) that were part of the reason the patient sought medical care were registered. Additionally, age, sex, vital signs, C-reactive protein, and blood cultures were registered. Patients that within 48 hours from admission fulfi lled any criteria for severe sepsis were compared with patients that did not. Odds ratios for severe sepsis were computed using univariable as well as multivariable logistic regression, controlling for age and gender as confounders. 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. P6 Systemic symptoms as markers for severity in sepsis J Edman-Wallér1, L Ljungström2, R Andersson3, G Jacobsson2, M Werner1 1Södra Älvsborg Hospital, Borås, Sweden; 2Skaraborg Hospital, Skövde, Sweden; 3Institute of Biomedicine, University of Gothenburg, Sweden Critical Care 2015, 19(Suppl 1):P6 (doi: 10.1186/cc14086) Methods This was a secondary analysis of a prospective study in 25 critically ill patients comparing diff erent measurement techniques. Temperature was measured intermittently with an axillary digital thermometer every 4 hours, and a urinary bladder thermistor catheter was used for continuous temperature measurement; the latter was considered the reference method. Fever (core temperature ≥38.3°C) episodes occurring within 60 minutes after each other were classifi ed as one episode. We compared the fever detection rate of both methods and calculated the diff erence in timing between both techniques. Introduction The objective of this study was to evaluate six general symptoms as markers for severe sepsis in patients with suspected bacterial infections. Severe sepsis is a common cause of death and morbidity. Early detection and treatment is critical for outcome. Clinical presentation varies widely and no single test is able to discriminate severe sepsis from uncomplicated infections or non-infectious emergencies. Apart from local symptoms of infection, the systemic infl ammatory reaction itself may give rise to general symptoms such as muscle weakness and vomiting. f Results Twenty-nine episodes of fever were detected in 10 patients (seven male) with a median APACHE II score of 10 (IQR 3 to 24) and a median SOFA score of 10 (IQR 8 to 11). Median duration of a fever episode was 1 hour 24 minutes (IQR 47 minutes to 2 hours 59 minutes) and median maximum temperature was 38.4°C (IQR 38.3 to 38.7). Median axillary temperature was 0.7°C (IQR 0.3 to 0.9) lower than core temperature. Using intermittent, non-invasive measurement, 27 out of 29 fever episodes (93.1%) remained undetected. Methods We present an observational, consecutive study. Data from ambulance and hospital medical records were analyzed. The survey included 290 patients (mean age: 70.6 years; median age: 74 years; male: 47%) who were admitted to a 550-bed secondary care hospital, receiving intravenous antibiotics for suspected community-acquired infections. General symptoms (fever/shivering, dyspnea, muscle Conclusion Intermittent, non-invasive temperature measurement failed to detect most of the fever episodes as measured by a bladder thermistor catheter and should not be used to screen for elevated body temperature in critically ill patients. Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study g y S Laukemann1, N Kasper1, N Kasper1, A Kutz1, S Felder1, S Haubitz2, B Müller1, P Schuetz1 Figure 1 (abstract P9). 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) Introduction Collection of blood cultures is routinely performed in patients with suspicion of infection in the emergency department (ED) despite a low yield of positive culture results. To increase sensitivity, diff erent clinical prediction rules and blood biomarkers have been put forward. Herein, we validated the performance of diff erent promising clinical prediction rules alone and in combination with novel blood biomarkers to predict blood culture positivity. Methods This is an observational cohort study including consecutive medical patients with suspected infection and collection of ED admission blood cultures. Five clinical prediction rules were calculated and admission concentrations of procalcitonin (PCT), C-reactive protein, neutrophil–lymphocyte count ratio (NLCR), lymphocyte count, white blood cell count, and red blood cell distribution width were measured. True blood culture positivity was assessed by two independent physicians. We used logistic regression models with area under the curve (AUC) to establish associations between clinical prediction rules and blood culture positivity. Figure 1 (abstract P9). y Results Of 1,083 included patients, 106 (9.8%) cultures were positive. Of the clinical prediction rules, the Shapiro rule performed best (AUC 0.733) followed by the Metersky rule (AUC 0.609). The best biomarkers were PCT (AUC 0.796), NLCR (0.692) and lymphocyte count (AUC 0.671). Combination of the Shapiro rule and PCT showed the best combination result (AUC of combined model 0.822). Limiting blood cultures to either the Shapiro rule ≥4 points or PCT >0.11 μg/l would reduce negative sampling by 25.6% while still identifying 100% of positive cultures. Using a Shapiro rule ≥3 points or PCT >0.25 μg/l limit would reduce negative sampling by 42.1% while still identifying 96.2% of positive cultures. Conclusion The four analyzed scores presented good calibration, but discrimination seems better in SAPS III. Given the diffi culty of calculating PSI, and its low discrimination (similar to CURB-65), we prefer to use the CURB-65 score in routine clinical practice. 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. P6 S3 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia The SMR for APACHE II was 0.87 and 0.85 for the SAPS III. Figure 1 shows the ROC curve for the four scores, the best observed discrimination obtained was for SAPS III score (AuC 0.79) and the worst was obtained for CURB-65 score (AuC 0.7). The Hosmer–Lemeshow test showed acceptable calibration for the four predictive systems (P > 0.05). Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study S Laukemann1, N Kasper1, N Kasper1, A Kutz1, S Felder1, S Haubitz2, B Müller1, P Schuetz1 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study Pre-exposure to mechanical ventilation and endotoxin infl uence bacterial growth and immune response during experimental ventilator-associated pneumonia Table 1 (abstract P10) Sternal infection P value Insulin 9/85 0.001 Current smoker 4/272 0.037 COPD 11/197 <0.001 Transfusion >3 19/302 0.001 J Sperber1, A Nyberg1, M Lipcsey2, A Larsson2, J Sjölin2, M Castegren2 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Uppsala University, Uppsala, Sweden Introduction Overproduction of nitric oxide (NO) is correlated with adverse outcomes in sepsis. NO is additionally a central part of the innate immune system defense against pathogens causing ventilator- associated pneumonia (VAP), which can complicate the clinical course during mechanical ventilation (MV). We hypothesized that pre-exposure to MV and systemic infl ammation from endotoxin each would infl uence bacterial growth in lung tissue, based on an altered immune response in experimental pneumonia. We used a porcine Pseudomonas aeruginosa VAP model with ventilatory and infl ammatory pre-exposures before inoculation to evaluate bacterial growth, development of lung damage, total NO production and infl ammatory cytokine response. Conclusion Postoperative deep sternal wound infections have statistical signifi cant correlation with the following parameters: transfusion with >3 red blood cell units, history of COPD, insulin dependence and when the patient is a current smoker. Also there is a tendency for correlation with CBP time >120 minutes (P = 0.056). References 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg 2007;2:23 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 2. Schimmer C, et al. Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann Thorac Surg. 2008;86:1897-904. y Methods Three groups of mechanically ventilated pigs were subjected to experimental VAP for 6 hours with intrapulmonary 1 × 1011 CFU P. aeruginosa at baseline. Two groups were pre-exposed to MV for 24 hours before bacterial inoculation: MV + Etx (n = 6, received endotoxin 0.063 μg × kg–1 × hour–1) and MV (n = 6, received saline in equivalent volume). One group, Un (n = 8), started the experiment unexposed to both MV and endotoxin, directly from the initiation of VAP. Postmortem lung tissue samples rendered bacterial cultures. References 1. Hoenig M, et al. Procalcitonin fails to predict bacteremia in SIRS patients: a cohort study. Int J Clin Pract. 2014;68:1278-81. y 2. Hoeboer SH, et al. Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin. J Infect. 2012;64:484-93. y 2. Hoeboer SH, et al. Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin. J Infect. 2012;64:484-93. q y Results A total of 35 patients (3.44%) were complicated by deep sternal wound infections. No statistical correlation was found with age >75, gender, DM, BMI >30, steroids, emergent operation, prolonged ventilation, CBP time >120 minutes, reintubation and NIV. Factors with statistical signifi cant correlation are presented in Table 1. P10 Predisposing factors for deep sternal wound infection after cardiac surgery Predisposing factors for deep sternal wound infection after cardiac surgery F Ampatzidou, M Sileli, A Madesis, K Antoniou, A Baddur, G Kechagioglou, T Asteri, G Drossos General Hospital G. Papanikolaou Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P10 (doi: 10.1186/cc14090) Conclusion Combination of clinical parameters combined in the Shapiro rule together with admission levels of PCT allows reduction of unnecessary blood cultures with minimal false negative rates. References 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. Introduction The aim of our study was to investigate perioperative risk factors associated with deep sternal wound infections in complicated cardiac surgery. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. y Methods A total of 1,017 patients underwent cardiac surgery in a 2-year period. We investigate the correlation between deep sternal S4 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 factors might be useful to know whether or not adult febrile patients have bacteremia. wound infection with the following 14 preoperative characteristics and perioperative parameters: age >75, female gender, diabetes mellitus (DM), insulin dependence, body mass index (BMI) >30, current smokers, COPD, cardiopulmonary bypass time (CBP) >120 minutes, use of steroids, emergency operation, prolonged mechanical ventilation (>48 hours), reintubation, transfusion with more than 3 units of red blood cells, and the postoperative use of non-invasive ventilation (NIV). The chi-square test was used for statistical analysis. wound infection with the following 14 preoperative characteristics and perioperative parameters: age >75, female gender, diabetes mellitus (DM), insulin dependence, body mass index (BMI) >30, current smokers, COPD, cardiopulmonary bypass time (CBP) >120 minutes, use of steroids, emergency operation, prolonged mechanical ventilation (>48 hours), reintubation, transfusion with more than 3 units of red blood cells, and the postoperative use of non-invasive ventilation (NIV). The chi-square test was used for statistical analysis. P11 Risk factors for bacteremia in adult febrile patients in emergency settings g A Mikami1, Y Natori1, F Omata2, S Ishimatsu1 1St Luke’s International Hospital, Tokyo, Japan; 2St Luke’s Life Science Institute, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P11 (doi: 10.1186/cc14091) Results The animals pre-exposed to endotoxin (MV + Etx) displayed higher bacterial growth (CFU × g–1) (P <0.05), lower PaO2/FiO2 (P <0.05) and lower nitrate levels (P <0.01) than the unexposed animals (Un). Plasma TNFα levels were higher in Un than in both pre-exposed groups MV + Etx and MV (P <0.01). There were no signifi cant diff erences between the two pre-exposed groups. Critical Care 2015, 19(Suppl 1):P11 (doi: 10.1186/cc14091) Introduction Blood culture is a critical procedure for detecting potentially life-threatening bloodstream infections (BSI). At the same time, early diagnosis and appropriate treatment of BSI are the key factors in order to improve prognosis. The purpose of the current analysis was to identify risk factors for bacteremia in adult febrile patients in emergency settings. g Conclusion Mechanical ventilation for 24 hours with concomitant endotoxin exposure enhances bacterial growth and lung damage during P. aeruginosa VAP, compared with inoculation without any pre- exposure to MV or endotoxin. The greater bacterial clearance in the unexposed animals was associated with higher NO production and higher levels of pro-infl ammatory cytokines. Methods We conducted a retrospective case–control study within a population of adult patients visiting the emergency department at a community hospital (St Luke’s International Hospital, Tokyo, Japan) and who underwent two sets of blood culture testing between 2003 and 2012. Among a total of 13,582 patients, 1,322 (10%) were detected as bacteremia. We included in this study 179 randomly selected patients from the bacteremia group and 321 randomly selected patients from the negative blood culture group to serve as the comparison group. Multivariate logistic regression was used to evaluate the relationship between clinical characteristics factors and bacteremia. Pre-exposure to mechanical ventilation and endotoxin infl uence bacterial growth and immune response during experimental ventilator-associated pneumonia NO production was measured with urinary nitrate levels over 6 hours of VAP. retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 2. Schimmer C, et al. Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann Thorac Surg. 2008;86:1897-904. P15 ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign g p p g J Melville, S Ranjan, P Morgan East Surrey Hospital, Redhill, UK Critical Care 2015, 19(Suppl 1):P15 (doi: 10.1186/cc14095) Introduction The aim of this study was to evaluate the eff ect of the Surviving Sepsis Campaigns on mortality rates, before and after the second surviving sepsis publication, and to assess whether patients with sepsis being admitted to the ICU had a lower APACHE II score on admission. Patients with sepsis, who require ICU care, have an extremely poor prognosis. It has been shown that the mortality rates range from 20.7% (severe sepsis) to 45.7% (septic shock) [1]. The surviving sepsis campaign was initiated in 2002. The fi rst, second and third publications were published in 2004, 2008 and 2012 respectively [2]. g Results According to elevation of CT grades, severity of illness was signifi cantly associated with high score (APACHE II: 10.5 to 4.0, 12.8 to 4.2, 16 to 4.2, SOFA: 2.6 to 1.5, 2.9 to 1.9, 6.8 to 3.7, CRP: 17.8 to 10.6, 22.4 to 10.1, 33.3 to 11.9) and also duration of mechanical ventilation and length of hospital stay were longer (duration of mechanical ventilation: 10.9 to 6.6, 11.5 to 6.7, 15.8 to 7.2, length of hospital stay: 23.4 to 10.6, 27.9 to 21.4, 48.7 to 36.2). y Methods A retrospective case note review was performed, looking at a sample of 5,954 patients who were 18 years or older who had been admitted to East Surrey Hospital (ESH) ICU between 1 January 2005 and 31 October 2014. The total number of patients with sepsis was 941. We compared results before and after the second publication of the surviving sepsis campaign, looking at mortality rates, age of patients, admission length prior to ICU transfer, APACHE II score and the length of stay on the ICU. Conclusion Novel classifi cation of CNF based on CT fi ndings showing the extension of fl uid collection is a useful indicator of the disease severity and predicting clinical outcome. These fi ndings may infl uence the strategy for the success of percutaneous catheter drainage. y Results From the beginning of 2005 to the end of 2008, the mortality rates for septic patients was 51.9% compared with 41.3% from the beginning of 2009 to end of October 2014. P15 this study. Cervical spaces were subdivided into three components according to the concept of interfascial planes. The extension of acute fl uid collection in cervical spaces was classifi ed into three grades: Grade I, fl uid collection confi ned to one component; Grade II, fl uid collection spreading into two or three components; and Grade III, fl uid collection spreading into four components or mediastinum. We analyzed association with CT grades and severity of illness (SOFA score, APACHE II score, CRP). All patients underwent percutaneous catheter drainage either ultrasonography guided or CT guided. We compared treatment outcome of CNF with CT grades. 1. Estaban A, et al. Crit Care Med. 2007;35:1284-9. 2. Alberti C, et al. Intensive Care Med. 2002;28:108-21. ICU mortality rates in patients with sepsis compared with patients without sepsis p J Melville, S Ranjan, P Morgan East Surrey Hospital, Redhill, UK Critical Care 2015, 19(Suppl 1):P14 (doi: 10.1186/cc14094) Introduction The aim of the study was to evaluate the diff erence in mortality rates between those admitted to the ICU with and without sepsis, and to assess the proportion of patients who had sepsis. Septic patients are one of the key groups of patients admitted to ICUs around the world. Septic patients have an extremely poor prognosis with published mortality rates ranging from 20.7% (severe sepsis) to 45.7% (septic shock) [1]. With septic patients making up roughly 21% of patients admitted to ICUs, it is important to assess whether these rates of mortality hold true to a district general ICU and to assess the extent of the diff erence in prognosis between patients with and without sepsis [2]. g y p y Conclusion Patients with sepsis admitted to ESH ICU had a 20% relative decrease in mortality after the second publication of surviving sepsis guidelines. The original aim of the campaign was to reduce mortality from sepsis by 25% in 5 years [3]. This decrease was not due to a signifi cant diff erence between the sets of patients. The decreased time to admittance to ICU may be due to improved recognition of the need for ICU care. Overall the surviving sepsis campaign has had a signifi cantly benefi cial eff ect on mortality rates in patients with sepsis. References 1. Estaban A, et al. Crit Care Med. 2007;35:1284-9. Methods We performed a retrospective case note review, looking at a sample of 5,954 patients 18 years or older who were admitted to East Surrey Hospital (ESH) ICU, which has an elective admissions rate of 3%, between 1 January 2005 and 31 October 2014. The total number of patients with sepsis was 941 compared with 5,013 without sepsis. We looked at mortality rates, APACHE II scores and length of stay on the unit. 2. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 3. Slade E, et al. Crit Care. 2003;7:1-2. Independent risk factors for long-term mortality in patients with severe infection Results From the beginning of 2005 to the end of October 2014, mortality rates in septic patients were 44.6% compared with 26.2% in nonseptic patients. Fisher’s two-tailed test showed a signifi cant diff erence (P <0.0001) between the mortality in septic and nonseptic patients. There was a signifi cant diff erence (Mann–Whitney) between APACHE II scores, with median scores of 18 and 13 in septic and nonseptic patients respectively. Septic patients had longer lengths of stay, with the mean and median 8.73 and 3.89 days respectively, compared with 4.90 and 2.5 in nonseptic patients. Septic patients made up 15.8% of all patients admitted to the ICU. J Francisco, I Aragão, T Cardoso J Francisco, I Aragão, T Cardoso Centro Hospitalar do Porto – Hospital Geral de Santo António, Porto, Portugal Critical Care 2015, 19(Suppl 1):P16 (doi: 10.1186/cc14096) Introduction The purpose of this study was to examine long-term mortality, 5 years after severe infection, and to identify independent risk factors associated with it. Methods A prospective cohort study developed at a tertiary care university-affi liated 600-bed hospital including all patients with severe infection admitted into intensive care, medical, surgical, haematology and nephrology wards, over a 1-year period (2008/2009). The outcome of interest was mortality 5 years following hospitalisation and its association with specifi c risk factors was studied through logistic regression. Conclusion Patients with sepsis admitted to ESH ICU made up a signifi cant minority of patients admitted to the ICU. Septic patients had a 70% relative higher mortality rate compared with nonseptic patients. The mortality rate of 44.6% fi ts with previously quoted mortality rates in septic shock. Patients with sepsis had a signifi cantly higher predicted mortality, recorded by their APACHE II score, which was statistically signifi cant. This also meant they needed longer ICU care, with the average length of stay almost doubled. Results There were 1,013 patients included in the study. Hospital mortality rate was 14% (n = 137) and 5-year mortality was 37% (n = 379). Factors independently associated with 5-year mortality were (adjusted odds ratio (95% confi dence interval)): age = 1.04 per year (1.03 to 1.05), cancer = 8.00 (3.06 to 20.88), chronic hepatic disease = 3.06 (1.06 to 8.87), chronic respiratory disease = 2.21 (1.06 to 4.62), haematologic Percutaneous drainage for patients with cervical necrotizing fasciitis with novel CT classifi cation based on extension of fl uid collection along the deep cervical space T Kiguchi, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P13 (doi: 10.1186/cc14093) Percutaneous drainage for patients with cervical necrotizing fasciitis with novel CT classifi cation based on extension of fl uid collection along the deep cervical space T Kiguchi, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P13 (doi: 10.1186/cc14093) Results In a multivariate logistic regression model, a statistically signifi cant independent eff ect was found for body temperature (BT) >38°C (OR = 2.58, 95% CI, 1.76 to 3.79, P <0.001), systolic blood pressure (SBP) <100 mmHg (OR = 1.72, 95% CI, 1.11 to 2.65, P = 0.01), CRP >10 mg/dl (OR = 3.03, 95% CI, 2.05 to 4.49, P <0.001) and PaCO2 <32 mmHg (OR = 2.3, 95% CI, 1.57 to 3.37, P <0.001). Receiver operating characteristic curve analysis revealed an area under the curve value of 0.725 for diff erentiating patients with bacteremia from negative culture. Introduction Cervical necrotizing fasciitis (CNF) is a rapidly evolving and life-threatening condition. Therefore, it is important for physicians to evaluate the severity of illness and to predict clinical outcome exactly in the early phase. We focused on extension of acute fl uid collection along the deep cervical space by CT fi ndings. The purpose of this study was to produce the CT grade and to analyze whether our CT grade is related to the clinical features and the responses to treatment of CNF. Methods Between June 2004 and December 2012, 42 patients diagnosed and treated for CNF in two institutions were included in p Methods Between June 2004 and December 2012, 42 patients diagnosed and treated for CNF in two institutions were included in Conclusion BT >38°C, SBP <100 mmHg, CRP >10 mg/dl and PaCO2 <32 mmHg are independently associated with bacteremia. These S5 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P15 ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign Fisher’s two-tailed test showed a signifi cant diff erence (P = 0.003) between the mortality before and after the second publication. The median ages before and after 2009 were 63.9 and 64.8 years. The time in hospital before admission to the ICU was greater before 2009 (6.15 days) compared with after 2009 (5.53 days). There was no signifi cant diff erence (Mann–Whitney test) between the APACHE II scores, with the mean and median score the same at 17.6 and 18 for both groups. The mean length of stay was 1 day longer after 2009 (8.07 days compared with 9.07 days). P17 Methods A prospective study design was used in order to investigate all sepsis patients either presenting to the emergency department or admitted to the ICU of a regional trauma centre. A total of 106 patients were recruited and all patients were considered eligible as per the SIRS and sepsis criteria [1]. The Sepsis-related Organ Failure Assessment score was determined over the fi rst 24 hours to assess organ function. Patients were assigned to groups as follows: sterile SIRS; uncomplicated sepsis; severe sepsis or septic shock as per the criteria. Assignment into groups was blinded and performed by an intensive care specialist independent of the study. Baseline demographics, clinical characteristics and outcomes were collected and surviving patients were sent a SF-12v2 survey at between 6 months and 2 years post hospital discharge. P18 Long-term health-related quality of life in survivors of sepsis: an epidemiological study P18 Long-term health-related quality of life in survivors of sepsis: an epidemiological study CE Battle1,2,3, G Davies1, M Vijayakumar3, PA Evans1 1NISCHR HBRU Morriston Hospital, Swansea, UK; 2College of Medicine, Swansea University, Swansea, UK; 3Ed Major Critical Care Unit, Morriston Hospital, Swansea, UK Critical Care 2015, 19(Suppl 1):P18 (doi: 10.1186/cc14098) Figure 1 (abstract P16). disease = 3.40 (1.64 to 7.04), Karnovsky Index <70 = 2.56 (1.63 to 3.71), infection by an ESKAPE pathogen = 1.65 (1.02 to 2.66) and severity of infection (reference is infection without SIRS): sepsis = 1.14 (0.7 to 1.83), severe sepsis = 1.18 (0.73 to 1.93), septic shock = 3.69 (1.78 to 7.65). The fi nal model had a very good discrimination for long-term mortality with an area under the ROC curve of 0.78 (Figure 1).ii disease = 3.40 (1.64 to 7.04), Karnovsky Index <70 = 2.56 (1.63 to 3.71), infection by an ESKAPE pathogen = 1.65 (1.02 to 2.66) and severity of infection (reference is infection without SIRS): sepsis = 1.14 (0.7 to 1.83), severe sepsis = 1.18 (0.73 to 1.93), septic shock = 3.69 (1.78 to 7.65). The fi nal model had a very good discrimination for long-term mortality with an area under the ROC curve of 0.78 (Figure 1). Conclusion The authors identifi ed several factors that were signifi cantly associated with increased long-term mortality in patients with severe infection. This information will help clinicians in the discussion of individual prognosis and clinical decision-making. Introduction Survivors of sepsis report persistent problems that can last years after hospital discharge. The main aim of this study was to investigate long-term health-related quality of life in survivors of SIRS and sepsis compared with Welsh normative data, controlling for age, length of stay and pre-existing conditions. The second aim was to investigate any diff erences in long-term health-related quality of life specifi cally with the patients categorised into three groups: SIRS, uncomplicated sepsis, and severe sepsis/septic shock. Conclusion The authors identifi ed several factors that were signifi cantly associated with increased long-term mortality in patients with severe infection. This information will help clinicians in the discussion of individual prognosis and clinical decision-making. References Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S6 Figure 1 (abstract P16). Figure 1 (abstract P17). Median ICU costs per patients and ICU cost per day according to study years. Figure 1 (abstract P16). Figure 1 (abstract P17). Median ICU costs per patients and ICU cost per day according to study years. intensivists to contribute a high standard of care within a restricted budget. The cost-eff ectiveness analysis should be evaluated in sepsis care cases. Direct intensive care costs of severe sepsis and septic shock patients in Thailand B Khwannimit, R Bhurayanontachai Songklanagarind Hospital, Hat Yai, Thailand Critical Care 2015, 19(Suppl 1):P17 (doi: 10.1186/cc14097) Introduction Costs of severe sepsis care from middle-income countries are lacking. This study investigated direct ICU costs and factors that could aff ect the fi nancial outcomes. fi Methods A prospective cohort study was conducted in the medical ICU of a tertiary referral university teaching hospital in Thailand over a 4-year period. y p Results A total of 897 patients, with 683 (76.1%) having septic shock. Overall ICU mortality was 38.3%. The median (interquartile range) ICU length of stay (LOS) was 4 (2 to 9) days. Community, nosocomial and ICU-acquired infection were documented in 574, 282 and 41 patients, respectively. The median ICU costs were €2,067.2 (986.3 to 4.084.6) per patient and €456.6 (315.3 to 721.8) per day. The ICU costs accounted for 64.7% of the hospital costs. In 2008 to 2011, the ICU costs signifi cantly decreased by 40% from €2,695.7 to €1,617, whereas the daily ICU costs decreased only 3.3% from €463.9 to €448.7 (Figure 1). The average ICU costs of patient with nosocomial and ICU-acquired infection were signifi cantly higher than patients with community-acquired infection. By multivariate logistic regression analysis, age, nosocomial or ICU infection, admission from emergency department, number of organ failures, ICU LOS, and fl uid balance in the fi rst 72 hours were independently associated with total ICU costs.i Results A total of 106 patients were included in the study. A mortality rate of 34% was recorded, leading to a fi nal response rate of 72% by the end of the data collection period. Quality of life was signifi cantly reduced in all patients when compared with local normative data (all P <0.0001). Reductions in the physical components of health-related quality of life were more pronounced in severe sepsis/septic shock patients when compared with uncomplicated sepsis and SIRS patients. Conclusion This is the fi rst observational study to specifi cally focus on the diff erent groups of SIRS and sepsis patients to assess long-term quality of life. Local population norms were used for comparison, rather than wide geographical norms that fail to refl ect the intricacies of a country’s population. Signifi cant reductions in quality of life were found in severe sepsis/septic shock patients compared with in uncomplicated sepsis and SIRS patients, when controlling for age, pre- existing conditions, hospital and ICU length of stay. Reference g Reference P21 P21 Global burden of sepsis: a systematic review C Fleischmann1, A Scherag1, NK Adhikari2, CS Hartog1, T Tsaganos3, P Schlattmann1, DC Angus4, K Reinhart1 1Jena University Hospital, Jena, Germany; 2University of Toronto, ON, Canada; 3University of Athens, Greece; 4University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P21 (doi: 10.1186/cc14101) Introduction Sepsis is a global healthcare challenge. However, comprehensive information on sepsis morbidity and mortality across the world is scarce. We aimed to estimate the global burden of sepsis and to identify knowledge gaps based on available evidence from observational epidemiological studies. g Methods We searched 15 international and national citation databases for population-level estimates on incidence rates of sepsis or severe sepsis per 100,000 person-years and case fatality rates in adult populations using consensus criteria and published in the last 40 years. No language or publication restrictions were applied. Studies were stratifi ed into four subgroups (setting: hospital or ICU for sepsis and severe sepsis) and meta-analyzed using metaprop of the R 3.0.2 package. Heterogeneity of the underlying eff ects across studies was expressed by the estimated τ, the square root of the between-study variance. y Conclusion Patients admitted to the ICU with severe CAP and immunosuppressive therapy have higher mortality, with no diff erences between HCAP and CAP. The delay in intubation as well as bacterial and inappropriate antibiotic treatment are factors that increase mortality. Results The search yielded 1,553 reports from 1979 to 2013, of which 37 met our criteria and 33 provided data for meta-analysis. The included studies were from 15 high-income countries in North America, Europe, Asia, and Australia. For these countries, the population incidence rate was 256 (95% CI, 182 to 360, τ = 0.43) hospital-treated sepsis cases and 151 (95% CI, 94 to 242, τ = 0.98) hospital-treated severe sepsis cases per 100,000 person-years, with large between-study heterogeneity. Restricted to the last decade, the incidence rate was 427 (95% CI, 281 to 648, τ = 0.24) sepsis cases and 331 (95% CI, 207 to 530, τ = 0.59) severe sepsis cases per 100,000 person-years. Hospital mortality was 15% for sepsis and 25% for severe sepsis during this period of time. There were no population-level sepsis incidence estimates from lower income countries. A tentative extrapolation from high-income-country data suggests global estimates of 30.7 million sepsis and 23.8 million severe sepsis cases, with potentially six million deaths each year. Analysis of the mortality rate in patients admitted to the ICU for severe community-acquired pneumonia Conclusion Severe sepsis and septic shock are conditions that consume large amounts of resources. Nonsurvivors had higher average spending than survivors. Patients admitted with septic shock had higher mortality than patients with severe sepsis with high mortality in relation to the prognostic indices adopted. The beginning of the antibiotics was longer in the nonsurvivors. We should adopt measures aimed at recognizing and earlier treatment of sepsis. If we improve our treatment, especially in septic shock, we will prevent deaths and decrement costs. Introduction The aim of the study was to analyze the factors associated with hospital mortality in patients with severe community-acquired pneumonia (CAP) who required ICU admission. Methods An observational, retrospective study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical, epidemiological and outcome variables. Quantitative variables were expressed as the mean and standard deviation. Qualitative variables are expressed as the percentage and absolute value. We applied the Mann–Whitney and Fisher’s exact test, as needed, with an alpha error of 5%. P21 p Results We analyzed 111 patients, 57.5 ± 17.7 years old, with 63.1% (70) males and APACHE II score on admission of 19.8 ± 17.7. ICU mortality was 29.7% (33) and in-hospital mortality was 32.4% (36). Ten percent of patients met criteria for medical care-associated pneumonia (HCAP); there were no signifi cant diff erences in mortality between HCAP and CAP (P = 0.075). Patients chronically taking immunosuppressive therapy had a signifi cantly higher mortality compared with the rest of the patients (47.8% vs. 28.4%, P = 0.07). The mortality rate was also higher in patients in whom NIV fail in the fi rst 24 hours (42.9% vs. 17.6% with P = 0.09). Patients who required intubation and mechanical ventilation in the fi rst 24 hours had a higher mortality rate (47.2% vs. 19%, P = 0.002). Regarding the etiology of pneumonia, in 11 patients the viral origin of infection was confi rmed (10 patients had H1N1 pneumonia and one patient CMV pneumonia), with a mortality rate signifi cantly lower than in patients with bacterial pneumonia (3.6% vs. 35.3%, P = 0.06). The use of the right antibiotic therapy at admission was associated with mortality (P = 0.0001). M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil Introduction Sepsis is a high-prevalence disease in ICUs, associated with high mortality and high costs, mainly in developing countries. The aim of this study is to demonstrate the ICU costs, in a private hospital, in patients admitted with severe sepsis and septic shock. p p p Methods A retrospective, observational, single-center study of patients admitted from November 2013 to March 2014 with severe sepsis and septic shock. The records data were taken from the Software Epimed, MV System, and IBM SPSS Statistics 21. The classifi cation was based on the Surviving Sepsis Campaign 2012. We included all 50 beds of an adult ICU, clinical and surgical. All patients older than 18 years with severe sepsis and septic shock were included. We evaluated the costs of patients during their ICU stay, and its relation to clinical presentation (severe sepsis and septic shock), antibiotic start time, permanence of ICU stay, and mortality. Only the fi rst episode per patient was recorded. Results From November 2013 to March 2014 were included 82 patients with criteria for severe sepsis and septic shock. The mean age of patients was 62.5 ± 21.8 years, divided equally between the genres. The overall mortality rate was 34.15%. The SAPS 3 was 56.43, with death probability set to Latin America 38.83%. Patients with severe sepsis had a mortality of 23.2% and those with septic shock had a mortality rate of 58%. The average total cost during ICU admission per patient was US$17,834 and the average daily cost was US$1,641. The daily cost in patients with severe sepsis and septic shock was US$1,263 and US$2,465 (P = 0.002), respectively, and in survivors and nonsurvivors was US$1,189 and US$2,512 (P = 0.001). The length of stay of patients Conclusion Our analyses underline the urgent need to implement global strategies to monitor sepsis morbidity and mortality – especially in low-income and middle-income countries. For further epidemiological studies, more consistent and standardized methodological approaches are needed to reduce between-study heterogeneity. In particular, further research on sepsis coding using administrative data seems necessary to derive sensitive and specifi c sepsis case identifi cations. P19 in the ICU was 11.09 days, being 11.3 days in patients with severe sepsis and 10.7 days in patients with septic shock (P = 0.785). The beginning of the antibiotics in nonsurvivors was 73.7 minutes and in survivors was 64.7 minutes (P = 0.757), with the earliest onset in patients with septic shock than in patients with severe sepsis (38.5 vs. 81.5 minutes, P = 0.141). P19 Analysis of the mortality rate in patients admitted to the ICU for severe community-acquired pneumonia C Joya-Montosa, MD Delgado-Amaya, H Molina-Diaz, E Curiel Balsera Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P19 (doi: 10.1186/cc14099) Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil Critical Care 2015, 19(Suppl 1):P20 (doi: 10.1186/cc14100) M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil g Reference Conclusion The ICU costs of severe sepsis management signifi cantly declined in Thailand. However, the ICU costs were a fi nancial burden accounting for two-thirds of the hospital costs. It is essential for Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. S7 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We included adult (age >18 years) ED patients presenting with severe sepsis/septic shock (sepsis with elevated lactate (>4 mmol/l)) or hypotension) from the prospective clinical ProCESS trial. We studied a subset of patients with microcirculatory videos obtained along with non-infected control patients. Using a sidestream dark-fi eld videomicroscope, we obtained image sequences from the sublingual mucosa and used video stabilization and frame averaging techniques to visualize slowly-moving leukocytes. We quantifi ed the number of rolling and adhered leukocytes present per 1 mm × 1 mm visual fi eld in a standardized 3-second clip. Furthermore, we extracted the total length of vessels candidate for counting of rolling/adhered leukocytes (vessels with an adequate focus). We report sample means with standard deviation and compare them with Student’s t test. how these occur and how we might prevent them. Our objective is to identify disparities by race, language, gender, socioeconomic status, insurance status and geography in acute sepsis care in emergency department (ED) or ICU settings in the published literature. p g p Methods We performed a systematic review of disparities in sepsis care. The search strategy and inclusion and exclusion criteria were defi ned a priori. A medical librarian searched the entire MEDLINE (PubMed), EMBASE and Cinahl databases prior to 2013. One author reviewed all abstracts and a second author reviewed 10% of all abstracts for agreement. Both reviewers independently reviewed each included article using an explicit study review tool. We included studies that met the following inclusion criteria: ED or ICU setting; disparities due to race, language, gender, socioeconomic status, insurance status or geography; process of care measures (antibiotics, lactate, i.v. fl uid resuscitation, central line placement, vasopressor use) or outcome measures (mortality, length of stay, complications, costs). We excluded studies involving organ-specifi c infectious conditions, pediatric populations, case reports, and review articles.i p Results We included a total of 64 patients with severe sepsis/septic shock and 32 non-infected controls. The mean number of adhered leukocytes per fi eld in the sepsis group was 2.1 (SD 2.3) compared with 0.4 (SD 0.8) in the non-infected group (P <0.001). This corresponded to a mean number of adhered leukocytes per unit vessel length of 0.16/mm (SD 0.22) and 0.03/mm (SD 0.06) for sepsis and non-infected groups, respectively (P <0.001). P23 S bli g y Methods This is a prospective observational study in patients scheduled for elective cardiac surgery. Serum samples were drawn prior to surgery, after connection to cardiopulmonary bypass (ischemia), after opening of cross-clamp (reperfusion) and after termination of surgery. The redox status of patients was measured using the bedside point of care RedoxSYS Diagnostic System™ (Luoxis, USA). Simultaneously the antioxidant capacity in serum samples were calculated in all perioperatively obtained serum samples. P24 P24 Time course of redox potential and antioxidant capacity in patients undergoing cardiac surgery C Stoppe1, G Schaelte1, S Kraemer2, C Benstoem2, D Bar-Or3, A Goetzenich2 1RWTH Aachen University, Aachen, Germany; 2RWTH Aachen University, University Hospital, Aachen, Germany; 3Swedish Medical Center, Trauma Research, Engelwood, CO, USA Critical Care 2015, 19(Suppl 1):P24 (doi: 10.1186/cc14104) References 1. Lagu T, Rothberg MB, Nathanson BH, et al. Variation in the care of septic shock: the impact of patient and hospital characteristics. J Crit Care. 2012;27:329-36. Lagu T, Rothberg MB, Nathanson BH, et al. Variation in the care of se 2. Barnato AE, Alexander SL, Linde-zwirble WT, Angus DC. Racial variation in the incidence, care, and outcomes of severe sepsis: analysis of population, patient, and hospital characteristics. Am J Respir Crit Care Med. 2008;177:279-84. 3. Dombrovskiy VY, Martin AA, Sunderram J, Paz HL. Occurrence and outcomes of sepsis: infl uence of race. Crit Care Med. 2007;35:763-8. Introduction Cardiac surgery regularly provokes infl ammation and oxidative stress which contribute to the development of organ failure and mortality of patients. While the assessment of single markers does not refl ect a comprehensive investigation of redox status, the measurement of oxidation–reduction potential (ORP) provides a reliable measure to assess the balance between total prooxidant and antioxidant balance in the blood. The aim of the present study was to investigate the overall redox potential in patients undergoing cardiac surgery. 4. Mayr FB, Yende S, Linde-zwirble WT, et al. Infection rate and acute organ dysfunction risk as explanations for racial diff erences in severe sepsis. JAMA. 2010;303:2495-503. 5. Plurad DS, Lustenberger T, Kilday P, et al. The association of race and survival from sepsis after injury. Am Surg. 2010;76:43-7. 5. Plurad DS, Lustenberger T, Kilday P, et al. The association of race and survival from sepsis after injury. Am Surg. 2010;76:43-7. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 For the rolling leukocytes, we observed a mean number of 27.8 (SD 19.4) in the sepsis group and 12.0 (SD 8.7) in the non-infected group (P <0.001) per fi eld. This corresponded to a mean number of rolling leukocytes per unit vessel length of 2.00/mm (SD 1.67) and 0.75/mm (SD 0.55), respectively (P <0.001). Results We identifi ed 778 abstracts; yielding 31 for inclusion (k = 0.95), 26 of 31 studies were excluded due to quality issues. Five articles met our inclusion criteria. Only one of the studies [1] contained data on process of care measures, showing that central venous monitoring was less likely to occur in older patients. Three studies [2-4] showed that Black patients had a higher incidence of sepsis, a higher hospitalization rate, and higher mortality rate. Plurad and colleagues [5] reported that Asian patients had increased incidence of post-traumatic sepsis. Overall, Black patients with sepsis were younger, had lower socioeconomic status and were more likely to be cared for in urban settings compared with their cohorts. p y Conclusion Our results show a higher number of rolling and adhered leukocytes in patients with severe sepsis/septic shock when compared with non-infected controls. This also applies when taking the total vessel length in the fi eld of view into consideration. This may hold potential as a useful tool in sepsis assessment. Conclusion We found little published data addressing whether disparities due to race, language, gender, socioeconomic status, insurance status or geography exist in the acute care of sepsis. As sepsis is a leading cause of in-hospital mortality, future research should determine whether such disparities exist. Specifi cally, prospective studies of the process of care in sepsis management may further elucidate additional factors that may contribute to these disparities. References P22 P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) Introduction Disparities in the incidence and outcomes of sepsis have been documented in observational studies but little is known about S8 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Fatty acid composition of erythrocytes in multiple organ dysfunction syndrome y y A Osipenko1, A Marochkov2 A Osipenko1, A Marochkov2 1A. Kuleshov Mogilev State University, Mogilev, Belarus; 2Mogilev Regional Hospital, Mogilev, Belarus Critical Care 2015, 19(Suppl 1):P25 (doi: 10.1186/cc14105) Results We found that severe septic patients showed lower CIV activity/ protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. Survivor severe septic patients (n = 130) showed lower CIV activity/protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. In addition, nonsurvivor severe septic patients (n = 68) showed lower CIV activity/protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. Besides, nonsurvivor severe septic patients showed lower CIV activity/protein quantity than survivor ones at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis.i p , g , Critical Care 2015, 19(Suppl 1):P25 (doi: 10.1186/cc14105) Introduction Change in fatty acid composition of erythrocytes and blood plasma in cases of various pathological conditions is evidence of lipid metabolism disorder and can indicate the reasons for and the degree of these disorders [1]. The aim of this study was to assess the FA composition of plasma and erythrocytes in patients with multiple organ dysfunction syndrome (MODS). g y y Methods The objects of study were 19 people with MODS (37.6 ± 8.3 years) of various etiologies. The blood of 17 healthy volunteers aged 38.4 ± 3.3 years served as control. The FA analysis was conducted using capillary gas–liquid chromatography. Quantitative evaluation of individual FA content was made as a mass percentage of their total (C14:0 to C22:6). Statistical analysis was performed using the Mann–Whitney U test (P <0.05). y p g Conclusion The major fi nding of our work, that represents the largest series of severe septic patients with data on OXPHOS function, was that survivor and nonsurvivor severe septic patients showed lower platelet CIV activity than healthy controls during the fi rst week of severe sepsis diagnosis. y Results Our data indicate that changes in blood plasma FA composition in patients with MODS are mainly caused by activation of lipolysis in fat depots and are accompanied by an increase of monounsaturated fatty acids, a decrease in saturated stearic acid and polyunsaturated fatty acids in the ratio. Sublingual leukocyte activation in patients with severe sepsis or septic shock BK Fabian-Jessing1, MJ Massey1, MR Filbin2, PC Hou3, H Kirkegaard4, HE Wang5, DM Yealy6, JA Kellum6, DC Angus6, NI Shapiro1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3Brigham and Women’s Hospital, Boston, MA, USA; 4Aarhus University Hospital, Aarhus, Denmark; 5University of Alabama at Birmingham, AL, USA; 6University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P23 (doi: 10.1186/cc14103) y Results All patients’ sera (n = 17) demonstrated a signifi cant increase of ORP upon start of myocardial ischemia (141.0 ± 4.8 mV vs. 157.9 ± 4.9 mV; P = 0.002) and compared with reperfusion (141.0 ± 4.8 mV vs. 158.6 ± 4.9mV; P <0.001, Figure 1A). In parallel, the antioxidant capacity signifi cantly decreased during surgery (0.505 ± 0.190 μC vs. 0.384 ± 0.120 μC; P = 0.022) corresponding to the increase of oxidative stress (Figure 1B). Introduction The objective of this study was to compare the number of rolling and adhered leukocytes in patients with severe sepsis/septic shock with non-infected controls. Microcirculatory fl ow alterations and endothelial cell dysfunction are elements of sepsis pathophysiology. Traditionally, microcirculatory emphasis has been on red blood cell vessel perfusion. However, assessment of interactions between white blood cells and endothelial cells may be another early diagnostic modality. Introduction The objective of this study was to compare the number of rolling and adhered leukocytes in patients with severe sepsis/septic shock with non-infected controls. Microcirculatory fl ow alterations and endothelial cell dysfunction are elements of sepsis pathophysiology. Traditionally, microcirculatory emphasis has been on red blood cell vessel perfusion. However, assessment of interactions between white blood cells and endothelial cells may be another early diagnostic modality. Conclusion This preliminary study is the fi rst to highlight the time course of overall redox potential and antioxidant capacity in cardiac surgery patients. Further studies are underway to evaluate the clinical signifi cance on outcome in cardiac surgery patients. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S9 Figure 1 (abstract P24). (A), (B) Perioperative time course of oxidative stress and antioxidant capacity. acids. In the test group of patients, as compared with the control group there was an elevated level (27.12 ± 0.78% vs. 25.80 ±0.77%, P <0.05) of saturated palmitic (C16:0) acid combined with the reduced (11.46 ± 0.52% vs. 13.95 ± 1.09%, P <0.001) level of linoleic (C18:2) acid. Reference 1. Kremmyda LS, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:195-218. P26 Lower platelet mitochondrial function in severe septic patients than in controls L Lorente1, M Martin2, J Blanquer3, J Solé-Violán4, L Labarta5, C Díaz6, A Jiménez1, E López-Gallardo7, J Montoya7, E Ruiz-Pesini7 1Hospital Universitario de Canarias, La Laguna, Tenerife, Spain; 2Hospital Universitario Nuestra Señora Candelaria, Santa Cruz, Tenerife, Spain; 3Hospital Clínico Universitario de Valencia, Spain; 4Hospital Universitario Dr Negrín, Las Palmas de Gran Canaria, Spain; 5Hospital San Jorge, Huesca, Spain; 6Hospital Insular, Las Palmas de Gran Canaria, Spain; 7Universidad de Zaragoza, Spain Critical Care 2015, 19(Suppl 1):P26 (doi: 10.1186/cc14106) Introduction The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the fi rst week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients). Introduction The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the fi rst week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients). Figure 1 (abstract P24). (A), (B) Perioperative time course of oxidative stress and antioxidant capacity. Methods This was a prospective, multicenter, observational study in six Spanish ICUs. We obtained blood samples from 198 severe septic patients at days 1, 4 and 8 of the severe sepsis diagnosis and from 96 sex-matched and age-matched healthy control individuals and determined platelet CIV activity/protein quantity. The endpoint of the study was 30-day mortality. Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection J Sole-Violan1, M López-Rodríguez1, E Herrera-Ramos1, J Ruiz-Hernández1, J Horcajada2, L Borderías3, J Blanquer4, J Ferrer1, O Rajas5, J Aspa5, F Rodríguez de Castro1, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital del Mar, Barcelona, Spain; 3Hospital San Jorge, Huesca, Spain; 4Hospital Clínico, Valencia, Spain; 5Hospital de la Princesa, Madrid, Spain Critical Care 2015, 19(Suppl 1):P27 (doi: 10.1186/cc14107) 1. Kremmyda LS, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:195-218. Fatty acid composition of erythrocytes in multiple organ dysfunction syndrome In conditions of increased level of monounsaturated palmitoleic (C16:1) and oleic (C18:1) FA in blood plasma (2.53 ± 0.40% vs. 1.55 ± 0.29%, P <0.001 and 25.18 ± 2.15% vs. 16.55 ± 1.17%, P <0.001, respectively), only the level of palmitoleic (C16:1) acid is increased in erythrocytes (0.56 ± 0.12% vs. 0.16 ± 0.12%, P <0.001). Despite the high content of oleic (C18:1) acid in blood plasma in case of MODS, in erythrocytes its relative level is not changed as compared with the control group. The disorder of lipid composition constancy in erythrocyte membranes is also manifested by change in the content of saturated palmitic (C16:0) and polyunsaturated linoleic (C18:2) fatty Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection J Sole-Violan1, M López-Rodríguez1, E Herrera-Ramos1, J Ruiz-Hernández1, J Horcajada2, L Borderías3, J Blanquer4, J Ferrer1, O Rajas5, J Aspa5, F Rodríguez de Castro1, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital del Mar, Barcelona, Spain; 3Hospital San Jorge, Huesca, Spain; 4Hospital Clínico, Valencia, Spain; 5Hospital de la Princesa, Madrid, Spain Critical Care 2015, 19(Suppl 1):P27 (doi: 10.1186/cc14107) Sublingual leukocyte activation in patients with severe sepsis or septic shock Conclusion The changes revealed in fatty acid composition of erythrocytes may indicate systemic modifi cations of cell membranes in MODS. Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort Results Patients who developed sepsis (n = 18) presented with signifi cantly higher platelet fi brinogen binding at T1 compared with patients who did not get infected (basal: P = 0.0014, upon stimulation: P <0.0035). At T1, ROC AUC for association of basal fi brinogen binding with the occurrence of sepsis was 0.79 (95% CI: 0.68 to 0.89). Elevated basal CD62P expression level was associated with increased 90-day mortality (P = 0.042, ROC AUC = 0.78 (0.64 to 0.88)). Kaplan–Meier survival curves illustrated that mortality was signifi cantly higher after stratifi cation based on T1 basal CD62P level (cutoff MFI >31.56, HR = 13.6, P = 8.23 × 10–6). Multivariate logistic regression analysis using clinical scores (SOFA, APACHE II, SAPS II, SAPS III) indicated that addition of CD62P level or of bound fi brinogen level signifi cantly improved prediction of mortality (odds ratio 1.078, P = 0.003) and sepsis (odds ratio 1.033, P = 0.0012), respectively. A Tridente, G Clarke, A Walden, A Gordon, P Hutton, J Chiche, P Holloway, G Mills, J Bion, F Stuber, C Garrard, C Hinds, GenOSept Investigators St Helens and Knowsley, Liverpool, UK Introduction Patients admitted to intensive care following surgery for faecal peritonitis present particular challenges in terms of clinical management and risk assessment that require close collaboration between surgical and intensive care teams [1]. We aimed at establishing whether dynamic assessment of trends in selected variables may be associated with outcomes, and therefore inform medical decision-making. g Methods We analysed trends in all 35 variables available for the fi rst week of ICU stay in 977 patients from 102 centres across 17 countries. The primary study outcome was 6-month mortality. Secondary outcomes were ICU, hospital and 28-day mortality. For each trend, Cox proportional hazards (PH) regression analyses, adjusted for age and gender, were performed for each endpoint. Trends found to be signifi cant in these analyses, after Bonferroni correction for multiple testing, were entered into a multivariate Cox PH model, to determine independent associations with mortality.i Conclusion Predisposition to severe infection in selected critically ill medico-surgical adults can be identifi ed on day 1 of admission based on circulating basally activated platelets. Levels of activated platelets may add incremental prognostic information to clinical scoring. Reference 1. de Stoppelaar SF, van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thromb Haemost. 2014;11:666-77. Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort p y Results The trends over the fi rst 7 days of ICU stay (primary analysis) retained as independently associated with 6-month outcome were worsening thrombocytopaenia (mortality HR = 1.02, 95% CI = 1.01 to 1.03, P <0.001) and changes in renal function (total daily urine output HR = 1.02, 95% CI = 1.01 to 1.03, P <0.001; renal SOFA subscore HR = 0.87, 95% CI = 0.75 to 0.99, P = 0.047), highest recorded level of bilirubin (HR = 0.99, 95% CI = 0.99 to 0.99, P = 0.02) and GCS SOFA subscore (HR = 0.81, 95% CI = 0.68 to 0.98, P = 0.028). Changes in renal function (total daily urine output and renal component of the SOFA score), GCS component of the SOFA score, total SOFA and worsening thrombocytopaenia were also independently associated with secondary outcomes. Dynamic trends over the fi rst 7 days of ICU stay in all other measured laboratory variables, physiological parameters or radiological fi ndings failed to be retained as independently associated with outcome on multivariate analyses. Furthermore, changes in respiratory support, renal replacement therapy and inotropic and/or vasopressor requirements appeared not to be independently associated with any of the primary or secondary outcomes. Secondary post hoc analyses on trends over the fi rst 3 and 5 days corroborated these fi ndings. P29 P29 Elevated basal levels of circulating activated platelets predict ICU-acquired sepsis and mortality: a prospective study N Layios CHU Sart Tilman, Liège, Belgium Critical Care 2015, 19(Suppl 1):P29 (doi: 10.1186/cc14109) P28 P28 Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort A Tridente, G Clarke, A Walden, A Gordon, P Hutton, J Chiche, P Holloway, G Mills, J Bion, F Stuber, C Garrard, C Hinds, GenOSept Investigators St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P28 (doi: 10.1186/cc14108) P30 Antiplatelet therapy does not infl uence outcome or host response biomarkers during sepsis: a propensity-matched analysis MA Wiewel1, SF De Stoppelaar1, LA Van Vught1, JF Frencken2, AJ Hoogendijk1, PM Klein Klouwenberg2, J Horn1, MJ Bonten2, MJ Schultz1, AH Zwinderman1, OL Cremer2, T Van der Poll1 1Academic Medical Center, University of Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P30 (doi: 10.1186/cc14110) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 have previously demonstrated that variants at SFTPA2 infl uence the severity of H1N1pdm infection. We have now studied genetic variants at diff erent genes, some of them previously associated with infections by infl uenza and/or other viruses. The purpose of this study was to analyze the role of genetic variants in the susceptibility and outcome of IVI. Conclusion Only deterioration in renal function, thrombocytopaenia and hyperbilirubinaemia over the fi rst 7 days of ICU stay were consistently associated with mortality at all endpoints. Reference 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. Methods In total, 136 white Spanish patients developed IVI (80.3% of them by H1N1pdm virus). The general population group consisted of 1,466 unrelated healthy volunteers. Patients and controls were analyzed for diff erent polymorphisms at 13 genes (FCGR2A, FCGR3A, FCGR3B, IL1RN, IL6, LTA, TIRAP, TLR1, TLR2, TLR3, TLR4, CCR5, IGHG2). Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection Introduction The role of genetic variability in the susceptibility and outcome of infl uenza virus infection (IVI) remains largely unknown. We Introduction The role of genetic variability in the susceptibility and outcome of infl uenza virus infection (IVI) remains largely unknown. We S10 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 9 Elevated basal levels of circulating activated platelets predict ICU-acquired sepsis and mortality: a prospective study N Layios CHU Sart Tilman, Liège, Belgium Critical Care 2015, 19(Suppl 1):P29 (doi: 10.1186/cc14109) IVI was detected in nasopharyngeal swabs using real-time PCR. The Hardy–Weinberg equilibrium was analyzed by Haploview v. 4.2. The comparisons of genotypes distribution based on susceptibility and severity were performed using the chi-squared test or Fisher’s exact test when needed. The relationship between severity in hospitalized patients and genotypes was evaluated by binary logistic regression models. Introduction Platelets are now considered to be immune and infl am- matory agents as well as key cells in coagulation, and as such have been implicated in the pathophysiology of sepsis [1]. Thrombocytopenia is associated with sepsis severity and poor prognosis, and hyperactivated platelets probably contribute to microvascular thrombosis and organ failure. In the present study, we evaluated platelet activation markers as potential predictive markers of sepsis and of mortality among four commonly encountered populations of patients admitted to ICUs. Results No associations were found between the diff erent genetic variants and susceptibility or severity of IVI. Variants at LTA, FCGR2A, IGHG2, TLR3 and CCR5, previously associated with severity of IVI were not replicated in our study. Methods Ninety-nine non-infected ICU patients were prospectively screened at day 1 (T1) and day 3 (T2) of admission after elective cardiac surgery, trauma, acute neurologic dysfunction or prolonged ventilation (>48 hours). A third sample was drawn when infection was diagnosed (Tx). We evaluated platelet activation by measuring the expression of P-selectin (CD62P) and fi brinogen binding on the cell surface before and after stimulation with major platelet agonists (ADP, collagen, and TRAP) through fl ow cytometry. Clinical scores were obtained at admission. Conclusion Our study does not suggest that polymorphisms at LTA, FCGR2A, IGHG2, TLR3 and CCR5 genes are associated with susceptibility or severity of IVI. 1. de Stoppelaar SF, van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thromb Haemost. 2014;11:666-77. P32 Methods We performed a prospective observational study in patients admitted with sepsis to the mixed ICUs of two hospitals in the Netherlands between January 2011 and July 2013. Cox proportional hazards regression was used to estimate the eff ect of antiplatelet therapy on mortality. To account for indication bias, a propensity score was constructed, and used to match antiplatelet therapy users to nonusers. Plasma biomarker levels, providing insight into hallmark host responses to sepsis, including activation of endothelial cells and the cytokine network, were determined during the fi rst 4 days after ICU admission. Mitochondrial dysfunction and ischemia in critical illness: an adipose tissue microdialysis study in 203 ICU patients M Theodorakopoulou, S Apollonatou, N Nikitas, D Vassiliadi, A Diamantakis, V Tsagkari, F Frantzeskaki, I Dimopoulou University Hospital of Athens, Greece Critical Care 2015, 19(Suppl 1):P32 (doi: 10.1186/cc14112) Introduction Ischemia and mitochondrial dysfunction have been implicated in critical illness. The potential of MD to diagnose and separate ischemia and mitochondrial dysfunction in ICU patients remains currently unknown. Introduction Ischemia and mitochondrial dysfunction have been implicated in critical illness. The potential of MD to diagnose and separate ischemia and mitochondrial dysfunction in ICU patients remains currently unknown. Results Of 1,070 sepsis patients, 297 (27.8%) were on antiplatelet therapy, including acetylsalicylic acid, clopidogrel and dipyridamole, prior to ICU admission. Antiplatelet users and nonusers diff ered signifi cantly with regard to several baseline characteristics, such as age, gender and cardiovascular disease. Antiplatelet therapy was not related to sepsis severity at presentation, the primary source of infection, causative pathogens, the development of organ failure or shock during ICU stay, or mortality up to 90 days after admission, in either the unmatched or propensity-matched analyses. Antiplatelet therapy did also not modify plasma concentrations of biomarkers.l Methods A retrospective, observational study of 203 mechanically ventilated patients studied over a 6-year period with MD including medical, surgical and trauma patients. Sepsis stages: SIRS (n = 24), severe sepsis (n = 46) and septic shock (n = 133). Median age 67 years (range: 17 to 92 years). Mortality was 53%. All subjects had a MD catheter placed in femoral adipose tissue upon admission to the ICU. Interstitial fl uid samples were collected six times per day, for 3 consecutive days, and were analyzed for glucose, lactate, pyruvate, and glycerol levels. The lactate to pyruvate (LP) ratio was calculated. Blood lactate was measured. P31 Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery S Ono1, T Ikeda1, T Kubo2, H Tsujimoto2, M Kinoshita2, T Ueno1 1Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan; 2National Defense Medical College, Tokorozawa, Saitama, Japan Critical Care 2015, 19(Suppl 1):P31 (doi: 10.1186/cc14111) Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery Antiplatelet therapy does not infl uence outcome or host response biomarkers during sepsis: a propensity-matched analysis l1 l 1 h 1 k 2 MA Wiewel1, SF De Stoppelaar1, LA Van Vught1, JF Frencken2, AJ Hoogendijk1, PM Klein Klouwenberg2, J Horn1, MJ Bonten2, MJ Schultz1, AH Zwinderman1, OL Cremer2, T Van der Poll1 1Academic Medical Center, University of Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P30 (doi: 10.1186/cc14110) Introduction Sepsis is a life-threatening condition, during which triggering of infl ammatory and coagulation cascades, together with endothelial damage, invariably leads to activation of platelets. Although platelets are essential components of primary hemostasis, uncontrolled platelet activation during sepsis may contribute to organ failure. The aim of this study was to investigate whether chronic antiplatelet therapy impacts on the presentation and outcome of, and the host response to, sepsis. S11 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery g g g y S Ono1, T Ikeda1, T Kubo2, H Tsujimoto2, M Kinoshita2, T Ueno1 1Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan; 2National Defense Medical College, Tokorozawa, Saitama, Japan Critical Care 2015, 19(Suppl 1):P31 (doi: 10.1186/cc14111) Introduction Programmed death-1 (PD-1) has been reported to be an immunoinhibitory receptor expressed by chronically stimulated T cells after T-cell activation. The present study was designed to evaluate the relationship between perioperative PD-1 expression on CD4+ T cells and the incidence of postoperative infectious complications in patients undergoing gastroenterological surgery. Conclusion Bedside subcutaneous adipose tissue MD is possible to diagnose and separate ischemia and mitochondrial dysfunction in general ICU patients. These two conditions are not so common; however, mitochondrial dysfunction seems to be associated with higher mortality rates. Methods This was a prospective observational study. The subjects of this study included 101 patients with gastroenterological disease who underwent elective abdominal surgery via laparotomy at the National Defense Medical College Hospital. Blood samples were taken on the preoperative day (Pre) and the fi rst postoperative day (POD1). We calculated CD4+ T-cell count and PD-1 expression on CD4+ T cells by fl ow cytometer. The occurrence of postoperative infectious complications was defi ned according to a combination of clinical fi ndings and the results of laboratory and other tests. The postoperative infectious complications in this study included incisional surgical site infections (SSIs), organ/space SSIs, enterocolitis, urinary tract infections, and pneumonia. Incisional and organ/space SSIs were diagnosed according to the defi nitions stated in the guidelines issued by the Center for Disease Control and Prevention. P32 Ischemia was defi ned as LP ratio >30 and pyruvate level <70 mmol, while mitochondrial dysfunction was defi ned as LP ratio >30 and pyruvate >70 mmol. y y Conclusion Pre-existing antiplatelet therapy does not infl uence clinical disease severity at presentation, nor the host response or outcome following sepsis. Acknowledgement This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (http://www. ctmm.nl), project MARS (grant 04I-201). py Results Analysis during the course of the 3-day period revealed three distinct patterns: no ischemia/mitochondrial dysfunction (n = 150 or 74%), ischemia (n = 27 or 13%) and mitochondrial dysfunction (n = 26 or 13%). On day 1, median blood lactate was higher in mitochondrial dysfunction (2.2 mmol/l) compared with both ischemia (1.3 mmol/l) and with no ischemia/mitochondrial dysfunction (1.3 mmol/l) (P = 0.004). Again on day 1, median interstitial fl uid lactate was higher in mitochondrial dysfunction (8.4 mmol/l), in comparison with ischemia (1.4 mmol/l) and with the group without ischemia/mitochondrial dysfunction (2.5 mmol/l) (P <0.001). Similar results were obtained with interstitial fl uid glycerol levels (P = 0.009). Median LP ratio was higher in ischemia (LP = 36), and mitochondrial dysfunction (LP = 33) compared with those without ischemia/mitochondrial dysfunction (LP = 17) (P <0.001). Median interstitial fl uid glucose was lower in ischemia (2 mmol/l) compared with both mitochondrial dysfunction (4 mmol/l) and with no ischemia/mitochondrial dysfunction (5 mmol/l) (P <0.001). ICU mortality was 77% in mitochondrial dysfunction, 52% in ischemia and 49% in the group without ischemia/mitochondrial dysfunction (P = 0.033). Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P33 (doi: 10.1186/cc14113) Introduction Pyruvate dehydrogenase (PDH) is a key component of aerobic metabolism. Multiple rodent studies have shown that PDH levels are low in sepsis. This leads to a shift to anaerobic metabolism, resulting in increased lactic acid. Alteration in PDH levels during sepsis, however, has never been studied in humans. The aim of this study was to identify whether PDH levels (activity and quantity) were altered in humans in sepsis. Results Postoperative infectious complications occurred in 30 of the 101 patients. CD4+ T-cell count was signifi cantly lower in the patients who developed postoperative infectious complications at POD1 compared with those from the patients who did not. In addition, PD-1 expression on CD4+ T cells was signifi cantly higher at Pre or POD1 in patients who developed postoperative infectious complications. Those results were similar for the incidence of organ/space surgical site infection. Preoperative PD-1 expression on CD4+ T cells tended to be higher in males than in females. We found there was a signifi cant negative correlation between preoperative PD-1 expression on CD4+ T cells and CD4+ T-cell count. Methods We conducted a case–control study at a single urban tertiary care center. We compared PDH levels between sepsis and healthy control subjects by measuring PDH levels in peripheral blood mononuclear cells via a novel assay. We measured PDH levels in control subjects at baseline and in sepsis subjects at 0, 24, 48 and 72 hours. Results There were 39 sepsis (age 67 ± 14 years, M ± SD) and 19 control (age 50 ± 12 years) subjects of similar gender (56% and 63% female, respectively) and race (79% and 68% Caucasian, respectively). PDH levels in the sepsis group were signifi cantly lower than the control Conclusion Perioperative CD4+ T-cell count or PD-1 expression on CD4+ T cells could be an early predictive marker for the development of postoperative infectious complications. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S12 group at all time points (Figures 1 and 2). After controlling for age, gender, race, and assay plate via multivariable linear regression, the eff ect of treatment group remained signifi cant. We were unable to control for comorbid illness, which was exclusively concentrated in the sepsis group. Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Figure 1 (abstract P33). Figure 2 (abstract P33). Figure 2 (abstract P33). Conclusion In the nonsurvivor or the severe patient with sepsis requiring steroid administration, the enhancement of C1INH activity was not observed, and the C1INH quantitative values were low. Further evaluation of the serial change of C1INH and the validity of C1INH replacement therapy in patients with septic shock may lead to a new strategy for management in sepsis. Expression of apolipoproteins L in neutrophils during sepsis I Akl1, C Lelubre1, M Piagnerelli1, P Biston1, P Uzureau1, H Fayyad Kazan2, B Badran3, M Ezzedine3, K Zouaoui Boudjeltia1, L Vanhamme4 1CHU de Charleroi-Hopital Andre Vesale, Montigny-Le-Tilleul, Belgium; 2Institut Jules Bordet, Université Libre de Bruxelles, Belgium; 3Doctoral School of Sciences and Technology, Platform of Research and Environmental Sciences, Beirut, Lebanon; 4Institute of Medicine and Molecular Biology IBMM, Charleroi, Belgium Critical Care 2015, 19(Suppl 1):P35 (doi: 10.1186/cc14115) Figure 2 (abstract P33). Figure 2 (abstract P33). group at all time points (Figures 1 and 2). After controlling for age, gender, race, and assay plate via multivariable linear regression, the eff ect of treatment group remained signifi cant. We were unable to control for comorbid illness, which was exclusively concentrated in the sepsis group.i Introduction Sepsis is characterized by a strong systemic infl ammatory reaction. The pathogenesis is driven by alterations in the immune system and is associated with high neutrophil counts related to a specifi c delay in apoptosis [1]. The apolipoproteins L (ApoLs) family comprises six members in humans (ApoL1 to ApoL6). In light of their deregulated expression in several pathologies, they are likely to be important molecular players of programmed cell death [2]. We analyzed ApoL expression in cohorts of septic and nonseptic ICU patients and healthy volunteers in order to test whether ApoLs could be involved in the neutrophil apoptotic program. Introduction Sepsis is characterized by a strong systemic infl ammatory reaction. The pathogenesis is driven by alterations in the immune system and is associated with high neutrophil counts related to a specifi c delay in apoptosis [1]. The apolipoproteins L (ApoLs) family comprises six members in humans (ApoL1 to ApoL6). In light of their deregulated expression in several pathologies, they are likely to be important molecular players of programmed cell death [2]. Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno We analyzed ApoL expression in cohorts of septic and nonseptic ICU patients and healthy volunteers in order to test whether ApoLs could be involved in the neutrophil apoptotic program. Conclusion PDH levels are signifi cantly lowered in humans during sepsis when compared with healthy controls, even when controlling for age, race and gender. Further research is needed to determine whether this fi nding persists after adjustment for comorbid disease, and whether lower PDH levels are associated with clinical outcomes. Methods By means of magnetic cell sorting, peripheral neutrophils were purifi ed from 20 healthy volunteers and 40 ICU patients with (n = 20) or without sepsis (n = 20). ApoL expression was analyzed at the mRNA and protein levels by real-time PCR and western blot analysis respectively. Apoptosis of purifi ed neutrophils was assessed using fl ow cytometry following 4 and 24 hours of in vitro incubation. We monitored the expression of C-reactive protein (CRP), an infl ammatory marker, and its correlation with ApoL expression in PMNs was studied by linear regression analysis.i Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Conclusion PDH levels are signifi cantly lowered in humans during sepsis when compared with healthy controls, even when controlling for age, race and gender. Further research is needed to determine whether this fi nding persists after adjustment for comorbid disease, and whether lower PDH levels are associated with clinical outcomes. P34 S i l h f C1 i hibi i i i h i li i Figure 1 (abstract P33). Figure 2 (abstract P33). Figure 1 (abstract P33). but also the plasma kallikrein–kinin system, fi brinolytic system and coagulation system. The biologic activities of C1INH can be divided into the regulation of vascular permeability and anti-infl ammatory functions. In recent years, hereditary angioedema (HAE), caused by an inherited defi ciency of C1INH, has been focused. During HAE attacks, vascular permeability was markedly increased, which leads to angioedema. In sepsis, signifi cant endothelial hyperpermeability is similarly observed systemically, but the role of C1INH has not been clarifi ed in the pathogenesis. The serial change of C1INH in patients with sepsis is not clear. The objective of this study was to clarify the serial change in C1INH in patients with sepsis and evaluate the impact of C1INH on their clinical course. Figure 1 (abstract P33). Methods We serially examined C1INH activity values (normal range 70 to 130%) and quantitative values (normal range 160 to 330 μg/ml) in patients with sepsis during the period between December 2012 and February 2013. We also analyzed their clinical course: prognosis, volume of infusion, body weight, urine volume, catecholamine administration, and steroid administration. Results The serial change of C1INH was evaluated in fi ve patients with sepsis (three male and two female; four survivors and one nonsurvivor; mean age, 68 ± 11 years). In the nonsurvivor, C1INH activity on admission value was 97.2% (normal range), and quantitative value was 133.1 μg/ml (below normal). In the patient with severe sepsis requiring fl uid resuscitation, catecholamine and steroid administration to maintain hemodynamics, C1INH activity value on admission was 94.4% (normal range), and quantitative value was 126.7 μg/ml (below normal range). His general condition was improved on day 6, and C1INH activity value and quantitative value increased (139.9%; above normal range, 250.1 μg/ml; normal range). In the other three patients with sepsis not requiring steroid administration, C1INH activity value on admission was 130.6 ± 8.7% (above normal range), and quantitative value was 215 ± 26.5 μg/ml (normal range). P34 Serial change of C1 inhibitor in patients with sepsis: a preliminary report T Hirose1, H Ogura1, K Jinkoo1, Y Nakamura1, H Hosotsubo1, T Shimazu1, E Kitano2, M Hatanaka2 1Osaka University Graduate School of Medicine, Suita, Japan; 2Kobe Tokiwa University, Kobe, Japan Critical Care 2015, 19(Suppl 1):P34 (doi: 10.1186/cc14114) Introduction C1 inhibitor (C1INH), belonging to the superfamily of serine protease inhibitors, regulates not only complement system, Results Our results showed a signifi cant downregulation in mRNA expression of ApoL1 (P <0.0001), ApoL2 (P = 0.0009), ApoL3 (P <0.0001) S13 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 correlates inversely with outcome. The aim of the study was to identify phenotypic and functional early markers of T cells and NK cells related to prognosis in the septic patient population. and ApoL6 (P = 0.0003) in purifi ed PMNs from ICU patients as compared with the healthy individuals. This downregulation was also validated at the protein level for ApoL1 and ApoL2, whereas ApoL 6 was upregulated in septic patients. We could not detect ApoL3 protein in any of the cohorts. This was accompanied by a signifi cant delay in PMN apoptosis in septic patients as compared with healthy volunteers (P <0.05) at 4 and 24 hours. We also showed a strong negative correlation in the three mixed groups between CRP and ApoL1 (R = –0.607), ApoL2 (R = –0.651), ApoL3 (R = –0.578) and ApoL6 (R = –0.506). and ApoL6 (P = 0.0003) in purifi ed PMNs from ICU patients as compared with the healthy individuals. This downregulation was also validated at the protein level for ApoL1 and ApoL2, whereas ApoL 6 was upregulated in septic patients. We could not detect ApoL3 protein in any of the cohorts. This was accompanied by a signifi cant delay in PMN apoptosis in septic patients as compared with healthy volunteers (P <0.05) at 4 and 24 hours. We also showed a strong negative correlation in the three mixed groups between CRP and ApoL1 (R = –0.607), ApoL2 (R = –0.651), ApoL3 (R = –0.578) and ApoL6 (R = –0.506). p g p p p p Methods We collected peripheral blood mononuclear cells from 47 patients with severe sepsis or septic shock at ICU admission (T0) and from 50 healthy controls. P34 Serial change of C1 inhibitor in patients with sepsis: a preliminary report On these subjects we evaluated frequency and absolute numbers of CD4+ and CD8+ T cells and of NK and B lymphocytes, the rates of regulatory CD4+CD25+Foxp3+ T cells (Tregs), the cytotoxic potential of CD4+, CD8+ T cells and of NK cells by evaluation of perforin (PER) and granzyme (GRA) expression and production of eff ector cytokines (namely IL-2, IL-17, IL-4, TNFα, IFNγ) by CD4+, CD8+ T cells and NK cells upon polyclonal stimulation. The markers were compared in patients with diff erent outcome. p p Conclusion The altered apoptotic fate of neutrophils in sepsis was correlated with the modifi cation of the expression profi le of ApoLs, a family of proteins thought to be involved in the apoptotic process. The role of these proteins in the sepsis-associated phenotype of neutrophils remains to be further elucidated. p pf Results Septic patients, compared with healthy donors, were characterized by global lymphopenia; we found increased frequencies of CD4+ T cells producing IL-2 (P = 0.0000000003), increased percentage of CD8 T cells producing IFNγ (P = 0.03), and reduced proportion of CD4+ T cells (P = 0.00007) and NK cells (P = 0.002) producing IFNγ. We also noticed an increased frequency of CD8+ T cells expressing PER (P = 0.00000025) and GRA (P = 0.01); moreover, the proportion of NK cells expressing GRA was also signifi cantly increased (P = 0.000019). To establish the prognostic value of these biological markers, we compared the cytokine expression by lymphocytes in septic patients that survived with those that died (D). We found that CD4+ and CD8+ TNFα-producing T cells were signifi cantly increased in D (P = 0.01 and P = 0.0001 respectively); similarly the percentage of CD8+ T cells producing IFNγ was more elevated in D (P = 0.006). The same was observed for IL-17 production by CD4+ T cells (P = 0.03) in D. On the contrary we observed a tendency to the reduction of circulating CD4+CD25+foxp3 (Tregs) in D (P = 0.08). References 1. Görgülü P, et al. Crit Care. 2011;15:R20. g 2. Vanhollebeke B, et al. Cell Mol Life Sci. 2006;63:1937-44. 2. Vanhollebeke B, et al. Cell Mol Life Sci. 2006;63:1937-44. P37 Results Seventy-three critically ill patients were included, 10 of whom developed an ICU-acquired infection. Compared with healthy subjects, whole blood leukocytes of patients were less responsive to ex vivo stimulation with LPS, as refl ected by strongly reduced TNFα, IL-1β and IL-6 levels in culture supernatants. However, results were not diff erent between patients who did and those who did not develop an ICU- acquired infection (Figure 1). Ex vivo and in vivo generation of neutrophil extracellular traps by neutrophils from septic patients N Takeyama, MH Huq, M Ando, T Gocho, MH Hashiba, H Miyabe, H Kano, A Tomino, M Tsuda, T Hattori, A Hirakawa N Takeyama, MH Huq, M Ando, T Goc Fujita Health University, Aichi, Japan Critical Care 2015, 19(Suppl 1):P36 (doi: 10.1186/cc14116) Introduction The primary aim of this study was to determine the diff erences in ex vivo generation of neutrophil extracellular traps (NETs) by neutrophils from septic and nonseptic patients. We further sought to examine plasma levels of cell-free DNA (cf-DNA) and histones to assess in vivo NET formation. p g Conclusion Septic patients are characterized by a peculiar immunophenotype which includes global lymphopenia and a specifi c pattern of cytokines. Some of the evaluated markers seem to individuate those with worse outcome; in particular, this group showed an infl ammatory phenotype with a higher expression of IFNγ, TNFα, IL- 17 and a tendency to a reduction of Tregs. Methods We isolated neutrophils from consecutive patients with sepsis (n = 17) and without sepsis (n = 18) admitted to the ICU. Neutrophils were activated by incubation with phorbol myristate acetate to induce release of NETs and NET formation was assessed by measuring the extracellular DNA level. Immunolabeling and fl uorescence imaging were also performed. Extracellular killing of bacteria by NETs was studied by co-culture of Escherichia coli and neutrophils in the presence of the phagocytosis inhibitor cytochalasin D. To assess in vivo NET formation, plasma levels of cf-DNA and histones were measured.i Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) Results The condition of the nonseptic patients was signifi cantly less severe than that of the septic patients. The SOFA score of septic patients and the nonseptic patients was 6 (3 to 18) and 2.5 (1 to 8), respectively (median (IQR), P = 0.02). The overall mortality rate was 29%. After stimulation with PMA, neutrophils isolated from septic patients released 4.08 ± 1.02% of their total DNA, whereas neutrophils from nonseptic patients released 29.06 ± 2.94% (P <0.0001). Immunofl uorescent staining of released DNA, elastase, and myeloperoxidase also revealed similar results. Neutrophils from nonseptic patients showed eff ective extracellular killing of E. coli through NETs, whereas neutrophils from septic patients did not (P <0.001). Plasma levels of cf-DNA and histones were higher in septic patients than in nonseptic patients (P <0.001). Introduction Critically ill patients show signs of immune suppression, which is considered to increase vulnerability to nosocomial infections. Whole blood stimulation is a frequently used functional test for immune suppression. We here aimed to assess the association between whole blood leukocyte responsiveness to lipopolysaccharide (LPS) and the subsequent occurrence of nosocomial infections in critically ill patients admitted to the ICU. g Conclusion The increase of the immature PMN count and immature/ total PMN ratio confi rmed recruitment of immature neutrophils from the bone marrow into the circulation. The ex vivo generation of NETs is downregulated in neutrophils isolated from patients with sepsis. However, it is unclear whether in vivo NET formation is also impaired during sepsis, so further investigation is necessary. Methods All consecutive critically ill patients admitted to the ICU between April 2012 and June 2013 with two or more systemic infl ammatory response syndrome criteria and an expected length of ICU stay of more than 24 hours were enrolled. Age-matched and gender-matched healthy individuals were included as controls. Blood was drawn the fi rst morning after ICU admission and stimulated ex vivo with 100 ng/ml ultrapure LPS for 3 hours. Tumor necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 were measured in supernatants. Alarming levels of heat shock proteins 72 and 90α in critically ill children The stimulated blood or plasma from septic patients was treated in vitro with 10 vol% polystyrene– divinylbenzene (PS-DVB)-based polymers (CG161, mean pore size 16 nm; CG300, mean pore size 30 nm) or left untreated. After adsorption, the plasma was separated and diluted with cell culture medium. The resulting conditioned medium was used to stimulate human umbilical vein endothelial cells (HUVEC) for 16 hours. HUVEC activation was assessed by the release of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)α, plasminogen activator inhibitor-1 (PAI- 1), as well as the expression of intercellular adhesion molecule (ICAM)- 1 and E-selectin. HUVEC were cultured at a shear stress of 5 dyne/ cm2 using the Ibidi perfusion system. Adhesion of monocytic THP-1 cells to HUVEC was studied after 4 hours of HUVEC stimulation with conditioned media. THP-1 cells were perfused over HUVEC at 1 dyne/ cm2 for 15 minutes, and adhering THP-1 were quantifi ed over time. Results The adsorbents CG161 and CG300 substantially decreased levels of TNFα, IL-1β, IL-6, IL-8 and IL-10 in LPS-stimulated blood. TNFα, a key stimulus for HUVEC, was reduced to 12% and 8% of the initial concentration by CG161 and CG300, respectively. Stimulation of HUVEC with the adsorbent-treated plasma resulted in signifi cantly diminished release of IL-6, IL-8, PAI-1 and decreased ICAM-1 and E-selectin expression, indicating reduced HUVEC activation. THP-1 adhesion was substantially decreased when HUVEC were stimulated with CG300-treated plasma as compared with untreated controls. Conclusion The fl ow model allows to study the eff ect of cytokine modulation on endothelial activation and to assess the interaction of activated endothelial cells with blood cells. Modulation of infl ammatory Methods Human whole blood was stimulated with LPS (100 ng/ ml) from Escherichia coli for 4 hours. The stimulated blood or plasma from septic patients was treated in vitro with 10 vol% polystyrene– divinylbenzene (PS-DVB)-based polymers (CG161, mean pore size 16 nm; CG300, mean pore size 30 nm) or left untreated. After adsorption, the plasma was separated and diluted with cell culture medium. The resulting conditioned medium was used to stimulate human umbilical vein endothelial cells (HUVEC) for 16 hours. HUVEC activation was assessed by the release of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)α, plasminogen activator inhibitor-1 (PAI- 1), as well as the expression of intercellular adhesion molecule (ICAM)- 1 and E-selectin. Alarming levels of heat shock proteins 72 and 90α in critically ill children HUVEC were cultured at a shear stress of 5 dyne/ cm2 using the Ibidi perfusion system. Adhesion of monocytic THP-1 cells to HUVEC was studied after 4 hours of HUVEC stimulation with conditioned media. THP-1 cells were perfused over HUVEC at 1 dyne/ cm2 for 15 minutes, and adhering THP-1 were quantifi ed over time. p j y y Methods Critically ill children with S (n = 16), SS (n = 15) or SIRS (n = 18) and H (n = 21) were enrolled in the study. ELISA was used to evaluate HSPs, chemiluminescence to measure ILs, and fl ow cytometry to evaluate nCD64 expression (IRB approved). p pp Results Patients in both septic groups had elevated HSP90α (P <0.0001), HSP72 (P <0.05), IL-6 (P <0.0001), IL-8 (P <0.02) and IL-10 (P <0.05) levels compared with H, whereas SS had increased HSP72, IL6 and TNFα compared with SIRS (P <0.05). SIRS patients presented increased HSP90α, IL-6 and IL-8 compared with H (P <0.05). Both HSPs were dramatically increased among nonsurvivors. In a logistic regression model, only HSP90α was independently associated with mortality (P <0.0001). HSP90α related positively (P <0.001) to nCD64, IL-8, IL-10, CRP, PRISM, PELOD, TISS, and LOS and negatively to HDL (P <0.001) and LDL (P <0.02). HSP72 also related negatively to HDL (P <0.001). i Results The adsorbents CG161 and CG300 substantially decreased levels of TNFα, IL-1β, IL-6, IL-8 and IL-10 in LPS-stimulated blood. TNFα, a key stimulus for HUVEC, was reduced to 12% and 8% of the initial concentration by CG161 and CG300, respectively. Stimulation of HUVEC with the adsorbent-treated plasma resulted in signifi cantly diminished release of IL-6, IL-8, PAI-1 and decreased ICAM-1 and E-selectin expression, indicating reduced HUVEC activation. THP-1 adhesion was substantially decreased when HUVEC were stimulated with CG300-treated plasma as compared with untreated controls. Conclusion Extracellular HSP72 and HSP90α are alarmingly elevated in critically ill children, especially in severe sepsis. HSP90α levels are independently associated with mortality, related to CD64, IL-8, IL-10, severity of illness, and outcome. Both HSPs are inversely related to the low LDL/low HDL septic metabolic pattern [2].i Acknowledgements This research has been co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. Investing in knowledge society through the European Social Fund. P40 P40 Alarming levels of heat shock proteins 72 and 90α in critically ill children M Fitrolaki1, H Dimitriou2, M Venihaki2, M Katrinaki2, G Briassoulis1 1University Hospital, Heraklion, Greece; 2University of Crete, Medical School, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P40 (doi: 10.1186/cc14120) P37 Specifi c patterns of T-cell cytokines as an early marker of outcome in septic patients A Franci, A Peris, F Liotta, F Annunziato, P Ruggiano, M Ferraro A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P37 (doi: 10.1186/cc14117) A Franci, A Peris, F Liotta, F Annunziato, P Ruggiano, M Ferraro A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P37 (doi: 10.1186/cc14117) Introduction The infl ammatory response of sepsis is developed in two phases, an infl ammatory phase (SIRS) and a phase more variable in frequency and intensity (CARS): this balance has an important eff ect on morbidity and mortality. Lymphopenia aff ects particularly T cells, and Conclusion The extent of reduced LPS responsiveness of blood leukocytes in critically ill patients on the fi rst day after ICU admission does not relate to the subsequent development of ICU-acquired infections. S14 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P38). Similarly reduced responsiveness of whole blood leukocytes to lipopolysaccharide. Figure 1 (abstract P38). Similarly reduced responsiveness of whole blood leukocytes to lipopolysaccharide. mediators with porous polystyrene-based polymers attenuates endothelial activation and reduces monocyte adhesion. mediators with porous polystyrene-based polymers attenuates endothelial activation and reduces monocyte adhesion. 39 ell-culture model to study endothelial activation in sepsis Alarming levels of heat shock proteins 72 and 90α in critically ill children Introduction The endothelium is a complex organ infl uenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic infl ammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anti- coagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the eff ect of adsorbent-based mediator modulation on endothelial activation. Introduction The endothelium is a complex organ infl uenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic infl ammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anti- coagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the eff ect of adsorbent-based mediator modulation on endothelial activation. Introduction Extracellular heat shock proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic infl ammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in the acute phase [1] of sepsis (S) or severe sepsis (SS). We determined serum HSP90α, HSP72 and neutrophil CD64 expression, IL-6, IL-8, IL-10, and TNFα in children with S or SS compared with SIRS (brain injury) or healthy children (H). Introduction Extracellular heat shock proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic infl ammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in the acute phase [1] of sepsis (S) or severe sepsis (SS). We determined serum HSP90α, HSP72 and neutrophil CD64 expression, IL-6, IL-8, IL-10, and TNFα in children with S or SS compared with SIRS (brain injury) or healthy children (H). Methods Critically ill children with S (n = 16), SS (n = 15) or SIRS (n = 18) and H (n = 21) were enrolled in the study. ELISA was used to evaluate HSPs, chemiluminescence to measure ILs, and fl ow cytometry to evaluate nCD64 expression (IRB approved). mediator modulation on endothelial activation. Methods Human whole blood was stimulated with LPS (100 ng/ ml) from Escherichia coli for 4 hours. Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome p p g g p Results Nineteen controls, six SIRS patients and 25 severe sepsis patients were studied. The percent expression of HLADR on CD14+ monocytes was signifi cantly diff erent between the three groups showing progressive decrease from controls (mean 90.5 ± 3.8%) to SIRS (mean 61.2 ± 5.9%) to severe sepsis (mean 39.2 ± 5.5%) patients (controls vs. severe sepsis, P <0.001; controls vs. SIRS, P = 0.006; SIRS vs. severe sepsis, P = 0.03). hsp70 and hsp90 MFI were signifi cantly diff erent between controls (mean 49.5 ± 4.9 and 33.5 ± 3.4 respectively), SIRS (mean 69.9 ± 16.5 and 46.5 ± 5.7 respectively) and severe sepsis patients (mean 33.3 ± 4.5 and 21.7 ± 2.7 respectively) (P <0.05 for all comparisons). Notably, the hsp level rose from controls to SIRS and fell from SIRS to severe sepsis patients. APACHE score increased signifi cantly (P = 0.023) in septic patients compared with SIRS.if Introduction Heat shock proteins (HSPs) have intracellular cyto pro tec- tive actions, while they act extracellularly as inducers of cytokines and stimulants for immune cells during stress. Their induction constitutes a highly conserved cellular defense mechanism against all kinds of stress. Our objective was to determine the intracellular as well as extracellular levels of HSP72 and HSP90α in patients with severe sepsis (SS) or systemic infl ammatory response syndrome (SIRS) admitted to a general ICU, compared with those of healthy individuals; to correlate their expression with severity of illness. i Conclusion There were a signifi cant diff erence in CD14/HLADR, a marker of immune paralysis, between controls and patients with SIRS or severe sepsis. hsp70 and hsp90 showed an initial stimulation followed by exhaustion as sepsis progressed.i Methods Eighty-two consecutively admitted patients in the ICU (35 SIRS, 47 SS) as well as 35 healthy controls (H) were fi nally enrolled in the study. Patients’ demographic characteristics, laboratory examinations and Acute Physiology and Chronic Health Evaluation (APACHE II) score were recorded on admission. HSP levels were determined intracellularly using four-color fl ow cytometry. Mean fl uorescence intensity (MFI) values for each HSP were measured and analyzed. Extracellular levels of HSPs were determined via ELISA. Acknowledgements This research was co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. P42 P42 Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1, A Pistiki2, T Christodoulopoulou1, M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) P42 Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1, A Pistiki2, T Christodoulopoulou1, M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1 A Pistiki2 T Christodoulopoulou1 p p p M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) p p p M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) Results Ninety-nine patients were included in the fi nal analysis. Eighteen patients developed severe sepsis or septic shock. They presented with signifi cantly higher levels of intermediate (CD14++/16+) and CD62L– monocytes and lower IL-2 levels at T1 compared with patients who did not get septic. ROC AUC for association of these parameters with the occurrence of sepsis were 0.78 (95% CI: 0.63 to 0.91), 0.72 (0.62 to 0.82) and 0.73 (0.65 to 0.82), respectively. High counts of these monocytic cells were also associated with increased 90- day mortality (P <0.01, ROC AUC = 0.87 (0.77 to 0.95), 0.79 (0.66 to 0.9)). Kaplan–Meier survival curves showed signifi cantly higher mortality after stratifi cation based on these cell counts at T1 (CD14++CD16+: cutoff >236.8 cells/μl, HR = 23.6 (P = 1.24 × 10–5); CD62L–: cutoff >95.4 cells/μl, HR = 6.67 (P = 7.6 × 10–4)). Multivariate logistic regression analysis using Introduction CD14/HLADR is an index of immune suppression. Heat shock proteins (hsp) regulate cell response to oxidative stress. Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome This abstract is part of the study ‘Heat Shock Proteins and Glutamine Alterations Related to Hormonal, Immunological, Infl ammatory and Molecular Response to Sepsis: A Combined Clinical and Experimental Study’. Results HSP expression diff ered signifi cantly between groups (Kruskal–Wallis), both intracellularly (HSP72 lower in SS, P <0.001), and extracellularly (higher levels of HSP90α (P <0.001) and HSP72 (P = 0.003) in SS). HSP72 and HSP90α intracellular expression was inversely correlated to severity of illness, as expressed by APACHE II score (Spearman’s, P = 0.003 and P = 0.025 respectively). Intracellular HSP72 was correlated to mortality when confounding factors were excluded from the analysis (logistic regression, P = 0.05). Extracellular HSP90α levels correlated with prolonged PT (P = 0.021) and INR (P = 0.008). Finally, in the SIRS group, intracellular levels of HSP90α were higher in nonsurvivors (P <0.001). Alarming levels of heat shock proteins 72 and 90α in critically ill children Conclusion The fl ow model allows to study the eff ect of cytokine modulation on endothelial activation and to assess the interaction of activated endothelial cells with blood cells. Modulation of infl ammatory S15 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 To evaluate the %HLA-DR expression on monocytes, the fresh whole blood was stained with anti-CD14-FITC, anti-HLA-DR-PE and CD45- PC5 while staining with anti-CD33-PE, anti-CD45-PC7, anti-hsp70-FITC and anti-hsp90-PE allowed evaluation of the MFI expression of hsps on CD33+ monocytes. Cells were then analyzed using fl ow cytometry. ANOVA with post hoc tests was used to compare CD14/HLADR cell counts and hsp70 and hsp90 levels among the three groups. P41 Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome K Apostolou1, K Vardas1, E Briassouli1, K Psara1, D Goukos1, E Mageira1, S Nanas1, C Routsi1, G Briassoulis2 1Evangelismos Hospital, National and Kapodistrian University of Athens, Greece; 2University Hospital, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P41 (doi: 10.1186/cc14121) Prospective immune profi ling in critically ill adults: before, during and after severe sepsis and septic shock Introduction Rethinking the host’s defense mechanisms during severe infection has led to the use of fl ow cytometry (FCM) and to the current concept of sepsis-induced immunosuppression. However, organ dysfunctions that develop in the period preceding severe sepsis as a consequence of surgery, trauma or burn might also trigger immune reprogramming predisposing to overwhelming infection. Our aim was to look for correlation of specifi c phenotypes among four commonly encountered populations of patients and the later occurrence of severe sepsis and septic shock. g Conclusion SS is characterized by high levels of extracellular HSPs. Intracellular HSP72 is highly expressed during the acute phase of stress in SIRS, while being downregulated in SS. HSP72 and HSP90α intracellular expression and extracellular level variations correlate with severity of illness and mortality.i Acknowledgements This research was co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. p p Methods In total, 114 non-infected patients were prospectively screened via FCM on days 1 (T1) and 3 (T2) of elective cardiac surgery, trauma, acute neurologic dysfunction and prolonged ventilation (>48 hours). A third sample was drawn when infection was diagnosed (Tx) and 7 days later (Tx + 7). Exclusion criteria included use of immunosuppressive agent(s). The broad panel of cell-specifi c antibodies focused on B, T lymphocytes (Tregs, Th17, NKT), NK cells, monocytes and neutrophils. Plasmatic levels of IL-2/IL-6/IL-7/TNFα/ IFNγ were also determined.i References References 1. Briassouli E, et al. Nutrition. 2014;30:1185-94. 2. Fitrolaki DM, et al. BMC Pediatr. 2013;13:31. References 1. Briassouli E, et al. Nutrition. 2014;30:1185-94. 2. Fitrolaki DM, et al. BMC Pediatr. 2013;13:31. P42 Macrophage phenotype in sepsis immunosuppression E Theodorakis, E Diamantaki, C Tsatsanis, D Georgopoulos, K Vap University of Crete, School of Medicine, Heraklion, Greece C l C (S l ) (d / ) Macrophage phenotype in sepsis immunosuppression E Theodorakis, E Diamantaki, C Tsatsanis, D Georgopoulos, K Vaporidi University of Crete, School of Medicine, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P44 (doi: 10.1186/cc14124) expression. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Introduction Sepsis is followed by profound, yet poorly characterized, innate immune system suppression. While low monocyte HLA-DR expression is observed in septic patients, its clinical signifi cance has not been established [1]. In vitro, repeated LPS stimulation induces a tolerant or M2 macrophage phenotype, characterized by decreased cytokine production [2], which could contribute to sepsis immunosuppression. The present study examines macrophage phenotype in a mouse model and in patients with sepsis immunosuppression. p p pp Methods Sepsis was induced in C57Bl6 mice by cecal ligation and puncture (CLP) followed by intratracheal instillation of Pseudomonas aeruginosa. Bronchoalveolar lavage fl uid (BALF), cells and serum, collected 12 hours after lung infection, were analyzed for bacterial load, cytokine levels and the classical M1 marker, iNOS. Peripheral blood monocytes isolated from septic adult patients admitted to the ICU on the 1st and 7th day after admission were analyzed by fl ow cytometry for the expression of HLA-DR and CD86 (co-stimulatory molecule and M1 marker), and for the M2 markers, CD163 and CD206. Additional blood samples from patients and healthy volunteers were exposed ex vivo to LPS prior to isolation and analysis of monocyte markers. p y y Results CLP-induced sepsis resulted in immunosuppression in mice, indicated by higher BALF bacterial load after infection in CLP than in sham-operated mice, and more severe injury on histology. Serum cytokines TNF and MIP2 were greater in CLP than in sham-operated mice. Although recruitment of CD11c+ alveolar macrophages post infection was threefold greater in CLP than in sham-operated mice, those macrophages expressed 40% lower levels of iNOS. Evidence of sepsis immunosuppression was present in most patients on the 7th day after ICU admission. P42 We evaluated the relationship of CD14/HLADR and hsp70/90 in patients with SIRS and severe sepsis versus healthy volunteers. Methods We evaluated 31 patients with SIRS or severe sepsis against a group of sex-matched healthy volunteers. Demographic data were obtained for all patients. APACHE score was calculated upon admission. Blood samples were collected upon diagnosis of SIRS or severe sepsis. S16 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 clinical scores indicated that addition of IL-2 levels at T1 signifi cantly improved prediction of sepsis (OR = 0.834, P = 0.02). Conclusion Predisposition to sepsis in selected critically ill medico- surgical adults can be identifi ed on day 1 of admission based on high counts of circulating intermediate and CD62L– monocytes and low levels of IL-2 (the latter provide incremental prognostic information). High counts of these specifi c monocytes correlate with higher 90-day mortality. clinical scores indicated that addition of IL-2 levels at T1 signifi cantly improved prediction of sepsis (OR = 0.834, P = 0.02). that septic patients display a strong correlation between mHLA-DR and mRNA-levels of HLA-DRA in whole blood [1]. mRNA-based HLA- DR monitoring by PCR would improve the clinical usage and facilitate conduction of multicentre studies. The primary focus in this study was to evaluate the correlation between mHLA-DR and HLA-DRA at diff erent time points during sepsis. In addition, we assessed the dynamic expression of both mHLA-DR and HLA-DRA, in relation to sepsis severity. Conclusion Predisposition to sepsis in selected critically ill medico- surgical adults can be identifi ed on day 1 of admission based on high counts of circulating intermediate and CD62L– monocytes and low levels of IL-2 (the latter provide incremental prognostic information). High counts of these specifi c monocytes correlate with higher 90-day mortality. y Methods Study patients (n = 54) were included at day 1 to 2 after hospital admission if blood cultures turned positive. Repeated sampling at days 1 to 2, 3, 7, 14 and 28 was performed. mHLA-DR was monitored by FCM and HLA-DRA by quantitative RT-PCR. Mixed models for longitudinal data were used after logarithmic transformation to calculate the interactional eff ects of time and severity on HLA-DR expression. P42 Low expression of CD86 and/or HLA-DR was observed in 71% of patients, and increased expression of M2 markers in 15% of patients. Upon LPS stimulation the normal decrease in M2 markers was absent in all patients on day 1, and partially restored in 50% of patients on day 7. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Conclusion Sepsis is associated with decreased monocyte expression of M1 markers and increased expression of M2 markers in septic mice and critically ill patients. Therefore, in addition to decreased HLA-DR expression, M2 macrophage polarization appears to be a component of sepsis-induced monocyte dysfunction, and should be considered for immune monitoring and targeted intervention. g g Acknowledgement Supported by GSRT research grant ExcellenceII-4620. R f References 1. Gomez H, et al. Crit Care Med. 2014;42:771. 2. Porta C, et al. Proc Natl Acad Sci U S A. 2009;106:14978. 2. Porta C, et al. Proc Natl Acad Sci U S A. 2009;106:14978. P45 Expression of mRNA levels of HLA-DRA in relation to monocyte HLA-DR: a longitudinal sepsis study SC Cajander, ET Tina, AB Bäckman, AM Magnuson, KS Strålin, BS Söderquist, JK Källman Örebro University, Örebro, Sweden Critical Care 2015, 19(Suppl 1):P45 (doi: 10.1186/cc14125) Expression of mRNA levels of HLA-DRA in relation to monocyte HLA-DR: a longitudinal sepsis study SC Cajander, ET Tina, AB Bäckman, AM Magnuson, KS Strålin, BS Söderquist, JK Källman Örebro University, Örebro, Sweden Critical Care 2015, 19(Suppl 1):P45 (doi: 10.1186/cc14125) Introduction Decreased monocyte surface HLA-DR (mHLA-DR) measured by fl ow cytometry (FCM) is an independent marker of immunosuppression in sepsis. In a previous report we demonstrated S17 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Correlation between mHLA-DR(FCM) and HLA-DRA(PCR) at day 1 to 2 (R = 0.78) and day 14 (R = 0.27). Both HLA-DR markers increased linearly on a log scale over time. The linear association was signifi cantly diff erent between the severe (n = 16) and nonsevere septic patients (n = 38) when measuring either mHLA-DR(FCM) or HLA-DRA(PCR). By pairwise comparison of means between the two severity groups, at every time point, the diff erences between groups were shown to be signifi cant at days 1 to 2 and 3 when monitoring mHLA-DR(FCM) and at days 1 to 2, 3 and 7 for HLA-DRA(PCR) (Figures 1 and 2).l used as a diagnostic criterion of sepsis. There are no published data to defi ne the role of dynamic control of EC in the process of intensive therapy as a prognostic marker and indicator of severity condition in critically ill patients. The aim was to determine the informative value of EC in the development of SIRS as a biomarker of sepsis and indicator of the severity condition and prognosis of outcome in the pathological process. Methods A total of 143 patients were enrolled in this study who were admitted to the ICU and had SIRS. All patients were divided into a septic group – patients with community-acquired pneumonia, complicated by sepsis – and two SIRS groups of noninfectious genesis – patients who had an acute cerebrovascular accident (CVA) and an acute myocardial infarction (AMI). The absolute EC was measured at admission and in the dynamics on days 3 to 5 of stay. HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) y Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) Introduction The aim of the present study was to evaluate the prognostic value of HLA-DR antigen expression in monocytes, in patients with community-acquired infections presenting with fever, as possible markers for the patients’ fi nal outcome. Table 1 (abstract P47). Dynamics of eosinophil count depending on outcome in groups i Methods A total of 81 patients (males = 46; females = 35) presenting with fever >38°C to the emergency room (ER) of the Department of Internal Medicine of the Patras University Hospital were enrolled in the study during a period of 12 months. Sera for monocyte HLA-DR expression were obtained from the patients on admission (day 1) and on days 3, 7 or discharge/death. Results were expressed as percentages of HLA-DR-positive monocytes, calculated by the coexpression of CD14 and HLA-DR antigens in the total CD14+ population. Additionally, the patients were evaluated using the Simplifi ed Acute Physiology Score (SAPS-II), the Sequential Organ Failure Assessment (SOFA) and the Mortality in Emergency Department Sepsis (MEDS) score on the same days while all the indicated clinical, laboratory and imaging procedures as required for fever’s diff erential diagnosis were followed. A questionnaire regarding demographic characteristics, comorbidities, medications used and patients’ survival was also completed. All statistical analyses were performed using SPSS v.21. Table 2 (abstract P47). Informational value of eosinophil count in assessment of disease outcome Results Lower mean HLA-DR monocyte antigen expression percen- tages were signifi cantly correlated to lower Glasgow Scale scores on all days of measurement. HLA-DR expression was signifi cantly negatively correlated to MEDS, SOFA and SAPS-II scores whereas patients who developed sepsis, severe sepsis, septic shock and MODS had signifi cantly lower HLA-DR values compared with the ones who did not. HLA-DR expression on day 1 was lower in patients who would develop SIRS and/or sepsis on days 3 and 7 (P <0.01). Additionally, HLA- DR expression was signifi cantly decreased in nonsurvivors (n = 33) compared with survivors (n = 48), whereas lower HLA-DR expression was correlated to longest duration of hospital stay at all time points (P <0.01). Conclusion EC may be an additional diagnostic marker which characterizes the nature of SIRS. Eosinopenia associated with prognosis of outcome in critical conditions. P45 y g Conclusion The correlation between fl ow cytometry and PCR-based HLA-DR monitoring is stronger in the early phase of sepsis. However, the linear associations over time, in relation to sepsis severity, display similar results for both HLA-DR markers. HLA-DRA(PCR) as a biomarker could be an alternative approach in monitoring immune status in sepsis but needs to be evaluated in relation to clinically relevant immunosuppression. Reference y y y Results The median EC was 75 cells/mm3 in septic patients on admission, which was signifi cantly lower than in patients with CVA (120 cells/mm3) and AMI (130 cells/mm3). Comparison of EC in septic patients between survivors and those who died showed signifi cant diff erences (Table 1). Receiver operating characteristic (ROC) analysis determined a value less than 80 cells/mm3 as the optimal diagnostic cutoff value with a high level of confi dence in the comparison of septic and noninfectious groups. Area under the ROC curves was 0.94, sensitivity of 80.8%, specifi city of 95.6%, P <0.0001. There was a signifi cant increase of EC in survivors, while the EC did not change signifi cantly among those who died in the dynamics. ROC analysis determined the cutoff values of EC, which indicated a high risk of an adverse outcome in septic patients (Table 2). 1. Cajander S, et al. Crit Care. 2013;17:R223. 1. Cajander S, et al. Crit Care. 2013;17:R223. HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) Conclusion Monocyte HLA-DR appears to be an early indicator for survival and infection progression and therefore it can be used as a predictive marker for the fi nal outcome of patients presenting in ER departments with fever. P48 Prevalence and clinical signifi cance of early endotoxin activity in septic shock patients M Bottiroli1, R Pinciroli1, G Monti2, M Mininni1, G Casella2, R Fumagalli2 1Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy; 2Anestesia Rianimazione 1, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P48 (doi: 10.1186/cc14128) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) Introduction The endotoxin activity (EA) assay is a useful test to risk stratify critically ill patients and assess for Gram-negative (GN) infection. However, the prevalence and signifi cance of early high levels of EA in patients with septic shock (SS) has yet to be elucidated. Introduction The idea of using the eosinophil count (EC) as a diagnostic marker for clarifying the nature of systemic infl ammatory response syndrome (SIRS) belongs to K Abidi, who showed that EC could be Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S18 Figure 1 (abstract P48). patients with systemic infl ammatory response syndrome (SIRS) and organ failure in ICU setting. Methods In total, 76 patients with SIRS and organ failure or who were suspected of sepsis during critical care were included. According to the levels of EAA, all patients were classifi ed into three groups (group L, EAA <0.4; group M, EAA ≥0.4 or EAA <0.6; group H, EAA ≥0.6). In order to evaluate the severity of illness, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score and catecholamine (CA) index were recorded. Blood samples were obtained to measure EAA levels, infl ammatory markers (procalcitonin, C-reactive protein and white blood cells), serum lactate level as an indicator of tissue hypoxia, and for blood culture. All patients were followed up for 6 months. APACHE II score, SOFA score, CA index, infl ammatory markers, serum lactate levels and blood culture results were examined for diagnosis of sepsis, severe sepsis, septic shock and for prognosis of 30-day mortality. Each value was also compared with EAA levels. Results Patient age was 69 ± 9.9 years (male: n = 48, female: n = 25). The total number of samples was 106 (group L/group M/group H: 35/35/36). Twenty-seven specimens were obtained from nonseptic patients and 83 specimens were obtained from septic patients. APACHE II score was highly correlated with SOFA score (P <0.05). In group H, the APACHE II score was signifi cantly higher (22.2 ± 0.8) than that in group M (18.4 ± 0.87) (P = 0.01). 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) Results A total of 107 consecutive patients were included. The overall median EA was 0.56 (0.44 to 0.71), with 46/107 (43%) patients testing positive for elevated EA (≥0.6). GN species were identifi ed in microbial cultures as the infective etiology in 49/107 (46%) patients, of which 28 (57%) developed bacteremia. GN infections were associated with higher levels of EA compared with other microbial causatives (0.61 (0.52 to 0.77) vs. 0.52 (0.38 to 0.64), P = 0.021). Patients with EA ≥0.6 showed signifi cantly higher lactate levels (2 (1 to 3) vs. 3.8 (1.7 to 6.4), P = 0.01), Sequential Organ Failure Assessment (9 (6 to 12) vs. 10 (8 to 14), P = 0.04) and inotropic score (20 (5 to 50) vs. 50 (16 to 100), P = 0.003) at inclusion. See Figure 1. Introduction Sepsis is a common reason for admission to ICUs throughout the world. During the past two decades, the incidence of sepsis in the USA has tripled and is now the 10th leading cause of death. As sepsis continues to impact negatively on critically ill patients, it is clear that early diagnosis and eff ective management could improve patient morbidity and mortality. Numerous studies have attempted to examine biomarkers and their ability to diagnose and prognosticate septic patients. Despite multiple eff orts, currently there are no reliable markers that can eff ectively improve our clinical eff ectiveness in diagnosing and managing septic patients. The purpose of our systematic review was to evaluate the diagnostic and prognostic value of various biomarkers used in septic patients. g Conclusion Elevated EA is a common fi nding in SS patients. In patients developing SS from a GN infection, higher levels of endotoxin activity could be measured within 24 hours. Furthermore, in our study, EA ≥0.6 identifi ed a subgroup of subjects at greater risk for worse clinical outcomes. We therefore propose use of the EA assay for the early identifi cation and risk stratifi cation of SS patients. Methods A systematic search of the literature was performed with MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials databases using terminology selected for biomarkers (through to and including November 2013). All articles involving neonates and not in English were excluded. Inclusion was agreed on by two independent reviewers of abstracts or full text. Biomarkers in sepsis: a systematic review Biomarkers in sepsis: a systematic review F Morriello1, J Marshall2 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) The SOFA score in group H was signifi cantly higher (9.9 ± 0.5) than that in group M (7.5 ± 0.6) and group L (7.9 ± 0.6) (P = 0.006). EAA levels were signifi cantly increased in septic patients (septic patients: 0.56 ±0.03, nonseptic patients: 0.42 ±0.05) (P = 0.011) and in the positive blood culture group (positive group: 0.66 ± 0.05, negative group: 0.48 ± 0.03) (P = 0.006). There was no relationship between EAA levels and other infl ammation markers or 30-day mortality. Conclusion In patients with suspected sepsis and positive blood culture, EAA levels were signifi cantly increased and had strong correlation with severity of disease. This result suggests that EAA indicates the state of sepsis regardless of the possibility of infection in patients with SIRS with organ failure. Figure 1 (abstract P48). Figure 1 (abstract P48). Methods We designed a prospective observational study including adult patients with clinically diagnosed SS. EA was measured on arterial blood by a chemiluminescent assay within the fi rst 24 hours from SS diagnosis. The fi nding of an EA value ≥0.6 was used as the cutoff for test positivity, as described elsewhere. In addition, laboratory, microbiological and clinical data were collected at inclusion. In-hospital follow-up was also conducted. Biomarkers in sepsis: a systematic review F Morriello1, J Marshall2 F Morriello1, J Marshall2 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) C-reactive protein and hemogram parameters for the nonsepsis SIRS and sepsis: what do they mean? p y B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) y gi Results Reduced BChE activity correlated with trauma severity and the resulting systemic infl ammation. Compared with serum levels of routinely measured infl ammatory biomarkers, changes in the BChE activity were detected signifi cantly earlier, suggesting that the BChE activity might serve as an early indicator of systemic infl ammation. yi Results Reduced BChE activity correlated with trauma severity and the resulting systemic infl ammation. Compared with serum levels of routinely measured infl ammatory biomarkers, changes in the BChE activity were detected signifi cantly earlier, suggesting that the BChE activity might serve as an early indicator of systemic infl ammation. Conclusion Our results suggest that BChE activity, measured using a POCT system, might play an important role in the early diagnosis of trauma-induced systemic infl ammation. Introduction The aim of this study was to investigate the laboratory parameters as an indicator of sepsis. Sepsis is one of the most common reasons of mortality and morbidity in the ICU [1]. Thus, it is important to distinguish sepsis from nonsepsis SIRS. CRP and hemogram parameters may be fast, easy and aff ordable alternatives in distinguishing sepsis from nonsepsis SIRS. Eosinophil count (EoC), lymphocyte count (LymC) and neutrophil–lymphocyte count ratio (NLCR) are used as sepsis indicators [1,2]. Conclusion Our results suggest that BChE activity, measured using a POCT system, might play an important role in the early diagnosis of trauma-induced systemic infl ammation. P53 Association between apoptosis and mortality in severe septic patients In the fully adjusted model, C C C C C d C d h d l L Lorente1, M Martín2, A González-Rivero1, J Ferreres3, J Solé-Violán4, L Labarta5, C Díaz6, A Jiménez1, J Borreguero-León1 1Hospital Universitario de Canarias, La Laguna, Tenerife, Spain; 2Hospital Universitario Nuestra Señora Candelaria, Santa Cruz, Tenerife, Spain; 3Hospital Clínico Universitario de Valencia, Spain; 4Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain; 5Hospital San Jorge, Huesca, Spain; 6Hospital Insular, Las Palmas de Gran Canaria, Spain Critical Care 2015, 19(Suppl 1):P53 (doi: 10.1186/cc14133) Introduction The apoptotic process, in which cells are actively elimi- nated by a programmed pathway, is increased in sepsis. Extrinsic and intrinsic apoptotic death cell pathways activate caspase-3, which leads to cell apoptosis. Cytokeratin 18 (CK-18), a protein present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK)-18 during apoptotic death. The novel objectives of this study were to determine whether there are associations between serum caspase-3 levels, serum CK-18 levels and mortality in septic patients. WBC, CRP, LymC, NeuC, NLCR and EoC were entered into the model. Results A total of 1,302 patients were categorized as nonsepsis SIRS (816, 62.7%) and sepsis (486, 37.3%). In the sepsis group, age, APACHE II, SOFA, mortality, length of ICU stay, CRP, NLCR and EoC were higher; LymC was lower than in the nonsepsis SIRS group (P <0.001 for each). Likelihood of sepsis (reference to nonsepsis SIRS) increased 2.62 (2.05 to 3.34), 2.02 (1.42 to 2.88) and 1.88 (1.36 to 2.60) times (OR (95% CI)) by the values of CRP >4.4 mg/dl, LmyC <500/mm3 and NLCR >15.7 respectively in mutually adjusted multivariate logistic regression (P <0.001 for each). y p p Methods A prospective, multicenter, observational study in six Spanish ICUs, including 216 patients with severe sepsis. We collected blood samples at the severe sepsis diagnosis moment to determine serum levels of caspase-3 (to assess the main executor of apoptosis) and CCCK- 18 (to assess the apoptosis level). The endpoint was 30-day mortality. Results We found that nonsurvivor (n = 76) in comparison with survivor (n = 140) septic patients showed higher serum levels of caspase-3 (0.41 ng/ml (0.14 to 0.52) vs. 0.11 ng/ml (0.10 to 0.25); P <0.001) and CCCK- 18 (448 (310 to 723) vs. 319 (236 to 445); P <0.001). P53 Association between apoptosis and mortality in severe septic patients Methods A total of 2,777 patients admitted to the ICU of two centers between 2006 and 2013 were evaluated retrospectively. The patients were diagnosed as SIRS(–), nonsepsis SIRS or sepsis at ICU admission by the consensus of two doctors in accordance with 1992 sepsis guidelines [3]. The patients who were under 18 years old, readmitted, immunosuppressive, SIRS(–) and whose laboratory values and outcomes were unknown were excluded. In total, 1,302 patients were divided into two groups as the nonsepsis SIRS group and the sepsis group. The patient’s age, gender, diagnoses (medical, elective and urgent surgery), APACHE II, SOFA, CRP, WBC, neutrophil count (NeuC), LymC, NLCR, EoC, platelet, mean platelet volume, length of ICU stay and mortality were recorded by a third doctor. In the fully adjusted model, WBC, CRP, LymC, NeuC, NLCR and EoC were entered into the model. Results A total of 1,302 patients were categorized as nonsepsis SIRS (816, 62.7%) and sepsis (486, 37.3%). In the sepsis group, age, APACHE II, SOFA, mortality, length of ICU stay, CRP, NLCR and EoC were higher; LymC was lower than in the nonsepsis SIRS group (P <0.001 for each). Likelihood of sepsis (reference to nonsepsis SIRS) increased 2.62 (2.05 to 3.34), 2.02 (1.42 to 2.88) and 1.88 (1.36 to 2.60) times (OR (95% CI)) by the values of CRP >4.4 mg/dl, LmyC <500/mm3 and NLCR >15.7 respectively in mutually adjusted multivariate logistic regression (P <0.001 for each). Methods A total of 2,777 patients admitted to the ICU of two centers between 2006 and 2013 were evaluated retrospectively. The patients were diagnosed as SIRS(–), nonsepsis SIRS or sepsis at ICU admission by the consensus of two doctors in accordance with 1992 sepsis guidelines [3]. The patients who were under 18 years old, readmitted, immunosuppressive, SIRS(–) and whose laboratory values and outcomes were unknown were excluded. In total, 1,302 patients were divided into two groups as the nonsepsis SIRS group and the sepsis group. The patient’s age, gender, diagnoses (medical, elective and urgent surgery), APACHE II, SOFA, CRP, WBC, neutrophil count (NeuC), LymC, NLCR, EoC, platelet, mean platelet volume, length of ICU stay and mortality were recorded by a third doctor. P52 P52 Serum cholinesterase activity as an early indicator of systemic infl ammation References 1. Abidi K, et al. Crit Care. 2008;2:R59. 2. Castelli GP, et al. Minerva Anestesiol. 2006;1-2:69-80. 3. Bone RC, et al. Chest. 1992;101:1644-55. P53 Association between apoptosis and mortality in severe septic patients Multiple logistic regression showed that serum caspase-3 levels >0.25 ng/ml were associated with mortality at 30 days (odds ratio = 6.51; 95% confi dence interval = 3.32 to 12.77; P <0.001), controlling for SOFA score and age. Kaplan–Meier survival analysis showed a higher risk of death in septic patients with serum caspase-3 levels >0.25 ng/ml than in patients with lower levels (hazard ratio = 3.80; 95% CI = 2.35 to 6.15; P <0.001). We found a positive association between serum levels of caspase-3 and CCCK-18 (ρ = 0.32; P <0.001).i Methods A prospective, multicenter, observational study in six Spanish ICUs, including 216 patients with severe sepsis. We collected blood samples at the severe sepsis diagnosis moment to determine serum levels of caspase-3 (to assess the main executor of apoptosis) and CCCK- 18 (to assess the apoptosis level). The endpoint was 30-day mortality. Conclusion CRP, LymC and NLCR may distinguish sepsis from nonsepsis SIRS. Thus, CRP and hemogram parameters may contribute to early diagnosis of sepsis. Results We found that nonsurvivor (n = 76) in comparison with survivor (n = 140) septic patients showed higher serum levels of caspase-3 (0.41 ng/ml (0.14 to 0.52) vs. 0.11 ng/ml (0.10 to 0.25); P <0.001) and CCCK- 18 (448 (310 to 723) vs. 319 (236 to 445); P <0.001). Multiple logistic regression showed that serum caspase-3 levels >0.25 ng/ml were associated with mortality at 30 days (odds ratio = 6.51; 95% confi dence interval = 3.32 to 12.77; P <0.001), controlling for SOFA score and age. Kaplan–Meier survival analysis showed a higher risk of death in septic patients with serum caspase-3 levels >0.25 ng/ml than in patients with lower levels (hazard ratio = 3.80; 95% CI = 2.35 to 6.15; P <0.001). We found a positive association between serum levels of caspase-3 and CCCK-18 (ρ = 0.32; P <0.001). P51 P51 C-reactive protein and hemogram parameters for the nonsepsis SIRS and sepsis: what do they mean? B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) Assessment was based on the biomarker’s ability to diagnose septic patients and its ability to predict mortality.i P49 Usefulness of endotoxin activity assay for early diagnosis of sepsis S Sekine, H Imaizumi, K Masumoto, H Uchino Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P49 (doi: 10.1186/cc14129) Results Of 5,257 articles identifi ed, all abstracts were screened, and 750 full-text articles were selected for review. These included primarily randomized controlled trials, cohort studies and postmortem studies. Of 49 biomarkers examined, 72% of the studies examined procalcitonin. Comparing the serum of septic patients with that of controls, most biomarkers were elevated in septic patients, even though only a few had high sensitivity (>85%) and high specifi city (>80%). It was often Introduction The purpose of this study was to evaluate the diagnostic and prognostic value of the endotoxin activity assay (EAA) of sepsis in S19 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 an important role in the infl ammatory response. Serum cholinesterase (butyrylcholinesterase (BChE)) is the major Ach hydrolyzing enzyme in plasma. The role of this enzyme during infl ammation has not yet been fully understood. Here, we describe a correlation between the BChE activity and the early systemic infl ammatory response upon traumatic injury. diffi cult to compare study group with control group as the control group patients were usually not healthy controls. Conclusion Overall the heterogeneity of studies, small sample size and the lack of true healthy controls infl uenced the ability to use the biomarker for prognostication of a septic patient. Furthermore, the lack of healthy control raises the question of redefi ning selection criteria in order to better study septic patients. Methods We measured BChE activity in patients with traumatic injury admitted to the emergency room using a point-of-care-test (POCT) system. In addition, we measured levels of routine infl ammation biomarkers during the initial treatment period. We used the Injury Severity Score to assess the trauma severity. Data were statistically analyzed using the Friedman test. Correlation analysis was performed using Spearman’s rank correlation test. P <0.05 was considered statistically signifi cant. P56 P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial A Kutz1, P Hausfater2, M Oppert3, C Alonso4, C Wissmann4, B Mueller1, P Schuetz1 1Kantonsspital Aarau, Switzerland; 2Hôpital Pitié-Salpêtrière and Univ- Paris 06, Paris, France; 3Klinikum Ernst von Bergmann, Potsdam, Germany; 4BRAHMS GmbH, Hennigsdorf, Germany Critical Care 2015, 19(Suppl 1):P56 (doi: 10.1186/cc14136) P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial A Kutz1, P Hausfater2, M Oppert3, C Alonso4, C Wissmann4, B Mueller1, P Schuetz1 P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial Results We included 87 patients (32 Group A, 25 Group B, 30 Group C). The serum concentration of S1P (mean ± SD) in the control group was 484.6 ± 152.6 μg/l and signifi cantly higher compared with Group A 239.4 ± 61.3 μg/l, Group B 248.6 ± 93.7 μg/l and Group C 141.6 ± 46.3 μg/l. We observed a negative correlation between S1P and SOFA score (Pearson r = –0.45, P <0.001, R2 = 0.2). The median SOFA score in our cohort was 6. We divided the cohort into two groups: SOFA score <6; and SOFA score >6. We tested the sensitivity and specifi city of S1P to indicate disease severity by ROC analysis. In our cohort the area under the curve (AUC) for S1P was 0.77 (CI 0.670 to 0.870) and therefore higher when compared with common markers of infl ammation (PCT, IL-6, CRP with AUC of 0.68 (0.560 to 0.796), 0.68 (0.554 to 0.786) and 0.67 (0.571 to 0.794), resp.).i 1Kantonsspital Aarau, Switzerland; 2Hôpital Pitié-Salpêtrière and Univ- Paris 06, Paris, France; 3Klinikum Ernst von Bergmann, Potsdam, Germany; 4BRAHMS GmbH, Hennigsdorf, Germany Critical Care 2015, 19(Suppl 1):P56 (doi: 10.1186/cc14136) Introduction Procalcitonin (PCT) is increasingly the standard in the emergency department (ED) for the diagnostic and prognostic workup of patients with suspected infections. Recently, B·R·A·H·M·S PCT direct, a new high-sensitive point-of-care test, has been developed for fast PCT measurement on capillary or venous blood samples with a measuring range of 0.1 to 10.0 μg/l. Methods This is a prospective, comparative international study conducted in three European EDs. P56 Consecutive patients with suspicion of bacterial infection were included. Duplicate determination of PCT was performed on two distinct B·R·A·H·M·S PCT direct test devices on capillary (fi ngertip) and venous whole blood (EDTA), and compared with the reference method (B·R·A·H·M·S PCT sensitive Kryptor or Elecsys B·R·A·H·M·S PCT, respectively). The diagnostic accuracy was evaluated by correlation and concordance analyses. Conclusion Our fi ndings suggest that S1P is a novel marker for severity of sepsis with severe sepsis and septic shock being associated with low levels of S1P. Moreover, blood concentrations of S1P might play a key role in sepsis pathophysiology. y gy Acknowledgements MSW and AN are equal contributors. Reference 1. Maceyka M, et al. Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol. 2012;22:50-60. y y Results A total of 303 patients were included over a 6-month period (60.4% male, median age 65.2 years). The correlation between capillary or venous whole blood and the reference method was excellent: r2 = 0.96 and 0.97, sensitivity 88.1% and 93.0%, specifi city 96.5% and 96.8%, concordance 93% and 95% respectively at a 0.25 μg/l threshold. No signifi cant bias was observed (–0.04 and –0.02 for capillary and venous whole blood) although there were 6.8% and 5.1% outliers, respectively. B·R·A·H·M·S PCT direct had a shorter time to result as compared with the reference method (25 vs. 147 minutes, diff erence 122 minutes, 95% CI = 110 to 134 minutes, P <0.0001). P55 Diagnostic accuracy and clinical relevance of an infl ammatory biomarker panel in early sepsis in adult critical care patients P Bauer, R Kashyap, S League, J Park, D Block, N Baumann, A Algeciras-Schimnich, S Jenkins, C Smith, O Gajic, R Abraham Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P55 (doi: 10.1186/cc14135) Diagnostic accuracy and clinical relevance of an infl ammatory biomarker panel in early sepsis in adult critical care patients Introduction Low awareness, late recognition and delayed treatment of sepsis are still common. CD64 is a marker of the innate immune response upregulated in sepsis. The primary goal of this prospective, double-blind study was to compare the diagnostic accuracy of neutrophil CD64 and other cellular markers, along with C-reactive protein (CRP) and procalcitonin (PCT) levels, in early sepsis. Conclusion This study found a high diagnostic accuracy and a faster time to result of the PCT direct in the ED setting. The B·R·A·H·M·S PCT direct may allow a more widespread use of PCT tests in outpatient clinics and smaller institutions. Methods Adult ICU patients, between 2012 and 2014 were eligible. The eight-color fl ow cytometric biomarker panel included CD64, CD163, HLA DR, CD15 and others. Diagnostic test results were compared with infection as the reference standard and sepsis as the target condition, using receiver operating characteristic curve analyses. Multivariable logistic regression was used to assess the relationship of sets of markers with the probability of sepsis, adjusting for other patient characteristics. Results A total of 219 patients were enrolled, 120 with sepsis, 99 served as controls. APACHE IV (median 70 vs. 57), SOFA (8 vs. 7), ICU (2 vs. 1) and hospital length of stay (6 vs. 4) were higher in the sepsis group. Serum cholinesterase activity as an early indicator of systemic infl ammation l AR Zivkovic, K Schmidt, J Bender, T Brenner, S Hofer Heidelberg University Hospital, Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P52 (doi: 10.1186/cc14132) l AR Zivkovic, K Schmidt, J Bender, T Brenner, S Hofer Heidelberg University Hospital, Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P52 (doi: 10.1186/cc14132) Conclusion The novel fi ndings of our study were that there is an association between serum caspase-3 levels, serum CK-18 levels and mortality in septic patients. There has been reported decreased apoptosis and increased survival in septic rats with the administration of caspase inhibitors; thus, it may be interesting to explore those agents in septic patients. Introduction Early diagnosis of systemic infl ammation, a generalised response to noxious stimuli, is fundamentally important for eff ective and goal-directed therapy. Various infl ammation biomarkers have been used in clinical and experimental practice. However, a defi nitive diagnostic tool for an early detection of systemic infl ammation remains to be identifi ed. Acetylcholine (Ach) has been shown to play Acknowledgements Funded by Grant FIS-PI-14-00220 from Instituto de Salud Carlos III (Madrid, Spain) and co-fi nanced by Fondo Europeo de Desarrollo Regional (FEDER). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S20 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Mortality was not diff erent. After adjustment for APACHE IV, CRP and PCT, CD64 molecules/neutrophil measure remained a signifi cant predictor of sepsis (OR = 1.852 for one-unit increase on the log scale, 95% CI = 1.083 to 3.168, P = 0.02, AUC = 0.90). See Table 1. P54 Sphingosine-1-phosphate is a new biomarker for severity in human sepsis MS Winkler1, A Nierhaus1, E Mudersbach1, M Holzmann1, A Bauer1, L Robbe1, C Zahrte1, G Daum2, S Kluge1, C Zoellner1 1University Medical Center Eppendorf, Hamburg, Germany; 2University Heart Center, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P54 (doi: 10.1186/cc14134) Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Cutoff for Sensitivity Specifi city Measure N AUC sepsis (%) (%) C-reactive protein (mg/l) 208 0.86 43 76.9 76.9 CD64 molecules/neutrophil 196 0.83 1,040.5 76.4 76.7 Procalcitonin (ng/ml) 216 0.82 0.74 73.1 73.2 %CD64+ neutrophils 196 0.81 49.96 74.5 74.4 Conclusion Neutrophil CD64 expression is an accurate predictor of early sepsis. Measure Introduction During sepsis a leading symptom is capillary leakage caused by endothelial damage, followed by multiorgan dysfunction. Sphingosine-1-phosphate (S1P) is a bioactive lipid with multiple functions. Cellular reactions depend on the S1P concentration in the blood and its binding to fi ve specifi c G-protein coupled receptors. S1P-regulated functions include: control of endothelial permeability; lymphocyte migration across microvessels depending on an S1P gradient; and control of vascular tone [1]. This clinical study will address the question of whether S1P blood concentrations are associated with sepsis severity.i p y Methods Following ethical approval we enrolled patients fulfi lling the ACCP/SCCM sepsis criteria into three groups (Group A: sepsis; Group B: severe sepsis; Group C: septic shock). A group of 20 healthy donors served as controls. Serum blood samples, laboratory data and clinical parameters are presented for day 1. The primary outcome variable was serum S1P concentration (μg/l) quantifi ed by mass spectrometry (Agilent®). The SOFA score was used to describe disease severity. Serum procalcitonin level correlates with endotoxin activity in patients with septic shock Serum procalcitonin level correlates with endotoxin activity in patients with septic shock B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) Introduction Endotoxin, a key component on the outer membrane of Gram-negative bacteria, is considered to be the most important toxin Introduction Endotoxin, a key component on the outer membrane of Gram-negative bacteria, is considered to be the most important toxin S21 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Procalcitonin as prognostic marker in severe sepsis of abdominal origin g A Gonzalez-Lisorge1, C Garcia-Palenciano1, G Ercole1, T Sansano-Sanchez1, M Campos Aranda2, F Acosta Villegas1 1Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 2Universidad de Murcia, Murcia, Spain Critical Care 2015, 19(Suppl 1):P58 (doi: 10.1186/cc14138) Introduction We evaluated the utility of procalcitonin (PCT) as a marker of outcome in severe sepsis of abdominal origin (SSAO). SSAO is one of the most prevalent pathologies in surgical ICUs (sICUs). Mortality from 19 to 70% has been reported. Biomarkers are basic tools for diagnosis, follow-up and outcome of sepsis. One of the most studied in last decades has been PCT. Its levels and kinetics could be useful to evaluate the outcome of septic patients. Conclusion Both biomarkers, procalcitonin and lactate provide diagnostic and prognostic information in ICU patients with sepsis, particularly when looking at biomarker kinetics. An evidence- based protocol incorporating both markers may further improve management of septic patients. p p Methods We studied all patients admitted to a sICU with SSAO, from 2007 to 2008. Data collected were: PCT levels on days 1, 3 and 7, gender, age, APACHE II on admission, positivity of surgical cultures, microorganisms isolated and sepsis origin (community or nosocomial). Results Sixty-nine patients were included. Mortality was 23%. Median age was 64.94 years. Median APACHE II was 16.43 points. At day 1, PCT levels were higher in survivors (S) than in exitus (E) (S: 29.22 ng/ ml vs. E: 14.93 ng/ml, P <0.05). PCT levels were infl uenced by gender (males: 27.74 ng/ml vs. females: 15.04 ng/ml, P <0.05), positive cultures (positive: 25.25 ng/ml vs. negative: 13.49 ng/ml, P <0.05) and isolation of Gram-negative microorganisms (Gram-negative: 27.53 ng/ml vs. Gram-positive: 14.77 ng/ml, P <0.05). Patients with community- acquired sepsis had higher levels of PCT on admission (37.53 ng/ml vs. 13.29 ng/ml, P <0.02). None of these factors had an infl uence on mortality. On day 3 PCT levels where higher in S (S: 20.65 ng/ml vs. E: 16.23 ng/ml, P <0.05). On day 7 PCT levels were higher in E (S: 3.54 ng/ ml vs. E: 12.88 ng/ml, P <0.05). PCT kinetics was diff erent depending on outcome. E patients presented persistently higher levels, whereas PCT in S decreased over time (P <0.05). PCT on day 7 best identifi ed outcome (AUC ROC 0.768). PCT ≥3.5 ng/ml predicted mortality (sensitivity 55%, specifi city 73%). References involved in the development of septic shock, and procalcitonin (PCT) serum concentration has been strongly associated with the severity of sepsis. The eff ect of elevated endotoxin activity (EA) on PCT serum level, organ function and mortality of patients with septic shock was evaluated on the day of admission to the ICU. 1. Charles PE, et al. BMC Infect Dis. 2008;8:38. 2. Rau BM, et al. Arch Surg. 2007;142:134-42. 3. Dahaba AA, et al. Minerva Anestesiol. 2009;75:447-52. 4. Karlsson S, et al. Crit Care. 2010;14:R205. Methods ICU patients with diagnosis of septic shock were consecutively added to the study group within the fi rst 24 hours. Serum PCT level and whole blood EA was immediately measured in all patients on admission (n = 157). Endotoxemia was defi ned as EA >0.4 EAU. Critical Care 2015, 19(Suppl 1):P59 (doi: 10.1186/cc14139) Introduction Blood lactate level is a routinely used biomarker for management of patients in septic conditions in the ICU. There is a lack of clinical research data comparing lactate with novel sepsis biomarkers, such as procalcitonin, in regard to the diagnostic and prognostic potential. Herein, we investigated the diagnostic and prognostic value of initial lactate and procalcitonin levels and their kinetics within the ICU stay for prediction of positive blood cultures and fatal outcome in a well characterized cohort of sepsis patients in a US critical care setting. Methods This is a retrospective, observational cohort study of adult patients with confi rmed severe sepsis or septic shock and with at least one procalcitonin and lactate measurement on admission to the ICU of Morton Plant Hospital (Clearwater, FL, USA). Logistic regression models were calculated to assess the association of biomarkers with blood culture positivity and fatal ICU outcome with area under the curve (AUC) as a measure of discrimination. Conclusion Septic shock with endotoxemia was associated with biochemical and clinical consequences including a higher PCT level, higher frequency of bacteremia, kidney failure, and death. Results The in-hospital mortality rate of the 1,075 included patients (age 68 years) was 23.8% (95% CI = 21.2 to 26.3%) and 18.4% of patients had positive cultures. In regard to the diagnostic value for bacteremic disease, initial procalcitonin had a higher discriminatory value (AUC 0.71) compared with initial lactate levels (AUC 0.52). In regard to prognosis, initial lactate level was the better mortality predictor (AUC 0.69) compared with procalcitonin (AUC 0.55), although both initial levels were signifi cantly lower compared with APACHE III (P >0.05 for both comparisons). When looking at biomarker kinetics, procalcitonin decrease was more strongly associated with fatal outcome compared with initial levels alone (AUC 0.66), but still lower compared with lactate kinetics (AUC 0.73). Procalcitonin or lactate clearance, or both, for risk assessment in patients with sepsis? Results from a prospective US ICU patient cohort i Results Endotoxemia was present in 61% of patients (group 1, n = 95, age 66 (57 to 75)), and in 39% of patients EA was low (group 2, n = 62, age 63 (55 to 76)). Median EA was 0.57 EAU (0.46 to 0.67) in group 1 and 0.27 EAU (0.17 to 0.36) in group 2 (P <0.001). The PCT level was six times higher in group 1 than in group 2 (19.6 ng/ml vs. 3.1 ng/ml, P <0.001) and was correlated with EA (P <0.001, R = 0.5). Median APACHE II score was 23 points (16 to 29) in group 1 and 19 (16 to 25) in group 2; but observed diff erence was not signifi cant. The severity of clinical status estimated by SOFA score was similar in both groups (10 (7 to 13) in group 1 and 11 (8 to 12) in group 2; NS). Forty-six percent of patients in group 1 and 27% in group 2 required renal replacement therapy (P = 0.01). ICU mortality of patients was 41%. The mortality rate was higher in group 1, compared with group 2, and Kaplan–Meier survival analysis of time to death showed statistical signifi cance between the two groups (P = 0.001, log-rank test). A Gram-negative pathogen as the primary source of infection was identifi ed in 64% of patients in group 1 and in 44% in group 2 (P = 0.004); bacteremia was detected in 26% of cases in group 1 and in 12% in group 2 (P = 0.02). Diagnostic value of procalcitonin and IL-6 for sepsis of older patients and other patients in the emergency department SH Woo College of Medicine, The Catholic University of Korea, Incheon St. Mary’s Hospital, Incheon, South Korea College of Medicine, The Catholic University of Korea, Incheon St. Mary’s Hospital, Incheon, South Korea Critical Care 2015, 19(Suppl 1):P60 (doi: 10.1186/cc14140) Introduction Emergency physicians are able to identify severely ill older patients with SIRS in the emergency department (ED). A previous study showed that elevated procalcitonin (PCT) and IL-6 level is a known marker of sepsis and predicts bacteremia and mortality. We assessed the diagnostic value of PCT and IL-6 in older patients and other patients with SIRS and sepsis in the ED. Methods This retrospective cohort study was conducted from January 2013 to December 2013. We enrolled 122 patients with SIRS, 55 were classifi ed as the older age group (>65 years of age). Measurement of serum PCT, IL-6, and white blood cell count was performed on initial admission to the ED. We analyzed these markers in older patients and other patients groups with sepsis. Results Of the 55 patients in the older group 33 (60%) patients had sepsis, and 40 (59.7%) patients of the other group had sepsis. PCT and IL-6 levels were signifi cantly higher in other patients with sepsis (P <0.001, P <0.001). But PCT and IL-6 levels were not higher in old age i Conclusion PCT on day 7 and PCT kinetics can be useful to predict outcome in SSAO. PCT is higher in community-acquired sepsis, when surgical cultures are positive, in Gram-negative isolations and in males. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S22 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 activity; and enhancement of sensitivity to antibiotics. In the case of severe infectious disease, antibiotics are often supplemented with administration of IVIg. patients with sepsis (P = 0.400, P = 0.169). The area under the receiver operating characteristic curve (AUC) for diagnosis of sepsis according to PCT and IL-6 was 0.823, and 0.772 for the other patients group. The diagnostic sensitivity, specifi city, positive predictive value, and negative predictive value of PCT for sepsis in other patients group were 79.5%, 81.5%, 86.1%, and 73.3% respectively, with a PCT cutoff value of 0.18 ng/ml. Procalcitonin levels in patients undergoing left ventricular assist device implantation Results The patient APACHE II score were 24.9 ± 8.2, the SOFA score 9.1 ± 3.7, and the survival rate after 28 days 83.4%. The values before IVIg administration were: procalcitonin 36.0 ± 463.3 (median 110) ng/ml, presepsin 4,548 ± 4,250 (median 3,337) pg/ml, CRP 15.6 ± 9.6 (median 14.7) mg/dl, and IL-6 13,860 ± 47,299 (median 630) pg/ml. All values were thus elevated. On the days after the completion of IVIg administration and on day 7, the level of almost all mediators (procalcitonin, presepsin, CRP, IL-6) decreased signifi cantly. In patients with suspected severe sepsis and septic shock, presepsin reveals valuable diagnostic capacity to diff erentiate sepsis severity compared with procalcitonin, IL-6, CRP, and WBC. Additionally, presepsin and IL-6 reveal prognostic value with respect to 30 days and 6 months all-cause mortality throughout the fi rst week of ICU treatment [1]. p M Holek, J Kettner, J Franeková, A Jabor M Holek, J Kettner, J Franeková, A Jabor Institute for Clinical and Experimental Medicine, Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P61 (doi: 10.1186/cc14141) Introduction Procalcitonin (PCT) is used for diagnosis of a bacterial infection. Several works described nonspecifi c elevation of PCT after cardiac surgery with cardiopulmonary bypass (CPB) caused by systemic infl ammatory response syndrome (SIRS) with various cutoff values for the presence of the infection (0.47 to 2.47 μg/l). However, in patients undergoing left ventricular assist device (LVAD), implantation data about PCT dynamics are lacking. y gi Conclusion The results of the present study found signifi cant decreases of procalcitonin, presepsin and IL-6 resulting from 3 days of immunoglobulin administration, but evidence is still limited and this needs to be confi rmed in larger studies. Methods PCT levels in 25 patients indicated for LVAD were prospectively assessed before surgery and during the postoperative period (days 1, 2, 14 and 30). Values were compared according to the presence of infectious complications (IC) and non-infectious complications such as acute renal failure (ARF) defi ned as injury by RIFLE criteria or necessity of right ventricular assist device (RVAD). Data were also analyzed using combined endpoints A (ARF, RVAD) and B (IC, ARF, RVAD). Values are presented as median with interquartile range (in μg/l). Diagnostic value of procalcitonin and IL-6 for sepsis of older patients and other patients in the emergency department SH Woo The diagnostic sensitivity, specifi city, positive predictive value, and negative predictive value of IL-6 for sepsis in other patients group were 67.5%, 81.5%, 84.4%, and 62.9% respectively, with a IL-6 cutoff value of 74.43 pg/ml. Methods The changes in sepsis markers (procalcitonin, presepsin, interleukin-6, C-reactive protein) followed by IVIg administration were investigated in severe sepsis or septic shock patients. The subjects were 410 patients admitted to an ICU with a diagnosis of severe sepsis or septic shock and from whom informed consent had been obtained for the present study. IVIg was administered intravenously for 3 days (5.0 g/ day) and measurements were undertaken before administration (day 1), on the day after completion of administration (day 4), and on day 7. The items measured were procalcitonin, presepsin, IL-6, and CRP. The eff ect of IVIg administration on these markers was then studied. The IVIg studied was polyethylene glycol-treated human immunoglobulin injection fl uid (2.5 g, 50 ml, one vial). f Conclusion PCT and IL-6 is useful predictive markers for diagnosing sepsis in adult patients (<65 years of age) with SIRS in the ED. But these markers are not useful for identifi cation of sepsis in older patients. Diagnostic and prognostic performance of PATHFAST Presepsin in patients with SIRS and early sepsis E Spanuth1 R Carpio2 R Thomae3 Diagnostic and prognostic performance of PATHFAST Presepsin i patients with SIRS and early sepsis E Spanuth1, R Carpio2, R Thomae3 1DIAneering GmbH, Heidelberg, Germany; 2Hospital Nacional Edgardo Rebagliati Martins-EsSalud, Lima, Peru; 3Mitsubishi Chemical Europe, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P63 (doi: 10.1186/cc14143) p , p , 1DIAneering GmbH, Heidelberg, Germany; 2Hospital Nacional Edgardo Rebagliati Martins-EsSalud, Lima, Peru; 3Mitsubishi Chemical Europe, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P63 (doi: 10.1186/cc14143) Introduction Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker. Introduction Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker. Methods Presepsin was determined at presentation (T0), after 8, 24 and 72 hours in 123 individuals admitted with signs of SIRS and/ or infection. Primary endpoint was death within 30 days. Presepsin was determined using the POC assay PATHFAST Presepsin (Mitsubishi Chemical, Japan). Conclusion PCT levels in patients undergoing LVAD implantation rise signifi cantly in the fi rst 2 days after surgery. Interestingly, this elevation is much higher than after routine cardiac surgery with CPB. Recent works suggest that PCT concentrations are aff ected by SIRS caused by contact with a nonphysiological surface. In the case of LVAD this immunological stimulation is long lasting and even more potent with additional RVAD or ARF treated with renal replacement therapy. In accordance with this hypothesis, our data show that the ability of PCT to detect infectious complication in LVAD patients is limited and its concentrations more probably correlate with postoperative complications in general. p Results Mean presepsin concentrations of the patient group at presentation and of the control group were 1,945 and 130 pg/ml, respectively (P <0.0001). Baseline presepsin diff ered highly signifi cant between patients with SIRS, sepsis, severe sepsis and septic shock. Table 1 (abstract P63). Presepsin levels during the course of the disease in survivors and nonsurvivors Table 1 (abstract P63). Presepsin levels during the course of the disease in survivors and nonsurvivors PSEP, median (IQR) (pg/ml) T0 T8 hours T24 hours T72 hours Survivors 590 622 574 533 (345 to 1,396) (367 to 1,912) (336 to 1,610) (324 to 1,246) Nonsurvivors 1,763 1,859 1,731 2,056 (705 to 6,616) (1,001 to 5,744) (809 to 4,586) (811 to 5,540) P value 0.0046 0.0005 0.0003 0.0013 P61 P61 Procalcitonin levels in patients undergoing left ventricular assist device implantation M Holek, J Kettner, J Franeková, A Jabor Institute for Clinical and Experimental Medicine, Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P61 (doi: 10.1186/cc14141) Reference 1. Behnes M, Bertsch T, Lepiorz D, et al. Diagnostic and prognostic utility of soluble CD 14 subtype (presepsin) for severe sepsis and septic shock during the fi rst week of intensive care treatment. Crit Care. 2014;18:507. p q g μg Results PCT levels were low before surgery (0.16, 0.10 to 0.35), increased signifi cantly within the fi rst (5.72, 2.18 to 9.75; P <0.001) and second (5.94, 2.54 to 11.99; P <0.001) day after operation and decreased on the 14th (0.27, 0.11 to 0.74) and 30th (0.10, 0.06 to 0.19) day. There was no signifi cant diff erence in PCT values between patients with or without IC as well as with or without RVAD. ARF increased PCT level signifi cantly only 14 days after LVAD implantation (0.68, 0.37 to 1.65 vs. 0.15, 0.11 to 0.34; P = 0.015). Subjects with endpoint A had signifi cantly higher PCT values on the second (19.53, 5.66 to 63.12 vs. 3.95, 2.33 to 8.85; P = 0.033), 14th (0.55, 0.31 to 1.44 vs. 0.15, 0. to 0.34; P = 0.020) and 30th (0.19, 0.11 to 0.29 vs. 0.08, 0.05 to 0.13; P = 0.016) day after operation. Patients with endpoint B had signifi cantly elevated PCT levels 2 (11.99, 3.23 to 24.16 vs. 3.95, 2.54 to 7.39; P = 0.027) and 14 (0.55, 0.28 to 0.90 vs. 0.13, 0.09 to 0.23; P = 0.005) days after surgery. Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury Y Nakamura, H Ishikura, J Tanaka, T Nishida, M Mizunuma, D Ohta, N Matsumoto, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P64 (doi: 10.1186/cc14144) g p p Methods We analyzed data from 14 trials and 4,211 ARI patients to study associations of admission PCT levels and setting specifi c treatment failure and mortality alone at 30 days. We used multivariable hierarchical logistic regression and conducted sensitivity analyses stratifi ed by clinical settings and ARI diagnoses to assess the results’ consistency. Introduction At the moment, we have few reports about the diagnostic accuracy of procalcitonin (PCT), presepsin (P-SEP) and CD64 as a diagnostic marker of sepsis for patients with acute kidney injury (AKI). This study aimed to clarify which is a more useful diagnostic biomarker for sepsis using PCT, P-SEP and CD64 with or without AKI in ICU patients. Methods This study was a single-center observational retrospective study. Blood samples were collected from 334 patients admitted to our ICU between April 2013 and March 2014. Then, we classifi ed the patients with or without AKI. In this study, we adopted RIFLE criteria for AKI diagnosis. After that, the patients in each group were classifi ed into the sepsis group and the nonsepsis group. We measured PCT, P-SEP and CD64 levels at the time of ICU admission and subsequently investigated the diagnostic accuracy of these biomarkers for detecting sepsis. y Results Overall, 864 patients (20.5%) experienced treatment failure and 252 (6.0%) died. The ability of PCT to diff erentiate patients with and without treatment failure was highest in the emergency department setting (treatment failure; area under the curve (AUC): 0.64 (95% confi dence interval (CI): 0.61, 0.67), adjusted odds ratio (OR): 1.85 (95% CI: 1.61, 2.12), P <0.001 – mortality; AUC: 0.67 (95% CI: 0.63, 0.71), adjusted OR: 1.82 (95% CI: 1.45, 2.29), P <0.001). In lower respiratory tract infections, PCT was a good predictor of identifying patients at risk for mortality (AUC: 0.71 (95% CI: 0.68, 0.74), adjusted OR: 2.13 (95% CI: 1.82, 2.49), P <0.001). In primary care and ICU patients no signifi cant associations of initial PCT levels and outcome were found. See Figure 1. Conclusion Admission PCT levels are associated with setting specifi c treatment failure and provide most prognostic information in ARI in the emergency department setting. p Results In this study we met 225 patients with non-AKI and 109 patients with AKI. Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury We conducted ROC analysis for diagnosing sepsis. In non-AKI patients, the AUC of PCT, P-SEP and CD64 were 0.904 (95% CI: 0.824 to 0.950), 0.892 (95% CI: 0.794 to 0.947) and 0.917 (95% CI: 0.842 to 0.958), respectively. In AKI patients, the AUC were 0.933 (95% CI: 0.859 to 0.970), 0.755 (95% CI: 0.642 to 0.840) and 0.905 (95% CI: 0.803 to 0.957), respectively. P64 P64 Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury Y Nakamura, H Ishikura, J Tanaka, T Nishida, M Mizunuma, D Ohta, N Matsumoto, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P64 (doi: 10.1186/cc14144) Introduction Whether the infl ammatory biomarker procalcitonin (PCT) provides prognostic information across clinical settings and diff erent acute respiratory tract infections (ARI) is poorly understood. Herein, we investigated the prognostic value of admission PCT levels to predict adverse clinical outcome in a large ARI population. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion CD64 and PCT were a useful biomarker for detecting sepsis for ICU patients with AKI. Conclusion CD64 and PCT were a useful biomarker for detecting sepsis for ICU patients with AKI. Twenty-four patients died during 30 days. The 30-day mortality was 19.5% in total, ranging from 10 to 32% between the fi rst and the fourth quartile of presepsin concentration. Nonsurvivors showed high presepsin values with increasing tendency during the course of the disease while in surviving patients this tendency was decreasing. See Table 1. Conclusion Presepsin demonstrated a strong relationship with disease severity and outcome. Presepsin provided reliable discrimination between SIRS and sepsis as well as prognosis and early prediction of 30- day mortality already at admission. Presepsin showed close association with the course of the disease. Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) g p T Ikeda, S Ono, T Ueno, H Tanaka, S Suda T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Introduction The potentially envisaged actions of intravenous immunoglobulin (IVIg) on severe infectious disease include: virus or toxin neutralizing action; opsonic eff ect; complement bacteriolytic S23 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P65 Prognostic value of procalcitonin in respiratory tract infections across clinical settings A Kutz, B Mueller, P Schuetz, for the IPD Study Group Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P65 (doi: 10.1186/cc14145) P65 Prognostic value of procalcitonin in respiratory tract infections across clinical settings A Kutz, B Mueller, P Schuetz, for the IPD Study Group Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P65 (doi: 10.1186/cc14145) P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin The aim of this study was to investigate a possible association between the etiology of BSIs and the serum PCT levels. f g Methods We randomly chose 425 patients from a 9-month epidemiologic study on the incidence of community-onset severe sepsis and septic shock in western Sweden 2011 to 2012. In total, 207 had severe sepsis and 218 had sepsis, mean age 71.2 versus 64.2 years; males 51%. Sampling was made on arrival in the ED. The NLCR was analyzed immediately, PCT later on plasma frozen at –80°C. A total of 122/425 patients had bacteremia, 72 (35%) in the severe sepsis group versus 50 (23%) in the sepsis group. Most common fi ndings were Escherichia coli (n = 33), Staphylococcus aureus (n = 24), streptococcal spp. (n = 33) and other enterobacteriacae spp. (n = 17).i y Methods ICU patients with clinical and laboratory signs of sepsis or septic shock, with documented BSIs and with both serum PCT and CRP measurements on the day of the positive blood sample (±1 day), were included. Illness severity was assessed by SOFA score on both admission and BSI day. Demographic, clinical, and laboratory data including PCT and CRP levels, as well as the white blood cell (WBC) count on the day of the BSI were recorded. PCT was measured by an electrochemiluminescence analyzer and CRP by the tholosimetric method (Roche, Switzerland). Results The NLCR shows signifi cantly higher sensitivity than PCT at recommended cutoff levels for bacteremia. Interestingly, this is true even for all 207 patients with severe sepsis, irrespective of bacteremia or not. Sensitivity fi gures with 95% confi dence interval: bacteremia (n = 122): NLCR 80% (0.73 to 0.87) versus PCT 66% (0.58 to 0.75), P = 0.01; severe sepsis with bacteremia (n = 72): NLCR 85% (0.77 to 0.93) versus PCT 70% (0.59 to 0.81), P = 0.03; and severe sepsis but no bacteremia (n = 135): NLCR 71% (0.65 to 0.77) versus PCT 61% (0.54 to 0.68), P = 0.03. Conclusion The NLCR can be used in the ED as a biomarker for bacteremia as well as severe sepsis and seems to perform as well as or even better than PCT in this setting. Rapid response, low cost and no need for extra sampling make it useful as a screening tool. P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin Introduction The objective of this study was to evaluate the neutrophil to lymphocyte count ratio (NLCR) versus procalcitonin (PCT) in diagnosing bacteremia in the emergency department (ED). The NLCR is a biomarker that appears early in the course of the acute infl ammatory response and reaches maximum levels within 4 hours after onset. An elevated NLCR has been shown to correlate to bacteremia at a cutoff level of >10 [1]. It is rapidly analyzed on a full blood cell count at low cost. The lowest recommended cutoff level for PCT is <0.5 ng/ml. Methods We randomly chose 425 patients from a 9-month epidemiologic study on the incidence of community-onset severe sepsis and septic shock in western Sweden 2011 to 2012. In total, 207 had severe sepsis and 218 had sepsis, mean age 71.2 versus 64.2 years; males 51%. Sampling was made on arrival in the ED. The NLCR was analyzed immediately, PCT later on plasma frozen at –80°C. A total of 122/425 patients had bacteremia, 72 (35%) in the severe sepsis group versus 50 (23%) in the sepsis group. Most common fi ndings were Escherichia coli (n = 33), Staphylococcus aureus (n = 24), streptococcal spp. (n = 33) and other enterobacteriacae spp. (n = 17). R l Th NLCR h i ifi l hi h i i i h PCT Introduction The objective of this study was to evaluate the neutrophil to lymphocyte count ratio (NLCR) versus procalcitonin (PCT) in diagnosing bacteremia in the emergency department (ED). The NLCR is a biomarker that appears early in the course of the acute infl ammatory response and reaches maximum levels within 4 hours after onset. An elevated NLCR has been shown to correlate to bacteremia at a cutoff level of >10 [1]. It is rapidly analyzed on a full blood cell count at low cost. The lowest recommended cutoff level for PCT is <0.5 ng/ml. E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) Introduction Early diff erentiation of bacterial from candidal bloodstream infections (BSIs) in the presence of sepsis or septic shock is crucial because of the need for appropriate treatment initiation. Clinical data, although limited, suggest a role for procalcitonin (PCT) [1-3]. References Introduction The purpose of the study was to assess the prognosis value of pro-adrenomedullin (pADM), C-reactive protein (CRP) and procalcitonin (PCT), lactate (LT), albumin (ALB), cholesterol (CHOL), white blood cell (WBC) and severity score in patients with severe sepsis or septic shock. 1. Martini A, et al. J Infect. 2010;60:425. 2. Petrikkos GL, et al. Eur J Clin Microbiol Infect Dis. 2005;24:272. 3. Charles PE, et al. Intensive Care Med. 2006;32:1577. P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin R f ( , ) Results A total of 64 ICU patients (mean age 58 ± 18 years, 39 males) with BSIs were included. SOFA sore was 9 ± 4 on ICU admission and 8 ± 4 on the day of BSI. In 30 of these patients Candida spp. were isolated in blood culture (candidemia group) whereas the remaining 34 had a bacterial etiology of BSI (bacteremia group). Serum PCT concentrations remained within normal ranges in most patients with candidemia whereas a wide range was observed in patients with bacteremia. Mean values of PCT and CRP levels were higher in the bacterial than in the candidemia BSI group: 18.5 ± 33.2 versus 0.73 ± 1.40 ng/ml, P <0.001 and 17.7 ± 10.3 versus 8.9 ± 8.0 mg/dl, P = 0.001, respectively. There was a signifi cant diff erence in WBC count between the two groups: 19,460 ± 10.174 versus 11,000 ± 5,440, P <0.001 for the bacteremia and candidemia BSI group, respectively. A ROC curve analysis of the predictive ability of PCT showed an AUC of 0.79 (P <0.001). When a cutoff point of 0.40 ng/ml was selected using Youden’s J statistic, a low value of PCT had in our sample a negative predictive value of 0.76 and a likelihood ratio (negative) of 0.30. 1. de Jager et al. Crit Care. 2010;14:R192. 1. de Jager et al. Crit Care. 2010;14:R192. Prognosis value of biomarkers in severe sepsis and septic shock MV De La Torre-Prados1, A García-de la Torre2, R Escobar-Conesa1, Conclusion A low serum PCT value could be considered as a diagnostic marker in distinguishing between BSIs of candidal or bacterial origin in ICU patients with varying severity of sepsis. R f g y Reference P66 Neutrophil to lymphocyte count ratio performs better than procalcitonin as a biomarker for bacteremia and severe sepsis in the emergency department LL Ljungström1, D Karlsson1, A Pernestig2, R Andersson3, G Jacobsson1 1Skaraborg Hospital Skövde, Sweden; 2School of Biosciences, University of Skövde, Sweden; 3Institute of Biosciences, Sahlgrenska Academy at the University of Gothenburg, Sweden Critical Care 2015, 19(Suppl 1):P66 (doi: 10.1186/cc14146) P68f P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) g y Reference . Schuetz P, Briel M, Christ-Crain M, et al. Procalcitonin to guide initiation and duration of antibiotic treatment in acute respiratory infections: an individual patient data meta-analysis. Clin Infect Dis. 2012;55:651-62. Figure 1 (abstract P65). Multivariate regression analysis for estimation of predictive value of PCT levels on admission stratifi ed by adverse events and mortality in diff erent settings and diagnoses. Treatment failure, ~mortality. ARI, acute respiratory tract infection; ECOPD, exacerbated chronic obstructive pulmonary disease; CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; Adj., adjusted; OR, odds ratio; CI, confi dence interval. Figure 1 (abstract P65). Multivariate regression analysis for estimation of predictive value of PCT levels on admission stratifi ed by adverse events and mortality in diff erent settings and diagnoses. Treatment failure, ~mortality. ARI, acute respiratory tract infection; ECOPD, exacerbated chronic obstructive pulmonary disease; CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; Adj., adjusted; OR, odds ratio; CI, confi dence interval. S24 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 diff erences were signifi cant in pADM (2.46 (1.21 to 4.89) vs. 1.68 (0.94 to 3.32) nmol/l; P = 0.012), LT (2.6 (1.6 to 3.94) vs. 1.6 (1.2 to 2.43) mmol/l; P <0.001) and ALB (2 (1.55 to 2.38) vs. 2.22 (1.96 to 2.7) g/dl; P = 0.001). Conclusion The protein pADM, LT and ALB showed good prognosis accuracy when measured on admission of septic patients to the ICU. diff erences were signifi cant in pADM (2.46 (1.21 to 4.89) vs. 1.68 (0.94 to 3.32) nmol/l; P = 0.012), LT (2.6 (1.6 to 3.94) vs. 1.6 (1.2 to 2.43) mmol/l; P <0.001) and ALB (2 (1.55 to 2.38) vs. 2.22 (1.96 to 2.7) g/dl; P = 0.001). Conclusion The protein pADM, LT and ALB showed good prognosis accuracy when measured on admission of septic patients to the ICU. P67 Prognosis value of biomarkers in severe sepsis and septic shock MV De La Torre-Prados1, A García-de la Torre2, R Escobar-Conesa1, J Perez-Vacas1, A Puerto-Morlán1, E Cámara-Sola1, A García-Alcántara1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Puerto Real University Hospital, Puerto Real, Cádiz, Spain Critical Care 2015, 19(Suppl 1):P67 (doi: 10.1186/cc14147) P69 Methods A prospective, observational study in adult patients with severe sepsis or septic shock in a polyvalent ICU. Demographics, severity scores (APACHE II and SOFA) and all of the biomarkers were studied within 24 hours from septic shock onset. Descriptive and comparative statistical analysis was performed using the statistical software packages SPSS v.15 and MedCalc® 9.2.1.0. P69 Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Estimating the duration of a central venous catheter at time of insertion g g Results A total of 2,148 patients were screened, and 85 (4%) fulfi lled entry criteria. The incidence of candidemia in these 85 patients was 8.2%, compared with 0.5% in the remaining 2,063 patients (relative risk 16.9%, 95% confi dence interval (CI) = 6.63 to 43.55). Baseline BDG was positive in 74 patients (87%), with a median number of positive tests of 3 (range 1 to 3) and a median value of 523 pg/ml (range 83 to 6,860). All seven patients with candidemia had positive baseline BDG (median value 523 pg/ml, range 203 to 3,660). The best cutoff of baseline BDG for the diagnosis of candidemia was 522 pg/ml (area under the ROC curve 0.883, 95% CI = 0.769 to 0.997), with sensitivity and specifi city of 86% and 88%, respectively. The cutoff value of 80 pg/ml had sensitivity and specifi city of 73% and 27%, respectively.f MJ Holmberg1, LW Andersen1, A Graver1, SB Wright1, D Yassa1, MD Howell2, MW Donnino1, MN Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2University of Chicago, IL, USA Introduction The aim of this study was to investigate whether clinicians can estimate, at the time of insertion, the length of time a central venous catheter (CVC) will remain in place, and to identify clinical variables which may predict CVC duration. CVC-related bloodstream infection is a known complication among critically ill patients. As infection rates may increase with duration of catheterization, more expensive antimicrobial-coated catheters may be used in patients with anticipated long duration of CVC use. i Conclusion This dynamic prediction rule was able to diff erentiate a group of ICU patients at high risk to develop candidemia, with a relative risk of 16.9. BDG is frequently positive in ICU patients. A cutoff value of 522 pg/ml was able to discriminate between candidemic and noncandidemic patients. A revision of the cutoff value for BDG in the ICU is needed. p g Methods We conducted a single-center, prospective study from January 2012 to November 2012. Clinicians prospectively estimated the anticipated duration of CVC at the time of line placement in an electronic procedure note. We collected demographics, past medical history, type of ICU, vital signs, laboratory values, SOFA score, mechanical ventilation and use of vasopressors at the time of placement. Continuous variables were compared with the Wilcoxon rank-sum test and categorical variables with the Fisher’s exact test. Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database y g S Fernandes1, D Bruno2, J Morgado3, C Calle4, C Ferreira5 1Centro Hospitalar Lisboa Norte CHLN, Lisbon, Portugal; 2Hospital Fernando Fonseca, Lisbon, Portugal; 3Centro Hospitalar de Lisboa Central, Lisbon, Portugal; 4Instituto Português de Oncologia, Lisbon, Portugal; 5National Institute of Legal Medicine and Forensic Sciences, Coimbra, Portugal Critical Care 2015, 19(Suppl 1):P70 (doi: 10.1186/cc14150) Results We enrolled 150 patients; median age was 65 (IQR: 52 to 74), 63 (42%) were female and mortality was 22%. Median time from CVC placement to removal was 5 (IQR: 3 to 8) days. The correlation between estimated CVC time and actual time was low (r = 0.36, P <0.001). Forty- eight (32%) patients had a long CVC duration. Clinician estimate had 46% sensitivity and 76% specifi city for predicting long duration of CVC. Of 30 variables tested, only temperature at the time of insertion was signifi cantly associated with long duration (OR: 1.30, 95% CI: 1.04 to 1.63, P = 0.02). The AUC for this model was 0.59 (95% CI: 0.49 to 0.69). Results We enrolled 150 patients; median age was 65 (IQR: 52 to 74), 63 (42%) were female and mortality was 22%. Median time from CVC placement to removal was 5 (IQR: 3 to 8) days. The correlation between estimated CVC time and actual time was low (r = 0.36, P <0.001). Forty- eight (32%) patients had a long CVC duration. Clinician estimate had 46% sensitivity and 76% specifi city for predicting long duration of CVC. Of 30 variables tested, only temperature at the time of insertion was signifi cantly associated with long duration (OR: 1.30, 95% CI: 1.04 to 1.63, P = 0.02). The AUC for this model was 0.59 (95% CI: 0.49 to 0.69). Conclusion Our results suggest a low correlation between clinician prediction at time of insertion and actual duration of CVC. We did not fi nd any good predictors of long duration of CVC. Given our relatively low sample size, we may have been underpowered. It may not be feasible to identify patients at the time of insertion who may benefi t from antimicrobial-coated catheters. Introduction Bloodstream infections in the ICU are a major trigger of morbidity and mortality. Several risk factors for bacteremia have been previously identifi ed, such as presence of a central venous catheter or invasive ventilation [1,2]. Iron is a key element for bacteria growth, and its metabolism is extensively altered by infl ammation. References following: dialysis, surgery, pancreatitis, and receipt of corticosteroids, other immunosuppressive agents or parenteral nutrition. Diff erent from the original description of the score which considered only the fi rst 7 days of ICU stay, we selected patients who fulfi lled these criteria at any time during the ICU stay. Once a patient fulfi lled these criteria, AFT (anidulafungin 200 mg followed by 100 mg daily) was initiated provided that the patients also presented with any of the following: fever, hypothermia, hypotension, leukocytosis, acidosis or elevated C-reactive protein. Blood cultures (days 1 to 2) and baseline serum BDG (days 1 to 3) were performed. Patients with candidemia were treated for ≥14 days, those without candidemia but ≥1 positive BDG (≥80 pg/ml) received AFT for ≥10 days, and patients with negative blood cultures and negative BDG discontinued anidulafungin.i 1. Tabah A, Koulenti D, Laupland K, et al. Characteristics and determinants of outcome of hospital-acquired bloodstream infections in intensive care units: the EUROBACT International Cohort Study. Intensive Care Med. 2012;38:1930-45. 2. Prowle JR, Echeverri JE, Ligabo EV, et al. Acquired bloodstream infection in the intensive care unit: incidence and attributable mortality. Crit Care. 2011;15:R100. Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA N 1 AL C l b 2 P E t 2 T G i 3 F S i ll 4 Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA N 1 AL C l b 2 P E 2 T G i 3 F S i ll 4 SA Nouer , AL Colombo , P Esteves , T Guimaraes , F Scapinello , B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Results We analyzed 246 consecutive episodes of severe sepsis (38%) or septic shock (62%). The 28-day mortality was 36.2%. The profi le of dead patients had a signifi cantly higher average age (65 (IQR: 75.5 to 57.5) vs. 63 (47 to 72); P <0.06), APACHE II (27 (22 to 30) vs. 23 (18 to 27); P <0.001) and SOFA (11 (9 to 12.75) vs. 9 (7 to 10); P <0.001). CRP (168.4 (106 to 285) vs. 165.4 (87.8 to 275) mg/dl; P = NS), PCT (6.5 (0.94 to 23.8) vs. 5.8 (0.97 to 19.59) ng/ml; P = NS) and WBC 14.7 (9.5 to 21.4) vs. 12.9 (5.5 to 17.5); P = NS) were increased in those who died, but CHOL (102 (75 to 134) vs. 108 (86 to 141) mg/dl; P = NS) had lower values. These Introduction Early initiation of antifungal therapy (AFT) improves the outcome in candidemic patients, but empiric AFT is not considered the standard of care. Methods We used a scoring system based on the presence of a central venous catheter and receipt of antibiotics, plus at least two of the S25 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P70 P70 Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database S Fernandes1, D Bruno2, J Morgado3, C Calle4, C Ferreira5 1Centro Hospitalar Lisboa Norte CHLN, Lisbon, Portugal; 2Hospital Fernando Fonseca, Lisbon, Portugal; 3Centro Hospitalar de Lisboa Central, Lisbon, Portugal; 4Instituto Português de Oncologia, Lisbon, Portugal; 5National Institute of Legal Medicine and Forensic Sciences, Coimbra, Portugal Critical Care 2015, 19(Suppl 1):P70 (doi: 10.1186/cc14150) Estimating the duration of a central venous catheter at time of insertion Pearson’s correlation coeffi cient was used to assess the correlation between estimated CVC time and actual time. Duration of CVC use was dichotomized into long (≥7 days) or short (<7 days), based on previous literature, and sensitivity and specifi city for predicting long duration was calculated. We performed a logistic regression analysis to identify variables associated with long CVC duration and calculated the area under the ROC curve (AUC). P72 Preventing catheter-related infections in ICUs: comparing catheter care techniques Preventing catheter-related infections in ICUs: comparing catheter care techniques S Ozden, R Iscimen, H Akalin, N Kelebek Girgin, F Kahveci, M Sinirtas Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P72 (doi: 10.1186/cc14152) Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database We aim to determine whether iron defi ciency is a risk or protective factor for bacteremia in the ICU. Methods We performed a retrospective analysis of patients included in the MIMIC-II database, an ICU database that collected data from patients admitted to the medical, surgical, coronary and cardiac surgery ICU of Boston’s Beth Israel Deaconess Medical Center during a period of 7 years. We performed logistic regression models to assess the association between iron and bloodstream infection. Results We included 3,980 patients, 2,988 with low serum iron (<60 ng/ ml) and 992 with normal/high serum iron (≥60 ng/ml). During their fi rst stay in the ICU, 351 (8.82%) patients developed bloodstream infections. Low serum iron was associated with increased odds of bloodstream infection (OR: 1.37; 95% CI: 1.04 to 1.80). After adjusting for age, gender, Simplifi ed Acute Physiology Score, presence of central venous catheter, ICU type, transfusions performed before iron measured, neoplastic disease, diabetes mellitus, hepatic disease, congestive heart failure and ferritin levels, low levels of iron were still associated with an increased odds of bacteremia (OR: 1.41; 95% CI: 1.03 to 1.9). In contrast, low serum iron was associated with a decreased risk of death in the hospital (adj OR: 0.73, CI: 0.57 to 0.95). P73 Results Among 1,746 ICU patients, CLA-BSI were diagnosed in 69 cases. The incidence index was 3.88/100 admissions to the ICU. CLA-BSI were diagnosed in 18% of the overall number (381) of device-associated healthcare-associated infections. Central line was used in 91.41 ± 4.4% patients during 19,819 patient-days and 18,155 central line-days. The median density of CLA-BSI/1,000 central line-days was 3.88/3.77/3.36 and 0.0 accordingly in years 2011/2012/2013 and 2014 (from January to November). The main pathogens of CLA-BSI were CN staphylococci (22%), Staphylococcus aureus (21%), and Enterobacteriaceae (29%). In this study, the density of CLA-BSI was about 50% lower (2.75 (2.0 to 6.06)) than in our previous study and in the INICC’s report (2014), but higher than in the CDC’s NHSN (2012) report. Comparison of three cutaneous antiseptic solutions for the prevention of catheter colonization in an ICU for adult patients: a multicenter prospective randomized controlled trial H Yasuda1, M Sanui2, T Komuro2, J Hatakeyama3, S Matsukubo4, S Kawano5, H Yamamoto6, K Andoh7, R Seo8, N Shime9, E Noda10, N Saito11 1Japanese Red Cross Musashino Hospital, Tokyo, Japan; 2Saitama Medical Center Jichi Medical University, Saitama, Japan; 3YokohamaCity Minato Red Cross Hospital, Kanagawa, Japan; 4Kurashiki Central Hospital, Okayama, Japan; 5The Jikei University School of Medicine, Tokyo, Japan; 6Toyonaka Municipal Hospital, Osaka, Japan; 7Sendai City Hospital, Miyagi, Japan; 8Kobe City Medical Center General Hospital, Hyogo, Japan; 9National Hospital Organization Kyoto Medical Center, Kyoto, Japan; 10Kyushu University Hospital, Fukuoka, Japan; 11Nippon Medical School Chiba Hokusoh Hospital, Tiba, Japan Conclusion The implementation of the infection control program and preventive interventions for patients with central venous catheters improved the safety and quality of healthcare in the ICU by reducing CLA-BSI rate. Critical Care 2015, 19(Suppl 1):P73 (doi: 10.1186/cc14153) Introduction The current CDC guideline for the prevention of intravascular catheter-related infections recommends skin preparation with a greater than 0.5% chlorhexidine with alcohol solution before central venous catheter (CVC) or peripheral arterial catheter (AC) placement. However, few studies investigated the superiority of 1% alcoholic chlorhexidine gluconate (1% CHG) over either 0.5% alcoholic chlorhexidine gluconate (0.5% CHG) or 10% aqueous povidone iodine (10% PVI) for the prevention of catheter colonization. The aim of this study is to compare the eff ectiveness of three skin antiseptic solutions for the prevention of intravascular catheter colonization. Reference 1. Kübler A, Duszyñska W, Rosenthal VD, et al. Device-associated infection rates and extra length of stay in an intensive care unit of a university hospital in Wroclaw, Poland: International Nosocomial Infection Control Consortium’s (INICC) fi ndings. J Crit Care. 2012;27:105.e5-10. Catheter-associated bloodstream infections in an ICU of a university hospital in Wroclaw, Poland: an international nosocomial infection control consortium’s fi ndings The main pathogens of CLA-BSI were CN staphylococci (22%), Staphylococcus aureus (21%), and Enterobacteriaceae (29%). In this study, the density of CLA-BSI was about 50% lower (2.75 (2.0 to 6.06)) than in our previous study and in the INICC’s report (2014), but higher than in the CDC’s NHSN (2012) report. 1. Safdar N, et al. Chlorhexidine-impregnated dressing for prevention of catheter-related bloodstream infection: a meta-analysis. Crit Care Med. 2014;42:1703-13. Preventing catheter-related infections in ICUs: comparing catheter care techniques S Ozden, R Iscimen, H Akalin, N Kelebek Girgin, F Kahveci, M Sinirtas Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P72 (doi: 10.1186/cc14152) Introduction Catheter-related bloodstream infections (CRBSI) are common and an important cause of morbidity and mortality in critical patients. Optimum approaches for preventing infections are presented in guidelines. This study aims to evaluate effi cacy of diff erent care techniques and education, to defi ne risk factors for decreasing ratio of CRBSIs and to analyze eff ects on morbidity and mortality. Introduction Catheter-related bloodstream infections (CRBSI) are common and an important cause of morbidity and mortality in critical patients. Optimum approaches for preventing infections are presented in guidelines. This study aims to evaluate effi cacy of diff erent care techniques and education, to defi ne risk factors for decreasing ratio of CRBSIs and to analyze eff ects on morbidity and mortality. Conclusion Low serum iron increases the risk of bloodstream infection in the ICU, and should be considered as a risk factor to stratify patients’ risk of bacteremia during ICU stay. S26 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods After ethics committee approval, patients admitted to the ICU, older than 18 years, who were thought to have a central venous catheter (CVC) for more than 48 hours, and whose fi rst catheter was inserted in the ICU were included in the study. Staff were educated before the study and periodically during the study. Catheter care and insertion were applied according to the guidelines. The study was planned as three sequences. In the fi rst group, catheter care was made with a sterile gauze pad. In the second and third groups, catheter care was made with chlorhexidine gluconate impregnated dressing. Also in the third group, a silver-coated needleless connector was inserted into the tip of venous catheters. median duration of catheter indwelling in the entire population was 3.7 days with an interquartile range of 2.0 to 6.7 days, with no signifi cant diff erence between the groups (P = 0.36). Thirteen catheters (5.1%) in the 10% PVI group were positive for catheter-tip colonization, whereas six catheters (2.2%) in the 1% CHG group and fi ve catheters (1.9%) in the 0.5% CHG group were positive (P = 0.07). P75 Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source B Civantos Martin, I Pozuelo Echegaray, C Guallar Espallargas, A Robles Caballero, C Briones, P Extremera Navas, P Millan Estañ, A Garcia de Lorenzo y Mateos Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P75 (doi: 10.1186/cc14155) Preventing catheter-related infections in ICUs: comparing catheter care techniques The probability of catheter colonization was signifi cantly higher in the 10% PVI group than each CHG groups (P = 0.028, log-rank test). The incidence of catheter colonization and CRBSI is compared between the three groups. Conclusion In this randomized controlled trial comparing the eff ective- ness of three cutaneous antiseptic solutions for the prevention of catheter colonization, either 0.5% or 1.0% CHG was superior to 10% PVI. median duration of catheter indwelling in the entire population was 3.7 days with an interquartile range of 2.0 to 6.7 days, with no signifi cant diff erence between the groups (P = 0.36). Thirteen catheters (5.1%) in the 10% PVI group were positive for catheter-tip colonization, whereas six catheters (2.2%) in the 1% CHG group and fi ve catheters (1.9%) in the 0.5% CHG group were positive (P = 0.07). The probability of catheter colonization was signifi cantly higher in the 10% PVI group than each CHG groups (P = 0.028, log-rank test). The incidence of catheter colonization and CRBSI is compared between the three groups. Conclusion In this randomized controlled trial comparing the eff ective- ness of three cutaneous antiseptic solutions for the prevention of catheter colonization, either 0.5% or 1.0% CHG was superior to 10% PVI. p Results Totally 105 patients were included in the study and every group included 35 patients. There was no diff erence between groups when evaluating reasons for catheter insertion. There was no statistically signifi cant diff erence according to emergent or elective catheterization, trying times, or catheter insertion side (P >0.05). CRBSI was determined in two patients in group 1, in one patient in group 2, and in no patient in group 3. In group 1 it was observed on the 4th and 11th days. In group 2 it was observed on the 18th day after catheterization. Before the study, a statistically signifi cant decrease was determined in CRBSI ratios before and after education (16.4/1,000, 12.9/1,000 catheter-days (P <0.001)). According to Group 1 a statistically meaningful decrease was assigned in CRBSI ratios in Groups 2 and 3 (4.84/1,000, 2.22/1,000, 0/1,000 catheter-days) (P <0.001, P <0.001, P <0.001). Catheter-associated bloodstream infections in an ICU of a university hospital in Wroclaw, Poland: an international nosocomial infection control consortium’s fi ndings i g W Duszynska1, VD Rosenthal2, A Litwin1, E Woznica1, A Kubler1 1University Hospital, Wroclaw, Poland; 2International Nosocomial Infection Control Consortium, Buenos Aires, Argentina Critical Care 2015, 19(Suppl 1):P74 (doi: 10.1186/cc14154) i g W Duszynska1, VD Rosenthal2, A Litwin1, E Woznica1, A Kubler1 1University Hospital, Wroclaw, Poland; 2International Nosocomial Infection Control Consortium, Buenos Aires, Argentina Critical Care 2015, 19(Suppl 1):P74 (doi: 10.1186/cc14154) Introduction Catheter-associated bloodstream infections are serious but potentially possible to reduce complication of treatment in the ICU. The aim of the study was to evaluate the frequency and etiology of central line-associated bloodstream infections (CLA-BSI) in ICU patients according to the International Nosocomial Infection Control Consortium (INICC) project. y Conclusion Continued education is important in preventing CRBSIs. Maximum precautions must be taken. Usage of antiseptic solutions with clorhexidine and chlorhexidine gluconate impregnated dressing decreased insertion side infections and usage of silver-coated needleless connectors reduced microorganism entry through the catheter lumen and provided a severe decrease in infection ratio. Reference j Methods A prospective, observational study was conducted in the 20-bed ICU of the University Hospital in Wroclaw from January 2011 to November 2014. CLA-BSI were diagnosed and evaluated according to protocols standardized by the INICC. The density of CLA-BSI/1,000 central line-days, the incidence index/100 admissions to the hospital, the central line utilization ratio (CL-UR) as well as the microbiological profi le of CLA-BSI were evaluated. The results were compared with our earlier published data and with the fi ndings of international reports. Methods A prospective, observational study was conducted in the 20-bed ICU of the University Hospital in Wroclaw from January 2011 to November 2014. CLA-BSI were diagnosed and evaluated according to protocols standardized by the INICC. The density of CLA-BSI/1,000 central line-days, the incidence index/100 admissions to the hospital, the central line utilization ratio (CL-UR) as well as the microbiological profi le of CLA-BSI were evaluated. The results were compared with our earlier published data and with the fi ndings of international reports. Results Among 1,746 ICU patients, CLA-BSI were diagnosed in 69 cases. The incidence index was 3.88/100 admissions to the ICU. CLA-BSI were diagnosed in 18% of the overall number (381) of device-associated healthcare-associated infections. Central line was used in 91.41 ± 4.4% patients during 19,819 patient-days and 18,155 central line-days. The median density of CLA-BSI/1,000 central line-days was 3.88/3.77/3.36 and 0.0 accordingly in years 2011/2012/2013 and 2014 (from January to November). Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source Methods This multicenter prospective randomized controlled trial was conducted in 15 Japanese ICUs from December 2012 to March 2014. Patients over 18 years of age undergoing CVC and AC placement in the ICU are randomized to have one of three skin antiseptic preparations before catheter insertion. After removal of the catheter, the distal tip is cultured using semiquantitative or quantitative techniques. The incidence of catheter colonization and catheter-related bloodstream infection (CRBSI) is compared between the three groups. Introduction Long experience in the treatment of neutropenic patients admitted to the ICU has taught us the importance of removing the permanent central venous catheter when infection is suspected, because of the great mortality associated. The problem usually comes when the origin of sepsis is not clear and we assume that mortality is Results A total of 997 catheters were placed, including 339 catheters using 1% CHG, 329 using 0.5% CHG, and 329 using 10% PVI. The S27 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 immune systems to suppress dissemination of infection by the netted chromatin decorated with antibacterial molecules. It was reported that NETs play an important role in various kinds of infections such as pneumonia. However, there is no report on NETs in patients of soft tissue infections. In this study, we evaluated NET production in pus and clarifi ed the role of NETs as a host defense in patients of soft tissue infections. not easy to avoid. It is important to know what happens to neutropenic patients admitted to the ICU because of sepsis from any source, in whom catheter infection cannot be excluded. Methods A retrospective, cohort, descriptive study was carried out between January 2013 and November 2014. Epidemiology data were collected from all neutropenic patients admitted to the ICU who came from hemato-oncology services and had an implanted central venous catheter. Microbiology results and data related to the catheter removal were described. Methods This study was conducted in the ICU of the Trauma and Acute Critical Care Center at Osaka University Hospital. We collected pus from the patients of soft tissue infections at the time when drainage or debridement was performed and when clinical improvement was observed. The smears of pus specimens were examined by immunohistochemistry to visualize the major NET components: DNA, neutrophil elastase, and histone H1. Use of nanotechnology-based surface antiseptic solutions in the ICU Y Kuplay, N Akgun, C Agalar, H Aydýn, O Alýcý, G Turan FSM Teaching and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P78 (doi: 10.1186/cc14158) Y Kuplay, N Akgun, C Agalar, H Aydýn, O Alýcý, G Turan FSM Teaching and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P78 (doi: 10.1186/cc14158) Introduction In our study, we aimed to compare the application of benzalkonium chloride (BC) – a nanotechnology-based product – for 24-hour periods and didecyl dimethyl ammonium chloride (DDAC) for 12-hour periods regarding effi ciency in application of surface antiseptics in the ICU.f Introduction In our study, we aimed to compare the application of benzalkonium chloride (BC) – a nanotechnology-based product – for 24-hour periods and didecyl dimethyl ammonium chloride (DDAC) for 12-hour periods regarding effi ciency in application of surface antiseptics in the ICU.f y Methods A retrospective observational study in a medical and surgical ICU in a tertiary care hospital on adult patients admitted from January 2010 to December 2013. The study enrolled 845 patients divided into 283 internal jugular CVC (IJC), 270 subclavian CVC (SCC) and 287 femoral CVC (FC) in which the catheters were inserted in the ICU by experienced physicians with at least 50 previously successful trials of central line insertion, using CVC bundle checklist. ICU length of stay, incidence of complications, APACHE II score adjusted severity and mortality were calculated for each group. Methods Two diff erent areas with eight beds at both sides of a common corridor in the ICU were named as areas A and B. BC was applied in area A with 24-hour periods and DDCA was applied in area B with 12- hour periods for surface cleaning. Samples were taken from a total of 20 diff erent surfaces including nurse-station desks, phones, keyboards, beds, bedside monitors and ventilators by the same infection control nurse every 24 hours from area A and every 12 hours from area B for 7 days. Swab samples were cultured on 5% sheep bloody agar and McConkey agar in the laboratory. Then the cultured mediums were incubated at 35 to 37°C in an aerobic environment for 18 to 24 hours. NCSS (Number Cruncher Statistical System) 2007 and PASS 2008 Statistical Software (UT, USA) programs were used for the statistical analysis.f Results Patient and catheter characteristics including the duration of catheterization were similar in all groups. Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source Concurrently, the patients’ clinical data and laboratory data of blood were recorded to analyze the relationship with NET production.i Results A total of 15 patients were included, mean age was 53 years old and 66% were male. The implanted catheter was removed in 80% of patients. Platelet transfusion was needed in 100% of patients before catheter removal and no complication was observed during catheter removal or in the insertion of a new one. In 53% of patients, catheter infection was confi rmed a posteriori. i p Conclusion Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source can be benefi cial for this kind of patient as it was found that in more than 50% of patients catheter infection was confi rmed a posteriori. Results A total of fi ve patients were included in this study and drainage of abscess or debridement of infection site was performed in all of the cases. Four patients of them were diagnosed as necrotizing soft tissue infections by Clostridium spp. (n = 1) and Bacteroides spp. (n = 3) and the other was diagnosed as cervical abscess by Streptococcus spp. In all cases, no NETs but neutrophils were identifi ed in the fi rst pus: however, NETs appeared in the later smears as the patients’ condition was getting better. Eff ect of insertion route on risk of central line-associated bloodstream infection in critically ill patients R Alhubail, N Hassan g g Conclusion These results suggested that NETs also worked as an immune system against soft tissue infections. Drainage or debridement of infection focus might promote NET production. KFSH-D, Dammam, Saudi Arabia Introduction Femoral, jugular or subclavian central venous catheterization (CVC) is routinely performed during the care of the critically ill. These invasive procedures contribute to additional morbidity, mortality, and costs derived from the interactions between traumatic, infectious and other complications. The aim of this study is to determine whether the subclavian, jugular or femoral central venous access (CVA) routes have an eff ect on the incidence of CLABSI in critically ill patients and to compare between these routes regarding major complications and ICU mortality. P78 Use of nanotechnology-based surface antiseptic solutions in the ICU The rate of CLABSI in the IJC, SCC and FC groups was 5.8 versus 7.2 versus 3.45 per 1,000 catheter- days respectively with P = 0.35. ICU mortality was 134 (47%) cases of the IJC group, 108 (39%) cases of the SCC group and 113 (39%) cases of the FC group. There was no signifi cant diff erence between the three groups of CVC in the incidence of CLABSI rate in the critically ill patients, and a slight increase in ICU mortality in the IJC group compared with the other two groups. Pneumothorax occurred in six (2.2%) cases of SCC and 11 (3.8%) cases of IJC with no signifi cant diff erence between the two groups as the P value was 0.3.f Results There were no statistical diff erences between two groups (Table 1). Conclusion Site of insertion of CVC does not appear to aff ect the rate of CLABSI among critically ill patients. Pneumothorax was recorded in SCC and IJC groups with no statistical preference to either group. Table 1 (abstract P78). Isolated pathogen ratio percentage A (BC) B (DDCA) P value First day 25 20 1.000 Second day 5 15 0.605 Third day 30 20 0.715 Fourth day 65 50 0.527 Fifth day 45 60 0.527 Sixth day 25 25 1.000 Seventh day 60 45 0.527 Table 1 (abstract P78). Isolated pathogen ratio percentage Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials h 1 k2 Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials E Choi1, J Park2 1Yeungnam University College of Medicine, Daegu, South Korea; 2Uijeongbu St. Mary’s Hospital, Uijeongbu, South Korea Critical Care 2015, 19(Suppl 1):P81 (doi: 10.1186/cc14161) Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials Results We observed a signifi cant reduction in DSSI rates in cardiac surgery following implementation of a multimodal ICP from 3.1% in 2010 down to 0.23% in November 2014 (Figure 1). Introduction Whole-body skin decolonization with chlorhexidine in critically ill patients reduces multidrug-resistant bacterial colonization, and catheter-related bloodstream infection (BSI). We performed a meta-analysis of randomized controlled trials to determine whether daily bathing with chlorhexidine decreased hospital-acquired BSIs in critically ill patients. Introduction Whole-body skin decolonization with chlorhexidine in critically ill patients reduces multidrug-resistant bacterial colonization, and catheter-related bloodstream infection (BSI). We performed a meta-analysis of randomized controlled trials to determine whether daily bathing with chlorhexidine decreased hospital-acquired BSIs in critically ill patients. Conclusion Implementing a multimodal ICP signifi cantly reduced the incidence of DSSI in our hospital but it remains diffi cult to identify which interventions were most eff ective. Reference 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. Figure 1 (abstract P79). Incidence of DSSI. Figure 1 (abstract P79). Incidence of DSSI. y Methods We searched the MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials databases to identify randomized controlled trials that compared daily bathing with chlorhexidine and a control (daily bathing with soap and water or nonantimicrobial washcloths, or implementation of MRSA screening and isolation) in critically ill patients. The primary outcome was hospital-acquired BSIs. Secondary outcomes were adverse eff ects of chlorhexidine and the incidence of identifi ed pathogens. i Results This meta-analysis included four studies. The overall incidence of hospital-acquired BSIs was signifi cantly lower in the chlorhexidine group compared with the control 0.80 (95% CI, 0.71 to 0.90; P <0.001; I2 = 29.4%). Gram-positive (RR = 0.59, 95% CI, 0.44 to 0.79, P = 0.000; I2 = 46.0%) and MRSA-induced (pooled RR = 0.64; 95% CI, 0.47 to 0.88, P = 0.006; I2 = 0.0%) bacteremias were signifi cantly less common in the chlorhexidine group. Chlorhexidine did not aff ect Gram-negative bacteremia or fungemia. Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU y GM Plantier1, CE Bosso1, BN Azevedo2, AC Correa3, AL Silva3, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil; 3Santa Casa de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P80 (doi: 10.1186/cc14160) Introduction Urinary catheter insertion is a common procedure in ICUs and can be an important cause of infection in the hospital environment [1,2]. We aimed to analyze the eff ect of chlorhexidine on long-term urinary catheter insertion and urinary tract infection (UTI) during a 5-year period in patients admitted to a coronary ICU. y p p y Methods Analysis of patients admitted to a coronary ICU of a medium- sized hospital in Brazil from January 2010 to May 2014. The institutional protocol of periprocedural antisepsis was changed from iodine-based antiseptic to chlorhexidine in 2012. The UTI diagnosis was based on urine culture (>105 colony-forming units per ml of urine) associated with at least one clinical/laboratory abnormality (fever >38°C, urination urgency, increased urinary frequency, dysuria, or suprapubic or lumbar pain). The UTI rate represents the urinary tract infections associated with long-term urinary catheter (patient with UTI associated with long-term urinary catheter divided by patients with long-term urinary catheter × 1,000). Results The urinary tract infection rates were 4.8 (year 2010: patients·day–1 (n: 2,511), long-term urinary catheter·day–1 (n: 1,455), device usage rate (958%)), 4.4 (year 2011: patients·day–1 (n: 2,529), long- term urinary catheter·day–1 (n: 1,140), device usage rate (45%)), 0.0 (year 2012: patients·day–1 (n: 2,660), long-term urinary catheter·day–1 (n: 783), device usage rate (29%)), 0.0 (year 2013: patients·day–1 (n: 2,573), long- term urinary catheter·day–1 (n: 960), device usage rate (37%)), and 0.0 (year 2014: patients·day–1 (n: 1,070), long-term urinary catheter·day–1 (n: 444), device usage rate (42%)). y y Methods Analysis of patients admitted to a coronary ICU of a medium- sized hospital in Brazil from January 2010 to May 2014. The institutional protocol of periprocedural antisepsis was changed from iodine-based antiseptic to chlorhexidine in 2012. The UTI diagnosis was based on urine culture (>105 colony-forming units per ml of urine) associated with at least one clinical/laboratory abnormality (fever >38°C, urination urgency, increased urinary frequency, dysuria, or suprapubic or lumbar pain). P79 P79 Reduction of deep surgical site infections in cardiac surgery by introducing a multimodal infection control program A Rutten, JP Ory, L Jamaer, A Van Assche, J Dubois Jessa Ziekenhuis, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P79 (doi: 10.1186/cc14159) y g y Results The urinary tract infection rates were 4.8 (year 2010: patients·day–1 (n: 2,511), long-term urinary catheter·day–1 (n: 1,455), device usage rate (958%)), 4.4 (year 2011: patients·day–1 (n: 2,529), long- term urinary catheter·day–1 (n: 1,140), device usage rate (45%)), 0.0 (year 2012: patients·day–1 (n: 2,660), long-term urinary catheter·day–1 (n: 783), device usage rate (29%)), 0.0 (year 2013: patients·day–1 (n: 2,573), long- term urinary catheter·day–1 (n: 960), device usage rate (37%)), and 0.0 (year 2014: patients·day–1 (n: 1,070), long-term urinary catheter·day–1 (n: 444), device usage rate (42%)). Introduction Deep surgical site infections (DSSI) are a major compli- cation after cardiac surgery with a high mortality rate and reported incidences between 0.5 and 5%. Implementing a comprehensive infection control program (ICP) reduces this incidence [1]. The incidence in our hospital varied from 3.1 to 3.8%, which was considered too high. We evaluated the impact of introducing a multimodal ICP on the incidence of DSSI. Methods We noticed a too high incidence of DSSI after cardiac surgery during an observational 3-year period (Figure 1). In February 2013 we introduced a bundle of interdisciplinary infection control measures. Medical and nursing staff of all involved departments took part in developing and implementing these guidelines. Besides emphasizing the importance of existing guidelines (antiseptic shower, hair removal by clipper, strict hand hygiene, prophylactic antibiotics, limiting OR traffi c, tight glycemic control (80 to 110 mg/ dl), and so on), new strategies were introduced. The most important new strategies were nasal decolonization with mupirocin twice daily 48 hours perioperatively, preoperative antiseptic skin preparation twice (chlorhexidine gluconate 0.5%), applying topical skin adhesive to the sternal wound postoperatively and in the case of CABG procedures maintaining a strict barrier between the vein harvesting procedure and the chest procedure. Conclusion The use of chlorhexidine in the periprocedural antisepsis of urinary catheterization contributed to the decrease of urinary tract infections associated with long-term urinary catheter in patients admitted to the coronary ICU. P80 Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU GM Plantier1, CE Bosso1, BN Azevedo2, AC Correa3, AL Silva3, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil; 3Santa Casa de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P80 (doi: 10.1186/cc14160) References 1. Silva E, et al. Crit Care, 2004;8:R251-60. 2. Vincent JL, et al. JAMA. 2009;302:2323-9. Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU The UTI rate represents the urinary tract infections associated with long-term urinary catheter (patient with UTI associated with long-term urinary catheter divided by patients with long-term urinary catheter × 1,000). 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. P77 P77 Role of neutrophil extracellular traps against soft tissue infections N Yamamoto1, M Ojima2, S Hamaguchi1, T Hirose2, R Takegawa2, N Matsumoto2, T Irisawa2, M Seki1, O Tasaki3, T Shimazu2, K Tomono1 1Division of Infection Control and Prevention, Osaka University Graduate School of Medicine, Suita, Japan; 2Osaka University Graduate School of Medicine, Suita, Japan; 3Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Critical Care 2015, 19(Suppl 1):P77 (doi: 10.1186/cc14157) Critical Care 2015, 19(Suppl 1):P77 (doi: 10.1186/cc14157) Introduction Neutrophils work as the frontline of defense against infections and neutrophil extracellular traps (NETs) are one of the S28 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The eff ect of a good surface disinfectant should begin immediately and it should have a long-lasting disinfecting eff ect on the surface. DDAC is an effi cient disinfectant used in medicine and the food industry to protect the surfaces. However, it may cause severe skin itching. BC, which is a nanotechnology-based product, leaves its active metabolites on the surface; it is applied by constituting a spongy layer. Since the effi ciency of BC lasts for 24 hours and it is applied to perform cleaning with 24-hour intervals, we think that it is preferable with regards to workforce gain and cost. Clinical validation of an electronic hand hygiene surveillance system y Methods A quasi-experimental study was done in three level III ICUs of a tertiary care hospital in Kolkata (January to April 2014). Data were collected on existing hand hygiene compliance rate, ventilator- associated pneumonia (VAP) rate, catheter-related bloodstream infection (CRBSI) rate, catheter-related urinary tract infection (CAUTI) rate, standardized mortality ratio (SMR) and average ICU length of stay in the abovementioned units. Root cause analysis was done and interventions were developed to improve hand hygiene compliance and was implemented (July to October 2014). Comparison was done between preintervention and postintervention periods. y P Levin, R Razon, MJ Cohen, CL Sprung, S Benenson Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2015, 19(Suppl 1):P84 (doi: 10.1186/cc14164) P Levin, R Razon, MJ Cohen, CL Sprung, S Benenson Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2015, 19(Suppl 1):P84 (doi: 10.1186/cc14164) Introduction Good hand hygiene (HH) is critical to infection control in the ICU. Electronic HH surveillance systems are purported to improve HH practices. Such a system was recently trialed in our ICU. The system is based on radiofrequency transponders in three locations: bracelets worn by ICU personnel; on all HH product dispensers; and above each patient’s bed. By correlating input from these three sources the system detects whether HH was performed before and after each patient contact. In the event that HH is not performed, the bracelet alerts the user (by vibration) in real time. This study represents a clinical validation of the system. Results In the preintervention period (January to April 2014) the hand hygiene compliance among the caregivers was found to be 40%, VAP rate (8.77), CRBSI rate (3.42), CAUTI rate (5.27), SMR (1.14) and average ICU length of stay was 6 days ± 5.85 SD (median 4.5). Interventions were developed and implemented as follows: education and awareness – road shows; positive reinforcement; secret watch nurse; e-ICU – electronic surveillance; ring the bell once every hour – baseline hand hygiene; visual reminders; availability of alcohol-based hand rub, soap and water and sinks; random hand swabs; and compliance audits. In the postintervention period (July to October 2014) data showed a signifi cant improvement in the hand hygiene compliance (75%). Further analysis showed an association with decrease in the incidence of VAP rate (4.71), CAUTI rate (3.51), CRBSI rate (2.65), SMR (1.05) and average ICU LOS 5.05 days ± 4.03 SD (median 4). Improving hand hygiene compliance leads to improved health outcome: an analysis y V Rao, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India p Conclusion Introduction of infection control bundle in the ICU reduced the incidence of nosocomial MDRO transmission and infection, which resulted in the reduction of anti-MRSA antibiotics and carbapenems use in critically ill patients. p p y p Critical Care 2015, 19(Suppl 1):P82 (doi: 10.1186/cc14162) Introduction Hand hygiene is the single most eff ective but least practiced action in breaking the chain of transmission of microbes. Studies have shown a correlation between the compliance of hand hygiene and its impact on the health outcome. Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials h 1 k2 The overall incidence of adverse events, such as skin rashes, was similar in both groups. Conclusion Implementing a multimodal ICP signifi cantly reduced the incidence of DSSI in our hospital but it remains diffi cult to identify which interventions were most eff ective. Reference 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. S29 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Daily bathing with chlorhexidine was associated with a reduction in the rates of hospital-acquired BSI without signifi cant complications in critically ill patients. It also decreased the incidence of Gram-positive hospital-acquired BSIs, especially MRSA. Results Admission to the ICU comprised 363 patients in the fi rst period and 380 patients in the second period. The incidence of transmission was decreased from 48 (13.2%) to 21 (5.5%) in methicillin-resistant Staphylococcus aureus (MRSA), from 16 (4.4%) to zero (0%) in multidrug- resistant Acinetobacter baumannii. The incidence of nosocomial infection by MDRO was also decreased from 23 (6.3%) to 17 (4.5%) in pneumonia, from fi ve (1.4%) to two (0.3%) in urinary tract infection, and from 12 (3.3%) to one (0.3%) in surgical site infection. The incidence of antibiotic use for MDRO infection was decreased from 41 (11.3%) to 24 (6.3%) in anti-MRSA antibiotics, and from 19 (5.2%) to eight (2.1%) in carbapenems. Clinical validation of an electronic hand hygiene surveillance system y Methods ICU staff (nurses and physicians) were followed by a trained observer over 60-minute periods. Each movement and contact during the period was documented. HH opportunities were determined according to WHO criteria and actual HH performance recorded. Observer and electronic data were compared for number of opportunities, HH performance and compliance. A satisfaction questionnaire was distributed to all users. Paired Student’s t test was used for comparison of the observer and electronic data. Results Observations were made over 56 time periods that included 836 HH opportunities and 485 occasions when HH was performed. The observer recorded 10.9 ± 7.6 HH opportunities/hour compared with 6.8 ± 6.9 for the electronic system (P <0.001). HH performance occurred on 8.7 ± 3.9 occasions/hour versus 6.0 ± 3.1 occasions/hour as recorded by the electronic system (P <0.001). Overall HH compliance was 62.5 ± 17.7% versus 57.5 ± 21.0% respectively (P = 0.523). On comparison of specifi c observation periods, there was poor correlation between compliance as recorded by the observer and electronic system (r = 0.03, P = 0.915). Satisfaction questionnaires were completed by 41 personnel. Satisfaction with the system was low or very low for 21/41 (61%). System inaccuracy (either bracelet alerts without cause, or lack of bracelet alerts when HH was required) was the most common reason for dissatisfaction (31/41, 76%), followed by physical discomfort from the bracelet (18/41, 44%). Conclusion Improved hand hygiene compliance can be attributed to decreased incidence of VAP, CRBSI, CAUTI, SMR and average ICU LOS. This does defi nitely impact the overall clinical outcome. However, continued surveillance of hand hygiene compliance and regular audits is of utmost importance to make the change sustainable. P83f Eff ects of infection control bundle to prevent nosocomial infection in the ICU A Matsushima, M Kawanami, S Fujimi, N Inadome, N Kubo, T Yoshioka Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P83 (doi: 10.1186/cc14163) Eff ects of infection control bundle to prevent nosocomial infection in the ICU in the ICU A Matsushima, M Kawanami, S Fujimi, N Inadome, N Kubo, T Yoshioka Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P83 (doi: 10.1186/cc14163) Introduction Multidrug-resistant organism (MDRO) infections in critically ill patients are often life-threatening. To prevent nosocomial infections of MDRO, we made an infection control bundle in our ICU in 2013. In this study we evaluated the eff ect of our infection control bundle to prevent nosocomial MDRO transmission and infection. Conclusion The electronic HH system consistently underestimated both HH opportunities and HH performance. The main reason for dissatisfaction with the system was inaccuracy of bracelet alerts. These data suggest that for an electronic system to be accepted by ICU staff , it has to be highly accurate and comfortable for the user. Methods Our infection control bundle consists of preemptive contact precaution to all care, active surveillance culture and isolation of patients with MDRO. This bundle was applied to all patients admitted to our ICU since 2013. The study period to evaluate the eff ects of the bundle was from April 2012 to March 2014, and we divided it into two periods; fi rst period (before introduction of the bundle) and second period (after introduction of the bundle). We compared the incidence of nosocomial transmission and infection of MDRO between the two periods. MDRO was defi ned as bacteria that were resistant to more than three kinds of antibiotics. Nosocomial transmission was defi ned when MDRO was detected later than 48 hours after admission. Nosocomial infection was diagnosed according to the National Nosocomial Infection Surveillance Manual. P86 A survey of UK acute clinicians’ knowledge of personal protective requirements for infectious diseases and chemical, biological, and radiological warfare agents AR Bond1, A Buckingham2, J Schumacher1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P86 (doi: 10.1186/cc14166) Evaluation of the microbial tightness of closed system transfer devices by simulating airborne and touch contamination University of Bonn, Germany Introduction The use of intravascular catheter devices is often associated with serious bloodstream infections due to microbial contaminations. To minimize risk of such infections NIOSH recommends S30 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 the use of closed system transfer devices (CSTDs). To evaluate the microbial tightness of CSTDs we developed two methods which simulate the bioburden in ambient air of operating rooms and ICUs. the use of closed system transfer devices (CSTDs). To evaluate the microbial tightness of CSTDs we developed two methods which simulate the bioburden in ambient air of operating rooms and ICUs. for clinicians treating patients exposed to infectious diseases and CBRN agents, ideally in a simulation setting. Further research into whether the required levels of PPE are readily available to clinicians would be pertinent. Methods The methods simulate airborne and touch contamination. We tested the microbial tightness of the integrated Safefl ow® valve of a Mini-Spike® which is used for drug admixture. The airborne contamination was done in an exposure chamber in which a nebulizer distributed defi ned B. subtilis spore aerosols [1]. A Mini-Spike® was inserted into a vial of 0.9% sodium chloride solution (NaCl). A nebulizer with a suspension of 4.8 × 105 CFU spores of B. subtilis per ml was used to generate an aerosol for 1 minute. The volume of B. subtilis suspension nebulized per minute was 0.278 ml. This corresponds to 1.34 × 103 aerosolized spores in the exposure chamber, which has a volume of 0.24 m3 (5.6 × 103 CFU per m3 air). The used concentration was 100 times higher than the microbial burden found in hospitals [2]. After nebulization the valve was disinfected and NaCl was withdrawn into a syringe at certain time intervals. The NaCl was incubated on tryptic soy agar at 37°C for 48 hours. Results were documented as CFU. For touch contamination, a Mini-Spike® was attached to a vial of NaCl. The valve of the Mini-Spike® was contaminated with 105 CFU Staphylococcus aureus. The subsequent procedure was done as described above. g References 1. Dunkelberg H, Fleitmann-Glende F. Measurement of the microbial barrier eff ectiveness of sterilization containers in terms of the log reduction value for prevention of nosocomial infections. Am J Infect Control. 2006;34:285-9. 1. Dunkelberg H, Fleitmann-Glende F. Measurement of the microbial barrier eff ectiveness of sterilization containers in terms of the log reduction value for prevention of nosocomial infections. Am J Infect Control. 2006;34:285-9. 2. Qudiesat K. Assessment of airborne pathogens in healthcare settings. AJMR. 2009;3:66-76. Results During the study period, 40 patients with TB were admitted to the ICU; 75% male and median age of 52 years (IQR 37.5 to 62.8). Overall, 22 (55%) patients died in the hospital, of whom 16 (40%) died in the ICU. Comorbid illness was identifi ed in 32 (80%) patients, with HIV infection being the most common, present in 15 (37.5%) patients. The main reason for ICU admission was respiratory failure (70%), followed by sepsis/septic shock (22.5%). Twenty-eight (70%) patients had isolated pulmonary disease, four (10%) had isolated extrapulmonary disease and eight (20%) had association of pulmonary and extrapulmonary disease. Mycobacterial cultures were positive in 31 (77.5%) patients; three patients presented monoresistant strains. Twenty-nine (72.5%) patients required mechanical ventilation and 21 (52.5%) required vasopressor infusion in the ICU; two patients were treated with ECMO. Thirty-four (85%) patients received antituberculosis therapy. The median length of stay was 11.5 (IQR 3.25 to 28.5) days in the ICU and 40.5 (IQR 21.0 to 62.8) days in the hospital. The presence of at least one comorbidity, smoking, age, sepsis/septic shock on admission, high SAPS II and APACHE II score, positive direct examination and PCR in respiratory samples, the need for mechanical ventilation or vasopressor infusion were signifi cantly associated with mortality (P <0.05). There was no association between mortality and HIV status, site of TB disease, concomitant acute disease or development of hospital infections. 2. Qudiesat K. Assessment of airborne pathogens in healthcare settings. AJMR. 2009;3:66-76. R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Introduction To describe the characteristics of the patients with tuberculosis (TB) requiring intensive care and to identify the factors associated with in-hospital mortality in an ICU in Portugal. Results Out of nine tested valves, none showed transmission of B. subtilis spores after airborne contamination. Three out of nine tested valves were contaminated with S. aureus after touch contamination. Conclusion Our study shows that both methods are suitable for evaluating the microbial tightness of CSTDs. References Methods A retrospective cohort study between January 2007 and July 2014 of all patients with TB admitted to the ICU of the Infectious Diseases Department of Centro Hospitalar de São João. Comorbid diagnoses, clinical features, radiological and laboratory investigations and outcomes were reviewed. The primary outcome was the in- hospital mortality. A univariate analysis was performed to identify risk factors for death. References References 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West Africa. http://www.cdc.gov/vhf/ebola/outbreaks/guinea/. 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West Africa http://wwwcdc gov/vhf/ebola/outbreaks/guinea/ 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West p g g 2. Brinker A, et al. Personal protection during resuscitation of CBW victims. A survey among medical fi rst receivers in the UK. Prehosp Disaster Med. 2009;24:525-8. A survey of UK acute clinicians’ knowledge of personal protective requirements for infectious diseases and chemical, biological, and radiological warfare agents AR Bond1, A Buckingham2, J Schumacher1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P86 (doi: 10.1186/cc14166) Introduction We conducted a survey to assess clinicians’ knowledge of personal protective equipment (PPE) requirements for infectious diseases and biochemical warfare agents. A safe level of PPE is essential when treating patients with highly infectious diseases or those contaminated with hazardous substances. The recent Ebola virus disease (EVD) outbreak in West Africa has highlighted that, although uncommon, contagious diseases with high mortality rates can be a threat to healthcare systems at local, national, and international levels [1]. Chemical, biological, radiological or nuclear (CBRN) contamination presents similar risks. Conclusion In this cohort we found a high mortality rate in the TB patients requiring intensive care. The risk factors for mortality due to severe TB are mainly related to the severity of organ failure, patient characteristics and burden of disease and not to HIV status or site of TB disease. Methods A validated, hand-delivered, multiple-choice questionnaire [2] was used to assess intensive care, emergency medicine, and anesthetics specialist registrars’ knowledge of respiratory and skin protection needed during a resuscitation scenario with advanced life support. Participants selected the PPE required for the biological hazards: EVD, severe acute respiratory syndrome (SARS), inhalational anthrax, plague and smallpox; and the biochemical hazards: sarin, hydrogen cyanide, phosgene and mustard gas (dichlordiethyl sulfi de). Responses were compared with UK national recommendations and a previous survey in 2009 [2]. Tuberculosis in the ICU: a retrospective cohort study Tuberculosis in the ICU: a retrospective cohort study R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 Introduction Changes in infection agents and their sensitivity to antibiotics are the main cause of severity of surgical infections. In spite of development and introduction of new drugs and methods of treatment, the number of patients with sepsis increases, so the problem in diagnosing and treatment is still far from resolution. Introduction Changes in infection agents and their sensitivity to antibiotics are the main cause of severity of surgical infections. In spite of development and introduction of new drugs and methods of treatment, the number of patients with sepsis increases, so the problem in diagnosing and treatment is still far from resolution. Methods A comparative retrospective analysis of 52 histories of patients with sepsis, which were treated in the Department of Surgical Infections in 1999 and 2013. g g Methods A comparative retrospective analysis of 52 histories of patients with sepsis, which were treated in the Department of Surgical Infections in 1999 and 2013. Results The number of patients with sepsis in 2013 was raised 2.7 times, in comparison with 1999. Mortality decreased from 79% in 1999 to 55% in 2013. In most cases sepsis was accompanied with immunosuppressive disorders, such as diabetes, oncology, alcohol and drug addiction, and HIV infection. We analyzed crops of discharge from the wound and blood cultures in 52 patients with sepsis. Crops of wound were taken during the initial surgical intervention, then every 3 to 7 days, as well as the surgical interventions being repeated. Blood cultures were performed in the presence or suspected diagnosis of sepsis, in accordance with the classifi cation Bone criteria. In comparison of spectrum of infection agents, Staphylococcus aureus is still leading (1999 – 36.6% of isolates, 2013 – 25%), and the percentage of MRSA was 0% in 1999 and 37.5% in 2013. The frequency of Gram-negative fl ora has increased: E. coli (8.5%/20%), P. aeruginosa (8.5%/12%), Klebsiella pneumoniae (0%/16%) and Acinetobacter spp. (0%/16%). Speaking about the resistance of microorganisms, there is still a high percentage of sensitivity to aminoglycoside antibiotics (79.4%/75%), glycopeptides (77.2%/71%), carbapenems (88.4%/78%) and also to the combination therapy (71.8%/62.4%), but also a reduction in sensitivity to the group of beta-lactam antibiotics (58.2%/32.5%) and fl uoroquinolones (64.6%/36.4%). Conclusion Cutaneous mucormycosis is less common than other clinical forms, most frequently seen in inmunocompetent patients but potentially lethal if treatment is not rapid. Patients at risk are those with disruption of the normal protective cutaneous barrier. Low-pathogenicity mycoplasma species induce immunoparesis and are highly prevalent amongst patients with ventilator-associated pneumonia TJ Nolan1, N Gadsby2, TP Hellyer3, K Templeton2, R McMullan4, J McKenna5, J Rennie1, CT Robb1, TS Walsh1, AG Rossi1, AJ Simpson3, A Conway Morris6 1University of Edinburgh, UK; 2NHS Lothian, Edinburgh, UK; 3Newcastle University, Newcastle, UK; 4Queen’s University, Belfast, UK; 5Belfast Health and Social Care Trust, Belfast, UK; 6University of Cambridge, UK Critical Care 2015, 19(Suppl 1):P89 (doi: 10.1186/cc14169) Introduction Ventilator-associated pneumonia (VAP) remains a signifi cant problem within ICUs. There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms of this immunoparesis remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent amongst patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. Conclusion The number of patients with sepsis has increased; the mortality of sepsis has decreased. The frequency of S. aureus isolation is still high, MRSA is the same. The frequency of Gram- negative fl ora isolation has increased, especially K. pneumoniae and Acinetobacter spp. The resistance of microorganisms to beta-lactams and fl uoroquinolones is rising but the sensitivity to aminoglycosides, glycopeptides, and carbapenems is still maintained. y Methods Two cohorts [1,2], totalling 159 patients, were recruited from 12 UK ICUs; all patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage. VAP was defi ned as growth of organisms at >104 CFU/ml on conventional culture. Thirty- six healthy donors underwent lavage for comparison. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. Additionally, healthy donor monocytes and macrophages (MDM) were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. P91 2. Hellyer T, et al. Thorax. 2014 [Epub ahead of print]. Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 In these patients, if signs are of sepsis it is very important to suspect the possibility of infection by Mucor and initiate empiric antifungal treatment with surgery to avoid high mortality. Surprisingly, in our series, determination of procalcitonin showed high levels in spite of not having value in fungal infection. 1. Conway Morris A, et al. Thorax. 2010;65:201-7. Infectious events and prescription of antimicrobials in the coronary ICU CE Bosso1, SV Ferreira2, GE Valerio2, JV Moraes2, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P91 (doi: 10.1186/cc14171) Introduction The eff ectiveness of initially used antimicrobials represents an important factor in infectious events in coronary intensive care units (CICU) [1]. This study aimed to analyze the prevalence of infectious events and the prescribed antimicrobial in CICU. Introduction The eff ectiveness of initially used antimicrobials represents an important factor in infectious events in coronary intensive care units (CICU) [1]. This study aimed to analyze the prevalence of infectious events and the prescribed antimicrobial in CICU. Results Mycoplasmataceae were found in 48% of patients with VAP, compared with 14% of patients without VAP (P <0.0001). Patients with sterile lavage had a similar prevalence to healthy donor lavage (10 vs. 8%, P = 0.54). The commonest organism identifi ed was M. salivarium. Human blood monocytes and MDM incubated with M. salivarium displayed impaired cytokine responses to lipopolysaccharide and MDM demonstrated impaired phagocytosis. Methods We analyzed the data of 2,005 patients admitted to the CICU for 3 years. The infectious events were based on general characteristics, main sites and outbreaks of infectious events in addition to the main microorganisms and pathogens. The prescription of antimicrobials was analyzed based on the isolated or associated use of antimicrobials. We also analyzed the adequacy of initial empirical antimicrobial according to the microbiological evidence. The general characteristics of events – that is, time, evidence of infection, infections by multidrug-resistant pathogens – are also presented. y Conclusion This study demonstrates a high prevalence of Mycoplasmataceae amongst patients with VAP, with a markedly lower prevalence amongst patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The commonest organism found, M. salivarium, is able to profoundly impair the functions of key immune cells and thus suggests that Mycoplasmataceae may contribute to VAP pathogenesis. Results The prevalence of infection was 4% (n = 81). Ventilator- associated pneumonia was 35% (n = 28), whereas urinary and primary bloodstream associated with catheters was 14% (n = 11) and 9% (n = 7), respectively. There was 82% (n = 66) evidence of microbiological infection. The main pathogens and microorganisms found were P88 Cutaneous mucormycosis in the ICU Cutaneous mucormycosis in the ICU EH Herrero, M Sánchez, A Agrifoglio, L Cachafeiro, MJ Asensio, B Galván, A García de Lorenzo Hospital La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P88 (doi: 10.1186/cc14168) Results Ninety-eight clinicians (anesthetics n = 51, emergency medicine n = 21, intensive care medicine n = 26) completed surveys. The best knowledge (76% correct) was for SARS, with less knowledge for anthrax, plague, EVD, and smallpox (60%). We found limited knowledge for chemical warfare agents (20 to 30%). Sixty to 80% of all incorrect responses were over-rated. There was no diff erence in knowledge compared with previous published results [2]. Introduction Mucormycosis is a devastating disease most commonly seen in immunosuppressed individuals. It has the propensity to disseminate in humans and cause rhinocerebral, pulmonary, gastrointestinal, and cutaneous infections. This study focuses on cutaneous mucormycosis, incidence, epidemiologic characteristics and mortality in intensive care medicine. Conclusion Despite national and regional training since previous surveys [2], the results indicate that further training on PPE is required S31 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We present a descriptive study in an ICU between the years 2001 and 2013 on the incidence of patients with cutaneous mucormycosis. Sociodemographics, comorbidities and laboratory data were recorded. Clinical data were collected to calculate the APACHE score. The main outcome was to analyze the epidemiological characteristics of patients with cutaneous mucormycosis and mortality. Results Seven patients were identifi ed with cutaneous mucormycosis between the years 2001 and 2013. The mean age of patients was 52 ± 4, with an APACHE score of 19 ± 9, and 57% died. All patients were admitted for trauma-related injury suff ering blast, abrasive injuries or burns. Mortality among patients with signs of sepsis was 100%, and only in one of them was empirically antifungal therapy started; in the others antibiotic treatment was directed. Among patients without signs of sepsis, the survivor was treated with amputation where mucoral infection was isolated. Procalcitonin rose in all patients with signs of sepsis. P90 Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 IV Avdoshin, LG Akinchits, ES Konstantinova, MA Shatil, ON Dobrydin, NA Bubnova Saint-Petersburg City Hospital of St. George, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P90 (doi: 10.1186/cc14170) Analysis of Gram-negative rod bacteremia in the surgical and medical ICU Analysis of Gram-negative rod bacteremia in the surgical and medical ICU D Adukauskiene, D Valanciene Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P92 (doi: 10.1186/cc14172) Introduction The aim was to analyze the Gram-negative bacteremia profi le and the predisposing factors for length of stay in the surgical and medical ICU and outcome. Methods Retrospective data analysis of patients during 4 years treated in a surgical and medical ICU with positive blood culture for Gram- negative rod. g Results Gram-negative rod monobacteremia (n = 160) cultures revealed: Escherichia coli (n = 52, 32.5%), Acinetobacter spp. (n = 47, 29.4%), Klebsiella spp. (n = 22, 13.7%), Enterobacter spp. (n = 20, 12.5%), Proteus spp. (n = 11, 6.9%), anaerobes (n = 3, 1.9%) and other Gram-negative rods, including Stenotrophomonas maltophilia, Haemophilus infl uenzae, Neisseria meningitidis, Achromobacter spp. and Actinobacillus limirensi (n = 5, 3.1%). Both E. coli and Acinetobacter spp. were responsible for the vast majority of Gram-negative rod monobacteremia (n = 99, 61.8%, P = 0.0128). Also most often (n = 50, 72.5%, P = 0.049) primary bacteremia was caused by E. coli and Acinetobacter spp. Separate group’s multidrug resistance was found: E. coli in 12 (23.1%) cases, Acinetobacter spp. in 45 (95.7%, P = 0.02), Klebsiella spp. in nine (40.9%), Enterobacter spp. in 11 (55.0%), Proteus spp. in six (54.6%) cases. The vast majority of patients with multidrug-resistant bacteremia were aged over 65 years (n = 64, 77.1%, P = 0.042), stayed in the ICU less than 14 days (n = 70, 84.3%, P = 0.039), and had lethal outcome (n = 74, 89.2%, P = 0.03). Patients who stayed in the ICU less than 14 days presented with primary Gram-negative rod bacteremia (n = 67, 57.7%, P = 0.03), need for mechanical ventilation (n = 90, 77.6%, P = 0.043) and lethal outcome (n = 112, 96.6%, P = 0.01). Lethal outcome was confi rmed in patients with primary Gram-negative rod bacteremia (n = 55, 79.7%, P = 0.03), MDR strain (n = 74, 89.2%, P = 0.03), presence of shock (n = 120, 75.0%, P <0.001), mechanical ventilation (n = 133, 74.3%, P <0.001), cancer chemotherapy (n = 18, 90.0%, P = 0.03), and chronic obstructive pulmonary disease (n = 13, 100%, P = 0.03). Conclusion The carriage of MDRO in ICU-admitted patients is important, especially for ESBL-GN. Is carriage of multidrug-resistant organisms a risk factor for nosocomial infections in an infectious diseases ICU? Introduction The objective was to evaluate whether asymptomatic carriage of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and extended-spectrum beta- lactamase producing Gram-negative bacilli (ESBL-GN) on admittance to the ICU of the University Hospital of Infectious Diseases Cluj-Napoca, Romania is a risk factor for infection due to these multidrug-resistant organisms (MDRO) during hospitalization. fi Conclusion We conclude that infection is prevalent even in CICU, and that the microbiological profi le is quite diverse, as well as the antibiotics. This allows us to better understand the profi le of this kind of unit. Reference g g p Methods A prospective study during a 6-month period (June to November 2014), including all adult patients admitted to our ICU. All patients were screened on admittance for nasal MRSA, intestinal VRE and ESBL-GN carriage. Patients admitted with any localization of infections due to these organisms were excluded. Patients were monitored for developing nosocomial infections due to MDRO during hospitalization. We evaluated previous colonization as a risk factor for future infections. We used bioMerieux selective chromogenic media for MDRO for screening and Vitek2Compact for identifi cation. Statistical analysis was performed with chi-square test and univariate analysis. 1. Silva E, et al. Crit Care. 2004;8:R251-60. y p q y Results From 119 screened adult patients, 65 women (54.6%), average age 67 years, we had at screening on admittance into the ICU: 14 positive MRSA (11.8%), 63 positive ESBL-GN (52.9% – 41 strains of Escherichia coli, 26 strains of Klebsiella sp., 11 strains of Proteus mirabilis and one strain of Enterobacter cloacae) and 35 positive VRE (29.4% – 33 strains of Enterococcus faecium and two strains of Enterococcus faecalis) without concomitant infection with these MDRO. The average duration of ICU stay was 7.32 days. During hospitalization, 14 patients (11.8%) developed nosocomial infections due to MDRO. Colonization with MDRO preceded nosocomial infections in: one of four patients with MRSA-positive blood cultures (P = 0.96), seven of eight patients with ESBL-GN infections (P = 0.10) and three of four patients with VRE urinary tract infections (P = 0.14). Although not statistically signifi cant, owing to the low number, most patients who developed infections with ESBL-GN had previous intestinal colonization. 1. Silva E, et al. Crit Care. 2004;8:R251-60. P93 Gram-positive bacteria (n = 24, 30%; Staphylococcus aureus (n = 16, 20%), Enterococcus faecalis (n = 4, 5%), Staphylococcus epidermidis (n = 3, 4%)), Gram-negative (n = 43, 53%; klebsiella sp. (n = 13, 16%), Pseudomonas aeruginosa (n = 7, 9%), Escherichia coli (n = 7, 9%)) and fungi (n = 5, 6%; candida sp. (n = 2, 3%), Candida albicans (n = 1, 1%), Candida dubliniensis (n = 1, 1%)). The commonly prescribed antimicrobials were piperacillin/tazobactam (n = 32, 40%), vancomycin (n = 30, 37%), polymyxin B (n = 23, 28%), cefepime (n = 16, 20%), meropenem (n = 12, 15%), cefuroxime (n = 8, 10%), ciprofl oxacin (n = 6, 7%), tigecycline (n = 6, 7%), ampicillin (n = 5, 6%), clindamycin (n = 4, 5%), chloramphenicol (n = 4, 5%), oxacillin (n = 4, 5%) and others (n = 32, 28%). There was 75% (n = 46) infection during hospitalization in the unit. Approximately 32% of infections were caused by multidrug-resistant pathogens, although there was effi ciency of 81% in the proper use of initial antimicrobials. Conclusion We conclude that infection is prevalent even in CICU, and that the microbiological profi le is quite diverse, as well as the antibiotics. This allows us to better understand the profi le of this kind of unit. Reference Is carriage of multidrug-resistant organisms a risk factor for nosocomial infections in an infectious diseases ICU? M Lupse1, M Flonta2, L Herbel2, A Petrovan2, A Binder2, N Todor3, A Cioara1 1University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Teaching Hospital of Infectious Diseases, Cluj-Napoca, Romania; 3‘Ion Chiricuta’ Institute of Oncology, Cluj-Napoca, Romania Critical Care 2015, 19(Suppl 1):P93 (doi: 10.1186/cc14173) p g References S32 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P96 admission; 4.8% required the extrarenal depuration technique. In total, 497 patients (44.1%) were coronary, 49.5% male, mean age 66.18 (SD ±12.6), CI (64.88 to 67.48); mean APACHE II 9.74 (SD ± 6.1), CI (9.1 to 10.3); and a mean time stay of 3.62 days (SD ±4.7), CI (3.1 to 4.1). Mortality in this group was 3.7%. In 61.9% of cases the diagnosis of admission was AMI, 16% arrhythmia and 11.6% unstable angina. Of patients, 629 were polyvalent (55.8%), 53.85% male, mean age 58.05 (SD ±17.2), CI (56.7 to 59.4); mean APACHE II 14.6 (SD ±9.1), CI (13.8 to 15.3); and a mean time stay of 4.64 days (SD ±7.7), CI (4 to 5.25). Mortality was 11.6%. In 33.2% the cause of income was digestive, 23.2% acute or chronic exacerbated respiratory failure, 12.4% severe sepsis/septic shock and 10.1% postoperative cardiovascular surgery. The incidence density (ID) of catheter-related bacteremia was 5.5, 92.8% from the fourth day of ICU admission; ID of ventilator-associated pneumonia (VAP) was 5.94, 88.9% since the fourth day; and ID of urinary tract infections (UTI) related to urinary catheter was 2.88, 80% of them since the fourth day. From all patients who developed intra-ICU infections, mean APACHE II in admission was 21.3 (SD ±9.6) with a mean time stay of 23.4 days (SD ±12.9) and a mortality percentage of 19.6%. P95 Emergence of isolates that are intrinsically resistant to colistin in critically ill patients: are we selecting them out? MN Sivakumar, M Hisham, V Nandakumar, T Gopinathan Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P95 (doi: 10.1186/cc14175) Introduction Poor infection control practices along with irrational usage of antibiotics lead to emergence of multidrug-resistant (MDR) organisms. Increasing use of colistin for treating MDR infections leads to selection of organisms that are intrinsically resistant to colistin. There are limited Indian literatures which evaluated the incidence of intrinsically resistant isolates to colistin in critically ill patients. Our study aimed to investigate the incidence of true pathogen or colonizer with the prior antibiotic exposure and patient’s clinical outcome. Conclusion Being a broad-spectrum bactericidal agent usable both orally and parenterally with low toxicity profi les and lesser prevalence of cross-resistance with other antimicrobials, fosfomycin can be an alternative to other broad-spectrum agents to treat uncomplicated infections as well as in the case of infections with MDR organisms where treatment options are very few. This study possibly reveals a much- needed solution for the rising carbapenem resistance and also for the treatment of infections with such MDR pathogens, thereby bringing down the length of stay in hospital, cost of therapy and suff ering on the part of the patients. Methods The prospective, cross-sectional study was carried out from March 2013 to April 2014. Clinical samples included culture positivity for isolates intrinsically resistant to colistin from patients who were admitted to the ICU or had a prior ICU stay in the same admission. Methods The prospective, cross-sectional study was carried out from March 2013 to April 2014. Clinical samples included culture positivity for isolates intrinsically resistant to colistin from patients who were admitted to the ICU or had a prior ICU stay in the same admission. Results A total of 93 unusual Gram-negative rods were isolated from 76 patients. This included 19.4% (n = 18) Serratia marcescens, 12.9% (n = 12) Stenotrophomonas maltophilia, 14% (n = 13) Burkholderia cepacia, 24.7% (n = 23) Proteus mirabilis, 17.2% (n = 16) Morganella morganii, 9.7% (n = 9) Elizebethkingia meningoseptica and 2.1% (n = 2) Providencia species. A total of 68.4% (n = 52) patients had prior exposure to either colistin or carbapenems or both. In total, 71% (n = 66) of the total isolates from patients had previous antibiotic exposure. 1. Rit K, et al. IJCP. 2013;24:451-5. 2. Garbati MA, et al. J Infect Dev Ctries. 2013;7:213-6. Analysis of Gram-negative rod bacteremia in the surgical and medical ICU The incidence of nosocomial infections with MDRO in the ICU is high. ESBL-GN intestinal colonization could be a risk factor for nosocomial infections but further studies are needed to confi rm this. Patient epidemiology in a level II hospital ICU and how main nosocomial infections aff ect morbidity and mortality M Muñoz, E Yuste, O Moreno, R Fernandez, R Ramirez Hospital Universitario San Cecilio, Granada, Spain Critical Care 2015, 19(Suppl 1):P94 (doi: 10.1186/cc14174) Introduction We describe the type of patient and the main nosocomial infections in a level II hospital ICU unit, 18 beds (12 polyvalent-general, six coronary). Introduction We describe the type of patient and the main nosocomial infections in a level II hospital ICU unit, 18 beds (12 polyvalent-general, six coronary). y Methods We used the ENVIN-HELICS database and made statistical calculations for all patients admitted to the ICU between 1 October 2012 and 30 September 2013 using SPSS v.15. g Results Patients admitted (1,126): 65.1% were male; mean age 61.72 (SD ±15.8), CI (60.7 to 62.7); mean APACHE II 12.6 (SD ±8.42), CI (12.12 to 13.11); and a mean time stay of 4.84 days (SD ±6.26). In total, 68.9% were provided from the community. A total of 44.1% were coronary, 2.84% trauma and 53.02% medical–surgical patients. A total of 29.8% had antibiotic therapy in the ICU, 20% had it before incoming. In total, 18.38% were treated with artifi cial airway (MV, tracheostomy). In total, 54.09% used a urinary catheter and 38.8% needed a central venous catheter. Fifteen percent of patients had some kind of surgery before Conclusion E. coli and Acinetobacter spp. – the most often pathogens of Gram-negative rod bacteremia – were mostly multidrug resistant. Multidrug-resistant bacteremia was related to age, length of stay less than 14 days, and lethal outcome. Predisposing factors for shorter length of stay: primary bacteremia, mechanical ventilation, lethal outcome, and for lethal outcome: primary bacteremia, multidrug resistance, presence of shock, mechanical ventilation, cancer chemotherapy, chronic obstructive pulmonary disease. S33 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P95 Among the total 93 intrinsically resistant isolates to colistin, 37.6% (n = 35) of isolates from all clinical sources (endotracheal, pus, urine and blood samples) were true pathogens and the remaining 62.3% (n = 58) were colonizers. There was a statistically signifi cant increase in length of ICU stay and duration of hospitalization in the presence of true pathogen. Conclusion Selection pressure due to extensive use of higher antibiotics may lead to emergence of intrinsically resistant isolates, which narrows the therapeutic options in the ICU. Our study emphasizes the paramount importance of establishing clinical relevance of these organisms before treating them as true pathogens. This calls for judicious use of higher antibiotics, implementation of an antibiotic stewardship program and strict infection control practices. f Kolkata A Chakraborty, S Roy, S Chakraborty, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P96 (doi: 10.1186/cc14176) Introduction In the era of rising prevalence of serious infections caused by multidrug-resistant (MDR) organisms and the paucity of in-fl ow of newer antimicrobial agents, the relatively older antibiotics that had been left out of clinical practice for various reasons are now being increasingly considered as the potential agents to combat such infections. Fosfomycin, known for almost four decades, has a broad spectrum of activity against several Gram-negative and Gram-positive bacteria. Methods This study, conducted in the Microbiology Department of Medica Superspecialty Hospital between July and November 2014, was aimed at testing the in vitro sensitivity of fosfomycin against isolates identifi ed from various clinical specimens from diff erent parts of Kolkata. After confi rming the identity and antibiogram by Microscan Autoscan 4, the isolates were tested for fosfomycin sensitivity by the Epsilometer test. MIC values were interpreted in accordance with the currently recommended Clinical and Laboratory Standards Institute (CLSI) criteria for urinary tract isolates of Escherichia coli and Enterococcus faecalis and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria for Enterobacteriaceae and Staphylococcus aureus. y Conclusion In our ICU the main cause of admission was the polyvalent patient, who is younger and has more severity with not much diff erence in mean time of stay compared with the coronary patient. The intra-ICU infections provide an increase of morbi-mortality risk and consumption of resources. p y Results Out of the 1,895 isolates tested, fosfomycin displayed an overall in vitro susceptibility against 90%, but only 64% against MDR strains. Among the MDR organisms nearly 78% of E. coli and 70% of Klebsiella spp. and 40% of MRSA isolates showed provisional MICs in the sensitive range while among the sensitive strains fosfomycin showed around 92% susceptibility. Our study results were comparable with the results obtained from an Indian study published from CMC Vellore in 2013 showing a fosfomycin susceptibility of around 75% among MDR uropathogenic E. coli. P97 Results A total of 93 unusual Gram-negative rods were isolated from 76 patients. This included 19.4% (n = 18) Serratia marcescens, 12.9% (n = 12) Stenotrophomonas maltophilia, 14% (n = 13) Burkholderia cepacia, 24.7% (n = 23) Proteus mirabilis, 17.2% (n = 16) Morganella morganii, 9.7% (n = 9) Elizebethkingia meningoseptica and 2.1% (n = 2) Providencia species. A total of 68.4% (n = 52) patients had prior exposure to either colistin or carbapenems or both. In total, 71% (n = 66) of the total isolates from patients had previous antibiotic exposure. Among the total 93 intrinsically resistant isolates to colistin, 37.6% (n = 35) of isolates from all clinical sources (endotracheal, pus, urine and blood samples) were true pathogens and the remaining 62.3% (n = 58) were colonizers. There was a statistically signifi cant increase in length of ICU stay and duration of hospitalization in the presence of true pathogen. Antibiotic synergy testing for multidrug-resistant Gram-negative pathogens in a Greek ICU E Douka, E Perivoliot, E Kraniotaki, M Nepka, C Routsi, K Fountoulis, A Skoutelis, S Zakynthinos Evangelismos General Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P97 (doi: 10.1186/cc14177) Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma p A Chakraborty, S Roy, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P99 (doi: 10.1186/cc14179) A Chakraborty, S Roy, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P99 (doi: 10.1186/cc14179) Introduction Urinary tract infection (UTI) is one of the most common bacterial infections in humans. Gram-negative organisms being the most common causative agent, the rising prevalence of resistance to a number of antibiotics and more importantly the production of extended spectrum beta-lactamase (ESBL) by these organisms is a growing concern worldwide. As the scenario is no better in community isolates, the choice of empirical antimicrobials for such infections becomes a great challenge for the clinicians. fi Results Against 59 MDR A. baumannii strains, the synergy eff ect of CRB/ COL was 55.9%, RIF/COL 38.9%, CRB/GEN 22%, CRB/AMK 20.3% and TG/COL 16.9%, respectively. Against 41 K. pneumoniae strains, synergy rates were: CRB/COL 43.9%, CRB/GEN 31.7%, PIP/TAZ/GEN 29.2% and TG/COL 24.4% respectively. Against 64 P. aeruginosa strains, synergy rates were: AMK/PIP/TAZ 64.6%, AMK/AZT 64.6%, AMK/CEF 58.3%, CRB/ COL 52%, AMK/CRB 25%. Conclusion The most eff ective combination for both the A. baumannii and K. pneumoniae strains tested was CRB/COL. The next most eff ective combination was RIF/COL and CRB/GEN respectively. No competitive eff ect was observed for RIF/COL combination in all cases tested. The most eff ective combinations for P. aeruginosa strains were AMK plus PIP/TAZ or AZT or CEF. The next most eff ective combination was CRB/ COL. We recommend implementation of an antibiotic synergy test for MDR pathogens as a routine antimicrobial test in the hospitals’ microbiology laboratories, especially for critically ill patients, since some combinations seem to excel. Further studies are needed for the correlation of these combinations with clinical effi cacy. Methods In this retrospective observational study we aimed at knowing the prevalence of ESBL production by organisms causing UTI in the community and to study the antibiogram of such isolates. Urine samples from patients with suspected UTI in the community were cultured for uropathogen by routine microbiological methods and susceptibility testing was done on Microscan Autoscan 4 (Siemens). y Results Out of 527 isolates of Enterobactereaceae, 314 (59.58%) were ESBL producers from the community samples compared with 315 (67.30%) from hospital samples, with Escherichia coli being the most commonly isolated pathogen. Enterobacter spp. P100 Is it possible to predict multidrug-resistant organism colonization and/or infection at ICU admission? F Callejo-Torre1, JM Eiros2, S Ossa-Echeverri1, P Olaechea3, F Alvarez-Lerma4, M Palomar5, Envin-Helics Study Group1 1Hospital Universitario de Burgos, Spain; 2Hospital Clínico Universitario de Valladolid, Spain; 3Hospital de Galdakao-Usansolo, Galdakao, Spain; 4Hospital del Mar, Barcelona, Spain; 5Hospital Arnau de Villanova, Lleida, Spain Critical Care 2015, 19(Suppl 1):P100 (doi: 10.1186/cc14180) y Methods We retrospectively assessed two hypothetical empirical antibiotic treatment algorithms for VAP on an 18-bed ICU. Data on consecutive episodes of microbiologically confi rmed VAP were collected over a period of 22 months and divided into a derivation (1 February 2013 to 30 November 2013) and validation (1 December 2013 until 15 November 2014) cohort. We constructed two algorithms in the derivation cohort. One is a local ecology-based algorithm (LEBA), according to clinical risk factors for MDR and susceptibility results in our hospital. The other is a Gram stain-based algorithm (GSBA). The selection of antibiotics on GSBA was directed against pathogens predicted from the results of bedside Gram staining of tracheal aspirates collected just before antibiotic therapy. Subsequently, LEBA and GSBA were retrospectively reviewed and compared with actually prescribed antibiotics in the validation cohort.i p Critical Care 2015, 19(Suppl 1):P100 (doi: 10.1186/cc14180) Introduction We tried to develop a predictive model for patients colonized/infected by any multidrug-resistant organism (MDRO-C/I) at ICU admission based on risk factors easy to obtain (not depending on complex clinical records), being aware that foreseeing MDRO-C/I at ICU admission is key for appropriate empirical treatment and infection control. p Results The fi rst 50 VAP episodes made up the derivation cohort and the subsequent 50 VAP episodes the validation cohort. Antibiotic coverage rates by applying LEBA and GSBA were identical (96% vs. 96%). GSBA proposed more narrow spectrum therapy as compared with LEBA (P <0.001). GSBA recommended carbapenems in signifi cantly less episodes than LEBA (P <0.001) and the same episodes as actually prescribed initial therapy (P = 1). However, there was signifi cant increase of antibiotic coverage rates in GSBA compared with the actually prescribed initial therapy (96% vs. 78%, P = 0.015). Conclusion Antibiotic coverage rates on GSBA were comparable with LEBA. The use of GSBA would result in a signifi cant reduction of the administration of broad-spectrum antibiotics. Bedside Gram staining may be useful to guide appropriate initial antibiotic therapy for VAP. Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma showed highest prevalence (80%) of ESBL production from the community samples. Among the ESBL producing strains from the community, the sensitivity to ciprofl oxacin, levofl oxacin and nitrofurantoin was 18%, 21% and 44% respectively while in the non-ESBL producers the sensitivity rates were 52%, 51% and 73% respectively. P98 Development of antibiotic treatment algorithms based on Gram stain to restrict use of broad-spectrum antibiotics in the treatment of ventilator-associated pneumonia: a retrospective analysis J Yoshimura, T Kiguchi, A Matsushima, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P98 (doi: 10.1186/cc14178) P98 Development of antibiotic treatment algorithms based on Gram stain to restrict use of broad-spectrum antibiotics in the treatment of ventilator-associated pneumonia: a retrospective analysis J Yoshimura, T Kiguchi, A Matsushima, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P98 (doi: 10.1186/cc14178) p y Conclusion Organisms producing the ESBL phenotype present with an added possibility of being resistant to other broad-spectrum antimicrobial agents which are commonly prescribed in the community to empirically treat such infections. This makes the choice of empirical antibiotic much more challenging in the community, drawing errors in judgment. A possibility of frequent overcorrection lies on the other side of the coin. This study also shows the possible need for empirical institution of class I carbapenems as one of the treatment options and outpatient parenteral antimicrobial therapy. Introduction Ventilator-associated pneumonia (VAP) is a common and serious problem in ICUs. Several studies have been conducted to determine the eff ectiveness of Gram stain of tracheal aspirates for diagnosing VAP. However, the eff ectiveness for predicting causative microorganisms and guiding appropriate initial antibiotic therapy has not been evaluated. The purpose of this study is to determine whether Gram stain of tracheal aspirates can guide appropriate initial antibiotic therapy for VAP. Antibiotic synergy testing for multidrug-resistant Gram-negative pathogens in a Greek ICU Introduction The emergence of multidrug-resistant (MDR) pathogens is a major cause of infection-related mortality among critically ill patients. The synergistic eff ect between commonly used antibiotics against diffi cult to treat nosocomial MDR Gram-negative strains, if present, could provide a viable option as an alternative therapy. The aim of this study was to investigate the potential of antibiotic synergy against MDR A. baumannii, K. pneumonia and P. aeruginosa strains, isolated from critically ill patients in a Greek ICU. y Methods We tested 59 A. baumannii, 41 K. pneumoniae and 64 P. aeruginosa strains, isolated during the period 2010 to 2013. All strains were resistant to carbapenems and showed reduced susceptibility or resistance to tigecycline or colistin (MIC >2), in accordance with CLSI guidelines. We evaluated double-drug combinations of carbapenem (CRB)/colistin (COL), tigecycline (TG)/COL, rifampicin (RIF)/COL, CRB/ gentamicin (GEN), CRB/amikacin (AMK) for A. baumannii, TG/COL, S34 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 CRB/COL, piperacillin–tazobactam (PIP/TAZ)/GEN, CRB/GEN for K. pneumoniae and AMK/(PIP/TAZ), AMK/aztreonam (AZT), AMK/cefepime (CEF), AMK/CRB and CRB/COL for P. aeruginosa strains. In order to perform synergy tests, the E-test methodology (BioMerieux, Marcy l’E’toile, France) was used. Synergy was defi ned as a fraction inhibitory concentration (FIC) index ≤0.5, additive eff ect 0.5 to 1, indiff erent or antagonistic eff ect >2 (Lorian defi nition). CRB/COL, piperacillin–tazobactam (PIP/TAZ)/GEN, CRB/GEN for K. pneumoniae and AMK/(PIP/TAZ), AMK/aztreonam (AZT), AMK/cefepime (CEF), AMK/CRB and CRB/COL for P. aeruginosa strains. In order to perform synergy tests, the E-test methodology (BioMerieux, Marcy l’E’toile, France) was used. Synergy was defi ned as a fraction inhibitory concentration (FIC) index ≤0.5, additive eff ect 0.5 to 1, indiff erent or antagonistic eff ect >2 (Lorian defi nition).f P103 Novel infl uenza A antibodies reduce severity of secondary pneumococcal pneumonia after infl uenza infection in mice KF Van der Sluijs, F Van Someren Greve, MD De Jong, MJ Schultz, NP Juff ermans Novel infl uenza A antibodies reduce severity of secondary pneumococcal pneumonia after infl uenza infection in mice KF Van der Sluijs, F Van Someren Greve, MD De Jong, MJ Schultz, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P103 (doi: 10.1186/cc14183) y Methods Data were collected prospectively from 69,894 patients admitted consecutively (stay >24 hours) to 147 Spanish ICUs participating in the National Surveillance Study of Nosocomial Infections in ICU registry (ENVIN) during April to June 2006 to 2010. Univariable and multivariable analysis was performed for both objectives but we used only easy-to-obtain variables for the predictive model exclusively from those admitted in 2010 (n = 16,950, 2/3 for analysis and 1/3 for subsequent validation). Introduction Secondary bacterial pneumonia after infl uenza infection can cause severe disease with a high mortality. Recently, a new group 2 infl uenza A antibody (AT10_002) has been developed, which binds to multiple H3 and H7 subtypes. In a mouse model of primary infl uenza infection, treatment with AT10_002 as a fusion antibody protects against lethal infection, and reduces loss of bodyweight [1]. We hypothesized that treatment with AT10_002 reduces weight loss, lung injury and bacterial outgrowth, in a mouse model of viral infection followed by secondary pneumococcal infection. y q Results In the 2006 to 2010 period, 1,046 were C/I by MRSA (note that relative risks are not included due to space limitations). First objective: previous antibiotic, APACHE II score >18, skin-soft tissue or postsurgical superfi cial skin infections, trauma or medical patient, age >65 (especially >75), urinary catheter and admitted from a long- term care facility were independent risk factors for MRSA-C/I in ICU. Multicolonization increased signifi cantly the risk of MRSA-C/I, and immunodefi ciency and gender male emerged as protective factors. Second objective: independent risk factors on ICU admission were male gender, trauma critical patient, urgent surgery, admitted from other ICU, community or long-term facility, being immunosuppressed and skin-soft tissue infection. All confi gured the risk model for which, although showing good discrimination (AUC-ROC, 0.77; 95% CI, 0.72 to 0.82), sensitivity (67%) and specifi city (76.5%) were insuffi cient for the ICU setting. P101 P101 Methicillin-resistant Staphylococcus aureus in the ICU: risk factors and a predictive model to detect it at ICU admission F Callejo-Torre1, JM Eiros2, S Ossa-Echeverri1, P Olaechea3, M Palomar4, F Alvarez-Lerma5, Envin-Helics Study Group1 1Hospital Universitario de Burgos, Spain; 2Hospital Clínico Universitario de Valladolid, Spain; 3Hospital de Galdakao-Usansolo, Galdakao, Spain; 4Hospital Arnau de Villanova, Lleida, Spain; 5Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P101 (doi: 10.1186/cc14181) g y p y Results Among 41 patients, 15 (37%) underwent FMS. There was no diff erence in age, sex, underlying disease, target temperature management, and prophylactic antibiotic use between two groups. Mean duration of ECMO was 4 days in both groups. The incidence of bacteremia was none in the group with FMS and fi ve (19%) in the group without FMS. Within fi ve cases of bacteremia, three were caused by enterobacterium. Introduction Being capable of predicting MRSA on ICU admission is crucial to enhance infection control and to avoid inappropriate empirical treatment. Two objectives were studied: to describe risk factors for MRSA colonization/infection (MRSA-C/I) once admitted to the ICU; and to develop a predictive model at ICU admission, based on easy-to-obtain admission factors. Conclusion FMS may be protective against bacteremia for OHCA patients undergoing ECMO. Protective eff ect of a fecal incontinence management system against bacteremia for out-of-hospital cardiac arrest patients undergoing extracorporeal membrane oxygenation S Kikuta, R Miki, S Ishihara, S Nakayama Hyogo Emergency Medical Center, Chuo, Kobe, Hyogo, Japan Critical Care 2015, 19(Suppl 1):P102 (doi: 10.1186/cc14182) Introduction Recently, extracorporeal cardiopulmonary resuscitation (ECPR) has become a common measure against cardiopulmonary arrest. In cases with ECPR, we usually insert cannulae for extracorporeal membrane oxygenation (ECMO) via the femoral artery and vein. However, the cannulation site is often contaminated by feces due to incontinence. Moreover, patients tend to be compromised by hypothermia due to the target temperature management, so we often experience central line-associated bloodstream infection of patients undergoing ECMO. We investigated the protective eff ect of a fecal incontinence management system (FMS) against bacteremia in patients undergoing ECMO. Conclusion MDRO prediction at ICU admission could not be based merely on clinical–demographic risk factors. Taking into account local particularities and combining risk factors with a rapid laboratory test might be the most eff ective way forward. p g g Methods We studied 41 consecutive patients undergoing ECMO for out-of-hospital cardiac arrest (OHCA) between April 2010 and May 2014. Patients were divided into two groups according to the use or no use of FMS (Flexi-Seal™). Patients who died within 48 hours or from whom cannulae for ECMO were removed within 48 hours were excluded. Patients’ characteristics, underlying disease, target temperature management, prophylactic antibiotic use and incidence of bacteremia during admission were recorded and analyzed retrospectively. Results Among 41 patients, 15 (37%) underwent FMS. There was no diff erence in age, sex, underlying disease, target temperature management, and prophylactic antibiotic use between two groups. Mean duration of ECMO was 4 days in both groups. The incidence of bacteremia was none in the group with FMS and fi ve (19%) in the group without FMS. Within fi ve cases of bacteremia, three were caused by enterobacterium. g g Methods We studied 41 consecutive patients undergoing ECMO for out-of-hospital cardiac arrest (OHCA) between April 2010 and May 2014. Patients were divided into two groups according to the use or no use of FMS (Flexi-Seal™). Patients who died within 48 hours or from whom cannulae for ECMO were removed within 48 hours were excluded. Patients’ characteristics, underlying disease, target temperature management, prophylactic antibiotic use and incidence of bacteremia during admission were recorded and analyzed retrospectively. P102 (relative risk not shown due to space limitation): age 65 to 74, medical or surgical critical patient (especially urgent surgery), admitted from other ICU or long-term facility, immunosuppression and deep postsurgical skin or skin-soft tissue infections. Admitted from the community and female gender emerged as protective factors. Although the predictive model showed good discrimination (AUC-ROC = 0.775 (95% CI, 0.744 to 0.807)), sensitivity was only 67.4%. Validation with the remaining 4,952 patients (1/3) showed an AUC-ROC = 0.712 (95% CI, 0.665 to 0.759) and a P value on the Hosmer–Lemeshow goodness of fi t test of 0.855. Even creating a new model, including variables obtained after ICU admission (severity by APACHE score, mechanical ventilation, central venous, arterial or urinary catheter, immunodefi ciency, parenteral nutrition, ventricular derivation, extrarenal depuration, non-invasive ventilation, tracheotomy, enteral nutrition and nasogastric tube), prediction capability did not improve (AUC-ROC = 0.801 (95% CI, 0.774 to 0.828), sensitivity 71.4%). Protective eff ect of a fecal incontinence management system against bacteremia for out-of-hospital cardiac arrest patients undergoing extracorporeal membrane oxygenation S Kikuta, R Miki, S Ishihara, S Nakayama Hyogo Emergency Medical Center, Chuo, Kobe, Hyogo, Japan Critical Care 2015, 19(Suppl 1):P102 (doi: 10.1186/cc14182) P100 Results The fi rst 50 VAP episodes made up the derivation cohort and the subsequent 50 VAP episodes the validation cohort. Antibiotic coverage rates by applying LEBA and GSBA were identical (96% vs. 96%). GSBA proposed more narrow spectrum therapy as compared with LEBA (P <0.001). GSBA recommended carbapenems in signifi cantly less episodes than LEBA (P <0.001) and the same episodes as actually prescribed initial therapy (P = 1). However, there was signifi cant increase of antibiotic coverage rates in GSBA compared with the actually prescribed initial therapy (96% vs. 78%, P = 0.015). Methods Data were collected prospectively from admission to discharge of 16,950 patients admitted consecutively (at least >24 hours) to 147 Spanish ICUs of the ENVIN (National Surveillance Study of Nosocomial Infections in ICUs) registry, from April to June 2010. To create the predictive model, 11,998 (2/3) patients were used for univariable and multivariable logistic regression model and 4,952 (1/3) for subsequent validation. Results With a MDRO prevalence of 2.12% (359 MDROs at ICU admission were detected in 314 patients), 87.58% patients had only one MDRO, meanwhile 12.42% were MDRO-C/I by two or more simultaneously. Risk factors used in the development of the predictive model and independently associated with MDRO-C/I at ICU admission were Conclusion Antibiotic coverage rates on GSBA were comparable with LEBA. The use of GSBA would result in a signifi cant reduction of the administration of broad-spectrum antibiotics. Bedside Gram staining may be useful to guide appropriate initial antibiotic therapy for VAP. S35 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Source of MDR infections in an ICU: busting the myth R Agrawal Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Results In total, 1,499 patients were included, of whom 265 patients (18%) had a viral respiratory tract infection with at least one virus. In 17 patients, two viruses were found; two patients had an infection with three viruses. The most prevalent was parainfl uenzavirus-3 (5.7%); 17 patients (1.1%) had an infection with infl uenza. The lowest prevalence of viral infections occurred in September (12%), the highest in October and February (both 26%). Of the patients tested positive in TA, only 46% also tested positive in NP. The median cp values were not signifi cantly diff erent between TA and NP swabs (31.1 vs. 31.6, P = 0.75). Introduction MDR infections in the ICU are not nosocomial all the time, as perceived commonly. We performed a 2-year retrospective study to analyze the source of culture positivity in a medical ICU and to identify which types of infections are more prevalent. Methods The data of a 35-bed medical ICU were analyzed from November 2012 to October 2014. The source of culture positivity was divided into three groups: patients admitted from the ER to the ICU who were referred from other hospitals or direct admissions, the second group was patients admitted within the hospital but outside the ICU for the fi rst 48 hours, and the third group was ICU-acquired infections. We also analyzed the data for type of infections, whether Gram-negative, Gram-positive or fungal. Conclusion The prevalence of viral respiratory tract infections is high in unselected ICU patients. Testing tracheal aspirate in combination with nasopharynx greatly increased detection of viruses, and yields similar cp values. Whether these viral infections are associated with prolonged mechanical ventilation and worse outcomes remains to be determined. g , p g Results There were 1,051 cultures positive in a 2-year period. In total, 46.8% (n = 492) of cultures were already positive on admission, which denotes community-acquired and referred patients from other hospitals. A total of 31.1% (n = 327) of cultures were positive from patients admitted to general wards for more than 48 hours and then transferred to the ICU. Twenty-two percent (n = 232) of cultures were ICU-acquired infections. The data show community-acquired and hospital-acquired infections are the bulk of the culture load in an ICU. P103 Afterwards validation with the remaining 4,952 (1/3) showed AUC-ROC = 0.72 (95% CI, 0.65 to 0.79) and P value on the Hosmer–Lemeshow goodness of fi t test = 0.539. The model did not improve even after including more complex variables (AUC-ROC = 0.82; 95% CI, 0.77 to 0.86, sensitivity 63.64%, specifi city 78.48%). y y p Methods Male C57Bl/6 mice were intranasally inoculated with 400 TCID50 Infl uenza A (H3N2). Two days after infection, mice were injected with either AT10_002 i.v. (n = 8) or a control antibody (n = 7). After 7 days, both groups were intranasally inoculated with 5 × 103 S. pneumoniae type 3 and were sacrifi ced 18 hours later. Outcome measures were weight loss, wet lung weight, cell count in bronchoalveolar lavage fl uid (BALF), and colony-forming units (CFUs) in lung homogenate. Data are represented as medians, and treatment groups are compared using nonparametric tests.i p Results Mice receiving AT10_002 showed signifi cantly lower weight loss at the time of sacrifi ce compared with the control group (+1% vs. –12% change in weight; P = 0.0003). Also wet lung weight was lower (68 vs. 96 mg; P = 0.0003), cell counts in BALF were lower (4.9 × 105 vs. 7.0 × 105 cells/ml; P = 0.0037) and CFUs in lung homogenate were lower (33 vs. 25 × 104 CFUs/mg; P = 0.0003) compared with controls. Conclusion Early treatment with infl uenza antibody AT10_002 signifi cantly reduces weight loss, lung injury and bacterial outgrowth, i Conclusion Independent risk factors for MRSA-C/I in the ICU and at ICU admission are described. To predict MRSA-C/I at ICU admission we should not rely on clinical–demographic risk factors alone. Its combination with a rapid laboratory test could be the way to proceed in future studies. Conclusion Early treatment with infl uenza antibody AT10_002 signifi cantly reduces weight loss, lung injury and bacterial outgrowth, S36 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 hospitals – 17 high-volume care units) in the Czech Republic from 1 January 2011 to 5 November 2013. Patients were divided into two groups: survivors (n = 274) and nonsurvivors (n = 261). in a mouse model of infl uenza infection followed by secondary pneumococcal pneumonia. Reference in a mouse model of infl uenza infection followed by secondary pneumococcal pneumonia. P103 Reference g Results Survivors versus nonsurvivors were similar in: age 65.8 (64.2; 67.5) versus 66.5 (64.7; 68.3) P = 0.583, men 159 (58.0%) versus 160 (62.0%) P = 0.376, APACHE II score 27 (15 to 40) versus 28 (15 to 40) P = 0.737. Statistically signifi cant diff erences between survivors versus nonsurvivors were found in the parameter ‘Consensus initial antimicrobial therapy with microbial cultures’ 178 (79.5%) versus 128 (58.4%) P <0.001 and in the parameter ‘Administration antimicrobials within the fi rst hour’ 163 (59.9%) versus 171 (70.7%) P = 0.001. Administration of 30 ml/kg crystalloid for hypotension or lactate 4 mmol/l (3 hours) and application of vasopressors (6 hours) were in both groups without statistically signifi cant diff erences. 1. Wagner K, et al. Proc Natl Acad Sci U S A. 2014;111:16820-5. Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock 1 l 1 Š 2 l 1 Š k1 Introduction The early identifi cation of severe sepsis and septic shock and early implementation of the SSC bundles were associated with reduced mortality [1]. The failure to initiate appropriate antimicrobial therapy increased mortality of septic shock patients [2]. We hypothesized that the parameter ‘Consensus initial antimicrobial therapy with microbial cultures’ correlates with outcome of septic shock patients. Conclusion Antibiotic stewardship and strict adherence to infection control protocols in hospitals and guidelines for general practitioners can signifi cantly reduce the load of resistant organisms in the ICU. This may eventually improve patient outcomes and help in preserving the antibiotics for future generations. Methods We analyzed 535 consecutive patients with septic shock (sepsis-induced hypotension persisting despite adequate fl uid resuscitation) from the EPOSS database (Data-based Evaluation and Prediction of Outcome in Severe Sepsis), which was developed to monitor and assess treatment effi cacy in patient with severe sepsis and septic shock. Patients were admitted to participating ICUs (12 P105 P105 Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock P Szturz1, P Folwarczny1, J Švancara2, R Kula1, P Ševèík1 1University Hospital and Faculty of Medicine Ostrava University, Ostrava, Czech Republic; 2Institute of Biostatistic and Analyses, Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P105 (doi: 10.1186/cc14185) P105 Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock P Szturz1, P Folwarczny1, J Švancara2, R Kula1, P Ševèík1 1University Hospital and Faculty of Medicine Ostrava University, Ostrava, Czech Republic; 2Institute of Biostatistic and Analyses, Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P105 (doi: 10.1186/cc14185) Prevalence of viral respiratory tract infections in acutely admitted and ventilated ICU patients: a prospective multicenter observational study y F Van Someren Greve1, KF Van der Sluijs1, R Molenkamp1, AM Spoelstra-de Man2, OL Cremer3, RB De Wilde4, PE Spronk5, MD De Jong1, MJ Schultz1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3University Medical Center Utrecht, the Netherlands; 4Leiden University Medical Center, Leiden, the Netherlands; 5Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P104 (doi: 10.1186/cc14184) g p y gif Conclusion We found that correct choice of antibiotics improves outcome of septic shock patients. The choice of empirical antimicrobial therapy depends on complex factors related to the underlying disease, susceptibility of pathogens, patient’s history and clinical syndrome. Adequate initial antimicrobial therapy as an important factor of survival along with suitable initial fl uid resuscitation and application of vasopressors should be a priority for healthcare in human septic shock. References p p Critical Care 2015, 19(Suppl 1):P104 (doi: 10.1186/cc14184) Introduction The prevalence of viral respiratory tract infections in critically ill patients is uncertain, as well as the optimal diagnostic method to detect these. The aim of this study was to assess the prevalence of viral respiratory tract infections in mechanically ventilated patients, in both the upper and lower respiratory tract.i Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Source of MDR infections in an ICU: busting the myth R Agrawal This could be attributed to increased surveillance and adherence to infection control practices in the ICU which may not be followed stringently in other parts of the hospital. Overuse of broad-spectrum antibiotics in community and primary care hospitals has resulted in a spurt in growth of resistant infections. This has reached an alarming level in developing countries. Out of total cultures positive 78.3% (n = 822) were Gram-negative infections which included community-based and non-ICU infections. References 1. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 2. Kumar A, et al. Crit Care Med. 2006;34:1589-96. 1. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 2. Kumar A, et al. Crit Care Med. 2006;34:1589-96. Methods A prospective observational study was performed in fi ve ICUs in the Netherlands. From September 2013 to April 2014, consecutive acutely admitted, mechanically ventilated patients were included, regardless of diagnosis at admission. Nasopharyngeal (NP) swabs and tracheal aspirates (TA) were collected at intubation, and were tested via multiplex RT-PCR for the following viruses: infl uenza A and B, parainfl uenzaviruses, RSV, human metapneumoviruses, bocaviruses, coronaviruses, rhinoviruses, enteroviruses, parechoviruses and adenoviruses. Viral DNA/RNA copies were expressed by crossing-point (cp) values. Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Positive Agreement Sensitivity Specifi city culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93.8 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92.4 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88.4 (BAL/ETA) Conclusion Sensitivity and specifi city of the new molecular approach for these main bacteria found in VAP could enable targeted fi rst-line antibiotic therapy. In the future, the development of this approach will aim at obtaining a bedside diagnostic in only a few hours. P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay for rapid (<1 hour) detection of bacteria carrying carbapenem-resistance genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is to compare PCR with microbiological cultures in critically ill, abdominal Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Positive Agreement Sensitivity Specifi city culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93.8 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92.4 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88.4 (BAL/ETA) Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Introduction Antimicrobial resistance constitutes a growing global threat, driven in part by inappropriate antimicrobial prescribing [1]. Most hospitals implement antibiotic policies to promote antimicrobial stewardship. This audit examined the Royal Cornwall Hospital Trust (RCHT) Critical Care Department’s compliance with the current standard defi ned in our local antimicrobial policy. This states that all antimicrobial prescriptions are to have an indication and review date recorded [2]. Sequential strategies to improve compliance were introduced prior to re-auditing the eff ects. Methods The RCHT Critical Care Department utilizes the Phillips Care Vue electronic patient record. P107 C P107 the ICU stay. We obtained two rectal swab specimens and two drainage samples to perform PCR assay and classic culture tests. We used Cohen’s K to test concordance of results. We considered concordant those results of positive detection of carbapenemase-producing bacteria by both methods (even if a polymicrobial growth was observed by cultures) or negative results by both methods. Concordance was studied for rectal swab and drainage specimens. Antibiotic susceptibility testing was performed through a semiquantitative method. the ICU stay. We obtained two rectal swab specimens and two drainage samples to perform PCR assay and classic culture tests. We used Cohen’s K to test concordance of results. We considered concordant those results of positive detection of carbapenemase-producing bacteria by both methods (even if a polymicrobial growth was observed by cultures) or negative results by both methods. Concordance was studied for rectal swab and drainage specimens. Antibiotic susceptibility testing was performed through a semiquantitative method. P107 Concordance between a new molecular real-time approach and traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Karam4, F Meynier3, B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, Hopital Dupuytren, Limoges, France; 3bioMérieux SA, Grenoble, France; 4Service de Réanimation, Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/cc14187) Results Eight complete samples sets were collected from seven patients. Seven rectal swab specimens were negative for both PCR and cultures. In one patient a positive culture from carbapenem-resistant P. aeruginosa was detected from the rectal swab resulting negative to PCR. In one patient a positive culture from carbapenem-resistant A. baumanii was detected by drainage culture resulting negative to PCR. In two cases a positive result was observed from both PCR and cultures of rectal swab and drainage specimens. Vim and KPC genes were detected in one case and A. baumanii and K. pneumoniae with carbapenem resistance were isolated from cultures. A KPC gene was detected by PCR in the other case, and K. pneumoniae with carbapenem resistance was isolated from cultures. In all other cases a negative result was observed by both PCR and cultures. Cohen’s K of 0.71 (95% CI = 0.21 to 1) was observed for rectal swab and drainage specimens. Introduction Early microbiological documentation may reduce attributable mortality and excessive use of broad-spectrum antibiotics in ventilator-associated pneumonia (VAP). P107 C Using bronchoalveolar lavage (BAL) and endotracheal aspirates (ETA), we studied a new molecular biology-based approach to detect and quantify bacteria in less than 3 hours. This prospective multicenter trial aimed at comparing the microbiological results obtained using this molecular protocol (easyMAG® system) and semiquantitative culture in suspected VAP. y y q p Methods ETA and BAL samples were consecutively collected during 10 months in adult patients in four ICUs of France. The molecular method includes a preprocessing liquefaction for ETA before DNA extraction. DNAs were extracted using the easyMAG® system. Real- time PCR (qPCR) was run using the ABI7500FastDx PCR instrument. The results presented here concern: Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacteriaceae. Quantifi cation was performed using qPCR standard curves, by converting the cycle threshold to CFU/ ml. Conclusion We need more data to evaluate the performance of PCR for rapid detection of carbapenemase-producing bacteria from rectal swabs and drainage of critically ill surgical patients even though its concordance with cultures seems to be good. Results A total of 125 suspected VAP were included from 122 patients. In total, 125 BAL and 107 ETA were collected. Sex ratio (M/F) was 76%, and CPIS ≥6 was calculated in 74.6% of the suspected VAP patients. Mean ventilation duration before sampling was 6 days. Seventy-eight percent and 65% of the BAL and ETA culture were positive respectively. Correlations between molecular method and culture on BAL and ETA are reported in Table 1. Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) Data from this system were interrogated at three stages to assess our compliance with the trust’s antimicrobial policy. The fi rst data interrogation was performed prior to any intervention, and refl ected baseline antimicrobial prescribing habits. The second data interrogation was performed during a period of active antibiotic stewardship promotion. The third data interrogation was performed following the addition of a care bundle to the prescribing module of Care Vue. This daily tick-box prompt reminded clinicians to check that all antimicrobial prescriptions had an indication and review date recorded. The records of all of the patients admitted to the critical care department during the periods of data interrogations were assessed for antimicrobial indication and review date transcription.i References . Klevens, et al. Estimating healthcare associated infections. Public Health Rep. 2007;122:160-6. . Klevens, et al. Estimating healthcare associated infections. Public Health Rep. 2007;122:160-6. . Hecker, et al. Unnecessary use of antimicrobials. Arch Intern Med. 2003;163:972-8. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S37 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P107 Concordance between a new molecular real-time approach and traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Karam4, F Meynier3, B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, Hopital Dupuytren, Limoges, France; 3bioMérieux SA, Grenoble, France; 4Service de Réanimation Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/cc14187) Introduction Early microbiological documentation may red attributable mortality and excessive use of broad-spectrum antibio in ventilator-associated pneumonia (VAP). Using bronchoalveo lavage (BAL) and endotracheal aspirates (ETA), we studied a n molecular biology-based approach to detect and quantify bacteria less than 3 hours. This prospective multicenter trial aimed at compar the microbiological results obtained using this molecular proto (easyMAG® system) and semiquantitative culture in suspected VAP. Methods ETA and BAL samples were consecutively collected dur 10 months in adult patients in four ICUs of France. The molecu method includes a preprocessing liquefaction for ETA before D extraction. DNAs were extracted using the easyMAG® system. Re time PCR (qPCR) was run using the ABI7500FastDx PCR instrument. T results presented here concern: Staphylococcus aureus, Pseudomo aeruginosa and Enterobacteriaceae. Quantifi cation was perform using qPCR standard curves, by converting the cycle threshold to C ml. Results A total of 125 suspected VAP were included from 122 patie In total, 125 BAL and 107 ETA were collected. Sex ratio (M/F) was 76 and CPIS ≥6 was calculated in 74.6% of the suspected VAP patie Mean ventilation duration before sampling was 6 days. Seventy-ei percent and 65% of the BAL and ETA culture were positive respectiv Correlations between molecular method and culture on BAL and are reported in Table 1. Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ in VAP patients Positive Agreement Sensitivity Specifi c culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88 (BAL/ETA) Conclusion Sensitivity and specifi city of the new molecular appro for these main bacteria found in VAP could enable targeted fi rst- antibiotic therapy. References In the future, the development of this approach aim at obtaining a bedside diagnostic in only a few hours. P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay rapid (<1 hour) detection of bacteria carrying carbapenem-resista genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is compare PCR with microbiological cultures in critically ill, abdom surgical patients. Methods We performed an observational study at University Hospita Giaccone’ Palermo. We enrolled abdominal surgical patients admit to the ICU with suspected abdominal sepsis or developing sepsis dur P108 P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Results From the fi rst data interrogation, antimicrobial prescriptions had an indication and review date transcription in 57% and 60% of cases respectively. Following the awareness campaign, the indication and review date transcription rate increased to 78% and 85% respectively. A daily electronic prompt was then added to our care bundle list. The fi nal data interrogation, performed after this intervention, demonstrated that the transcription rates for both the indication and the review date had increased to 96%. Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay for rapid (<1 hour) detection of bacteria carrying carbapenem-resistance genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is to compare PCR with microbiological cultures in critically ill, abdominal surgical patients. Conclusion We have demonstrated that the use of a daily prompt within an electronic patient record can greatly improve compliance in recording the indication and review date for all antimicrobials. These data support the widespread implementation of an electronic prescribing system where daily reminders are integrated in an eff ort to improve compliance with antimicrobial stewardship. Methods We performed an observational study at University Hospital ‘P. Giaccone’ Palermo. We enrolled abdominal surgical patients admitted to the ICU with suspected abdominal sepsis or developing sepsis during Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S38 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Eventually, the IC was confi rmed for 403/835 patients (peritonitis: 177; candidaemia: 141; deep candidiasis: 61; mixed infection sites: 24). Candida albicans was the main pathogen (67%), then C. glabrata (16%). At inclusion, CIC were treated with caspofungin (Cas): 55%, and fl uconazole (Flu): 34%, whereas these antifungals were administered to 46% and 45% of SIC, respectively. Patients with SIC were more severe than those with CIC. The two main criteria for initiating empirically an AFT were a central venous catheter (79%) and severe septic shock (70%). The rate of change of the initial AFT was higher in the CIC group (49%) than in the SIC group (33%, P <0.0001). P111 Epidemiological cohort study of systemic antifungal therapy for suspected or confi rmed invasive candidiasis in the ICU: the Amarcand2 study J Constantin1, JF Timsit2, JP Gangneux3, JP Mira4, P Montravers2, H Dupont5, P Perrigault6, O Lortholary7, E Azoulay8, O Leroy9 1CHU Estaing, Clermont-Ferrand, France; 2Paris Diderot University/Bichat Hospital, Paris, France; 3Rennes University Hospital, Rennes, France; 4Cochin University Hospital, Paris, France; 5Amiens University Hospital, Amiens, France; 6Montpellier University Hospital, Montpellier, France; 7Necker University Hospital, Paris, France; 8Saint-Louis University Hospital, Paris, France; 9Tourcoing University Hospital, Tourcoing, France Critical Care 2015, 19(Suppl 1):P111 (doi: 10.1186/cc14191) P108 In the CIC group, it was mostly for changing the antifungal agent (de-escalation Cas  Flu in half of the patients) based on mycological tests results. In the SIC group, the AFT was modifi ed almost as often for changing the drugs (including 22% de-escalation Cas  Flu) as for stopping the AFT. The 28-day mortality of candidaemia was 42% in cases of C. glabrata, 40% in cases of C. albicans, and 20% in cases of C. parapsilosis. Among survivors, the median duration of treatment was 17 to 21 days according to the infection site in cases of CIC, and 10 days in cases of SIC. Epidemiological cohort study of systemic antifungal therapy for suspected or confi rmed invasive candidiasis in the ICU: the Amarcand2 study J Constantin1, JF Timsit2, JP Gangneux3, JP Mira4, P Montravers2, H Dupont5, P Perrigault6, O Lortholary7, E Azoulay8, O Leroy9 1CHU Estaing, Clermont-Ferrand, France; 2Paris Diderot University/Bichat Hospital, Paris, France; 3Rennes University Hospital, Rennes, France; 4Cochin University Hospital, Paris, France; 5Amiens University Hospital, Amiens, France; 6Montpellier University Hospital, Montpellier, France; 7Necker University Hospital, Paris, France; 8Saint-Louis University Hospital, Paris, France; 9Tourcoing University Hospital, Tourcoing, France l l d y Conclusion This is the largest PK study of 100 mg daily of MCF in severely burned critically ill patients. The inverse correlation between MCF exposure and % burned TBSA suggests that patients with large burned TBSA may need higher doses of MCF. Nevertheless, MCF levels in plasma and burn eschar tissues after the fi rst and multiple doses were above the MIC90 against most clinically important Candida species. g y p g Critical Care 2015, 19(Suppl 1):P111 (doi: 10.1186/cc14191) Introduction Prescription of antifungal treatments (AFT) in ICUs in case of suspected or confi rmed invasive candidiasis (SIC or CIC) has been challenged by diff erent guidelines. The study aimed to describe the epidemiology of the invasive candidiasis (IC), analyze the criteria for the AFT initiation, the AFT type, and its changes during patient follow-up. Methods A prospective observational multicenter cohort study. Consecutive adult patients with SIC or CIC and treated with systemic AFT were included between October 2012 and September 2013 in 104 French ICUs. References 1. World Health Organisation. Antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/ surveillancereport/en/. 1. World Health Organisation. Antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/ surveillancereport/en/. 2. Royal Cornwall Hospital Trust. Automatic stop/review date policy for antimicrobials. 2012. Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries gi q Results A total of 102 mechanically ventilated patients, 75 men and 27 women, were included in the study. All patients showed VAP caused by MDR bacteria. They were stratifi ed by outcome into survivors and nonsurvivors. ICU mortality was 55%. Gender, cause of admission, the causative microbe, colonization of bronchial secretions and secondary bacteremia had no correlation with outcome. Age and APACHE II score were higher in nonsurvivors (P <0.01 and P <0.05 respectively). The time-onset of pneumonia after admission was longer in patients with VAP caused by Klebsiella or Pseudomonas than those with VAP caused by acinetobacter (P <0.01). Patients with Klebsiella or Pseudomonas pneumoniae needed more time on mechanical ventilation than those with pneumonia from acinetobacter (P <0.01). Introduction Micafungin (MCF) is an echinocandin agent with broad activity against Candida spp., which are frequently isolated in blood and eschar cultures of burned patients, who present diff erent pharmacokinetics (PK) characteristics. Due to the limited information about its PK, we investigate MCF levels in plasma and burn eschar tissues in this population. p p Methods A PK study of MCF at standard dosage (100 mg/day). Cmax (end of the infusion) and Cmin (before next dose) plasma levels of MCF were obtained after fi rst dose and at steady state (days 4 and 5 of therapy); and on day 5 in eschars (1 to 3 hours after infusion). They were measured by HPLC. Spearman’s rho test was used for bivariate correlations between MCF exposure and patient’s clinical factors. Conclusion VAP caused by MDR bacteria is a leading cause of ICU death. Age and APACHE II score are signifi cant risk factors of death. References 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 2. Charles MP, et al. Australas Med J. 2013;6:430-4. Results There were 10 patients (eight men; age: 18 to 77 years). Patients’ characteristics and PK are shown in Table 1. A high interindividual variability was observed in the concentrations of MCF. Peak plasma concentrations after the fi rst and repeated doses of MCF were inversely correlated with % burned TBSA (Spearman’s ρ = –0.695 and –0.750 (P <0.05), respectively), but not with the time from burn injury. MCF concentrations in burn eschars were not correlated with % burned TBSA. MCF was well tolerated. One patient had candidemia. The crude mortality was 40%. P112 Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries A Agrifoglio1, MJ Asensio1, M Sánchez1, B Galván1, E Herrero1, L Cachafeiro1, E Perales1, S Luque2, A García de Lorenzo1 1La Paz/IdiPAZ University Hospital, Madrid, Spain; 2Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P112 (doi: 10.1186/cc14192) Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries A Agrifoglio1, MJ Asensio1, M Sánchez1, B Galván1, E Herrero1, L Cachafeiro1, E Perales1, S Luque2, A García de Lorenzo1 1La Paz/IdiPAZ University Hospital, Madrid, Spain; 2Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P112 (doi: 10.1186/cc14192) P113 P113 Tedizolid clearance by in vitro continuous renal replacement therapy model SJ Lewis, L Switaj, BA Mueller University of Michigan, Ann Arbor, MI, USA Critical Care 2015, 19(Suppl 1):P113 (doi: 10.1186/cc14193) Factors associated with survival of ICU patients with pneumonia caused by multidrug-resistant Gram-negative bacteria M Georgiadou, E Pappa, E Papandreou, H Pavlou, M Eforakopoulou KAT-EKA General Hospital Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P110 (doi: 10.1186/cc14190) Introduction Multidrug-resistant (MDR) bacterial pneumonia is associated with signifi cant morbidity and mortality in severely ill ICU patients. The assessment of factors associated with the onset and clinical course of MDR pneumonia may improve treatment eff ectiveness. The purpose of this study is to identify factors associated with outcome in mechanically ventilated patients with ventilator- associated pneumonia (VAP) caused by MDR bacteria. y Conclusion French ICU patients are treated with antifungal agents selected according to the candidiasis severity, contrary to ESCMID guidelines which recommend initiating with echinocandins regardless of severity. As recommended, the therapy was secondarily adapted to microbiological results. y Methods We studied retrospectively all mechanically ventilated patients treated in the A’ ICU of KAT General Hospital in Athens from 1 January 2011 to 31 December 2013 and showed ventilator-associated pneumonia from MDR Gram-negative bacteria. Standard demographic and clinical data, the causative organisms and outcome were recorded. For statistical signifi cance, chi-square and Student t tests were used. P112 P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration G Kennedy1, J Osborn1, S Flanagan2, N Alsayed3, S Bertolami1 1Cubist Pharmaceuticals, Lexington, MA, USA; 2Cubist Pharmaceuticals, San Diego, CA, USA; 3Cubist Pharmaceuticals, Zurich, Switzerland Critical Care 2015, 19(Suppl 1):P114 (doi: 10.1186/cc14194) Introduction Tedizolid phosphate, a novel oxazolidinone antibacterial prodrug recently approved by the US Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections, is available as oral (that is, tablets) and intravenous formulations. The clinical pharmacokinetics of tedizolid, the active moiety of tedizolid phosphate, are similar when orally administered tedizolid phosphate is given as powder in a capsule or as tablets. This suggests that crushing tablets prior to administration is unlikely to alter tedizolid pharmacokinetics, provided no drug is lost during administration. To determine whether the expected dose of tedizolid phosphate can be delivered via nasogastric (NG) tube in critically ill patients who have diffi culty swallowing, this study evaluated the stability and recovery of tedizolid phosphate 200 mg tablets after crushing, dispersion in water, and passage through an NG tube. Introduction Tedizolid phosphate, a novel oxazolidinone antibacterial prodrug recently approved by the US Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections, is available as oral (that is, tablets) and intravenous formulations. The clinical pharmacokinetics of tedizolid, the active moiety of tedizolid phosphate, are similar when orally administered tedizolid phosphate is given as powder in a capsule or as tablets. This suggests that crushing tablets prior to administration is unlikely to alter tedizolid pharmacokinetics, provided no drug is lost during administration. To determine whether the expected dose of tedizolid phosphate can be delivered via nasogastric (NG) tube in critically ill patients who have diffi culty swallowing, this study evaluated the stability and recovery of tedizolid phosphate 200 mg tablets after crushing, dispersion in water, and passage through an NG tube. for treatment of nosocomial pneumonia, common in critically ill patients with acute kidney injury. There are limited data on tedizolid disposition in continuous renal replacement therapy (CRRT). This study’s purpose was to assess continuous hemofi ltration (CHF) and continuous hemodialysis (CHD) infl uence on tedizolid clearance. Methods Validated, bovine blood-based, in vitro CHF and CHD models were used with six new HF 1400 (polysulfone) and six new Multifl ow 150 (AN 69) hemodiafi lters. P113 Tedizolid clearance by in vitro continuous renal replacement therapy model P113 Tedizolid clearance by in vitro continuous renal replacement therapy model SJ Lewis, L Switaj, BA Mueller University of Michigan, Ann Arbor, MI, USA Critical Care 2015, 19(Suppl 1):P113 (doi: 10.1186/cc14193) Introduction Tedizolid is an oxazolidinone antibiotic approved to treat acute bacterial skin and soft tissue infection and is under investigation Results In total, 870 patients were included and 835 evaluable, the IC was confi rmed at study inclusion for 291 and suspected for 544 patients. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S39 Table 1 (abstract P112). Clinical and pharmacokinetic characteristics of patients Table 1 (abstract P112). Clinical and pharmacokinetic characteristics of patients Plasma Plasma Days from SOFA MCF dose Cmax/Cmin Cmax/Cmin Burn eschar admission at the % burned (mg/kg after fi rst dose at steady state tissue on day 5 to the start beginning LOS in BICU Patient TBSA % FT ABSI body weight) (μg/ml) (μg/ml) (μg/g) of MCF of MCF (days) 1 35 20 9 1.3 8.6/0.8 7.4/1.0 2.3 38 1 75 2 40 35 8 2.0 8.5/1.1 9.4/1.8 <LQ 15 6 23 3 23 16 8 1.3 6.4/0.8 10.3/1.2 <LQ 12 2 17 4 70 40 12 1.1 3.9/0.5 4.5/0.8 0.4 8 6 43 5 23 12 7 1.3 7.5/1.8 8.0/1.4 0.6 10 5 19 6 70 60 11 1.2 3.4/0.5 5.0/0.9 1.5 12 5 70 7 80 70 12 1.1 3.8/0.4 4.0/0.4 0.2 34 2 61 8 60 50 10 1.4 4.8/0.5 4.3/1.0 0.2 34 2 90 9 44 34 10 1.3 4.5/1.1 9.1/2.3 0.2 8 6 34 10 34 28 9 1.3 4.1/0.7 5.4/1.0 0.7 10 5 35 Median 42 34.5 9.5 1.3 4.7/0.7 6.4/1.0 0.5 12 5.0 39 IQR 31.3 to 70.0 19.0 to 52.5 8 to 11.3 1.1 to 1.4 3.9 to 7.5/0.5 to 1.1 4.5 to 9.1/0.9 to 1.4 0.3 to 1.1 9.5 to 19.8 3.5 to 5.6 22.7 to 71.3 ABSI, Abbreviated Burn Severity Index; BICU, burn intensive care unit; FT, full thickness; IQR, interquartile range; LOS, length of hospital stay; LQ, limit of quantifi cation (<0.1 μg/ml); SOFA, Sequential Organ Failure Assessment; TBSA, total body surface area. CLTM appears modest relative to total body clearance and is unlikely to require dose adjustments. CRRT adsorption in the clinical setting is likely less than what we observed in this in vitro, continuously recirculating blood model. CLTM appears modest relative to total body clearance and is unlikely to require dose adjustments. P113 Tedizolid clearance by in vitro continuous renal replacement therapy model CRRT adsorption in the clinical setting is likely less than what we observed in this in vitro, continuously recirculating blood model. Figure 1 (abstract P113). P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration G Kennedy1, J Osborn1, S Flanagan2, N Alsayed3, S Bertolami1 1Cubist Pharmaceuticals, Lexington, MA, USA; 2Cubist Pharmaceuticals, San Diego, CA, USA; 3Cubist Pharmaceuticals, Zurich, Switzerland Critical Care 2015, 19(Suppl 1):P114 (doi: 10.1186/cc14194) P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration P115 Introduction In daptomycin (DAP), 1,061 mg hour/l of the area under the concentration–time curve (AUC)/MIC was required to obtain clinical success [1], and a trough serum concentration (Cmin) cutoff point of 24.3 g/ml was most signifi cantly associated with CPK elevation [2]. Reportedly, DAP at a recommended dosage of 8 mg/kg is removed in patients undergoing high-fl ow continuous venovenous hemodiafi ltration (CVVHDF) (blood fl ow and fi ltration rates were 150 ± 48 and 2 l/hour). In Japan, CVVHDF is preferentially performed with lower fl ow rates. Investigating eff ects of fl ow rate on DAP removal during continuous renal replacement therapy is essential to adjust therapeutic dosages. We aimed to investigate the pharmacokinetics of DAP in CVVHDF patients in this setting. 5 Antiviral prophylaxis inhibits cytomegalovirus reactivation in critical illness NJ Cowley1, A Owen1, J Millar1, SC Shiels1, RL Woolley2, NJ Ives2, H Osman1, P Moss2, JF Bion1 1University Hospital Birmingham, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P115 (doi: 10.1186/cc14195) Introduction Reactivation of latent cytomegalovirus (CMV) can lead to viraemia or CMV disease and has been detected in up to 30% of critically ill patients without prior history of immune suppression. However, the clinical importance of this observation remains unclear. We report a proof-of-concept randomised controlled trial of two antiviral drugs in intensive care patients to determine their impact on CMV reactivation. Methods We conducted a single-centre randomised controlled study of high-dose valaciclovir or low-dose valganciclovir prophylaxis, as compared with standard care, in CMV seropositive patients in the ICU at Queen Elizabeth Hospital Birmingham, UK. Patients were excluded if CMV seronegative. Study participants randomised to a study drug received either 450 mg valganciclovir daily enterally (or ganciclovir intravenously) or 2 g valaciclovir four times daily enterally (or aciclovir intravenously) for a period of up to 28 days. Blood was collected for CMV viral load during the 28-day study period. The primary outcome measure was reactivation of CMV in blood above 20 copies.ml–1 (assay detection limit) by day 28. Methods DAP (6 mg/kg) was administered intravenously every 48 hours to CVVHDF patients in the ICU. Blood and fi ltrate samples were collected at 0, 1, 1.5, 2, 5, 12, 24, and 48 hours after infusion. All collected samples were analyzed using HPLC according to the method of Tobin and colleagues [3]. P115 Maximum concentration (Cmax), elimination half- life (t1/2), area AUC, Cmin, volume of distribution (Vd), clearance (CL), fraction unbound, and sieving coeffi cient (Sc) were evaluated. Patient characteristics and CVVHDF parameters including blood, dialysate, and fi ltration fl ow rates were recorded. il Results Three patients were included in the study. Mean blood, dialysate, and fi ltration fl ow rates were 86.7 ± 11.5 ml/minute, 417 ± 29 ml/hour, and 417 ± 29 ml/hour, respectively, confi rming that CVVHDF was performed under low-fl ow setting. Cmax was 50.1 ± 12.7 mg/l (31.9, 70.5, 49.7 mg/l); t1/2, 35.1 ± 34.8 hours (18.6, 11.5, 70.5 hours); AUC, 889 ± 399 mg hour/l (471, 967, 1,260 mg hour/l); Cmin, 16.0 ± 10.3 mg/l (2.3, 24.7, 14.0 mg/l); Vd, 26.0 ± 20.9 l (23.8, 6.34, 47.9 l); CL, 9.47 ± 4.56 ml/minute (14.7, 6.35, 7.37 ml/minute); and fraction unbound, 5.8% (5.7, 4.1, 7.6%). Sc and CL of dialyzer were 0.08 ± 0.03 (0.11, 0.04, 0.07) and 1.20 ± 0.39 ml/minute (1.70, 0.88, 0.96 ml/minute), respectively. Results A total of 124 patients were randomised; 44 control, 34 valaciclovir, and 46 valganciclovir. Recruitment to the valaciclovir arm was halted early because of an imbalance in mortality (44% mortality vs. 19% in other arms). Independent blinded review of all deaths did not reveal any deaths attributable to unexpected causes. Fourteen patients were excluded from the primary analysis because of baseline CMV reactivation. CMV reactivation occurred in 30% (12/40) of the control arm but only 3% (1/39) in the valganciclovir arm (RR: 0.09 (95% CI: 0.01, 0.6)). When the two treatment arms were considered together, reactivation was observed in only 4% (3/70) (RR: 0.1 (95% CI: 0.04, 0.5)). See Figure 1. Conclusion DAP (6 mg/kg daptomycin every 48 hours) in patients receiving low-fl ow CVVHDF resulted in showing variability of AUC and avoiding accumulation. Owing to small case numbers, it needs further study. g Conclusion This is the fi rst study in critical care to assess the feasibility of antiviral prophylaxis to prevent CMV reactivation in a mixed population of critically ill patients. Low-dose valganciclovir was shown to suppress CMV reactivation as eff ectively as higher-dose valaciclovir. P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration Tedizolid’s transmembrane clearances (CLTM) during CHF and CHD were assessed by measuring sieving (SC) and saturation (SA) coeffi cients at various ultrafi ltrate (Quf) (1, 2, 3 l/ hour) and dialysate fl ow rates (Qd) (1, 2, 3 and 6 l/hour), using a blood fl ow rate (Qb) of 200 ml/minute. Tedizolid adsorption was tested in a 1 l recirculating CHF model at Quf of 2 l/hour and Qb of 200 ml/minute over 4 hours. Adsorption (%) was calculated after correcting for the dilution by CHF priming volume. Urea was added as a control in all experiments. Methods For each assay, run in triplicate, one 200 mg tablet of tedizolid phosphate was crushed, dispersed in water, drained under gravity through one of two types of NG tubes (type 1, Kangaroo Nasogastric Feeding Tube, 10 Fr 43" (109 cm); type 2, Salem Sump Dual Lumen Stomach Tube, 18 Fr/CH (6.0 m) 48" (122 cm)), and collected for recovery analysis by high-performance liquid chromatography with UV detection. To analyze the chemical stability of the crushed tablet dispersed in water, the aqueous preparation was assayed initially after dispersion and again after 4 hours at room temperature, without NG tube passage. The prespecifi ed limit for tedizolid phosphate in recovery samples was 90 to 110% of the dose. Limits were also specifi ed for levels of certain impurities. Results Urea SC and SA were ~1 in all experiments. In CHF, mean tedizolid SC ranged from 0.52 to 0.57 for HF1400 and from 0.50 to 0.54 for M150. CLTM did not diff er between fi lter types for Quf of 1, 2, and 3 l/hour. In CHD, mean tedizolid SA ranged from 0.46 to 0.56 for HF1400 and from 0.38 to 0.44 for M 150. Tedizolid CLTM with the HF1400 was higher than M150 values at Qd of 6 l/hours (P <0.02). Tedizolid exhibited irreversible adsorption within 10 minutes. See Figure 1. Results The average and individual recovery values of tedizolid phosphate were within 90 to 110% of the 200 mg dose when crushed tablets, dispersed in water at room temperature, were transferred through the 2 NG tubes (type 1: 95.8%; type 2: 93.6%). There was Conclusion Tedizolid’s CLTM is dependent on hemodiafi lter type and Qd for CHD and Quf in CHF. P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration At conventional CRRT rates, tedizolid S40 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Acknowledgements Research funded by the NIHR under the RfPB Programme (PG 1010 23225). Views expressed are of the authors and not necessarily the NHS, NIHR, or DOH. Acknowledgements Research funded by the NIHR under the RfPB Programme (PG 1010 23225). Views expressed are of the authors and not necessarily the NHS, NIHR, or DOH. no signifi cant change in recovery values after 4 hours of storage at room temperature (93.9% initially and 94.7% after 4 hours). Results for degradation products and impurities were also within specifi ed limits in NG recovery samples and in the 0-hour and 4-hour aqueous preparations. P116 p p Conclusion The stability and recovery of tedizolid phosphate were not infl uenced by crushing the tablets and passing through an NG tube. Therefore, administration of crushed tedizolid phosphate tablets to patients is unlikely to alter the pharmacokinetics of tedizolid compared with whole tablets. Pharmacokinetics of daptomycin in patients undergoing low-fl ow continuous venovenous hemodiafi ltration TI Ide1, Y Takesue1, K Ikawa2, S Nishi1 1Hyogo College of Medicine, Nishinomiya City, Japan; 2Hiroshima University, Hiroshima, Japan Critical Care 2015, 19(Suppl 1):P116 (doi: 10.1186/cc14196) PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? S De Winter1, J Wauters1, E Van Wijngaerden1, W Peetermans1, P Annaert2, J Verhaegen1, JB Gillet1, D Knockaert1, I Spriet1 1University Hospitals Leuven, Belgium; 2Catholic University Leuven, Belgium Critical Care 2015, 19(Suppl 1):P118 (doi: 10.1186/cc14198) Methods A total of four colistin-resistant (MIC ≥4) GNB were isolated from ICU patients with nosocomial MDR infections. All four isolates were Klebsiella pneumonia. Among these isolates three were from blood and one from endotracheal aspirate and all four isolates were sensitive to fosfomycin in vitro. All of these patients had multiple comorbidities with recent history of colistin exposure. Intravenous fosfomycin sodium (inj Fosmicin; Meiji, Japan) was started as a combination therapy with carbapenem. Introduction Studies in the ICU showed that a single amikacin dose of ≥25 mg/kg should be used in conditions of increased distribution volume (Vd) such as severe sepsis/shock [1]. However, no data are available for emergency department (ED) patients in the early phase of sepsis/septic shock. The purpose of this study was to determine whether a single amikacin dose of 25 versus 15 mg/kg results in PK/PD target attainment for ED patients. Results Among the three bacteremic patients, two recovered completely from sepsis as well as the patient with ventilator-associated pneumonia. There was clinical as well as microbiological cure with normalization of sepsis markers. The only one bacteremic patient who died during the course of therapy was later diagnosed to have azole- resistant fungemia as a superinfection. g p Methods ED patients with severe sepsis/shock were randomly treated with a single amikacin dose of 25 versus 15 mg/kg. Blood samples were collected at +1 (peak), +6 hours and +24 hours (trough) after the start of infusion. Primary outcome was PK/PD target attainment defi ned as a peak/MIC >8, corresponding with both actual MIC values documented from isolated pathogens, as well as EUCAST susceptibility breakpoints for Enterobacteriaceae and P. aeruginosa; that is, 8 mg/l. Noncompartmental analysis was used to calculate PK parameters. g p Conclusion Based on the evidence of clinical experience and available studies, intravenous fosfomycin therapy may be considered as the last option for the treatment of MDR GNB infection where there is documented colistin resistance and where there is literally no other choice of antibiotic therapy. PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? Results During a study duration of 20 months, 50 patients were enrolled in each dosing regimen resulting in 100 peak concentrations, 92 and 88 +6 hours and +24 hours concentrations respectively. Target attainment using local MIC values (median 2 mg/l, documented in 56 isolated Gram-negative pathogens) was achieved in 95% in both groups (P = 0.98). Using EUCAST susceptibility breakpoints, the target Table 1 (abstract P118) Introduction In vitro studies suggest that there is signifi cant adsorption of amikacin, netilmicin, gentamicin and tobramycin to polyacrylonitrile haemofi lters. This occurs rapidly and has the potential to substantially reduce the peak aminoglycoside concentration, which will reduce effi cacy [1]. However, whether signifi cant adsorption occurs in vivo is unknown. We therefore carried out a controlled in vivo study of the eff ect of amikacin adsorption by polyacrylonitrile fi lters during haemofi ltration, using a porcine model of acute renal failure. l, clearance. aMann–Whitney U <0.05, 15 versus 25 mg/kg ED patients Conclusion The EUCAST-based PK/PD target was only attained in 76% of patients treated with 25 mg/kg. However, in contrast to ICU patients, the majority of ED patients are treated for community-acquired infections, so MIC values are signifi cantly lower than the EUCAST susceptibility breakpoints, warranting PK/PD target attainment in both 25 and 15 mg/kg dosing regimens when local epidemiology is taken into account. i g Methods A porcine model of acute renal failure was created by bilateral ligation of the renal arteries and veins. Eight pigs underwent haemofi ltration using a 0.6 m2 polyacrylonitrile fi lter, blood fl ow 200 ml/ minute, ultrafi ltration rate 1,000 ml/hour. All ultrafi ltrate was returned to the pigs via a separate venous catheter so that any elimination of amikacin by haemofi ltration could only be due to adsorption. Another eight pigs underwent sham haemofi ltration in which blood was pumped around a haemofi ltration circuit without a haemofi lter and without ultrafi ltration. Both groups of pigs were given intravenous amikacin, 15 mg/kg body weight over 30 minutes, and blood samples were taken from the arterial limb of the haemofi lter circuit at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150, and 180 minutes after the start of the amikacin administration to assay amikacin concentrations. Results Post-distribution peak concentration of amikacin was slightly, but signifi cantly, lower in the CRRT group than that in sham group (55.0 ± 4.5 vs. 61.1 ± 5.9 mg/l, P <0.05).f Reference 1. Taccone et al. Crit Care. 2013;14:R53. Intravenous fosfomycin therapy in critically ill patients infected with colistin-resistant enterobacteriacae Conclusion This study shows that the eff ect of adsorption by polyacrylonitrile haemofi lters on in vivo amikacin peak concentrations is small, and less than would be expected from in vitro data. Introduction Carbapenem-resistant enterobacteriacae emerged in recent years as one of the most challenging groups of antibiotic- resistant pathogens. Polymyxins are considered as the last resort for the treatment of infections with carbapenem-resistant Gram-negative bacilli (GNB). Inadequate or extensive use of colistin leads to emergence of colistin resistance in GNB, jeopardizing treatment options in ICUs, potentially increasing mortality and morbidity and necessitating prudent use of alternative antibiotics. Fosfomycin, a phosponic acid derivative which acts primarily by disrupting bacterial cell wall synthesis, is a broad-spectrum antibiotic. Fosfomycin tromethamine is an oral formulation approved for the treatment of uncomplicated urinary tract infection caused by multidrug-resistant (MDR) bacteria. Recently fosfomycin is also available as a sodium/disodium formulation for intravenous use, which is showing promising result against MDR/ potentially drug-resistant pathogens. Acknowledgement This work was supported by a grant from the Hong Kong Research Grants Committee, CUHK 4644/08M. Reference 1. Tian Q, Gomersall CD, Ip M, Tan PE, Joynt GM, Choi GY. Adsorption of amikacin, a signifi cant mechanism of elimination by hemofi ltration. Antimicrob Agents Chemother. 2008;52:1009-13. References 1. Safdar N, et al. Antimicrob Agents Chemother. 2004;48:63-8. 2. Bhavnani SM, et al. Clin Infect Dis. 2010;50:1568-74. . Safdar N, et al. Antimicrob Agents Chemother. 2004;48:63-8. 3. Tobin CM, et al. J Antimicrob Chemother. 2008;62:1462-3. Figure 1 (abstract P115). CMV reactivation over time. Each line represents a single patient. Figure 1 (abstract P115). CMV reactivation over time. Each line represents a single patient. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S41 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 was attained in 76% versus 40% in the 25 versus 15 mg/kg group, respectively (P < 0.0001). Single-dose PK parameters are displayed in Table 1 and compared with the ones reported in the ICU [1]. P117 P117 Adsorption of amikacin during continuous venovenous haemofi ltration in a swine model of acute renal failure CD Gomersall, Q Tian, D Reynolds, M Ip, G Choi, G Joynt The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P117 (doi: 10.1186/cc14197) Table 1 (abstract P118) PK parameter 15 mg/kg ED 25 mg/kg ED 25 mg/kg ICU Peak (mg/l) 58 (47 to 70)a 91 (72 to 105)a 73 (62 to 90) Trough (mg/l) 6 (3 to 12) 5 (3 to 15) 7 (2 to 15) Vd (l/kg) 0.3 (0.3 to 0.5) 0.4 (0.2 to 0.6) 0.4 (0.3 to 0.5) Cl (ml/minute/kg) 1.6 (1 to 2.3)a 2.2 (1.4 to 3)a 1.9 (1.3 to 3.5) Cl, clearance. aMann–Whitney U <0.05, 15 versus 25 mg/kg ED patients. 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation Introduction Amikacin Inhale is an integrated drug–device combi- nation in development by Bayer HealthCare through a collaboration with Nektar Therapeutics, to improve clinical outcome in intubated and mechanically ventilated patients with Gram-negative pneumonia. It is available in two confi gurations: on-vent for intubated patients and hand-held for extubated patients to complete aerosolized antibiotic therapy. Amikacin Inhale is a smart system that consists of the pulmonary drug delivery system with a vibrating mesh nebulizer and the specially formulated Amikacin Inhalation Solution (400 mg every 12 hours for 10 days). The objectives of this study were to evaluate the performance of the Amikacin Inhale hand-held confi guration with supplemental O2 concentration supplied at diff erent fl ow rates and in diff erent orientations. We hypothesize that the delivered dose of amikacin will not signifi cantly change with increased O2 fl ow rate or varying orientation.i q Results Preventive administration of IT as an adjunct to systemic antibiotics was associated with a lower incidence of NP in group 1 (group 1 33.3%, group 2 66.7%, χ2 = 6,000; P = 0.014) and a shorter duration of ICU stay (group 1 8.0 ± 4.6 days vs. 17.1 ± 18.4 days, P = 0.03). The mortality did not diff er between groups: 11.1% in group 1 and 22.2% in group 2 (P ≥0.99). On day 3 Acinetobacter spp. (30.5%), K. pneumoniae (22.0%), B. cepacia (13.2%) and P. aeruginosa (34.3%) were detected in BAL, there were no diff erences between groups. In group 1 CPIS remained stable and APACHE II decreased. CPIS and APACHE II were lower in group 1 on day 5 (P = 0.0004). y Conclusion Early administration of IT as an adjunct to systemic antibiotics is eff ective in prevention of NP in multiple trauma patients: it promotes decrease of NP incidence and decrease of ICU stay. Methods In the hand-held confi guration of Amikacin Inhale, amikacin is aerosolized into a holding chamber. Amikacin aerosol is inhaled with ambient air entering the bottom of the chamber through the inhalation valve. Supplemental O2 may be supplied through the O2 port and mixes with ambient air entering through the inhalation valve. O2 concentration and delivered dose at the mouthpiece exit were characterized in vitro at various O2 fl ow rates (2 to 10 l/minute). O2 concentrations were measured every minute until the end of dosing. Early preventive administration of inhaled tobramycin in severe polytrauma Early preventive administration of inhaled tobramycin in severe polytrauma A Kuzovlev1, A Shabanov2, T Chernenkaya2, V Moroz1, A Goloubev1 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.V. Sklifosofsky Research Institute, Moscow, Russia Critical Care 2015, 19(Suppl 1):P121 (doi: 10.1186/cc14201) y Results Overall, nine cases with CNS infection were recorded aged from 22 to 74, all males. LP was performed between the second and 17th day (average 8.3 days) and the CSF analysis showed 40 to 6,000 cells – mainly PMNs, protein 161 mg% to 287 mg% and glucose from 3 to 58 mg/dl. They were all colonized with Acinetobacter baumannii sensitive only to colistin. CSF cultures were negative for all patients besides one, who grew A. baumannii. Of those, seven (77%) were receiving i.v. colistin, eight (88%) carbapenems, and eight (88%) glycopeptides, all in combination with other antibiotics. Median i.t. administration time was 9.1 days. All patients responded to i.v. and i.t. antibiotics but there was one case in which fever relapsed and increased number of cells in subsequent LP was observed which was attributed to colistin, which was withdrawn. All these patients survived, and were discharged to the ward. p y A Kuzovlev1, A Shabanov2, T Chernenkaya2, V Moroz1, A Goloubev1 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.V. Sklifosofsky Research Institute, Moscow, Russia Critical Care 2015, 19(Suppl 1):P121 (doi: 10.1186/cc14201) Introduction Nosocomial pneumonia (NP) occurs in 30 to 50% of multiple trauma patients. It is mostly caused by multiresistant Gram- negative bacteria. Use of inhaled antibiotics as adjuncts to systemic antibiotics presents a great outlook for the prevention of NP in multiple trauma patients. The aim of the study was to evaluate the effi cacy of early administration of inhaled tobtamycin (IT) as an adjunct to systemic antibiotics for the prevention of NP in polytrauma. Methods Fifty-four ICU mechanically ventilated patients with multiple trauma (ISS >30; car accident 55.6%; fall 29.6%; train accident 11.1%; domestic 3.7%) were enrolled in the single-center randomized trial. Groups were comparable in ISS, age, sex, type of trauma, and blood loss. Patients were randomized into two groups: Group 1 (n = 27), addition of IT to systemic antibiotics (ciprofl oxacin 800 mg/day; metronidazol 1,500 mg/day); Group 2 (n = 27), only systemic antibiotics (same regimen). Early preventive administration of inhaled tobramycin in severe polytrauma Inhaled tobramycin (300 mg twice daily via nebulizer) and systemic antibiotics were administered within the fi rst 24 hours Conclusion Patients treated with the abovementioned regime showed clinical and biochemical improvement. The above drug combination turned out to be successful in neurosurgical ICU patients with CNS infection. Intrathecal administration of colistin, vancomycin and amikacin for central nervous system infections in ICU neurosurgical patients P Alexandropoulos, S Georgiou, V Chantziara, A Tsimogianni, E Chinou, V Karagiannisa, G Michaloudis Introduction Central nervous system (CNS) infections in ICU patients after neurosurgery are a diffi cult and life-threatening complication demanding immediate action. In many cases intravenous (i.v.) administration of antibiotics is not suffi cient; thus, intrathecal (i.t.) administration is required. g pi pp 2 Results The mean O2 concentration ranged from 36 to 70% over 2 to 10 l/minute and was ≥40% at ≥3 l/minute. The delivered dose did not change substantially with increasing enriched O2 fl ow rate (72 to 82% of nominal dose). At 0° and 45° orientations, the delivered dose of amikacin was 74 to 80% and 73 to 76% of the nominal dose (400 mg), respectively. Methods From January 2013 to November 2014 all cases with CNS infections were recorded. Inclusion criteria were the presence of fever ≥38.5°C, increased infl ammatory markers, compatible lumbar puncture (LP) fi ndings (increased number of polymorphonuclear leukocytes, increased protein and low glucose compared with serum levels) and no evidence of other site of infection. All subjects were receiving appropriate i.v. antibiotic treatment based on cultures. Intrathecal administration of 300,000 iu colistin, 25 mg vancomycin and 25 mg amikacin was performed taking under consideration that neurosurgical patients in the ICU have CNS infection attributed to Gram-negative bacteria or/and to Staphylococcus species. Conclusion Amikacin Inhale was shown in vitro to be suitable for extubated patients who require supplemental O2. The delivered dose was independent of supplemental O2 and device orientation. Reference 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. S42 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P120 after ICU admission. After obtaining the results of bronchoalveolar lavage microbiology, the antibiotic regimen was switched according to the sensitivity. The primary outcome measure was new onset of NP and duration of ICU stay. Microbiological, X-ray, CPIS, signs of sepsis and oxygenation index were used as objective indicators of the clinical progress. The secondary outcome measure was 30-day mortality. Diagnosis of NP was made according to the standard clinical and CPIS criteria. The data were statistically analyzed by SPSS 11.5 (M, σ, Newman–Keuls test; chi-square-test P <0.05). after ICU admission. After obtaining the results of bronchoalveolar lavage microbiology, the antibiotic regimen was switched according to the sensitivity. The primary outcome measure was new onset of NP and duration of ICU stay. Microbiological, X-ray, CPIS, signs of sepsis and oxygenation index were used as objective indicators of the clinical progress. The secondary outcome measure was 30-day mortality. Diagnosis of NP was made according to the standard clinical and CPIS criteria. The data were statistically analyzed by SPSS 11.5 (M, σ, Newman–Keuls test; chi-square-test P <0.05). P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation N Kadrichu1, K Corkery1, T Dang1, P Challoner2 1Novartis Pharmaceuticals, San Carlos, CA, USA; 2Nektar Therapeutics, San Francisco, CA, USA Critical Care 2015, 19(Suppl 1):P120 (doi: 10.1186/cc14200) P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation A ventilator was connected to the set up to simulate patient breathing. The delivered dose was measured at the exit of the mouthpiece. Drug distribution within the test setup compartments was analyzed using HPLC. The in vitro delivered amikacin dose was also measured at nebulizer orientations of 0° and 45° (n = 3 per orientation) using a simulated breathing profi le with no supplemental O2. P122 Intrathecal administration of colistin, vancomycin and amikacin for central nervous system infections in ICU neurosurgical patients P Alexandropoulos, S Georgiou, V Chantziara, A Tsimogianni, E Chinou, V Karagiannisa, G Michaloudis Saint Savvas Oncology Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P122 (doi: 10.1186/cc14202) Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Bl 1 N Ni 1 M B i l1 P S h 2 E Ull 3 I S Wid 1 Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Blum1, N Nigro1, M Briel1, P Schuetz2, E Ullmer3, I Suter-Widmer1, B Winzeler1, R Bingisser1, H Elsaesser3, D Drozdov2, B Arici2, SA Urwyler1, J Refardt1, P Tarr4, S Wirz4, R Thomann5, C Baumgartner6, H Duplain7, D Burki8, W Zimmerli3, N Rodondi6, B Mueller2, M Christ-Crain1 1University Hospital Basel, Switzerland; 2Medical University Clinic, Kantonsspital Aarau, Switzerland; 3Kantonsspital Baselland/Liestal, Liestal, Switzerland; 4Kantonsspital Baselland/Bruderholz, Bruderholz, Switzerland; 5Bürgerspital, Solothurn, Switzerland; 6Inselspital, Bern University Hospital, Bern, Switzerland; 7Hôpital du Jura, Site de Delémont, Delémont, Switzerland; 8Viollier SA, Basel, Switzerland Results We included nine trials enrolling 2,637 patients. Eight trials were of unclear risk of bias and one was classifi ed as having low risk of bias. In trials comparing heparin with placebo or usual care, the risk ratio for death associated with heparin was 0.88 (95% CI = 0.77 to 1.00, I2 = 0%, 2477 patients, six trials). In trials comparing heparin with other anticoagulants, the risk ratio for death was 1.30 (95% CI = 0.78 to 2.18, I2 = 0%, 160 patients, three trials). In trials comparing heparin with placebo or usual care, major hemorrhage was not statistically signifi cantly increased (risk ratio 0.79, 95% CI = 0.53 to 1.17, I2 = 0%, 2,392 patients, three trials). In one small trial of heparin compared with other anticoagulants, the risk of major hemorrhage was signifi cantly increased (2.14, 95% CI = 1.07 to 4.30, 48 patients). Important secondary and safety outcomes, including minor bleeding, were sparsely reported. Critical Care 2015, 19(Suppl 1):P125 (doi: 10.1186/cc14205) Critical Care 2015, 19(Suppl 1):P125 (doi: 10.1186/cc14205) Introduction Clinical trials yielded confl icting data about the benefi t of adding systemic corticosteroids for community-acquired pneumonia (CAP). We evaluated whether short-term corticosteroid treatment reduces time to clinical stability in patients hospitalized for CAP. Methods This randomized, placebo-controlled multicenter trial compared prednisone 50 mg for 7 days with placebo in patients hospitalized with CAP. The primary endpoint was time to clinical stability. Results Overall, 802 patients were randomized in seven Swiss hospitals from December 2009 to May 2014. Time to clinical stability was shorter in the prednisone group compared with placebo (3.0 vs. 4.4 days, HR = 1.33, 95% CI = 1.15 to 1.50, P <0.001). The prednisone group as compared with the placebo group had a shorter time to hospital discharge (6 vs. i References 1. Alejandria MM, Lansang MD, Dans LF, Mantaring III JBlas. Intravenous immunoglobulin for treating sepsis, severe sepsis and septic shock. Cochrane Database Syst Rev. 2013;9:CD001090. doi:10.1002/14651858.CD001090.pub2. 2. Department of Health. Clinical guidelines for immunoglobulin use. London: Department of Health; 2011. y p 2. Department of Health. Clinical guidelines for immunoglobulin use. London: Department of Health; 2011. Methods We included randomized controlled trials from MEDLINE, EMBASE, CENTRAL, Global Health, Scopus, Web of Science, the International Clinical Trials Registry Platform (inception to April 2014), conference proceedings, and reference lists of relevant articles. Two reviewers independently identifi ed and extracted trial-level data from randomized trials investigating unfractionated or low molecular heparin administered to patients with sepsis, severe sepsis, septic shock or DIC associated with infection. Internal validity was assessed in duplicate using the Risk of Bias tool. Our primary outcome was mortality. Safety outcomes included hemorrhage, transfusion and thrombocytopenia. Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis g Conclusion The use of IVIg does not appear to aff ect mortality in sepsis. There was also no statistical benefi t or harm demonstrated by using IVIg. This also holds true whether IVIg is given either according to the guidelines or not; however, stricter adherence to the guidelines does have fi nancial implications. Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) Introduction Septic shock is characterized by systemic infl ammation coupled with upregulation of coagulation. Heparin is an inexpensive and widely available anticoagulant with anti-infl ammatory properties. The objectives our study were to evaluate the effi cacy and safety of heparin in patients with sepsis, septic shock or disseminated intravascular coagulation (DIC) associated with infection. Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Bl 1 N Ni 1 M B i l1 P S h 2 E Ull 3 I S Wid 1 7 days (HR = 1.19, 1.04 to 1.38), P = 0.012) and a shorter duration of intravenous antibiotic treatment (4 vs. 5 days (diff erence, –0.89 days, –0.20 to –1.57 days), P = 0.011). All-cause mortality, ICU stay, recurrent pneumonia and rehospitalization rate were similar in both groups. Incidence of pneumonia-associated complications until day 30 tended to be lower in the prednisone group (2.8% vs. 5.6%, OR = 0.49, 0.23 to 1.02, P = 0.06). The prednisone group had a higher rate of in-hospital hyperglycemia needing insulin treatment (19.4% vs. 10.9%, OR = 1.96, 1.31 to 2.93, P = 0.001). Other adverse events compatible with corticosteroid use were rare and similar in both groups. Conclusion Heparin in patients with sepsis, septic shock, and DIC associated with infection may be associated with decreased mortality; however, the overall impact remains uncertain. Safety outcomes have been under-reported and require further study. Large randomized trials are needed to evaluate the effi cacy and safety of heparin in patients with sepsis, severe sepsis, and septic shock. P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis R Zarychanski1, AM Abou-Setta1, S Kanji2, AF Turgeon3, A Kumar1, DS Houston1, E Rimmer1, BL Houston4, L McIntyre2, AE Fox-Robichaud5, PC Hebert6, DJ Cook5, DA Fergusson2 1University of Manitoba, Winnipeg, MB, Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Université Laval, Québec City, QC, Canada; 4University of Toronto, ON, Canada; 5McMaster University, Hamilton, ON, Canada; 6Centre hospitalier de l’Université de Montreal (CHUM) – Hopital Notre-Dame, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) Reference 1. Karaiskos I, Galani L, Baziaka F, Giamarellou H. Intraventricular and intrathecal colistin as the last therapeutic resort for the treatment of multidrug-resistant and extensively drug-resistant Acinetobacter baumannii ventriculitis and meningitis: a literature review. Int J Antimicrob Agents. 2013;41:499-508. S43 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis R Zarychanski1, AM Abou-Setta1, S Kanji2, AF Turgeon3, A Kumar1, DS Houston1, E Rimmer1, BL Houston4, L McIntyre2, AE Fox-Robichaud5, PC Hebert6, DJ Cook5, DA Fergusson2 1University of Manitoba, Winnipeg, MB, Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Université Laval, Québec City, QC, Canada; 4University of Toronto, ON, Canada; 5McMaster University, Hamilton, ON, Canada; 6Centre hospitalier de l’Université de Montreal (CHUM) – Hopital Notre-Dame, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) guidelines for prescription, with a mortality rate of 25%. Nine patients did not meet the national guidelines for prescription, with a mortality rate of 44%. The diff erence in mortality rates between the two groups did not reach statistical signifi cance (P = 0.6). There was no signifi cant diff erence in APACHE II scores between the two groups. There was also no diff erence in mortality between those receiving IVIg and those who did not. We also found no diff erence in those receiving single or double doses of IVIg. P126 Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) P124 P124 Use of intravenous immunoglobulin to treat sepsis in a general ICU Y Drakeford, J Kelly, P Morgan, J Melville, A Holland Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2015, 19(Suppl 1):P124 (doi: 10.1186/cc14204) Use of intravenous immunoglobulin to treat sepsis in a general ICU Y Drakeford, J Kelly, P Morgan, J Melville, A Holland Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2015, 19(Suppl 1):P124 (doi: 10.1186/cc14204) Introduction Sepsis is a major cause of admission to the ICU, and a leading cause of death for ICU patients. Intravenous immunoglobulin (IVIg) is indicated in the treatment of some patients with sepsis, although the evidence for this remains controversial. The use of IVIg is regulated due to its high cost, and prescription guidelines have been revised by the NHS, coordinated by the National Demand Management Programme for Immunoglobulin. Conclusion Prednisone treatment for 7 days in hospitalized patients with CAP shortens time to clinical stability, time to hospital discharge and duration of intravenous antibiotic treatment without an increase in complications. Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model T Masuda1, C Mitaka2, M Khin Hnin Si2, K Kido2, Y Qi2, T Uchida2, S Abe2, T Miyasho3, M Tomita4 1Tokyo Medical and Dental University, Tokyo, Japan; 2Tokyo Medical and Dental University Graduate School, Tokyo, Japan; 3Rakuno Gakuen University, Hokkaido, Japan; 4Tokyo Medical and Dental University Hospital of Medicine, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P127 (doi: 10.1186/cc14207) g p gi Results The mortality rate of 28 days in the L-group was 32.8%, and was 18.8% in the H-group. Mean arterial pressure increased signifi cantly (P <0.01) in the H-group compared with the L-group. WBC counts in the L-group increased and in the H-group decreased (P <0.01) during PMX- DHP treatment. Platelet counts in both groups decreased signifi cantly (P <0.01).There was no signifi cant diff erence between before and after PMX-DHP in IL-6 levels. On the other hand, IL-1ra decreased signifi cantly before and after PMX-DHP. Also, IL-6 and IL-1ra in the L-group were signifi cantly higher than those in the H-group at the start of PMX- DHP. PCT values in the L-group were increased compared with the H-group at the start of PMX-DHP (P <0.01). PCT in the L-group increased signifi cantly (P <0.01), but no signifi cant changes in the H-group. PAI- 1 showed no signifi cant changes before and after PMX-DHP and no changes in both groups at the start of PMX-DHP. Introduction Direct hemoperfusion with a polymyxin B immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock [1]. However, the mechanisms of action behind PMX-DHP are not fully understood. Therefore, the purpose of this study was to elucidate mechanisms of action behind PMX-DHP in a rat model of cecal ligation and puncture. Introduction Direct hemoperfusion with a polymyxin B immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock [1]. However, the mechanisms of action behind PMX-DHP are not fully understood. Therefore, the purpose of this study was to elucidate mechanisms of action behind PMX-DHP in a rat model of cecal ligation and puncture. Methods Sprague–Dawley rats were anesthetized and were mechanical ventilated after tracheostomy. The right internal carotid artery was cannulated with a catheter for continuous measurement of the arterial pressure and heart rate. P128 y p Results RecAP administration resulted in a terminal elimination half- life and plasma clearance of 49 to 58 hours and 2.8 to 3.4 l/hour after single ascending doses, respectively, and 63 to 66 hours and 3.1 to 3.8 l/hour after multiple ascending doses. Peak recAP concentrations and AP activity levels were reached at the end of the 1-hour infusion and showed a rapid decline with about 10% of the maximum concentration remaining at 4 hours and less than 5% at 24 hours post start. Although the maximal concentration and total systemic exposure of recAP and AP activity increased slightly more than dose proportionally this is of no signifi cance in the estimated therapeutic dose range. RecAP treatment was generally well tolerated and anti-drug antibodies could not be detected in serum. P129 Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network White blood cell counts have an impact on septic patient outcome followed by polymyxin-B immobilized fi ber with direct hemoperfusion p H Tanaka, T Ikeda, S Ono, S Suda, T Ueno Tokyo Medical University, Hachioji Medical Center, Hachioji, Japan Critical Care 2015, 19(Suppl 1):P128 (doi: 10.1186/cc14208) Introduction The mortality rate of severe sepsis and septic shock is varied and high (25 to 70%). In our institute, the indication for polymyxin-B immobilized fi ber with direct hemoperfusion (PMX-DHP) has been that circulatory failure (systolic blood pressure <90 mmHg or required catecholamines and high lactacidemia) continued despite following early goal-directed therapy by the Surviving Sepsis Campaign guidelines 2012. Conclusion RecAP is characterized by a long serum terminal half-life, by stable serum AP levels and did not exert any safety concerns when administered to healthy volunteers. These results pave the way to investigate the potential of recAP as a new treatment option for sepsis- associated AKI in a phase II clinical trial, which will start at the end of 2014 [Clinical Trial Register:NCT02182440]. g Methods This study included 80 patients with severe sepsis or septic shock due to abdominal infection retrospectively. These subjects were divided into two groups: those with WBC counts <4,000 (L-group: 64 patients) and those with WBC counts >12,000 (H-group: 16 patients). Mean arterial pressure, WBC counts, platelet counts, interleukin-6 (IL-6), and plasminogen activator inhibitor-1 (PAI-1) were measured immediately before the initiation and after the completion of PMX- DHP. Statistical analysis was performed using the chi-squared test for background factors, with Wilcoxon’s rank-sum test for comparison within a group, and Mann–Whitney’s U test for comparison between groups. The signifi cance level was set at P <0.05. References 1. Pickkers P, et al. Crit Care. 2012;16:R14. , ; 2. Heemskerk S, et al. Crit Care Med. 2009;37:417-23.e1. Pharmacokinetics, safety and tolerability of h alkaline phosphatase in healthy volunteers 1 d2 3 l 2 Methods We conducted a retrospective audit of pharmacy records of IVIg prescriptions issued to ICU patients with severe sepsis and septic shock from 2009 to 2014 against national prescription guidelines. Microbiology results were examined to support prescriptions, and admission APACHE II scores and unit outcomes were examined. Results From 2009 to 2014, 644 patients were admitted to the ICU with severe sepsis and septic shock, with a mortality rate of 41%. Seventeen patients received IVIg. Of these, eight patients met the national Methods We conducted a retrospective audit of pharmacy records of IVIg prescriptions issued to ICU patients with severe sepsis and septic shock from 2009 to 2014 against national prescription guidelines. Microbiology results were examined to support prescriptions, and admission APACHE II scores and unit outcomes were examined. p p y E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) Results From 2009 to 2014, 644 patients were admitted to the ICU with severe sepsis and septic shock, with a mortality rate of 41%. Seventeen patients received IVIg. Of these, eight patients met the national Introduction Clinical trials showed renal protective eff ects of bovine intestinal alkaline phosphatase in critically ill patients with S44 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion PMX-DHP improved hemodynamics, acid–base balance, and creatinine levels through reducing cytokines and renal tubular cell death in a rat model of cecal ligation and puncture. These fi ndings suggest the preventive role of PMX-DHP in the development of sepsis- related acute kidney injury. sepsis-associated acute kidney injury (AKI) [1,2]. Recently, human recombinant AP (recAP) was developed as a pharmacological attractive replacement. We conducted a phase I clinical trial to evaluate tolerability, safety and pharmacokinetics of recAP in healthy volunteers. Methods In a randomized, double-blind, placebo-controlled phase I trial, healthy volunteers received via a 1-hour i.v. infusion a single dose of recAP (200, 500, 1,000 or 2,000 U/kg; n = 33) or multiple doses of recAP (500 or 1,000 U/kg; n = 18) on three consecutive days (n = 18). Serum recAP concentrations, AP activity levels and anti-drug antibodies were measured, and safety parameters were monitored. Reference 1. Ronco C, et al. Polymyxin B hemoperfusion: a mechanistic perspective. Crit. Care. 2014;18:309. 1. Ronco C, et al. Polymyxin B hemoperfusion: a mechanistic perspective. Crit. Care. 2014;18:309. Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model The right femoral vein was cannulated with a catheter for infusion of saline (10 ml/kg/hour) during the study period. The rats were randomized into three experimental groups: cecal ligation and puncture (CLP) + dummy column (Dummy- DHP) group (n = 10), CLP + PMX-DHP group (n = 10), and sham group (n = 4). Four hours after CLP, Dummy-DHP or PMX-DHP was performed for 1 hour. Blood was drawn from the right internal carotid artery, perfused through PMX column or dummy column, and returned to the right femoral vein. The heart rate, mean arterial pressure, arterial blood gases, and plasma concentrations of creatinine, lactate, potassium, and cytokines (IL-6 and IL-10) were measured at baseline and at 4, 5, and 8 hours after CLP. At the completion of the experiment, the rats were killed overdose of pentobarbital. The kidney, liver, and lung were harvested, and histopathologic examinations of these organs were performed. g g p Conclusion The mortality rate of the L-group tended to be higher than that of the H-group. Infl ammatory and anti-infl ammatory cytokines in the L-group were higher than those of the H-group. These results indicate that leukopenia (WBC <4,000) in severe sepsis patients leads to more severe outcome and hypercytokinemia than leukocytosis (WBC >12,000) in severe sepsis patients. P129 Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network E Sevketoglu1, D Yildizdas2, O Horoz2, H Kihtir1, T Kendirli3, S Bayraktar4, J Carcillo5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Istanbul, Turkey; 2Cukurova University Medical Faculty, Adana, Turkey; 3Ankara University Medical Faculty, Ankara, Turkey; 4Haseki Research and Training Hospital, Istanbul, Turkey; 5University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Results Hypotension and metabolic acidosis occurred in the CLP + Dummy-DHP group, whereas hemodynamics and acid–base balance were better maintained in the CLP + PMX-DHP group. Plasma concentrations of lactate, creatinine, potassium, and cytokines were signifi cantly higher in the CLP + Dummy-DHP group than in the CLP + PMX-DHP group at 8 hours. Renal tubular cell death was observed in the CLP + Dummy-DHP group, but not in the CLP + PMX-DHP group. Critical Care 2015, 19(Suppl 1):P129 (doi: 10.1186/cc14209) Introduction Thrombocytopenia-associated multiple organ failure (TAMOF) can lead to high mortality in critically ill children, possibly related to consequences of thrombotic microangiopathy. Plasma Introduction Thrombocytopenia-associated multiple organ failure (TAMOF) can lead to high mortality in critically ill children, possibly related to consequences of thrombotic microangiopathy. Plasma S45 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 APACHE scores before and after ECAC therapy were available for eight patients who died; APACHE score decreased >5 points in fi ve patients after single application of ECAC. exchange therapy may improve thrombotic microangiopathy [1]. The purpose of this observational cohort study is to describe whether there is an association between use of plasma exchange therapy and outcome in the Turkish TAMOF network. Conclusion ECAC can be used as adjuvant therapy in treatment of severe sepsis/septic shock/MOF. Our patients had high PM and four could be saved with use of ECAC. We could expect a better outcome if ECAC was used early (<24 hours) during treatment. However, future well-designed studies are needed to clarify the role of ECAC in patients with MOF/septic shock. Methods We performed a retrospective cohort analysis in patients with TAMOF at three diff erent pediatric ICUs comparing those who received plasma exchange (+) plus standard therapies with those who did not receive plasma exchange (–) and only received standard therapies. Results Among the 42 TAMOF patients enrolled, all had a primary or secondary sepsis diagnosis. Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network Fifteen received plasma exchange therapy (PE(+) group) and 27 received standard medical treatment without plasma exchange (PE(–) group). The mean age was 17.69 months (8.24 to 54.22) in the PE(+) group, and 13.46 months (6.47 to 20.55) in the PE(–) group. Age (P = 0.232), gender (P = 0.206), thrombocyte count (P = 0.09), OFI score (P = 0.111) and Pelod score (P = 0.177) on admission were not statistically diff erent between groups. The overall 28-day mortality was higher in the PE(–) group 70.37% compared with 26.67% in the PE(+) group (univariate P =0.006; multivariate controlling for Pelod, OFI, PRISM scores and neurological failure P = 0.048). Length of stay was increased in the PE(+) group (P = 0.004). Clinical experience of using a novel extracorporeal cytokine adsorption column for treatment of septic shock with multiorgan failure Methods We retrospectively evaluated 387 patients who received a broad-spectrum antimicrobial treatment for septic shock due to GNB between January 2009 and December 2012 in the ICU of 10 Japanese tertiary hospitals. After alignment of the treatment time phase for each patient, we divided the patients into two groups according to whether PMX treatment was performed within 24 hours after ICU admission (PMX group: n = 129 and non-PMX group: n = 258). The primary endpoint was 28-day mortality. Ruby Hall Clinic, Pune, India Introduction Severe sepsis and multiorgan failure (MOF) are major causes of death in the ICU. The extracorporeal cytokine adsorption column (ECAC; Cytosorb®, CytoSorbents Corporation, USA), a critical care focused therapeutic device, results in rapid in vitro and in vivo elimination of several key cytokines and prevents organ failure. Use of ECAC in patients with sepsis is a new area of research with insuffi cient data to promote large prospective RCTs. Studies published to date have shown promising results. We report our clinical experience with ECAC for severe sepsis/septic shock/MOF patients. Results The mean (SD) age and SOFA scores on ICU admission were 72.5 (12.5) years and 10.0 (3.4), respectively. The infection site was intra-abdominal (47.0%), pulmonary (17.6%), and urinary tract (27.8%). Two-thirds of all patients had bacteremia due to GNB. No diff erence in 28-day mortality was observed between the two groups (PMX: 33.9% vs. non-PMX: 33.1%, P = 0.87). In the Cox regression analysis adjusted for age, sex and facilities, the PMX treatment (hazard ratio = 0.87; 95% confi dence interval, 0.53 to 1.43) did not improve the outcome.f Methods A retrospective evaluation of ECAC in patients admitted to a tertiary ICU from November 13 to October 14 to analyze: clinical safety; selection of a subgroup of patients where it could be used; selection of timing for initiation; number of device fi lters required per patient; and selective markers to identify above initiation. Patients were managed with standard of care (SOC; antibiotics, vasopressors, i.v. fl uids, sepsis dosed steroids) and ECAC as adjuvant therapy. Vitals, APACHE II and SOFA scores were measured. i Conclusion No diff erence in mortality rate was observed after adjustment for the endotoxin adsorption therapy with PMX in the patients with septic shock due to GNB. Reference Introduction Mortality from septic shock in the ICU remains high, ranging from 30 to 50%. In particular, Gram-negative bacilli (GNB) account for 40% of the causative bacteria of severe sepsis, which progresses to multiorgan failure due to signifi cant infl ammation. Hemoperfusion with polymyxin B-immobilized fi ber (PMX) adsorbs endotoxin and can reduce the infl ammatory cascade of sepsis due to GNB. However, the clinical effi cacy of this treatment has not been demonstrated. We aimed to verify the effi cacy of endotoxin adsorption therapy by using PMX. Reference 1. Nguyen TC, Carcillo JA. Bench-to-bedside review: Thrombocytopenia- associated multiple organ failure – a newly appreciated syndrome in the critically ill. Crit Care. 2006;10: 235. Reference 1. Nguyen TC, Carcillo JA. Bench-to-bedside review: Thrombocytopenia- associated multiple organ failure – a newly appreciated syndrome in the critically ill. Crit Care. 2006;10: 235. Eff ectiveness of polymyxin B immobilized fi ber hemoperfusion in patients with septic shock due to Gram-negative bacillus infection: the PMXHP study N Saito1, K Sugiyama2, T Ohnuma3, T Kanemura4, M Nasu5, Y Yoshidomi6, H Adachi7, H Koami8, Y Tsujimoto9, A Tochiki10, Y Wagatsuma11, T Myumi12 1Chiba Hokusou Hospital, Nippon Medical School, Chiba, Japan; 2Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan; 3Saitma Medical Center, Jichi Medical University, Saitama, Japan; 4National Hospital Organization Disaster Medical Center, Tokyo, Japan; 5Urasoe General Hospita, Okinawa, Japan; 6Saga-ken Medical Center, Koseikan, Saga, Japan; 7Iizuka Hospital, Fukuoka, Japan; 8Saga University Hospital, Saga, Japan; 9Yamagata Prefectural Central Hospital, Yamagata, Japan; 10Tsukuba Medical Center Hospital, Ibaraki, Japan; 11University of Tsukuba, Ibaraki, Japan; 12University of Occupational and Environment Health, Fukuoka, Japan Conclusion The positive association found between use of plasma exchange therapy and improved survival supports the potential of this therapy in Turkish children with TAMOF. The positive, although less so, associated treatment eff ect observed after controlling for illness severity provides further rationale for performing a randomized controlled trial in the pediatric Turkish TAMOF network. Sample size calculations call for a 100-patient trial with a pre hoc interim analysis after enrollment of 50 TAMOF patients. p Critical Care 2015, 19(Suppl 1):P131 (doi: 10.1186/cc14211) Introduction Mortality from septic shock in the ICU remains high, ranging from 30 to 50%. In particular, Gram-negative bacilli (GNB) account for 40% of the causative bacteria of severe sepsis, which progresses to multiorgan failure due to signifi cant infl ammation. Hemoperfusion with polymyxin B-immobilized fi ber (PMX) adsorbs endotoxin and can reduce the infl ammatory cascade of sepsis due to GNB. However, the clinical effi cacy of this treatment has not been demonstrated. We aimed to verify the effi cacy of endotoxin adsorption therapy by using PMX. Impact of evolving cardiac catheterisation services on admissions to a regional ICU Results Nineteen ICU patients (14 men, fi ve women; 24 to 72 years; average ICU stay 10 days; average ventilator days 9) with APACHE II >17 (except one with dengue shock syndrome), SOFA score ≥11 (n = 16) and the majority having infection largely in the lung (n = 8; alone or with UTI and blood infection) followed by the abdomen (n = 4), UTI (n = 3) and others (n = 4) were given ECAC (total ECAC = 31). Predicted mortality (PM) was >40% in 16, >30% in two and <30% in two (tropical infections) patients. Duration of therapy was 6 hours (no. of ECAC = 18) and 8 hours (n = 4; no. of ECAC = 5) for the majority of patients. Overall, four patients (two with tropical infections and two with PM >40%) survived; three of them had were ECAC early (<24 hours of admission). The majority of patients (n = 11) who died could be given ECAC only once. Of patients who died, seven were given ECAC late (>24 hours). g EA Gorman, D Trainor Royal Victoria Hospital, Belfast, UK Critical Care 2015, 19(Suppl 1):P132 (doi: 10.1186/cc14212) EA Gorman, D Trainor Introduction National UK audit data demonstrate cardiac catheterisation services, including percutaneous coronary intervention and noncoronary interventions, are increasing [1-3]. National mortality rates post cardiac catheterisation are also increasing, refl ecting an increasing proportion of sicker patients undergoing interventional procedures [3]. National audit procedures do not evaluate patients admitted to intensive care post cardiac catheterisation. We aimed to Introduction National UK audit data demonstrate cardiac catheterisation services, including percutaneous coronary intervention and noncoronary interventions, are increasing [1-3]. National mortality rates post cardiac catheterisation are also increasing, refl ecting an increasing proportion of sicker patients undergoing interventional procedures [3]. National audit procedures do not evaluate patients admitted to intensive care post cardiac catheterisation. We aimed to S46 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P133). evaluate the impact of an evolving regional cardiac catheterisation service on a regional intensive care unit (RICU) serving a population of 1.8 million. Methods A retrospective review was carried out. Patients admitted from the regional cardiac catheterisation laboratory to the regional ICU, between September 2009 and September 2014, were identifi ed using validated RICU admission records. Clinical data were extracted from computerised patient records. Impact of the introduction of e-learning prior to a basic transthoracic echo course P Madhivathanan1, S Jain2, D Walker3 Introduction Deep venous thrombosis (DVT) is an increasing major cause of mortality and morbidity. There is a need for quick, easy, cheap, convenient and reliable diagnostic tools. The objectives were to ascertain the diagnostic concordance of emergency doctor- performed ultrasound (EDUS) of the lower extremities with specialist doctor-performed (radiologist or vascular surgeon) echo-Doppler (SDED), in the diagnosis of DVT, and to identify possible causes of nonconcordance. P Madhivathanan1, S Jain2, D Walker3 P Madhivathanan1, S Jain2, D Walker3 1Barts Health NHS Trust, London, UK; 2Homerton University Hospitals NHS Foundation Trust, London, UK; 3University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P133 (doi: 10.1186/cc14213) P Madhivathanan , S Jain , D Walker 1Barts Health NHS Trust, London, UK; 2Homerton University Hospitals NHS Foundation Trust, London, UK; 3University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P133 (doi: 10.1186/cc14213) Introduction Focused Intensive Care Echo accreditation is a nationally approved pathway for training and accreditation in basic transthoracic echocardiography (TTE) in the UK. Recently, an e-learning module, the Intensive Care Echo and Basic Lung Ultrasound (ICE-BLU), has been introduced to facilitate TTE learning [1]. Previous work from our group has shown that incorporating simulation-based teaching elements into a basic TTE course improves candidates’ satisfaction [2]. We assessed the impact of introducing the ICE-BLU e-learning programme prior to our simulation-based basic TTE course. Methods A prospective, multicentre study. Adult patients (>18 years old) with clinical suspicion of DVT, with high or moderate risk (on Wells scoring), or low risk with increased D-dimer levels, were eligible for the study. Emergency doctors performed two EDUS in femoral and popliteal areas (these results were blinded). After this, echo-Doppler was performed by specialist doctors. Both procedures were done within 24 hours of each other. The fi nal result was considered nonconcordant if one or both of the EDUS did not match with the SDED. These SDED were used as reference (as standard clinical practice). Methods Prior to the August 2014 course, all candidates were required to complete the ICE-BLU e-learning module. On the morning of the course, the candidates completed a questionnaire to assess the impact of the e-learning module. The survey included questions on the quality of content, user friendliness, whether the content was pitched at the right level and any problems faced whilst accessing the e-learning module. P133 p Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) Diagnostic concordance of emergency doctor-performed bedside ultrasonography versus specialist-performed echo-Doppler ultrasonography in the diagnosis of deep venous thrombosis of lower limbs 2. National Institute for Cardiovascular Outcomes. Myocardial ischaemia national audit project. Public report April 2012–March 2013. http://www.ucl.ac.uk/nicor/audits/minap/reports. 3. British Cardiovascular interventional society. National coronary interventional procedures. Public report. January 2012–December 2013. http://www.bcis.org.uk/resources/PCI_Audit_Report_2012_fi nal.pdf. DL Ly-Pen1, JP Penedo Alonso2, MS Sánchez Perez2, FR Roldán Moll2, MZ Zamorano Serrano2, LD Díaz Vidal2, SJ Justo3 1Southend University Hospital, Westcliff -on-Sea, UK; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) DL Ly-Pen1, JP Penedo Alonso2, MS Sánchez Perez2, FR Roldán Moll2, MZ Zamorano Serrano2, LD Díaz Vidal2, SJ Justo3 1Southend University Hospital, Westcliff -on-Sea, UK; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) References 1. http://www.e-lfh.org.uk/programmes/icu-echoultrasound. Accessed 1 Dec 2014. 2. Madhivathanan PR, et al. Simulation-based transthoracic echo teaching: a tertiary centre experience. Intensive Care Med. 2014;40 Suppl 1:S158-9. 1. British Cardiovascular Interventional Society. Audit returns for adult interventional procedures. January 2013–December 2013. http://www.bcis. org.uk/documents/BCIS_Audit_2013_for_web_Version_23-11-2014.pdf. Impact of evolving cardiac catheterisation services on admissions to a regional ICU p p Results A total of 170 patients were identifi ed (representing 2.9% of critical care admissions during this time). Baseline characteristics: 71.7% male, median age 66 (IQR 55 to 74), median APACHE score 18 (IQR 15 to 23). Seventy-one patients (41.7%) had an APACHE score >20. Fifteen patients (8.8%) were aged >80 years. Admissions increased yearly – 20 in 2010, 26 in 2011, 35 in 2012, 47 in 2013, 37 at the end of the third quarter of 2014 (projected 59 admissions by year end 2014). Median length of stay was 3.5 days (IQR 1.8 to 7.2). Average length of stay reduced yearly (9.14 days in 2010 to 5.01 days in 2014). ICU bed- days per year remained static over the 5-year period. Critical care and hospital mortality rates were 33% and 39% respectively. There was a trend towards increasing mortality yearly, and with increasing age and APACHE score. Conclusion Our survey has shown that the e-learning initiative was welcome by the candidates. We conclude that introduction of e-learning prior to a simulation-based basic TTE course enhances candidates’ satisfaction and feedback. Conclusion An evolving cardiac catheterisation service is having a signifi cant impact on intensive care services within a regional centre. Increasing mortality trends in this critical care population refl ects post- cardiac catheterisation mortality trends nationally. We suggest intensive care admissions post cardiac catheterisation should be included in the national audit, to allow forward planning of intensive care services and to promote quality improvement within this population. References Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) y Results From January 2014 to December 2014, 32 patients with septic shock (study group) and 20 patients with sepsis but no septic shock (control group) were recruited. The baseline characteristics were similar. Conventional echocardiographic measurements, including LVEF (59.53% vs. 60.67% in the control group, P = 0.450) and fractional shortening (31.98% vs. 32.98%, P = 0.323), did not diff er between the two groups. The study group had a greater degree of myocardial dysfunction measured by left ventricular global longitudinal strain (–14.6 vs. –17.6, P = 0.005, with a less negative value implying worse myocardial contractility). The hemodynamic profi les (cardiac index 3.49 l/minute/m2 vs. 3.41 l/minute/ m2 respectively, P = 0.764) were not statistically diff erent.i Introduction Myocardial depression is common among septic patients [1]. The aim of this study was to assess whether the values of cardiac index (CI) and velocity–time integral (VTI) calculated by echocardiography diff er between survivors and nonsurvivors of sepsis. Methods This was a prospective observational study. We included adult newly admitted septic patients, regardless of disease severity. Exclusion criteria were concomitant pregnancy or obstetric/gyne- co logical sepsis and co-existing or terminal diseases that may limit life expectancy. At the moment of recruitment, additional exclusion criteria included: concomitant pulmonary embolism, trauma or acute ischemic coronary disease; pericardial tamponade; aortic valve disease; tachyarrhythmias and absence of adequate echocardiographic windows. Echocardiographic evaluations were made within the fi rst 10 minutes of initiation of fl uid therapy in the emergency room. All measurements and images were obtained with a 1.5 to 3.5 MHz phased array transducer using a standard cardiac preset. CI is the quotient of the cardiac output (CO) divided by the body surface area. The CO is the product of the stroke volume by the heart rate. Stroke volume is calculated as the product between aortic VTI (measured using pulsed- wave Doppler) and aortic cross-sectional area. The latter is calculated in the long axis parasternal window using the left ventricular outfl ow tract diameter measurement. Impact of the introduction of e-learning prior to a basic transthoracic echo course We also analysed candidates’ feedback from our January and August 2014 courses (Figure 1). Results From September 2013 to September 2014, a total of 328 patients were enrolled. Fifty-one investigators from seven hospitals performed the EDUS. Each patient had the EDUS (femoral and popliteal areas) and SDED (also in femoral and popliteal areas). Of 328 pairs of US studies, 37 were nonconcordant between EDUS and SDED. Two EDUS were incomplete, so the concordance analysis was performed with 326 US studies, with 35 discordant. The percentage of agreement between EDUS and SDED was 89.26%. The kappa index was 0.76 (95% CI = 0.69 to 0.84), and this means a substantial agreement. Results The response rate of the survey was 100%. Eighty per cent of candidates completed the e-learning module. The e-learning module was rated high by most candidates (80%). However, nearly one-half of the candidates faced problems accessing the module, online. Analysis of candidates’ feedback (from the January and August 2014 courses) revealed that candidates’ overall impression was better with the introduction of e-learning prior to the course. g Conclusion There is substantial agreement between EDUS and SDED in the diagnosis of DVT, in routine clinical practice. This confi rms the results of previous papers. The largest nondiagnostic concordance in thrombus occurs in the early performances of emergency doctors, S47 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Comparing group I versus group II, the mortality rate was 45%, and there was a statistically signifi cant diff erence for temperature (P = 0.001), HR (P = 0.001) and WBC count (P = 0.01) on admission. Upon comparing survivors versus nonsurvivors in group I there was a statistical diff erence in HR on day 7 (P = 0.02), successful vasopressor withdrawal (P = 0.02), P/F ratio (P = 0.02) and ScVO2 on day 7 (P = 0.03). Regarding IL-1α, IL-1β, TNFα and troponin I there was no statistical signifi cant diff erence between groups I and II but IL-6, IL-10 and CRP showed statistically signifi cant diff erence on admission PV and CS. Pro- BNP shows statistically signifi cant diff erence in all CS samples between septic and nonseptic groups. Regarding echo upon comparing the survivors versus nonsurvivors, E’d/t on day 0 shows a statistically signifi cant diff erence between both groups. Reference 1. Angus DC. Epidemiology of severe sepsis in USA. Crit Care Med. 2001;29:1303-10. P135 P135 Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) Conclusion This is a fi rst study in the adult population to show that the use of speckle tracking imaging can diagnose signifi cant sepsis- induced myocardial dysfunction, which was not otherwise detectable by conventional echocardiography. P135 Cardiac abnormalities in patients with septic shock detected by speckle tracking echocardiography PY Ng1, WC Sin1, AK Ng2, WM Chan1 1Queen Mary Hospital, Hong Kong; 2Grantham Hospital, Hong Kong Critical Care 2015, 19(Suppl 1):P135 (doi: 10.1186/cc14215) P135 Cardiac abnormalities in patients with septic shock detected by speckle tracking echocardiography PY Ng1, WC Sin1, AK Ng2, WM Chan1 1Queen Mary Hospital, Hong Kong; 2Grantham Hospital, Hong Kong Critical Care 2015, 19(Suppl 1):P135 (doi: 10.1186/cc14215) Introduction Sepsis-induced myocardial dysfunction is a well- recognized condition and confers worse outcomes in septic patients. However, the diagnostic criteria remain poorly described. Echocardiographic assessment by conventional parameters such as left ventricular ejection fraction (LVEF) is often aff ected by ongoing changes in preload and afterload conditions. Novel echocardiographic technologies such as speckle tracking imaging have evolved for direct assessment of the myocardial function. In this study, we investigate the measurement of myocardial strain by speckle tracking imaging for the diagnosis of sepsis-induced myocardial dysfunction. g p y p Conclusion Diastolic dysfunction was seen in 90% of patients. Fever, HR, and WBC counts are still good early indicators for diagnosis of sepsis. Vasopressor withdrawal on the seventh day was a good predictor for survival. Admission serum IL-6, IL-10 and CRP from PV were better indicators for sepsis than IL-1, pro-BNP and troponin I. Admission TNFα and seventh-day IL-6 levels were highly prognostic for mortality. CS samples proved that NT pro-BNP is a good indicator for sepsis diagnosis and a good predictor for survival. TNFα from CS samples was also a good predictor of mortality. SAPS II and a slower E’d/t on admission was a good predictor of mortality. R f Methods This is a prospective, case–control study at a university- affi liated tertiary care adult medical ICU. Consecutive patients admitted with a diagnosis of septic shock meeting the international consensus criteria were included. Patients with other causes of myocardial dysfunction were excluded. They are compared with age-matched, gender-matched, and cardiovascular risk factor-matched controls, who were admitted to hospital for sepsis but did not develop septic shock. Conventional echocardiographic parameters, as well as speckle tracking imaging of myocardial function, were obtained within 24 hours of diagnosis. Offl ine analyses of endocardial tracings were performed by two independent operators. Reference Impact of the introduction of e-learning prior to a basic transthoracic echo course SAPS II and seventh-day SOFA are good predictive scores for mortality in sepsis. especially until the fi fth performance. After the sixth one, the incidence of mismatches falls dramatically. It seems advisable to mentor the training programmes with at least fi ve shadowed performances in order to lower the incidence of mistakes. P136 Evaluating the eff ect of sepsis and septic shock on myocardial functions by echocardiography and serum biomarker level in peripheral veins and coronary sinus M Soliman1, A Alazab1, R El Hossainy1, M Nirmalan2, H Nagy1 1Cairo University, Cairo, Egypt; 2University of Manchester, UK Critical Care 2015, 19(Suppl 1):P136 (doi: 10.1186/cc14216) P138 Speckle tracking imaging for evaluation of eff ects of positive end-expiratory pressure level on right ventricular function M Türker, A Pirat, A Camkiran Firat, B Pirat, A Sezgin, G Arslan Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P138 (doi: 10.1186/cc14218) Figure 2 (abstract P139). Values of PEEP above which LS was not present. Introduction Positive end-expiratory pressure (PEEP) is commonly used to correct hypoxemia in the ICU. However, PEEP may impair right ventricular functions by increasing its afterload. Speckle tracking imaging (STI) is a new echocardiography strain analysis technique that provides direct assessment of myocardial contractility during systole and diastole. The aim of this study was to evaluate the eff ects of diff erent PEEP levels on right ventricular functions by using STI in patients undergoing coronary artery bypass grafting surgery. Methods After ethics committee approval and patients’ written consent, we prospectively analyzed 20 CABG surgery patients. After initiation of mechanical ventilation and before sternotomy, 5, 10, and 20 cmH2O PEEP were applied in 5-minute intervals consequently. After stabilization at each PEEP level, four-chamber and two-chamber images of the right ventricle were recorded using TEE. The right ventricle diameter, velocity, longitudinal strain, SR, and fractional area change (RVFAC) were calculated and evaluated from the recorded images. PEEP at which LS disappeared or reappeared were compared using the Wilcoxon signed-rank test to assess the infl uences of anatomical side and PEEP increase or decrease. The values of PEEP at disappearance of LS for the right lung were not statistically signifi cantly diff erent from the left lung (P = 0.844 for increases, P = 0.938 for decreases). The values of PEEP at which LS disappeared obtained during increases were not statistically signifi cantly diff erent from values obtained during decreases (P = 1.000 for left lung, P = 0.875 for right lung; Figure 1). From data pooled from both sides and protocols, the median value of PEEP at which LS disappeared as a false positive sign of pneumothorax was 25 cmH2O (interquartile range = 20 to 30 cmH2O). At PEEP = 10 cmH2O, no patient showed absence of LS, whereas at PEEP = 35 cmH2O, all patients showed absence of LS (Figure 2). g Results The mean age of study patients (85% male) was 59.7 ± 10.5 years. Intraoperative mean, systolic, and diastolic arterial blood pressures and heart rate were similar at the three PEEP levels. P139 Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP J Golub1, A Markota1, A Stožer2, G Prosen3, A Bergauer1, F Svenšek1, A Sinkovič1 1University Medical Centre Maribor, Slovenia; 2University of Maribor, Slovenia; 3Community Health Centre, Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P139 (doi: 10.1186/cc14219) P140 Lung ultrasound in quantifying lung water in septic shock patients L De Geer, A Oscarsson, M Gustafsson Linkoping University Hospital, Linkoping, Sweden Critical Care 2015, 19(Suppl 1):P140 (doi: 10.1186/cc14220) Evaluating the eff ect of sepsis and septic shock on myocardial functions by echocardiography and serum biomarker level in peripheral veins and coronary sinus M Soliman1, A Alazab1, R El Hossainy1, M Nirmalan2, H Nagy1 1Cairo University, Cairo, Egypt; 2University of Manchester, UK Critical Care 2015, 19(Suppl 1):P136 (doi: 10.1186/cc14216) Introduction Sepsis is a leading cause of death in critically ill patients despite the use of modern antibiotics and resuscitation therapies. Biomarkers and cardiovascular changes have an important place in this process. Myocardial depression occurs in 40% of septic patients. Methods Twenty patients (group I) with sepsis or septic shock were included and 10 patients (group II) served as the nonseptic group. Group I morbidity and mortality at day 28 in the ICU were targeted as the endpoint. Laboratory investigations, APACHE IV, SAPS II and SOFA scores were calculated. Biomarkers IL-1α, IL-1β, IL-6, IL-10, TNFα, CRP, NT-proBNP and troponin level were estimated on admission and day 7 in the peripheral vein (PV) and coronary sinus (CS). Transthoracic echocardiography and tissue Doppler imaging was done on admission and on day 7. Results In 3 months, 58 patients were included. The average age was 46.6 years, and 36 were male. Overall mortality was 14%. We included 16 patients with sepsis syndrome, 27 patients with severe sepsis and 15 patients with septic shock. Severe sepsis patients presented with higher values of CI, when compared with sepsis syndrome and septic shock patients (3.46, 3.08 and 2.92 l/minute/m2, respectively, P = NS). The same occurred with VTI (19.27, 18.81 and 16.74 cm for severe sepsis, sepsis syndrome and septic shock, respectively; P = NS). Mean values of CI were lower in nonsurvivors of sepsis (2.51 vs. 3.35 l/minute/ m2, P = 0.018). Mean values of VTI were also lower in nonsurvivors (14.83 vs. 19.01 cm, P = 0.022). Conclusion In our study, nonsurvivors of sepsis presented with lower values of both CI and VTI in the emergency department. Therefore, CI S48 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P139). Absence of LS during increasing and decreasing PEEP. Figure 2 (abstract P139). Values of PEEP above which LS was not present. Figure 1 (abstract P139). Absence of LS during increasing and decreasing PEEP. and VTI may be good markers of sepsis severity and mortality in newly admitted patients. In addition, further studies are warranted to assess the role of CI and VTI as therapeutic targets. Reference 1. Silva E, Pedro Mde A, Sogayar AC, et al. Brazilian Sepsis Epidemiological Study (BASES study). Crit Care. 2004;8:R251-60. P138 Compared with 5 and 10 cmH2O PEEP, mean RVFAC signifi cantly decreased at 20 cmH2O PEEP (P = 0.001). Right ventricle velocity reduced with incremental PEEP increases (P <0.05). Mean SR values decreased at 20 cmH2O PEEP when compared with 5 cmH2O PEEP (P = 0.03). Mean right ventricle diameter measurements decreased with incremental PEEP increases; however, this decrease was signifi cantly diff erent between 20 cmH2O PEEP and other two PEEP levels (P = 0.01). The mean right ventricle strain value signifi cantly decreased at 20 cmH2O PEEP when compared with other two PEEP levels (P <0.001 for both). p g Conclusion According to this study, higher PEEP levels correlate with disappearance of LS without pneumothorax. Absence of LS in patients with high PEEP should be interpreted with caution and other signs of pneumothorax should be sought before therapeutic interventions are attempted. Conclusion Compared with 5 and 10 cmH2O PEEP levels, right ventricle functions in terms of strain, SR, right ventricle diameter, and RVFAC were signifi cantly impaired at 20 cmH2O PEEP level. P140 Variations in near-infrared spectroscopy-derived oxygen downslope during a vascular occlusion test in critically ill patients: relationship with outcome p A Donati, E Damiani, A Carsetti, V Monaldi, E Montesi, P Pelaia A Donati, E Damiani, A Carsetti, V Monaldi, E Montesi, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P141 (doi: 10.1186/cc14221) Università Politecnica delle Marche, Ancona, Italy y Critical Care 2015, 19(Suppl 1):P141 (doi: 10.1186/cc14221) Results No changes in mean arterial pressure were observed. The perfused small vessel density tended to decrease at t1 and normalize at t2 (Figure 1) in the hyperoxia group. These variations appeared early after 2 minutes of FiO2 changes. A signifi cant increase in lactate levels over time (from 1.1 (0.9 to 1.7) at t0 to 1.4 (1.1 to 1.9) mmol/l at t2, P = 0.01) was seen in the hyperoxia group. Introduction Near-infrared spectroscopy (NIRS) with a vascular occlusion test (VOT) can be used to extrapolate information regarding the tissue oxygen extraction rate. We explored the meaning of variations in tissue oxygen saturation downslope (StO2down) during a VOT in critically ill patients. Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Figure 1 (abstract P142). y p Methods In this prospective observational study, NIRS (thenar eminence) was applied every day in 93 patients admitted to the ICU. A VOT was performed using a 40% StO2 target. The slope of the desaturation curve was assessed separately for the fi rst part (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between Down2 – Down1 was calculated.if Results No signifi cant diff erences were seen in StO2 Down1 or Down2 between ICU survivors (n = 76) and ICU nonsurvivors (n = 17) over Figure 1 (abstract P141). Figure 1 (abstract P142). Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Normobaric hyperoxia and the microcirculation in critically ill patients: a prospective observational study Normobaric hyperoxia and the microcirculation in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, E Montesi, S Ciucani, M Rogani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P142 (doi: 10.1186/cc14222) Conclusion LUS non-invasively and user-independently quantifi es lung water in concordance with, but does not replace, invasive measurements. Further studies are needed establish the role of LUS as a monitoring and diagnostic tool in septic shock patients. References Introduction It is well known that oxygen acts as a vasoconstrictor. We evaluated the impact of normobaric hyperoxia on the sublingual microcirculation in critically ill patients. Introduction It is well known that oxygen acts as a vasoconstrictor. We evaluated the impact of normobaric hyperoxia on the sublingual microcirculation in critically ill patients. 1. Frassi F, et al. J Card Fail. 2007;13:830-5. 1. Frassi F, et al. J Card Fail. 2007;13:830-5. 2. Prosen G, et al. Crit Care. 2011;15:R114. Methods Forty mechanically ventilated (FiO2 ≤50%) patients with hemodynamic stability were enrolled in a prospective observational study. The fi rst 20 patients underwent a 2-hour period of hyperoxia (FiO2 = 100%), and 20 patients were studied as controls (no FiO2 variations). The sublingual microcirculation (three sites) was evaluated with sidestream dark-fi eld imaging at baseline (t0), after 2 hours of hyperoxia (t1), and 2 hours after return to baseline (t2). Continuous video recording was also performed during FiO2 variations on one and the same area (2-minute video). 3. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP The values of S49 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 guidelines [3]. The median number of B-lines was 15 (IQR 10 to 30) and the median (IQR) NT-proBNP, EVLWI and oxygenation index (OI) were 7,800 ng/l (3,690 to 15,050), 11 ml/kg (IQR 8 to 18) and 9.2 (5.7 to 15.7), respectively. None of the characteristics diff ered signifi cantly between survivors and nonsurvivors. The number of B-lines correlated to EVLWI (ρ = 0.45, P = 0.04; r2 = 0.20, P = 0.04), but not to NT-proBNP (ρ = –0.42, P = 0.06), OI (ρ = 0.25, P = 0.31) or ICU length of stay (ρ = 0.14, P = 0.57). On Bland–Altman analysis, mean diff erences and 95% limits of agreements between B-lines and EVLWI was 4.9 (–14.5 to 24.5), and 5.9 (–3.5 to 15.3) when assessing observer agreement. The ICC between methods was 0.52 (95% CI = –0.17 to 0.81) and 0.90 (95% CI = 0.73 to 0.92) between observers. the fi rst 10 days in the ICU, while Down2 – Down1 was higher in ICU nonsurvivors (Figure 1). Patients in the upper quartile of mean Down2 – Down1 showed the highest 90-day mortality (P = 0.014).l Conclusion ICU nonsurvivors tended to show a fl attening in the last part of the desaturation curve during a VOT, suggesting a reduced tissue oxygen extraction. This may depend on microvascular dysfunction and/or cellular hypometabolic status. P142 Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP Introduction Quantifi cation of lung ultrasound (LUS) artifacts (B-lines) is used to assess pulmonary congestion in emergency medicine and cardiology [1,2]. We investigated B-lines in relation to extravascular lung-water index (EVLWI) from invasive transpulmonary thermodilution in septic shock patients. Our aim was to evaluate the role of LUS in an intensive care setting. 1University Medical Centre Maribor, Slovenia; 2University of Maribor, Slovenia; 3Community Health Centre, Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P139 (doi: 10.1186/cc14219) Methods Twenty-one patients admitted with septic shock to a general ICU underwent LUS of eight zones, four per hemithorax, within 24 hours after ICU admission. EVLWI was calculated simultaneously by transpulmonary thermodilution using a pulse-contour continuous cardiac output system, and NT-proBNP and clinical data were collected. Two physicians blinded to other data independently quantifi ed the number of B-lines. Spearman’s rho was used to test the correlation of B-lines to EVLWI and clinical data, and linear regression and Bland– Altman analysis were used to assess the agreement between B-lines and EVLWI. Interobserver variability was tested using Bland–Altman analysis and intraclass correlation coeffi cient (ICC). Introduction The objective of our study was to estimate correlation between PEEP and disappearance of lung sliding (LS) due to lung overdistension in the absence of pneumothorax. Methods We performed a prospective study from September 2013 to May 2014 in adult patients with respiratory failure who required mechanical ventilation, lung CT and recruitment manoeuvre. Lung CT was used as the gold standard to exclude pneumothorax. A staircase recruitment manoeuvre was used with 5 cmH2O increases of PEEP from baseline to 35 cmH2O and decreases in reverse order. The duration of each step was 1 minute. Lung ultrasound was performed to evaluate LS at each step in one intercostal window in the highest point of left and right hemithoraces by physicians trained in lung ultrasound and blinded to changes in PEEP.i Results Fourteen patients (67%) were male, the median age was 62 years (IQR 55 to 68) and eight (38%) patients had cardiac comorbidities. In median, SAPS 3 was 64 (IQR 60 to 74), ICU length of stay was 3 days (IQR 2 to 8) and seven patients (33%) died within 30 days of ICU admission. All patients were mechanically ventilated and treated according to Results In all, eight patients were included; fi ve (62.5%) males, mean age 70.1 ± 7.4 years. Mean auto-PEEP was 0.7 ± 0.4 cmH2O. Prospective nonrandomized observational study of the use of an impedance threshold device in patients with spontaneous respiration and hemodynamic instability Conclusion The length of time to lactate normalization in the severe burn patient is a marker of survival and a simple parameter to guide the endpoint of resuscitation; however, the percentage of lactate clarifi ed in the fi rst 24 hours is not valid. Introduction The use of an impedance threshold device (ITD) in cardiac arrest victims has been shown to increase the systolic arterial pressure (SAP) by increasing venous return [1]. There are limited studies concerning the use of ITD in patients with spontaneous respiration and hemodynamic instability. The purpose of this study is to evaluate changes of hemodynamic parameters with the use of ITD in patients with spontaneous respiration and hemodynamic instability. Lactate in the burn patient This may refl ect a tissue hypometabolic status, which may be better elicited in the fi nal part of the ischemic challenge. Healthy volunteers (n = 27) were studied as controls. The slope of the desaturation curve was assessed separately for the fi rst (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between, Down2 – Down1, was calculated. Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P <0.05); no diff erence was seen between ICU survivors (n = 7) and nonsurvivors (n = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P <0.05); no diff erence was seen between ICU survivors (n = 7) and nonsurvivors (n = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Conclusion Tissue oxygen extraction was reduced in septic patients. Nonsurvivors showed a fl attening in the last part of the desaturation curve during a VOT, while the fi rst part of the StO2 downslope did not show any diff erence between survivors and nonsurvivors. This may refl ect a tissue hypometabolic status, which may be better elicited in the fi nal part of the ischemic challenge. y Results During a period of 2 years we studied 143 patients; their mean age was 46.98 ± 19.38 years, mean TBSA burn injury of the study population was 22.82 ± 20.25. The fl ame was the most frequent mechanism. A total of 83 patients were in mechanical ventilation and 13.6% of them developed ARDS. The mortality range in the study group was 17%. Serum lactate at admission ≥2 mmol/l was associated with 31.3% mortality versus 6% in patients with a serum lactate at admission <2 mmol/l (P <0.05). Length of time to lactate normalization variable is associated with mortality (P <0.02). The average lactate normalization time was 4.6 days in nonsurvivors while in survivors it was 2.02 days. A relation does not exist between the lactate clearance and mortality in all patients. P144 P144 Prospective nonrandomized observational study of the use of an impedance threshold device in patients with spontaneous respiration and hemodynamic instability C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P144 (doi: 10.1186/cc14224) P143 Near-infrared spectroscopy for assessing the tissue oxygen extraction rate during sepsis: relationship with outcome C S ll S d S C l Introduction Microcirculatory dysfunction impairs tissue oxygenation during sepsis. We applied near-infrared spectroscopy (NIRS) to evaluate the tissue oxygen extraction rate in sepsis and its relationship with outcome. Methods A prospective observational study; 14 septic patients underwent NIRS monitoring (thenar eminence) with a vascular occlusion test (using a 40% StO2 target) at admission to the ICU. Figure 1 (abstract P141). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S50 Figure 1 (abstract P143). 10 minutes after the intervention. Endpoint of the study was a change of patient’s SAP after application of ITD. Results The SAP of patients that were included in the study increased 15 to 22 mmHg (P <0.05). Heart rate remained unchanged. Eighty percent of patients declared good to very good tolerance from ITD application. Conclusion In this observational study of patients with spontaneous respiration and hypotension, ITD increased the SAP, while it seems that it was well tolerated by patients. Additional and larger studies will be needed in the future in order to investigate the benefi ts of ITD when used to patients with spontaneous respiration and hemodynamic instability. Reference 1. Pirrallo RG, et al. Eff ect of an inspiratory impedance threshold device on hemodynamics during conventional manual cardiopulmonary resuscitation. Resuscitation. 2005;66:13-20. 1. Pirrallo RG, et al. Eff ect of an inspiratory impedance threshold device on hemodynamics during conventional manual cardiopulmonary resuscitation. Resuscitation. 2005;66:13-20. Lactate in the burn patient p EH Herrero, M Sánchez, L Cachafeiro, A Agrifoglio, B Galván, MJ Asensio, A García de Lorenzo Hospital La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P145 (doi: 10.1186/cc14225) Introduction Severe burns result in rapid loss of intravascular volume due to development of a severe capillary leak and hypovolemic shock. It is widely accepted that traditional markers, such as blood pressure and urinary output, are useful but do not suffi ciently refl ect global perfusion, regional microcirculation or reversal shock. Blood lactate concentration is widely used in ICUs as a reliable prognostic marker of global tissue hypoxia. Our aim is to determine whether the percentage of lactate clarifi ed in the fi rst 24 hours is valid as a guide for resuscitation. Methods We prospectively studied 143 consecutive burn patients admitted to our Burn Unit. Sociodemographics and comorbidities data were recorded. Clinical data were collected to calculate the Acute Burn Severity Index. Resuscitation according to the Parkland formula was guided by a urinary output of 0.5 to 1 ml/hour and the results of monitoring the blood pressure. Crystalloid solution (Ringer´s acetate) was given exclusively during the fi rst 24 hours. Early surgical excision of burn eschar and early coverage of excised burn wounds with autografts was performed. Initial and subsequent serum lactate levels were measured to calculate lactate clearance according to the formula: lactate basal – lactate at 24 hours / lactate basal × 100. The primary outcome was mortality. Figure 1 (abstract P143). Healthy volunteers (n = 27) were studied as controls. The slope of the desaturation curve was assessed separately for the fi rst (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between, Down2 – Down1, was calculated. Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P <0.05); no diff erence was seen between ICU survivors (n = 7) and nonsurvivors (n = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Conclusion Tissue oxygen extraction was reduced in septic patients. Nonsurvivors showed a fl attening in the last part of the desaturation curve during a VOT, while the fi rst part of the StO2 downslope did not show any diff erence between survivors and nonsurvivors. Is the shock index a universal predictor in the emergency department? A cohort study Results Twenty-three patients underwent VA ECMO for refractory CS in the study period. Etiologies of CS were: 11 acute myocarditis, fi ve acute myocardial infarction and seven acute decompensation of chronic cardiomyopathy. The median time of ECMO was 10 days (4 to 15). Thirteen patients died during hospital stay and 10 survived. Three patients were bridged to LVAD and two to heart transplant; eight were bridged to recovery. The main cause of ICU death was multiple organ dysfunction (12/13). Nonsurvivors showed signifi cantly higher LAC0 (5 (2 to 6) vs. 8 (5 to 11), P = 0.021). Lactate clearance at 48 hours was not signifi cantly diff erent between survivors and nonsurvivors (79%, 95% CI = 67 to 86 vs. 60%, 95% CI = 32 to 72, P = 0.08). However, LAC48 was predictive for ICU mortality (AUC 0.82; 95% CI = 0.64 to 1.0; P = 0.011). ROC curve analysis identifi ed the accuracy was highest by setting the lactate <2 mmol/l. Patients that did not normalize lactate (LAC <2 mmol/l) after 48 hours despite hemodynamic restoration had poorer outcome at 30 days, as is shown in the Kaplan–Meier curve (log- rank P = 0.006) (Figure 1). Introduction The shock index (SI; heart rate/systolic blood pressure) is a widely reported tool to identify acutely ill patients at risk for circulatory collapse in the emergency department (ED). Because old age, diabetes, essential hypertension, and β-/Ca2+ channel-blockers might reduce the compensatory increase in heart rate and mask blood pressure reductions in shock or pre-shock states, we hypothesized that these factors weaken the association between SI and mortality, reducing the utility of SI to identify patients at risk. Methods This was a cohort study from Odense University Hospital of all fi rst-time visits to the ED between 1995 and 2011 (n = 111,019). The outcome was 30-day mortality. We examined whether age ≥65 years, diabetes, essential hypertension, and use of β-/Ca2+ channel-blockers modifi ed the association between SI and mortality. The prognostic value of SI ≥1 was evaluated with diagnostic likelihood ratios. Conclusion Failing to normalize patient’s LAC in the fi rst 48 hours of VA ECMO assistance for CS is a predictor of ICU mortality. Targeting LAC level <2 mmol/l at 48 hours post ECMO institution might be a reasonable goal for these patients. Results We observed a 30-day mortality of 3%. Blood lactate normalization following venoarterial ECMO institution for refractory cardiogenic shockfi Methods A 5-month prospective nonrandomized observational study that included 50 adult patients with spontaneous respiration and hypotension in the emergency room and the wards of multiple causes except trauma. After measurement of the SAP and verifi cation of hypotension (SAP ≤90 mmHg), a mask-style ITD was added. Hemodynamic parameters were evaluated every 1 minute and for M Bottiroli, D Decaria, T Maraffi , S Nonini, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P146 (doi: 10.1186/cc14226) Introduction Venoarterial (VA) ECMO is used to support patients with refractory cardiogenic shock (CS). Elevated lactate level (>2 mmol/l) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S51 Figure 1 (abstract P146). an inotropic score as a predictor of mortality and morbidity among children who diagnosed septic shock. Figure 1 (abstract P146) Methods A multicenter retrospective chart review was performed in two pediatric ICUs. A total of 93 children with septic shock were recruited. Hourly doses of following inotropes were recorded for the fi rst 48 hours after admission: dopamine, dobutamine, adrenaline and noradrenaline. The inotrope score for every hour, minimum, maximum and mean values for the fi rst 24 hours, and subsequent 24 hours were calculated. In our analysis, the inotrope score was calculated as described by Wernovsky. We expanded this formula to include norephinephrine as follows: Wernovsky Inotrope Score = dopamine dose (μg/kg/minute) + dobutamine dose (μg/kg/minute) + 100 × epinephrine dose (μg/kg/ minute). Our adjusted inotrope score = Wernovsky Inotrope Score + 100 × norepinephrine dose (μg/kg/minute).f p p μg g Results Forty-two of 93 patients died. Age and sex were not diff erent between survivors and nonsurvivors. Signifi cantly higher mean and maximum inotropic score for the fi rst 24 hours and fi rst 48 hours were found in nonsurvivors than those of survivors (P <0.05). Using 15 as a cutoff point for predicting mortality, the sensitivity and specifi city were 69.76% and 50.98% respectively. The association between Prism scores and minimum, mean and maximum inotrope scores were statistically signifi cant for 0 to 24 hours, 25 to 48 hours and 0 to 48 hours. Mean 0 to 24 hours and maximum 0 to 48 hours inotrope scores were weakly associated with prolonged ICU stay (P = 0.047, P = 0.042 respectively). There were no signifi cant relationships between inotrope scores and receiving mechanical ventilation.i Figure 1 (abstract P146). Is the shock index a universal predictor in the emergency department? A cohort study With SI <0.7 as reference, a SI of 0.7 to 1 was associated with an adjusted OR of 2.9 (CI 2.7 to 3.2) for 30-day mortality while the adjusted OR for SI ≥1 was 10.3 (CI 9.2 to 11.5). ORs for SI ≥1 were reduced (but still signifi cant) in patients who were older, hypertensive, or on β-/Ca2+ channel-blockers, whereas diabetes had no eff ect. The OR for SI ≥1 in patients ≥65 years was 8.2 (CI 7.2 to 9.4) compared with 18.9 (CI 15.6 to 23.0) in younger patients. β-/Ca2+ channel-blocked patients had an OR of 6.4 (CI 4.9 to 8.3) versus 12.3 (CI 11.0 to 13.8) in nonusers, and the OR for hypertensive patients was 8.0 (CI 6.6 to 9.4) versus 12.9 (CI 11.1 to 14.9) in nonhypertensive patients. The OR for SI ≥1 of 9.3 (CI 6.7 to 12.9) in diabetics did not diff er from the OR of 10.8 (CI 9.6 to 12.0) in nondiabetic patients. A SI of 0.7 to 1 was associated with ORs signifi cantly greater than 1 (range: 2.2 to 3.1) with no evident diff erences within the subgroups. A SI measurement ≥1 was associated with lower positive likelihood ratios in patients ≥65 years, with hypertension, diabetes or using β-/Ca2+ channel-blockers (range 4.9 to 6.5) compared with patients not exposed to these factors (range 7.6 to 11.6). Blood lactate normalization following venoarterial ECMO institution for refractory cardiogenic shockfi is an indicator of end-organ hypoperfusion. We hypothesize that the lactate (LAC) normalization had prognostic value in this cohort of patients. Methods We performed a retrospective observational study on patients admitted to the ICU for refractory CS from January 2010 to November 2014. Patients with postcardiotomy and/or post-transplant CS were excluded. Demographics, clinical, hemodynamic and biochemical values were collected. LAC was measured on arterial blood before ECMO institution (LAC0) and after 48 hours (LAC48). Lactate clearance was calculated as follows: (LAC0 – LAC48) / LAC0 × 100. Data were analyzed by comparative statistic; sensibility and specifi city were tested with ROC. Conclusion The mean and maximum inotropic scores in the fi rst 24 hours and 0 to 48 hours are an independent predictor of mortality in critically ill children with septic shock. P148 Is the shock index a universal predictor in the emergency department? A cohort study AK Kristensen1, JG Holler1, J Hallas2, A Lassen1, N Shapiro3 1Odense University Hospital, Odense, Denmark; 2University of Southern Denmark, Odense, Denmark; 3Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P148 (doi: 10.1186/cc14228) P149 Risk factors for severe vasodilatory shock after cardiac surgery J Almeida, F Galas, J Fukushima, E Almeida, A Gerent, E Osawa, C Park, R Nakamura, A Leme, M Sundin, R Kalil Filho, F Jatene, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P149 (doi: 10.1186/cc14229) Introduction Vasodilatory shock is a well-known complication in patients who undergo cardiac surgery with cardiopulmonary bypass (CPB) and its occurrence is associated with higher morbidity and mortality. Despite that, clinical characteristics of vasoplegic shock and its spectrum of severity are poorly described. The aim of this study was to compare patients who developed mild to moderate vasoplegic shock with patients who developed a severe form and to identify predictive factors for the severe form of vasoplegic shock. Results Heterogeneity of LS eff ects was registered in a number of values. The most signifi cant positive eff ect which allowed one to evaluate myocardial reserve was demonstrated by decrease of PPA (93.5% of patients) and increase of EF (77.6% of patients). The most signifi cant changes were also noted in decrease of TrI, PH and MI (in 53.2%, 36.6% and 36% of patients, respectively). In 69.2% of patients with noncoronarogenic dilated cardiomyopathy the eff ect of LS exposure was marked. In the majority of patients with ischemic cardiomyopathy the eff ect was moderate. In case of the absence of LS-positive eff ect, perioperative use of mechanical circulatory support was considered. Conclusion Preoperative use of LS allows one to evaluate myocardial reserves and prepare high-risk patients with CHF for surgery. Our fi ndings may serve as one of the additional criteria to choose the type of surgical treatment: reconstructive surgery (with or without perioperative mechanical circulatory support) or heart transplantation. Methods We performed an observational study in 300 patients who underwent cardiac surgery with CPB and presented within the fi rst 24 hours after surgery with refractory hypotension and used a vasopressor agent. Severe vasoplegic shock was defi ned as a requirement of norepinephrine higher than 1 μg/kg/minute or the use of two or more vasopressors. Baseline characteristics, laboratorial, clinical and intraoperative data, such as amount of fl uids, bleeding, blood transfusion, inotropes and length of CPB were collected at ICU admission. Logistic regression was performed using severe vasodilatory shock as the outcome. Results There were 46 (15%) patients who develop the severe form of vasodilatory shock within 24 hours after cardiac surgery. Reference Reference 1. Jochberger S, et al. Intensive Care Med. 2009;35:489-97. 1. Jochberger S, et al. Intensive Care Med. 2009;35:489-97. P149 In a univariate analysis, patients with the severe form were more likely to be older, to receive more blood transfusion and inotropic agents, to have higher levels of serum lactate, lower hemoglobin concentration and lower SvO2 at the end of the procedure, lower cardiac output index, higher heart rate and higher levels of reactive C protein at ICU admission. These patients also experienced more postoperative organ dysfunction, had a longer length of ICU stay and higher mortality. There were no diff erences between patients regarding amount of fl uids and length of CPB. In a multivariate analysis we identify age (OR = 1.04, 95% CI = 1.01 to 1.08, P = 0.016), intraoperative use of epinephrine (OR = 5.49, 95% CI = 2.42 to 12.43, P <0.001), higher serum lactate at the end of the procedure (OR = 1.04, 95% CI = 1.01 to 1.06, P = 0.001) and intraoperative blood transfusion (OR = 5.06, 95% CI = 2.19 to 11.69, P <0.001). P151 Levosimendan versus dobutamine in cardiac surgery MD Delgado-Amaya, EC Curiel-Balsera, CJ Joya-Montosa Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P151 (doi: 10.1186/cc14231) Introduction Early studies suggested a signifi cant increase in survival in patients treated with levosimendan compared with dobutamine or placebo (LIDO, RUSSLAN and CASINO trials). However, two subsequent studies (SURVIVE and REVIVE II) have not confi rmed these fi ndings. Methods A prospective observational study of all patients undergoing cardiac surgery at Malaga’s Regional Hospital from March 2009 to March 2013. We analyzed patients who used levosimendan compared with those that used dobutamine in the fi rst hours after cardiac surgery, discarding patients in which neither of these two drugs were used or surgical cases that arrived at the ICU with both inotropics. We analyzed demographic variables as well as clinical complications in the ICU and overall perioperative mortality of patients. We performed a second analysis using the propensity score, obtaining the probability of patients being treated with either drug, pairing each patient who received levosimendan with its nearest neighbor receiving dobutamine. Results We collected 875 patients: 331 received one of the two drugs, 50 received both drugs and 494 did not receive any drug. ICU mortality was 7.2% (levosimendan group) and 12.5% (dobutamine group), P = 0.1. After adjustment for severity and type of surgery, the use of levosimendan in the postoperative period was not a protective factor for ICU mortality (P = 0.18, OR = 0.5, 95% CI = 0.18 to 1.3). In the matched sample, mortality was 7.4% (levosimendan group) and 5.9% (dobutamine), P = 0.73. After logistic regression adjusted for severity, measured with EuroSCORE and type of surgery, levosimendan was not a protective factor for ICU mortality (P = 0.8, OR = 1.2, 95% CI = 0.26 to 5.45).f Conclusion This study demonstrated that older patients, intraoperative blood transfusion and utilization of epinephrine were independently associated with a more severe form of vasodilatory shock after cardiac surgery with CPB. Also, we identifi ed that a higher lactate at the end of the procedure was an independent predictive factor for this severe form of shock. Is an inotrope score a predictor of mortality and morbidity in children with septic shock? E Sevketoglu1, A Anil2, S Kazanci1, O Yesilbas1, M Akyol1, S Bayraktar3, N Aksu4, S Hatipoglu1, M Karabocuoglu5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Bakýrkoy, Turkey; 2Izmir Katip Celebi University Medicine Faculty, Izmir, Turkey; 3Haseki Research and Training Hospital, Istanbul, Turkey; 4Tepecik Research and Training Hospital, Izmir, Turkey; 5Sisli Memorial Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P147 (doi: 10.1186/cc14227) E Sevketoglu1, A Anil2, S Kazanci1, O Yesilbas1, M Akyol1, S Bayraktar3, N Aksu4, S Hatipoglu1, M Karabocuoglu5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Bakýrkoy, Turkey; 2Izmir Katip Celebi University Medicine Faculty, Izmir, Turkey; 3Haseki Research and Training Hospital, Istanbul, Turkey; 4Tepecik Research and Training Hospital, Izmir, Turkey; 5Sisli Memorial Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P147 (doi: 10.1186/cc14227) Introduction Inotropes and vasoactive drugs in septic shock are commonly used to maintain cardiac output, tissue perfusion and oxygenation. We undertook this study with the purpose of evaluating S52 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion SI is independently associated with 30-day mortality in a broad population of ED patients. Old age, hypertension and β-/Ca2+ channel-blockers weaken this association, but the association remains prognostic. SI ≥1 suggests substantial risk of 30-day mortality in all ED patients. for the surgical intervention (2 to 4 days before surgery). In total, 44.9% of the patients had CHF caused by noncoronarogenic dilated cardiomyopathy and 55.1% by ischemic cardiomyopathy. Indication for LS therapy was left ventricular ejection fraction (EF) <35% (28 ± 6%). Seventy percent of patients had mitral insuffi ciency (MI), grades II to IV, 63% tricuspid insuffi ciency (TrI), grades II to III, 84% pulmonary hypertension (PH), 47% arterial hypertension, grades II to III, and 27% of the patients had left ventricular aneurysm. Mean level of BNP was 1,803 ± 124 pg/ml. LS was administered as i.v. infusion in doses of 0.025 to 0.1 μg/kg/minute without previous bolus injection. Mean duration of infusion was 27.5 ± 5.3 hours. After infusion all patients underwent control assessment of values. All patients were operated: 25 (23.3%) underwent reverse cardiac remodeling, 63 (58.9%) myocardium revascularization (MR) with mitral or aortic valve replacement, 17 (15.9%) MR and/or resection of left ventricular aneurism and two (1.9%) heart transplantation. Levosimendan in the emergency department: a useful tool to improve cellular perfusion Introduction Levosimendan was originally developed for the treat ment of decompensated heart failure in situations for which conventional therapy is not suffi cient. It is an eff ective calcium- sensitising drug with vasodilatory and inotropic eff ects and improves cardiac contractility. Trials have shown positive outcome benefi t with the use of levosimendan [1]. We reviewed the usage levosimendan at our institution and outcome of these patients. Introduction Levosimendan is an inotropic seldom used in emergency departments (EDs). It has shown improved mortality in patients with shock [1] with few adverse eff ects [2]. This work denotes the experience of levosimendan use in the ED of Hospital Universitario Mayor between 2012 and 2014. p Methods We reviewed the use of levosimendan at Harefi eld from January 2013 through December 2013. Patient demographics, logistic EuroSCORE (Figure 1), diagnosis, surgical or intervention details, inotropic support, dosage and duration of levosimendan use, length of stay in the ICU, cost (Table 1) and patient outcome were collected. Results Levosimendan was used in 30 patients, 23 (77%) male and seven (23%) female. Median age was 69 (59 to 72.8). Levosimendan was used post cardiac surgery, post angioplasty and in patients with ventricular assist devices (VAD) and extracorporeal membrane oxygenator (ECMO). Most of the patients received a standard regimen of 12.5 mg administered at a dose of 0.1 μg/kg/minute for 24 hours. Concurrent noradrenaline was used in most of the patients ranging from 0.02 to 0.2 μg/kg/minute. The median length of stay in the ICU was nine (6 to 14.5) days for survivors and 23.5 (7.5 to 36) days in nonsurvivors. Sixteen patients (55%) survived and were discharged from the hospital. Methods We reviewed the use of levosimendan at Harefi eld from January 2013 through December 2013. Patient demographics, logistic EuroSCORE (Figure 1), diagnosis, surgical or intervention details, inotropic support, dosage and duration of levosimendan use, length of stay in the ICU, cost (Table 1) and patient outcome were collected. Methods We present a retrospective study which analyzes the eff ect, after 24 hours, of levosimendan administration on perfusion parameters of 166 ED patients. Patients had to have shock diagnosis of any cause. Diff erences between the initial and fi nal mean value of the following parameters were evaluated: lactate, central venous oxygen saturation (ScvO2) and venoarterial diff erence of CO2 (DvaCO2). Data were stratifi ed according to levosimendan categories (initial or rescue). Reference Reference Preoperative treatment with levosimendan helps to evaluate myocardial reserves in cardiosurgical patients with chronic heart failure Conclusion In our environment, we have observed diff erences in the use of levosimendan compared with dobutamine (higher rate of men undergoing CABG, diabetes and worse EF). After homogenizing the sample of patients by propensity score, an eff ect on mortality is discarded and we observed a signifi cant need for use of norepinephrine and a nonsignifi cant trend for prolonged mechanical ventilation and renal failure requiring renal replacement therapy, both probably related with the greatest need for vasopressors observed. Introduction The aim of the study was to assess the possibility of preoperative levosimendan (LS) administration in myocardial reserve evaluation and choice of the method of surgical treatment in patients with chronic heart failure (CHF). Introduction The aim of the study was to assess the possibility of preoperative levosimendan (LS) administration in myocardial reserve evaluation and choice of the method of surgical treatment in patients with chronic heart failure (CHF). Methods LS was used in 107 (female and male) patients (mean age 53 ± 3 years) as a component of CHF therapy to prepare them S53 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P152 Reference 1. Follath F, et al. Lancet. 2002;360:196-202. Levosimendan in the emergency department: a useful tool to improve cellular perfusion In addition, association between diff erent variables with mortality was sought. Diff erences were considered statistically signifi cant at probability levels below 0.05.f y , ( ) p Results Levosimendan was used in 30 patients, 23 (77%) male and seven (23%) female. Median age was 69 (59 to 72.8). Levosimendan was used post cardiac surgery, post angioplasty and in patients with ventricular assist devices (VAD) and extracorporeal membrane oxygenator (ECMO). Most of the patients received a standard regimen of 12.5 mg administered at a dose of 0.1 μg/kg/minute for 24 hours. Concurrent noradrenaline was used in most of the patients ranging from 0.02 to 0.2 μg/kg/minute. The median length of stay in the ICU was nine (6 to 14.5) days for survivors and 23.5 (7.5 to 36) days in nonsurvivors. Sixteen patients (55%) survived and were discharged from the hospital. p y Results There were no diff erences in APACHE II values between patients who received levosimendan as initial therapy from those who received it as a rescue measure. A total of 41 patients fulfi lled lactate normalization requirements (lactate <2.0 or clearance >50%) (Table 1). Forty-four patients reached normal values of SvcO2 and 37 patients of DavCO2 after levosimendan initiation. There were no associations between the normalization of lactate, SvO2 and DvaCO2 and diff erent types of shock. Twenty-nine patients who received initial therapy with levosimendan normalized their lactate values and 12 who received it as a rescue therapy (P <0.05). Sixty-three patients developed hypotension, and none had adverse eff ects requiring discontinuation of the drug. Hospital mortality was 47.7%. Variables associated with mortality in the study group were lactate value at admission (OR = 1.3, 95% CI = 1.0 to 1.7), the use of vasopressin after start levosimendan (OR = 7.5, 95% CI = 1.9 to 28.6) and the use of norepinephrine before starting (OR = 10.8, 95% CI = 1.9 to 60.7). Figure 1 (abstract P152). Logistic EuroSCORE of all patients. Table 1 (abstract P153). Perfusion variables before and after levosimendan Variable Initial Final Lactate (mmol/l) 3.3 (0.5 to 18) 2.38 (0.4 to 17)* SvcO2 (%) 56.3 (23 to 85) 65.2 (37 to 91)* DvaCO2 (mmHg) 8.2 (–16 to 26) 7.3 (–2 to 21) P <0.01. McNemar test. Table 1 (abstract P153). Perfusion variables before and after levosimendan Variable Initial Final Figure 1 (abstract P152). Logistic EuroSCORE of all patients. Figure 1 (abstract P152). Levosimendan in the emergency department: a useful tool to improve cellular perfusion Logistic EuroSCORE of all patients. Conclusion Levosimendan use in the ED, as initial or rescue therapy, normalizes lactate values and improves the SvcO2 after 24 hours, without an increase in adverse eff ects. f Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Levosimendan Adrenaline Milrinone Maximum dose (μg/kg/minute) 0.2 1 0.7 Cost per vial (£) 894 2.30 16 Cost per 24 hours (£) 894 7 112 Conclusion We have successfully used this drug in high-risk patients during the perioperative period with good results without major complications. Levosimendan seems to reduce catecholamine requirement, the need for mechanical circulatory support, and the duration of critical care, which can justify the cost of this drug. It can be also useful in weaning patients from short-term VAD and ECMO. Larger studies are required in this area. Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Levosimendan Adrenaline Milrinone P153 Levosimendan in the emergency department: a useful tool to improve cellular perfusion P153 Levosimendan in the emergency department: a useful tool to improve cellular perfusion G Devia, J Torres, S Lopez Hospital Universitario Mayor, Bogotá, Colombia Critical Care 2015, 19(Suppl 1):P153 (doi: 10.1186/cc14233) Levosimendan: use, cost-eff ectiveness and outcome in a tertiary cardiothoracic centre Levosimendan: use, cost-eff ectiveness and outcome in a tertiary cardiothoracic centre A Ranjan, N Bhudia, I McGovern, C Walker, L Kuppurao Royal Brompton & Harefi eld NHS Foundation Trust, Harefi eld, London, UK Critical Care 2015, 19(Suppl 1):P152 (doi: 10.1186/cc14232) A Ranjan, N Bhudia, I McGovern, C Walker, L Kuppurao Royal Brompton & Harefi eld NHS Foundation Trust, Harefi eld, London, UK Critical Care 2015, 19(Suppl 1):P152 (doi: 10.1186/cc14232) P153 References 1. Landoni G, et al. Minerva Anestesiol. 2010.76:276-86. 2. Mebazaa A, et al. JAMA. 2007.297:1883–91. Conclusion We have successfully used this drug in high-risk patients during the perioperative period with good results without major complications. Levosimendan seems to reduce catecholamine requirement, the need for mechanical circulatory support, and the duration of critical care, which can justify the cost of this drug. It can be also useful in weaning patients from short-term VAD and ECMO. Larger studies are required in this area. Introduction The inotropic agents used in the ICU are dobutamine and milrinone; unfortunately, they have shown signifi cant side eff ects when used for myocardial depression during septic shock. Our objective is to describe Mn behavior in septic shock. S54 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 adaptive threshold for low amplitude QRS detection; QRS fi lter with an extended frequency range; management of VT alarm priorities. Conclusion A majority of the FPs was linked to user settings and patient conditions. The algorithm changes resulted in a clear reduction of ECG algorithm-related FP alarms, while the magnitude of the reduction depends strongly on the settings at the bedside. References 1. ECRI Institute. 2013;42:354-9. 2 Drew BJ et al PLoS ONE 2014 9: e110274 Methods We reviewed 72 clinical records of patients with diagnosis of septic and mixed shock who received Mn through January to December 2013. Demographic, hemodynamic, metabolic and gasometric data were recorded before and after Mn infusion. The PiCCO monitoring system was used. Data were expressed as mean and standard deviation. The statistical analysis used Student’s t test. P <0.05 was considered signifi cant. adaptive threshold for low amplitude QRS detection; QRS fi lter with an extended frequency range; management of VT alarm priorities. Conclusion A majority of the FPs was linked to user settings and patient conditions. The algorithm changes resulted in a clear reduction of ECG algorithm-related FP alarms, while the magnitude of the reduction depends strongly on the settings at the bedside. References 1. ECRI Institute. 2013;42:354-9. 2. Drew BJ, et al. PLoS ONE. 2014.9: e110274. 1. ECRI Institute. 2013;42:354-9. P155 Admission to the ICU with cerebrovascular event (CVE) and trauma was related to arrhythmia (P = 0.021, P = 0.032, respectively). There was a signifi cant relationship between VIP and sepsis presence (P <0.001, P <0.001). Atrial fi brillation was the most frequent type of arrhythmia (53%), and the most frequently used medication was diltiazem (28.5%). The patients who had arrhythmias had a longer ICU stay (P = 0.021). The mortality rate for all patients was 48.1%. There was no statistically signifi cant relationship between arrhythmia and mortality (P >0.05). False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 y j 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA , , ; , , , Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) Introduction The ECRI Institute has identifi ed alarm fatigue as the number one health technology hazard [1]. A recent study on 461 ICU patients investigated 2,558,760 alarms [2]. In total, 88.8% of the annotated 12,671 arrhythmia alarms were false positives (FPs). It was concluded that the excessive number of alarms is ‘a complex interplay of inappropriate user settings, patient conditions, and algorithm defi ciencies’. Nine conditions causing alarms, four of which were ECG algorithm related, were reported [2]. In this study, we investigated a new algorithm in which improvements targeting three of the reported four ECG-related conditions were implemented: low amplitude QRS; wide QRS; nonactionable ventricular tachycardia (VT). gi p y y Conclusion Older age, higher APACHE II scores, trauma, CVE, VIP and sepsis increases arrhythmia risk in critically ill patients. Atrial fi brillation is most common and the most preferred treatment for all arrhythmias is diltiazem. References Results Seventy-two patients were studied: 36.5% were women, mean and SD of age, APACHE II, mechanical ventilation days and long ICU stay were: 67 ± 16 years, 18.5 ± 5.9 points, 14.9 ± 12.9 and 24.5 ± 21.9 days, respectively. A total 20.3% of the patients received dobutamine. Thirty- nine percent presented mixed shock. Global mortality was 23%. After Mn infusion: cardiac index (CI) increased: 3.1 ± 1 to 3.3 ± 1.1, cardiac rate increased: 82.4 ± 14.4 to 88.3 ± 18 and ScvO2 increased: 71.1 ± 10.3 to 76.1 ± 7.3 (P <0.05). PaCO2 arteriovenous diff erence and lactate were reduced: 7.36 ± 3.3 to 6.04 ± 3.6 and 18.7 ± 14.9 to 13.1 ± 9.1 (P <0.05). CVP, MAP, RVSI, VSTI, EVLWI and base excess showed no signifi cant diff erence. Mn initial average dose was 0.35 ± 0.13. NE before and after Mn infusion showed no signifi cant diff erence: 0.11 ± 0.20 versus 0.12 ± 0.22. References 1. Kanji S, et al. Crit Care Med. 2008;36:1620-4 1. Kanji S, et al. Crit Care Med. 2008;36:1620-4. j , ; 2. Annane D, et al. Am J Respir Crit Care Med. 2008;178:20-5. 2. Annane D, et al. Am J Respir Crit Care Med. 2008;178:20-5. P156 Arrhythmia incidence and risk factors in critically ill patients G Tasdemir, N Kelebek Girgin, A Aydin Kaderli, E Cizmeci, R Iscimen, F Kahveci, A Aydinlar Uludag University Medical Faculty, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P156 (doi: 10.1186/cc14236) Introduction Cardiac arrhythmias may be observed at any time during the ICU stay. The prognosis may suff er due to these arrhythmias. In our study, we aimed to evaluate incidence and risk factors of arrhythmias occurring in patients in the ICU. Introduction Cardiac arrhythmias may be observed at any time during the ICU stay. The prognosis may suff er due to these arrhythmias. In our study, we aimed to evaluate incidence and risk factors of arrhythmias occurring in patients in the ICU. Conclusion Mn optimizes cardiovascular performance in septic shock and mixed shock, without aff ecting hemodynamic variables and global tissue perfusion. In addition, we observed that the IC, ScvO2, PaCO2 arteriovenous diff erence and lactate are related variables. References Methods Patients treated in the ICU were included in the study if they fulfi lled the following: age >18, no cardiac valvular disease, no cardiac surgery in the recent 6-month period, no history of myocardial infarction (MI), need for mechanical ventilation, and one or more organ failure. Demographic, hemodynamic and laboratory parameters, APACHE II score, presence of sepsis, acute renal failure, MI, and VIP during the ICU stay were recorded. Therapies used for arrhythmia and response to therapies were also recorded. 1. Holmes CL, et al. Vasoactive drugs for vasodilatatory shcok in ICU. Curr Opin Crit Care. 2009;15:398-402. 2. Jentzer JC, et al. Pharmacotherapy update on the use of vasopressors and inotropes in the intensive care unit. J Cardiovasc Pharmacol Ther [Epub ahead of print].l p p Results Two hundred and fourteen patients were included in the study. Twenty-six percent (n = 56) of patients had arrhythmias. Incidence was higher in females (P = 0.045). Average age of arrhythmic patients was 69 (19 to 86), and they were older than nonarrhythmic patients (P <0.001). APACHE II scores were higher in arrhythmic patients (P = 0.001). 3. Deep A, et al. Evolution of haemodynamics and outcome of fl uid-refractory septic shock in children. Intensive Care Med. 2013;39:1602-9. 3. Deep A, et al. Evolution of haemodynamics and outcome of fl uid-refractory septic shock in children. Intensive Care Med. 2013;39:1602-9. High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery N De Mey, I Brandt, C Van Mieghem, K De Decker, G Cammu, L Foubert OLV AALST, Aalst, Belgium Critical Care 2015, 19(Suppl 1):P158 (doi: 10.1186/cc14238) N De Mey, I Brandt, C Van Mieghem, K De Decker, G Cammu, L Foubert OLV AALST, Aalst, Belgium Introduction Hs-cTn is the new standard cardiac biomarker for the diagnosis of myocardial necrosis. We conducted a prospective study to compare the course and values of Hs-cTn and CK-MB after CABG and OPCAB. We also evaluated the relationship between values >10 × 99th percentile URL of CK-MB and Hs-cTn as a possible marker for perioperative myocardial infarction. g g Results The cTnI concentrations measured in the control group ranged from 0.4 to 17.2, mean 2.1 (95% CI: 1.6 to 2.6) ng/l, without age dependency. Men revealed higher levels than women, means (IQR) were 2.8 (1.2 to 2.6) and 1.1 (0.7 to 1.3) ng/l. The CLSI nonparametric method revealed a 99th percentile value of 16 ng/l. The quantifi cation of cTnI above the LoD (1.0 ng/l) and below the 99th percentile was possible in 79 of 120 individuals. The imprecision profi le according to NCCLS revealed 20%, 10% and 5% CVs at concentrations of 2, 3 and at 20 ng/l, respectively. The discharge diagnosis was NSTEMI in 71 patients. The cTnI median values at 0, 3 and 6 hours were 46, 166 and 399 ng/l, respectively. AUC values at 0, 3 and 6 hours were 0.923, 0.964 and 0.969 for hs-cTnT and 0.919, 0.962 and 0.958 for cTnI, respectively. cTnI revealed AUC values of absolute changes from admission to 3 hours and from admission to 6 hours were 0.920 and 0.931, respectively.i Methods All adult patients undergoing cardiac surgery between February and August 2014 were included. Exclusion criteria were urgent surgery, GFR <60 ml/minute/1.73 m2, CK-MB >4 μg/l and/or Hs-cTn >14 ng/l at baseline (BL). Hs-cTn and CK-MB were measured before induction (BL), upon arrival in the ICU and at fi xed times after arrival. Patients with perioperative AMI as defi ned by the third universal defi nition of AMI were excluded for post hoc analysis [1]. Results Of the 93 patients admissible for inclusion, 40 in the CABG and 14 in the OPCAB group met all inclusion criteria in this preliminary dataset. High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery CK-MB values are higher from ICU arrival up to T24 versus baseline in both CABG and OPCAB (P <0.0001) with a peak at T3. For Hs-cTn, ICU up to T48 values are higher (P <0.01) in CABG with a peak Conclusion The PATHFAST cTnI demonstrated complete fulfi llment of the analytical criteria for high-sensitive cTn assays: The imprecision (CV) at the manufacturer-recommended 99th percentile value was 5%. The quantifi cation of cTnI in was possible in 65.8% of healthy individuals. The examination of the diagnostic characteristics revealed complete concordance with the hscTnT assay for detection of NSTEMI and for assessment of absolute changes of cTn values (rise and/or fall) in NSTEMI patients. PATHFAST cTnI showed highly sensitive detection of NSTEMI with increasing sensitivity at admission and after 3 to 6 hours, not going along with decreased specifi city. The PATHFAST cTnI might be useful at the point-of-care setting for early rule-in and rule-out diagnosis of NSTEMI. Figure 1 (abstract P158). Figure 1 (abstract P158). Analytical and diagnostic characteristics of the high-sensitivity PATHFAST troponin I assay p y E Spanuth1, R Thomae2, E Giannitsis3 E Spanuth1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P159 (doi: 10.1186/cc14239) E Spanuth1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P159 (doi: 10.1186/cc14239) Conclusion Among ADHF patients, the coupling of pulmonary hypertension and RVD carries an incremental risk, having a portentous impact on the survival rate. Introduction The POC PATHFAST cTnI assay (Mitsubishi Medience, Japan) has shown promising analytical validity. We thought to evaluate whether the assay could be classifi ed ‘highly-sensitive’. P158 yi g y Methods cTnI was determined using PATHFAST in 120 healthy individuals (60 men and 59 women, 21 to 69 years old, median 42 years). Cardiac disorders were excluded by cardiac magnetic resonance imaging. The diagnostic characteristics were investigated by determination of cTnI and cTnT (Roche hs-cTnT) in 181 patients admitted to the chest pain unit at presentation, and 3 and 6 hours later. The results were related to the discharge diagnoses. Pulmonary hypertension, right ventricular dysfunction and acute heart failure: a portentous consortium y Methods The false alarm rate of the new algorithm (GE Carescape, 2012) was compared with that of the algorithm evaluated in the study (GE Solar, 2003) [2] on the collected ECG waveform data. User settings such as QRS detection sensitivity (high/normal) were not available. Therefore, normal sensitivity was assumed for both versions. With the old algorithm, 10 patients with low QRS amplitudes gave a signifi cantly higher number of FPs than were reported [2]. For those patients, both sensitivity modes were tested with the old algorithm. Sixty-six percent of patients with a pacemaker did not have the pacemaker mode selected [2]. Outlier patients in which false alarms were due to user settings (20 patients with a pacemaker) or patient condition (four patients with a bundle branch block) rather than algorithm defi ciency were excluded. HD Michalopoulou, HM Michalopoulou, P Stamatis, E Kostetsos, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P157 (doi: 10.1186/cc14237) Introduction Pulmonary hypertension and right ventricular dysfunc- tion (RVD) are frequently encountered in patients with acute heart failure. We sought a better understanding of the coupling between RVD and pulmonary hypertension in the setting of acute decompensated heart failure (ADHF) as it might improve the prognostic stratifi cation and infl uence the survival rates. l Methods Echocardiography was performed in 329 patients with ADHF and right ventricular function was assessed by measuring the right ventricular fractional area, and a right ventricular ejection fraction (RVEF) <35% was taken as the cutoff value for RV systolic dysfunction. The systolic pulmonary pressure (PASP) was calculated from the tricuspid regurgitation signal applying the modifi ed Bernoulli equation, Results Improved algorithm resulted in 66% reduction of FP alarms. When using the high-sensitivity mode for the 10 patients with low QRS, FP reduction was 18%. No compromises regarding detection of true events were found. The 24 outlier patients contributed to 81.3% of FP alarms. The algorithm changes responsible for the reduced FPs were: S55 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 at T6, and from T3 to T48 in OPCAB (P <0.05) versus baseline (Figure 1). In CABG patients CK-MB levels are higher versus OPCAB from ICU up to T12 (P <0.03), and from ICU to T48 for Hs-cTn levels (P <0.02). Pulmonary hypertension, right ventricular dysfunction and acute heart failure: a portentous consortium In 39 CABG patients (97.5%) and 10 OPCAB patients (71.4%) all individual Hs-cTn values are above 140 ng/l (= 10 × 99th percentile of URL). at T6, and from T3 to T48 in OPCAB (P <0.05) versus baseline (Figure 1). In CABG patients CK-MB levels are higher versus OPCAB from ICU up to T12 (P <0.03), and from ICU to T48 for Hs-cTn levels (P <0.02). In 39 CABG patients (97.5%) and 10 OPCAB patients (71.4%) all individual Hs-cTn values are above 140 ng/l (= 10 × 99th percentile of URL).i and pulmonary hypertension was considered as PASP >35 mmHg. Based on the values of PASP and RVEF the study group was classifi ed into four subgroups: group 1, normal PASP/preserved RVEF; group 2, high PASP/preserved RVEF; group 3, normal PASP/ low RVEF; group 4, high PASP/low RVEF. The primary endpoint was all-cause mortality. The median follow-up was 18 months. Survival analysis was performed according to the Cox regression method, adjusted for age, gender, LV function, estimated glomerular fi ltration rate, troponin I, hemoglobin, serum sodium and BNP levels. Conclusion Both CK-MB and Hs-cTn levels increase signifi cantly after cardiac surgery. Postoperative Hs-cTn levels exceed the 10 × 99th percentile of URL in nearly all CABG patients. Our data show an important discrepancy between the 10 × 99th percentile for both biomarkers, and suggest that a diff erent defi nition for postoperative AMI may be needed when Hs-cTn is used. Results Pulmonary hypertension was found in 78% of the patients (median PASP: 53 mmHg). As compared with the patients with normal PASP the patients with pulmonary hypertension were more likely to be in New York Heart Association functional class (NYHA) III or IV (86% vs. 49%, P <0.001), had a lower RVEF (23 ± 9% vs. 32 ± 8%, P <0.001), and had signifi cantly higher BNP levels (280 ± 107 pg/ml vs. 540 ± 320 pg/ ml, P <0.001). In a Cox model, compared with patients with normal right ventricular function and without pulmonary hypertension (group 1), the adjusted hazard ratio for mortality was 3.1 (95% CI: 1.6 to 4.2, P <0.01) in group 2, 0.3 (95% CI: 0.2 to 1.9, P = 0.3) in group 3 and 4.2 (95% CI: 1.9 to 6.1, P <0.001) in group 4. y Reference y Reference 1. Thygesen et al. J Am Coll Cardiol. 2012;60:1581-98. P160 P160 Combining therapeutic hypothermia and primary coronary intervention in comatose survivors of ventricular fi brillation due to ST-elevation myocardial infarction R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) Combining therapeutic hypothermia and primary coronary intervention in comatose survivors of ventricular fi brillation due to ST-elevation myocardial infarction R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) y R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) Introduction Primary percutaneous coronary intervention (PPCI) is the preferred reperfusion strategy for ST-elevation acute myocardial infarction (STEMI). In comatose survivors of cardiac arrest, mild induced hypothermia (MIH) improves neurological recovery. Figure 1 (abstract P158). S56 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Of the 3,835 nontraumatic patients treated in the ED, 3,196 were adults. In this adult population, 500 ECGs were performed in patients whose symptoms suggest ACS. The median age was 62.3 years and the sex ratio was 1.16. Clinical presentation was chest pain (31%), dyspnea (14%), palpitations (5%), disturbance of consciousness (3%) or others (47%). Fifty-six (11.2%) were diagnosed as ACS, including 20 ST-elevation myocardial infarction (STEMI), 28 non-STEMI and eight unstable angina. Of the 20 STEMI patients, eight (40%) and fi ve (25%) were diagnosed as STEMI complicated by right ventricular and posterior wall ischemia respectively, which means that these complications could have been missed by standard 12-lead ECG. Methods A total of 112 patients undergoing PPCI and MIH were compared with 32 comparable consecutive patients who underwent PPCI but no MIH. We hypothesized that combining both methods lead to better survival rate. MIH was induced (propofol, fentanyl, saline 4 ml/kg BW, 2°C) and maintained for 24 hours, targeting 32 to 34°C. Spontaneous rewarming was allowed (0.5°C).if g Results There were no signifi cant diff erences between the MIH and Control group in general characteristics, cardiac arrest circumstances and angiographic features. Except for decreases in heart rate during MIH, there was no diff erence between MIH and no MIH groups in arterial pressure, peak lactate (7.7 vs. 6.2 mmol/l; P = 0.36), need for vasopressors (57% vs. 41%; P = 0.09), aortic balloon counterpulsation (13% vs. 22%; P = 0.19), repeat cardioversion/defi brillation (17% vs. 25%; P = 0.30). P160 There was lower incidence of inotropic use (36% vs. 59%; P = 0.01) and use of antiarrhythmics (11% vs. 53%; P = 0.002). There was no diff erence in FiO2 during mechanical ventilation and in renal function. See Table 1. y Conclusion Eighteen-lead ECG with synthesized right-sided and posterior precordial leads was an effi cient method to diagnose ACS in a Caucasian population within 10 minutes of ED arrival. It is particularly performant to detect right ventricular ischemia early, which can modify acute therapeutic strategy. References 1. Thygesen K, et al. Circulation. 2007;116:2634-53. 2. Katoh T, et al. J Nippon Med Sch. 2011;78:22-9. 3. Tamura A, et al. Am J Cardiol. 2014;114:1187-91. References 1. Thygesen K, et al. Circulation. 2007;116:2634-53. 2. Katoh T, et al. J Nippon Med Sch. 2011;78:22-9. 3. Tamura A, et al. Am J Cardiol. 2014;114:1187-91. Table 1 (abstract P160). Survival after 12 months MIH Control P CPC 1/2 50 (45%) 5 (15%) 0.002 CPC 3/4 0 0 NS Cardio 20 (18%) 6 (19%) 0.95 CNS 18 (16%) 17 (53%) 0.00001 Table 1 (abstract P160). Survival after 12 months Conclusion Hospital survival with CPC 1/2 was signifi cantly better in the MIH group (45% vs. 15%; P = 0.01). Our study clearly demonstrates that PPCI and MIH are feasible and may be combined safely in comatose survivors of ventricular fi brillation in STEMI setting. Such strategy improves survival with good neurological recovery. References Introduction After an acute myocardial infarction with ST-segment elevation (STEMI) treated with percutaneous coronary intervention (PCI), the left ventricle (LV) can undergo negative remodeling (R–). We aimed to investigate whether global longitudinal strain (SGL) of the left ventricle (LV) predicts remodeling. e e e ces 1. HACA Study Group. Mild therapeutic hypothermia to improve the neurological outcome after cardiac arrest. N Engl J Med. 2002;346:549-56. 2. Knafelj R, Radsel P, Ploj T, Noc M. Primary percutaneous coronary intervention and mild induced hypothermia in comatose survivors of ventricular fi brillation with ST-elevation acute myocardial infarction. Resuscitation. 2007;74:227-34. g g ; 2. Knafelj R, Radsel P, Ploj T, Noc M. Primary percutaneous coronary intervention and mild induced hypothermia in comatose survivors of ventricular fi brillation with ST-elevation acute myocardial infarction. Resuscitation. 2007;74:227-34. Methods Transthoracic echocardiography with speckle tracking imaging (TTE-STI) was performed 2 to 3 days after primary PCI and 6 months later in patients with diagnosis of STEMI. P160 LV R– criteria were: LVEF increase ≤5% and end-diastolic volume increase ≥15%. Logistic regression and ROC curve analysis was used for the statistical analysis. Results Eighty-three patients (56 ± 11 years) with STEMI at any LV localization and subjected to primary PCI were studied during 2012: LV P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) y y Methods A consecutive registry of patients seen in 2013 (January to October) in the ICU of a hospital without a HU. The total transfer time is considered from the call to the Emergency Coordination Center until arrival at the HU. In turn, this time is divided into activation time, arrival time of the relocation team, patient preparation time and transfer time. Methods A consecutive registry of patients seen in 2013 (January to October) in the ICU of a hospital without a HU. The total transfer time is considered from the call to the Emergency Coordination Center until arrival at the HU. In turn, this time is divided into activation time, arrival time of the relocation team, patient preparation time and transfer time. In the case of primary PCI, the door-to-balloon time was estimated by adding to the total transfer time the initial assessment and completion time of catheterization and balloon infl ation. The times are expressed in minutes, as the median and interquartile range. In the case of primary PCI, the door-to-balloon time was estimated by adding to the total transfer time the initial assessment and completion time of catheterization and balloon infl ation. The times are expressed in minutes, as the median and interquartile range. Introduction Diastolic dysfunction as evaluated by tissue Doppler imaging (TDI), particularly by E/E’ ratio (peak early diastolic transmitral velocity/peak early diastolic mitral annular velocity) and mitral annular E’-wave, is common and crucial in critical illness. Our prospective observational study assessed the prognostic signifi cance of TDI variables versus cardiac biomarkers, B-type natriuretic peptide (BNP), troponin-T (TnT) and investigated determinants of plasma BNP rise, in septic shock. g Results During 10 months of 2013, we treated 162 STEMI. Of these, 104 had evidence of reperfusion (64%). Primary PCI was performed in 24 patients (23%), of which 10 were transferred from the hospital to the HU. Fibrinolytic therapy was used in 62 patients (59%), of these 20 (32.2%) required rescue PCI. The transfer time for primary PCI was 0:39:44 (0:31:41 to 0:44:32) minutes. The transfer time for rescue PCI was 0:38:56 (0:37:25 to 0:51:29) minutes. Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) p y gi Results Mean ± SD APACHE II score was 21.22 ± 7.28, mean ± SD admission SOFA score was 10.25 ± 2.76. Hospital mortality was 55%. Nonsurvivors had increased E/E’ (15.56 ± 1.48; P <<0.00001) and reduced E’ 6.32 ± 0.68 cm/second (P <<0.00001) compared with survivors, who exhibited inverse correlations with an E/E’ signifi cantly lower (9.30 ± 2.88) and higher E’ (9.01 ± 0.85 cm/second). In contrast, BNP and TnT levels displayed remarkably lower statistical signifi cance in nonsurvivors (P = 0.005, P = 0.007 respectively). The ROC curves had an area under the curve of 0.98 for the E’, and 0.92 for the E/E’. Vasopressor management (noradrenaline dose) (P = 0.0001), fl uid balance (P <0.001) and E/E’ (P = 0.00004) were independent predictors of plasma BNP concentration. Introduction The goal of the study was to analyse cerebral blood fl ow in pregnancy complicated by preeclampsia. Introduction The goal of the study was to analyse cerebral blood fl ow in pregnancy complicated by preeclampsia. Methods This was a prospective study. I group: 45 patients, 17 to 38 years (mean age 27.5 ± 5.3 years) with verifi ed diagnosis of severe preeclampsia; control group: 72 healthy women with normal pregnancy, third trimester, 19 to 34 years (mean age 24.5± 4.3 years). Exclusion criteria: potentially haemodynamically signifi cant stenosis; congestive heart disease; arrhythmia; large changes in haemorheology; diabetes mellitus; and craniospinal trauma and syncope. Study of cerebral fl ow was improved by the method of transcranial dopplerography (TCD). All patients underwent duplex scan of extracranial portions of brachiocephalic arteries and transcranial duplex scan in the area of middle cerebral artery (MCA) (segment M1). During duplex scan of brachiocephalic arteries lumen, the presence of extravasal causes for basic blood fl ow disturbances was estimated. We determined lumen of large basilar arteries and quantitative features of blood fl ow in MCA. Synthesized 18-lead electrocardiogram as routine myocardial ischemia detection in an emergency department: a preliminary evaluation in Europe Figure 1 (abstract P162). Clinique Notre-Dame de Grâce, Gosselies, Belgium q g Critical Care 2015, 19(Suppl 1):P161 (doi: 10.1186/cc14241) Introduction Standard 12-lead electrocardiogram (ECG) is, with biomarkers, the most accurate method in the diagnosis of acute coronary syndrome (ACS). However, posterior (V7-V8-V9) and right (V3R-V4R-V5R) derivations are not systematically performed due to the time-consuming procedure involved, despite major therapeutic implications (fl uid loading instead of nitrates use in right ventricular involvement) and published guidelines [1]. Recently, an 18-lead ECG system, standard 12-lead ECG and six additional synthesized leads (assessing posterior and right ventricular areas) in only one recording procedure has been developed. The reliability of this material (ECG 2550; Nihon Kohden Co. Ltd, Japan) was already validated in this indication in an Asian population [2,3]. Methods We conducted a prospective, observational study with patients admitted to our emergency department (ED), during a 6-month period. Requirement for ECG was guided by physician’s discretion according to patient’s history. All patients with chest pain, dyspnea, palpitations, disturbance of consciousness, malaise or abdominal complaint underwent synthesized 18-lead ECG within 10 minutes of ED arrival. The aim of the study was to evaluate the eff ectiveness of the synthesized 18-lead ECG as an ischemia triage tool in the ED, and particularly the ability to early detect a right ventricular involvement. Figure 1 (abstract P162). S57 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 R– patients (n = 35, 42%) and no LV R– patients (n = 48, 58%). Diabetes mellitus (41% vs. 19%; P <0.001) and TnI levels (1.2 ± 2.1 μg/l vs. 0.4 ±0.3 μg/l; P = 0.005) showed higher incidence in LV R– patients. SGL was –12.5 ± 5.6% in no LV R– patients and –6.5 ± 3.4 in LV R– patients. In the regression analysis just LV SGL and SL in left anterior descending territory remained signifi cant, OR: 1.85 (1.24 to 2.76) (P <0.001) and OR: 1.63 (1.15 to 2.31) (P <0.001), respectively. P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) The door-to-balloon time estimated for primary PCI was 80 minutes. Methods Twenty-seven mechanically ventilated adult patients admitted to our ICU were evaluated within 72 hours of evolving septic shock. Patients underwent two transthoracic echocardiographies within 72 hours of the onset of septic shock: shortly after diagnosis and 24 hours later (confi rmatory), alongside relevant measurements of cardiac biomarkers. Peak mitral infl ow E and A velocity waves were recorded using pulsed-wave Doppler at the mitral valve tips from the apical four-chamber view, peak early (E’) and late (A’) diastolic myocardial velocities were obtained by TDI at the septal mitral annulus in the apical four-chamber view. E/E’ was calculated. P ≤0.01 was reported as statistically signifi cant. Conclusion Times for interhospital transfer of patients with STEMI who had undergone urgent catheterization are within the range considered optimal. In the case of primary PCI, times are lower than the 90 to 120 minutes recommended practice guidelines. Synthesized 18-lead electrocardiogram as routine myocardial ischemia detection in an emergency department: a preliminary evaluation in Europe The analysis of ROC curves revealed that at the cutoff level of –12.46%, SGL identifi es LV R– with a sensibility of 81% and a specifi city of 86% (AUC = 0.88: 95% CI: 0.79 to 0.96; P <0.001) (Figure 1).i fl ow velocity (Vps), maximal end-diastolic velocity (Ved), time-adjusted maximal velocity (TAMX), resistance index (RI), pulsative index (PI), and systolic/diastolic ratio (S/D) were determined. Signifi cance of mean value diff erences were calculated using the STATISTICA 6.0 program with determination of Student’s t criteria with normal spread in the group. g p Results All haemodynamic values in the M1 segment of MCA in preeclamptic patients were decreased in comparison with the same values in healthy pregnant women with diff erent signifi cance: PI (mean 0.77 vs. 0.84, P <0.01); RI (mean 0.52 vs. 0.54, P <0.05); Vps (mean 90.22 vs. 104.74 cm/second, P <0.001); Ved (mean 43.25 vs. 48.53 cm/second, P <0.001); TAMX (mean 61.48 vs. 67.30 cm/second, P <0.01); and S/D (mean 2.02 vs. 2.06, P <0.05). Found pathophysiological changes of cerebral haemodynamics were consistent with a dopplerographic pattern of diminished perfusion and are typical for vascular segments, which are located proximally to the zone of abnormally high haemodynamic resistance: prestenotic arterial segments, episodes of arterial hypertension and distal vasoconstriction. Conclusion SGL assessment in the fi rst days after primary PCI is useful in the prediction of LV R– independently of the myocardial infarction localization. P164 Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) Analysis of the interhospital transfer times in patients with ST-elevation acute coronary syndrome for undergoing urgent coronariography Analysis of the interhospital transfer times in patients with ST-elevation acute coronary syndrome for undergoing urgent coronariography FM Acosta Diaz, O Moreno, M Muñoz, R Fernandez, J Soto, M Colmenero San Cecilio Universitary Hospital, Granada, Spain Critical Care 2015, 19(Suppl 1):P163 (doi: 10.1186/cc14243) y Conclusion With TCD we obtained a possibility to determine and estimate changes in cerebrovascular fl ow in pregnant patients with severe preeclampsia. This enhances diagnostic possibilities of some serious pregnancy complications, and gives us deep understanding of some components of pathogenesis and increased treatment effi cacy. y p p Critical Care 2015, 19(Suppl 1):P163 (doi: 10.1186/cc14243) Introduction The aim was to analyze the related assistance times to transfer patients with ST-elevation acute coronary syndrome (STEMI) referred to another hospital with a hemodynamics unit (HU) for performing emergency catheterization (primary or rescue PCI). P165 P168 Results A total of 207 male and 144 female patients, APACHE II score 21 ± 7, 62 ± 14 years old, one to 126 TPTDs per patient. Diagnosis: cirrhosis/liver failure n = 112 patients (31.9%), sepsis 55 (15.7%), ARDS 46 (13.1%), GI aff ection 21 (6.0%), cardiogenic shock 19 (5.4%), various 98 (27.9%). Patients with liver failure were slightly younger than the other patients (58 ± 11 vs. 64 ± 15 years; P <0.001). All other baseline characteristics were comparable including APACHE II (20 ± 7 vs. 21 ± 8; NS), SAPS (39 ± 12 vs. 41 ± 14; NS), height (172 ± 9 vs. 170 ± 9 cm; NS) and weight (76 ± 20 vs. 73 ± 17 kg; NS). Among haemodynamic parameters, preload markers GEDVI (753 ± 168 vs. 790 ± 226 ml/m2; P = 0.182) and CVP (14.4 ± 8.8 vs. 14.9 ± 7.1 mmHg; P = 0.250) were comparable. Despite comparable preload parameters, the following parameters were signifi cantly diff erent: patients with acute or chronic liver failure had signifi cantly higher cardiac index (4.3 ± 1.3 vs. 3.3 ± 1.3 l/minute/ m2; P <0.001), stroke volume index (50 ± 15 vs. 37 ± 15; P <0.001), pulse pressure (75 ± 19 vs. 65 ± 21 mmHg; P = 0.021) and cardiac power index (0.7 ± 0.24 vs. 0.60 ± 0.28 W/m2; P <0.001). By contrast, MAP (77 ± 15 vs. 80 ± 15 mmHg; P = 0.045), SVRI (1,305 ± 638 vs. 1,877 ± 898 dyns/ cm5m2; P <0.001) and heart rate (84 ± 19 vs. 92 ± 22/minute; P <0.001) were signifi cantly lower in patients with liver failure. P168 Room temperature transpulmonary thermodilution (TPTD) with increased indicator 20 ml TPTD bolus compared with standard TPTD with 15 ml iced saline: a prospective observational study W Huber1, E Maendl1, A Beitz1, M Messer1, T Lahmer1, B Henschel1, S Rasch1, C Schnappauf1, RM Schmid1, ML Malbrain2 1Klinikum rechts der Isar, Technical University of Munich, Germany; 2Ziekenhuis Netwerk, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P168 (doi: 10.1186/cc14248) Introduction Use of ice-cold saline is assumed to provide best accuracy of TPTD to obtain the cardiac index (CI), global end-diastolic volume (GEDVI) and extravascular lung-water (EVLWI). However, room- temperature injectate might facilitate TPTD outside the ICU. Accidental intra-arterial injection: an under-reported preventable never event femoral TPTD (Pulsion Medical Systems, Germany). Results Fifteen female and 16 male patients, APACHE II score 21 ± 7. Mean values of CI (4.02 ± 0.98 vs. 3.96 ± 0.91 l/minutem2; P <0.001), GEDVI (800 ± 166 vs. 796 ± 163 ml/m2; P = 0.011) and EVLWI (10.3 ± 3.7 vs. 9.7 ± 3.6 ml/kg; P <0.001) were slightly higher when measured at room temperature compared with cold saline. Mean bias and PE values were 0.06 ± 0.37 l/minutem2 and 18.6% for CI, 4 ± 81 ml/m2 and 20.2% for GEDVI and 0.5 ± 1.1 ml/kg and 22.7% for EVLWI. Bias values in case of femoral compared with jugular indicator injection were not signifi cantly diff erent for CI (0.04 ± 0.41 vs. 0.11 ± 0.30 l/minutem2; P = 0.161) and EVLWI (0.56 ± 1.19 vs. 0.42 ± 1.07 ml/kg; P = 0.492). Bias for GEDVI-room was signifi cantly lower for femoral CVC compared with jugular indicator injection (–6.0 ± 81.1 vs. 18.9 ± 78.3 ml/m2; P = 0.008). Conclusion Compared with previous data using 15 ml room- temperature injectate, our data with 20 ml room-temperature injectate in general provide acceptable bias and percentage error when compared with standard TPTD with 15 ml iced saline. This also applies for femoral CVC room-temperature TPTD which might also be related to a new PiCCO-2 algorithm correcting for femoral CVC site. Reference M Mariyaselvam, A Hutton, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P166 (doi: 10.1186/cc14246) Introduction Depending upon the medication administered, accidental administration of medication into the arterial line can cause devastating complications. This wrong-route injection is a never event in the UK but may be under-reported especially when occurring in the unconscious patient who may not notice associated pain temporally. Under-reporting may occur because resultant complications may be delayed a number of hours and the accountable healthcare worker may not recognise or choose not to report the error. In 2008 the UK National Patient Safety Agency (NPSA) reported only 76 incidents related to poor sampling technique but few wrong route arterial injections. Of these 21% suff ered moderate to severe harm [1]. The NPSA suggests that training and the use of clear labelling alongside red arterial tubing and standard red lock caps be used to prevent arterial sampling errors. Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka By the transtemporal approach in the MCA M1 segment, peak systolic Conclusion Diastolic dysfunction as evaluated by E/E’ and E’ constitutes a major independent predictor of outcome in septic shock, compared with cardiac biomarkers, suggesting that echocardiographic techniques assessing diastolic dysfunction in sepsis may replace cardiac biomarkers for mortality prediction. Fluid balance, vasopressor management and diastolic dysfunction are independent predictors of BNP elevation in septic shock. Our fi ndings should be confi rmed by an extended prospective study. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S58 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P166 the PiCCO-2 device with the latest algorithm correcting GEDVI for femoral TPTD (Pulsion Medical Systems, Germany). Accidental intra-arterial injection: an under-reported preventable never event Methods In 2014, we conducted a national postal survey of ICUs in the UK to attempt to determine the rate of accidental intra-arterial injections. The survey was sent to the clinical director of every ICU and they were asked whether they were aware of any unintentional arterial line injection having occurred in their hospital in the last 5 years. 1. Huber W, et al. J Crit Care. 2014;29:1133. 1. Huber W, et al. J Crit Care. 2014;29:1133. y Results Of the 56 ICUs that responded, 16 (28.5%) reported that they had personally seen an accidental injection into the arterial line. P169 Transpulmonary thermodilution-derived haemodynamics in patients with liver failure: a prospective study in 351 patients W Huber, A Breitling, B Henschel, S Mair, S Goetz, J Tschirdewahn, J Frank, A Beitz, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2015, 19(Suppl 1):P169 (doi: 10.1186/cc14249) Conclusion Despite the arterial line safety recommendations made by the NPSA in 2008, we demonstrate that intra-arterial injection is still a problem and that it remains under-reported. Our incidence is likely to be an underestimate as it relies on the recollections of a single individual in each institution. Medical errors can be mitigated by consideration of human factors and system engineering to improve patient safety. A focus on clinical awareness, colour coding and training may lead to improvements; however, institutions and clinical directors also bear a responsibility to prevent never events and a number of engineered solutions are now available such as needle-free non-injectable arterial sampling devices to protect the healthcare environment and make this error impossible [2,3]. Introduction Patients with acute or chronic liver failure are considered to have an altered pattern of haemodynamics. Nevertheless, there is a lack of studies systematically investigating haemodynamics in patients with liver failure. Therefore, it was the aim of this study to compare transpulmonary thermodilution (TPTD)-derived haemodynamics of 112 patients with acute or chronic liver failure with 239 patients without liver failure. Acknowledgement Funding from Eastern Academic Health Science Network, UK. References Acknowledgement Funding from Eastern Academic Health Science Network, UK. Methods We analyzed a prospectively maintained database including 6,016 TPTD measurements in 351 patients. To account for diff erent numbers of TPTDs in diff erent patients, comparison of fi rst measurements of patients with and without liver failure was the primary endpoint. Statistics: Wilcoxon test for unpaired samples; IBM SPSS Statistics 22. 1. Rapid response alert 06. NPSA; 2008. 1. Rapid response alert 06. NPSA; 2008. 2. Mariyaselvam et al. Anaesthesia. 2014;70:51-5. 3. Mukhopadhyay et al. Crit Care. 2010;14:R7. Reference 1. Romano M, et al. Care Med. 2002;30:1834-41. Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report Measures were performed by a single trained operator, who was blind to CVP values. Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. Measures were performed by a single trained operator, who was blind to CVP values. Results Forty-eight patients were included. A poor correlation was found between CVP and IJV and EJV circumference and area in mechanically ventilated patients. A strong correlation was found between CVP and EJV circumference (r: 0.74; P = 0.0004; 95% CI: 0.421 to 0.897) and area (r: 0.702; P = 0.0012; 95% CI: 0.35 to 0.88) in spontaneously breathing patients. Conventional receiver-operating characteristic curves were generated to assess the utility of EJV circumference and area to predict low (≤8 mmHg) versus high (>8 mmHg) CVP values. AUC for EJV circumference and area was 0.935 (P <0.0001; 95% CI: 0.714 to 0.997) and 0.87 (P <0.0001; 95% CI: 0.63 to 0.98) respectively (Figure 1). Conclusion These results highlight a potentially evolving role of Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report Introduction There are few methods of cardiac output (CO) estimation validated in children. The aim of this study is to investigate the reliability of an uncalibrated pulse contour method of CO estimation, the pressure recording analytical method (PRAM), in pediatric patients scheduled for diagnostic right and left heart catheterization, compared with the oxygen-direct Fick method. yg Methods Cardiac index (CI) was simultaneously estimated by Fick, and PRAM applied to pressure signals recorded invasively from a femoral catheter. All measurements were performed in steady-state condition. PRAM CI measurements were obtained for 10 consecutive beats simultaneously during the Fick CI estimation. Agreement between Fick and PRAM was assessed using the Bland–Altman method. Correlation coeffi cient, bias, and percentage of error were calculated. fi Results Forty-three CI measurements were performed in 43 patients. The data showed good agreement between CIFick and CIPRAM: r2 = 0.98; bias –0.0074 l/minute/m2; limits of agreement from –0.22 to 0.22 l/minute/m2. The percentage error was 8%. Figure 1 shows the Bland–Altman plot. Figure 1 (abstract P171). Figure 1 (abstract P170). Bland–Altman plot of the cardiac index measured with Fick versus PRAM. Figure 1 (abstract P170). Bland–Altman plot of the cardiac index measured with Fick versus PRAM. Figure 1 (abstract P171). Nonetheless, the external jugular vein (EJV) circumference and area have not been evaluated. Considering the role of EJV visual assessment in the clinical estimation of CVP, we hypothesized that EJV ultrasound evaluation could be used to reliably estimate CVP. Conclusion PRAM provides reliable estimates of cardiac output in hemodynamically stable pediatric cardiac patients compared with the Fick method. Reference Conclusion PRAM provides reliable estimates of cardiac output in hemodynamically stable pediatric cardiac patients compared with the Fick method. f Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. Measures were performed by a single trained operator, who was blind to CVP values. Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. P168 A recent study [1] showed acceptable bias and percentage error (PE) for CI-room derived from TPTD with 15 ml room temperature saline compared with CI-cold using 15 ml iced saline for TPTD. However, GEDVI-room and EVLWI-room had borderline PE values close to 30%, and the bias of GEDVI-room markedly increased with higher values of GEDVI and in case of femoral CVC. Since imprecision of TPTD-room might be reduced by a larger volume of injectate, it was the aim of our study to compare CI, GEDVI and EVLWI derived from TPDT using 20 ml room temperature injectate with standard TPTD with 15 ml iced saline. Conclusion Our data derived from a large TPTD database demonstrate markedly diff erent haemodynamics in patients with cirrhosis or acute liver failure with the only exception of static preload markers GEDVI and CVP. These fi ndings should be considered in instable patients with liver failure. Methods In 31 patients 236 sets with two 20 ml TPTDs with 21°C and subsequently two standard TPTDs with 4°C saline were obtained using Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S59 Figure 1 (abstract P171). P170 P170 Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report J Alonso Iñigo1, F Escribá1, J Carrasco1, J Encarnación1, M Fas2, M Barberá1 1Hospital Universitario y Politécnico La Fe, Valencia, Spain; 2Hospital Universitario de la Ribera, Alzira, Spain Critical Care 2015, 19(Suppl 1):P170 (doi: 10.1186/cc14250) P172 Do intravascular hypovolaemia and hypervolaemia result in changes in pulmonary blood volume? g p y JJ Vos1, TW Scheeren1, SA Loer2, A Hoeft3, JK Wietasch1 1University of Groningen, University Medical Center Groningen, the Netherlands; 2Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, the Netherlands; 3University of Bonn, Germany Critical Care 2015, 19(Suppl 1):P172 (doi: 10.1186/cc14252) Introduction Hypovolaemia is generally believed to induce centrali sa- tion of blood volume. Therefore, we evaluated whether hypovolaemia and hypervolaemia result in a change in central blood volume (that is, pulmonary blood volume (PBV)) and we explored the eff ects on the distribution between PBV and circulating blood volume (Vd circ). Introduction Hypovolaemia is generally believed to induce centrali sa- tion of blood volume. Therefore, we evaluated whether hypovolaemia and hypervolaemia result in a change in central blood volume (that is, pulmonary blood volume (PBV)) and we explored the eff ects on the distribution between PBV and circulating blood volume (Vd circ). Methods Twenty anesthetized Landrace/Large-White pigs (19 ± 2 kg, 10 to 15 weeks) were subjected to a fi xed hemorrhage (50% over 30 minutes). The pigs were randomly allocated into two groups (n = 10 per group). In group A, ITD was the only treatment for hypotension, while in group B, an intravenous administration of 1 l Ringer lactate was applied for treatment of hypotension. Hemodynamic parameters were continuously assessed for the fi rst 30 minutes after blood loss. Methods After local District Governmental Animal Investigation Committee approval, blood volume was altered in both directions randomly in steps of 150 ml (mild) to 450 ml (moderate) either by haemorrhage, retransfusion of blood, or infusion of colloids in six Foxhound dogs. The anaesthetised dogs were allowed to breathe spontaneously. Blood volumes were measured using the dye dilution technique: PBV was measured as the volume of blood between the pulmonary and aortic valve, and Vd circ by two-compartmental curve fi tting [1,2]. The PBV/Vd circ ratio was used as a measure of blood volume distribution. A linear mixed model was used for analysing the infl uence of blood volume alterations on the measured haemodynamic variables and blood volumes. yi Results Mean systolic arterial pressures (SAPs) 30 minutes after the intervention in each group were as follows: group A 80 ± 5 mmHg and group B 90 ± 4 mmHg. Maximum SAPs during the assessment period were: group A 89 ± 2 mmHg and group B 128 ± 5 mmHg. p P Balsorano, S Romagnoli, A De Gaudio P Balsorano, S Romagnoli, A De Gaudio AOUC Careggi, Florence, Italy AOUC Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P171 (doi: 10.1186/cc14251) AOUC Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P171 (doi: 10.1186/cc14251) Introduction Although recognized as a questionable indicator of the intravascular volume, central venous pressure (CVP) is integrated in many therapeutic algorithms for hemodynamic resuscitation of critically ill patients [1]. In an attempt to simplify CVP estimation, several clinical and ultrasonographic approaches have been suggested [2-5]. p y g Conclusion These results highlight a potentially evolving role of EJV circumference and area in the hemodynamic management S60 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of spontaneously breathing patients. An important aspect of the suggested approach is its simplicity, requiring basic technical skills and making it suitable in any scenario where an ultrasound machine is available. P175 Relation between global end-diastolic volume and left ventricular end-diastolic volume A Pironet1, P Morimont1, S Kamoi2, N Janssen1, PC Dauby1, JG Chase2, B Lambermont1, T Desaive1 1University of Liège, Belgium; 2University of Canterbury, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P175 (doi: 10.1186/cc14255) (Figure 1). Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. References 1. Anesthesiology. 1994;81:76-86. 2. Intensive Care Med. 2001;27:767-74. Figure 1 (abstract P172). Figure 1 (abstract P172). 1University of Liège, Belgium; 2University of Canterbury, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P175 (doi: 10.1186/cc14255) Introduction Measurement of global end-diastolic volume (GEDV) is provided by cardiovascular monitoring devices using thermodilution procedures. The aim of this study was to assess the relation between this clinically available index and left ventricular end-diastolic volume (LVEDV), which is typically not available at the patient bedside. Methods Measurements were performed on six anaesthetised and mechanically ventilated pigs. Volume loading via successive infusions of saline solution was fi rst performed and was followed by dobutamine infusion. These two procedures provided a wide range of LVEDV values. During these experiments, GEDV was intermittently measured using the PiCCO monitor (Pulsion AG, Germany) during thermodilutions and LVEDV was continuously measured using an admittance catheter (Transonic, NY, USA) inserted in the left ventricle. Results Table 1 presents the linear correlations obtained between LVEDV and GEDV. These correlations are good to excellent, with r2 values from 0.59 to 0.85. However, the coeffi cients of the linear regressions present a large intersubject variability, which prevents the precise estimation of LVEDV using GEDV. Nevertheless, variations in LVEDV are well reproduced by the GEDV index. The variations in LVEDV actually equal 21 to 48% of those in GEDV. The coeffi cient b is always nonzero, indicating that some proportion of the GEDV index is actually not linked to LVEDV. Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. R f Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. Comparative study between fl uidless resuscitation with permissive hypotension using the impedance threshold device versus aggressive fl uid resuscitation with Ringer lactate in a swine model of hemorrhagic shock C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P174 (doi: 10.1186/cc14254) References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. Introduction Permissive hypotension, which results in avoidance of intravascular overpressure and thereby avoidance of platelet plug dislodgement early in the clotting mechanism, improves the results after trauma and hemorrhage. The research hypothesis is that augmentation of negative intrathoracic pressure with the use of an impedance threshold device (ITD) will improve hemodynamic parameters, without aff ecting permissive hypotension or causing hemodilution. On the other hand, aggressive resuscitation with Ringer lactate will cause hemodilution and intravascular pressures that are very high for permissive hypotension, capable of platelet plug dislodgement. 2. Intensive Care Med. 2001;27:767-74. P172 Do intravascular hypovolaemia and hypervolaemia result in changes in pulmonary blood volume? Mean pulse pressure was higher in the ITD group versus the fl uid resuscitation group (P <0.05). After the assessment period, mean hematocrit in group A was 24 ± 2%, while in group B it was 18 ± 1% (P <0.001). Conclusion In our study, the ITD increased SAP and pulse pressure without overcompensation. On the other hand, aggressive fl uid resuscitation led to a signifi cant increase of SAP >100 mmHg capable of clot dislodgement and in addition led to hemodilution. Results A total of 68 alterations in blood volume resulted in changes in Vd circ ranging from –33 to +31% (Figure 1). PBV decreased during mild and moderate haemorrhage, while during retransfusion PBV increased during moderate hypervolaemia only. The PBV/Vd circ ratio remained constant during all stages of hypovolaemia and hypervolaemia (Figure 1). P175 R l i 1. Anesthesiology. 1994;81:76-86. P175 However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) p S Hutchings1, P Hopkins1, A Campanile2 pp Methods This prospective, single-center study included 80 patients with sepsis from the Department of Critical Care Medicine of Zhejiang Hospital. Patients were randomly assigned to either Group A or Group B, with patients of in Group A fi rst taking the passive leg raising test and then taking the fl uid infusion test, while patients in Group B followed the opposite protocol. NICOM was used to continuously record hemodynamic parameters such as cardiac output (CO), heart rate (HR) and central venous pressure (CVP), at baseline1, PLR, baseline2, and volume expansion (VE). Fluid responsiveness was defi ned as the change in CO (ΔCO) ≥10% after VE. Introduction We performed an evaluation of three devices used for assessment of volume status in critically ill patients in our institution: transthoracic echocardiography (TTE) (CX50; Philips Ultrasound), bio reactance (NICOM; Cheetah Medical) and pulse contour-based thermodilution (PiCCO; Pulsion Medical). Methods Ten mechanically ventilated critically ill patients with PiCCO monitoring in situ and a good quality of images on transthoracic view were included. All study measurements were made in triplicate. A single trained cardiologist, blinded to the results from the other monitors, performed the TTE study. Diff erences among the three methods were assessed for signifi cance using one-way ANOVA, Spearman’s coeffi cient and Bland–Altman analysis. All statistical analyses were performed using Graph-pad Prism 5 and P <0.05 was taken as signifi cant. g ( ) Results CO increased during PLR (from 5.21 ± 2.34 to 6.03 ± 2.73 l/ minute, P <0.05); and after VE (from 5.09 ± 1.99 to 5.60 ± 2.11 l/minute, P <0.05). The PLR-induced change in CO (ΔCOPLR) and the VE-induced change in CO (ΔCOVE) were highly correlated (r = 0.80 (0.64 to 0.90)), while the CVP and ΔCOVE were uncorrelated (r = 0.12 (–0.16 to 0.32)). The areas under the ROC curves of ΔCOPLR and ΔCVP for predicting fl uid responsiveness were 0.868 and 0.514 respectively. ΔCOPLR ≥10% was found to predict fl uid responsiveness with a sensitivity of 86% and a specifi city of 79%.l g p p gi Results Ninety measurements were obtained. NICOM and TTE-derived stroke volume appeared well matched but PICCO-derived values showed signifi cant variation (F = 2.4, P = 0.09). References Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S61 Table 1 (abstract P175). Linear regressions between LVEDV and GEDV Subject a b (ml) r2 1 0.26 7.64 0.82 2 0.43 –47.10 0.66 3 0.21 –12.99 0.75 4 0.25 –11.42 0.59 5 0.41 –65.42 0.85 6 0.48 –65.75 0.68 LVEDV = a × GEDV + b. regards fl uid administration between PiCCO and echocardiography. NICOM appeared unreliable in this setting. regards fl uid administration between PiCCO and echocardiography. NICOM appeared unreliable in this setting. Conclusion The results show that GEDV and LVEDV are generally well correlated, but the correlation coeffi cients are subject specifi c. A preliminary calibration step (for instance using echocardiography) is thus necessary to infer LVEDV from GEDV. Introduction Fluid administration is always important and diffi cult during the therapy of patients with sepsis. Accurately predicting fl uid responsiveness and thus estimating whether the patient will benefi t from fl uid therapy seems particularly important. The present study intended to predict fl uid responsiveness in patients with sepsis using a bioreactance-based passive leg raising test, and to compare this approach with the commonly used central venous pressure (CVP) approach. P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) There was no correlation between TTE velocity time integer (VTI) and NICOM stroke volume variation (SVV) (r = 0.24, P = 0.20; Figure 1A) but a good correlation and small bias between TTE-VTI and PiCCO-SVV (r = 0.76, P <0.0001; Figure 1B). Applying the following indications for volume expansion (PiCCO and NICOM SVV >15% and TTE VTI variability >15%) we found an agreement in 71% of cases between TTE and PiCCO and in 42% of cases between echocardiography and NICOM. i Conclusion Bioreactance-based PLR could predict fl uid responsiveness in patients with sepsis, while CVP could not. P176 P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment H Barrasa, J Maynar, S Castaño, Y Poveda, P Garcia Domelo, A Tejero, G Baziskueta, A Quintano, B Fernández Miret, M Iturbe, S Cabañes, F Fonseca Alava University Hospital-Santiago, Vitoria, Spain Critical Care 2015, 19(Suppl 1):P180 (doi: 10.1186/cc14260) Introduction Dynamic parameters are becoming standard for fl uid responsiveness assessment. Cutoff values are diff erent in the literature. The aim was to assess the accuracy of diff erent preload parameters to predict fl uid responsiveness using pulse power analysis and to compare diff erent levels of hemodynamic response due to passive leg raising (PLR) against the eff ect of a fl uid challenge (FC). y Results We evaluated 27 patients, age 68 (95% CI: 61 to 74) and APACHE II score 22 (95% CI: 18 to 26). Seven patients were high responders, eight patients were moderate responders and 12 were nonresponders. DO2 was signifi cantly increased in high responders (37 ± 35%, P <0.01) as compared with moderate responders or nonresponders. Furthermore, nonresponders had a decrease in their DO2 (–10 ± 7%, P <0.01), while moderate responders showed no change in their DO2 (1.6% ± 10, P = 0.73) after fl uid challenge. We found no diff erences in changes in lactate levels and central venous oxygen saturation (ScvO2) between high responders, moderate responders and nonresponders. No diff erences in the changes of VCO2 or VO2/VCO2 ratio were found between high responders, mild responders and nonresponders too. Changes in DO2/VCO2 ratio were found to be signifi cantly increased only in high responders (47 ± 73% vs. –14 ± 31%, P = 0.02) and not in mild responders (15 ± 54% vs. –14 ± 31%, P = 0.15) as compared with nonresponders. g gfl g Methods A prospective study in a 17-bed mixed ICU. Patients were fully ventilated and CO monitored with LiDCOplus® and underwent a FC due to hypotension and/or hypoperfusion and preload dependence (SVV and/or PPV >10%). PLR was performed before FC. Hemodynamic data were recorded prePLR, postPLR and postFC with 0.5 l crystalloids. We compared diff erent cutoff values of increase in CO and SV (10 to 15%) to assess the ability of PLR, SVV, PPV and CVP to predict the response to FC. Statistical analysis: continuous variables expressed as mean ± SD. P180 P180 Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment H Barrasa, J Maynar, S Castaño, Y Poveda, P Garcia Domelo, A Tejero, G Baziskueta, A Quintano, B Fernández Miret, M Iturbe, S Cabañes, F Fonseca Alava University Hospital-Santiago, Vitoria, Spain Critical Care 2015, 19(Suppl 1):P180 (doi: 10.1186/cc14260) Global end-diastolic volume: a better indicator of cardiac preload in patients with septic shock L Mirea, R Ungureanu, D Pavelescu, I Grintescu g Clinical Emergency Hospital of Bucharest, Romania g y p , Critical Care 2015, 19(Suppl 1):P179 (doi: 10.1186/cc14259) g y p , Critical Care 2015, 19(Suppl 1):P179 (doi: 10.1186/cc14259) Introduction The aim of the study was to assess the value of the global end-diastolic volume (GEDV) evaluated by transpulmonary thermodilution as an indicator of cardiac preload comparing with stroke volume variation (SVV) in patients with septic shock. g p p Conclusion Our results support the idea that a reversible FC (PLR; CO cutoff 12.6%) is best at identifying responder patients to a FC. Dynamic parameters (SVV/PPV) are also eff ective when appropriate. Beat-to- beat SV and CO using pulse power analysis is a valid tool for these tests. Methods A prospective, observational study performed in an interdisciplinary ICU including 91 patients with septic shock. Hemodynamic monitoring was performed with a new calibrated pulse wave analysis method (VolumeView/EV1000; Edwards Lifesciences) in 37 patients (group EV1000) or with an uncalibrated method (FloTrac/ Vigileo; Edwards Lifesciences) in 54 patients (group Vigileo) during the fi rst 72 hours. All patients were receiving mechanical ventilation and vasopressors. Measurements were performed before and immediately after volume loading using 500 ml Ringer solution over a short period (<30 minutes).l p References p p Referencesl 1. De Backer D. Can passive leg raising be used to guide fl uid administration? Crit Care. 2006;10:170. 2. Pinsky MR. Functional haemodynamic monitoring. Curr Opin Crit Care. 2014;20:288-93. 3. Teboul JL, Monnet X. Pulse pressure variation and ARDS. Minerva Anestesiol. 2013;79:398-407. Conclusion Stroke volume produced by bioreactance appeared to be comparable with that measured by echocardiography but not with PiCCO. There was a good agreement between decision-making as 4. Monnet X, Teboul JL. Volume responsiveness. Curr Opin Crit Care. 2007;13:549-53. Figure 1 (abstract P176). Figure 1 (abstract P176). t P176) S62 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P178 Conclusion The transpulmonary thermodilution GEDV is a better indicator of cardiac preload than SVV in patients with septic shock. Acknowledgements This paper was cofi nanced from the European Social Fund, through the Sectorial Operational Programme Human Resources Development 2007–2013, project number POSDRU/159/1.5/S/138907 ‘Excellence in scientifi c interdisciplinary research, doctoral and postdoctoral, in the economic, social and medical fi elds – EXCELIS’; coordinator, The Bucharest University of Economic Studies. References P178 Acute changes of metabolic parameters after fl uid challenge T Nguyen, D De Bels, M Pustetto, P Cottignies, J Devriendt, C Pierrakos Brugmann Hospital, Brussels, Belgium Critical Care 2015, 19(Suppl 1):P178 (doi: 10.1186/cc14258) Introduction The detection of heart response to fl uid administration is still a challenge in clinical practice. Changes in metabolic parameters may be useful to detect changes in cardiac output (CO) after fl uid expansion. 1. Michard F, et al. Chest. 2003;124:1900-8. 2. Bendjelid K, et al. Br J Anaesth. 2013;111:573-9. Methods This is a prospective observational study in adult critically ill patients. CO was measured either by echocardiography or by a thermodilution method (PiCCO, Swan–Ganz catheter). Hemodynamic measurements and blood gas analysis were obtained before and after a fl uid challenge with either 1,000 ml crystalloid or 500 ml colloid. Arterial and central venous blood gas samples were taken simultaneously. Oxygen delivery (DO2), oxygen consumption (VO2) and carbon dioxide production (VCO2) were calculated according to well-known formulas. Patients were divided into three groups (high responders, mild responders and nonresponders) according to their change in CO (>20%, 10 to 20%, <10%, respectively). P181 P181 Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment Comparison before and after was done using a paired Student’s t test, and receiver operating characteristic (ROC) curves were generated by varying the discriminating threshold of each variable. p Conclusion Only signifi cant increases of CO (>20%), after fl uid administration, lead to improved oxygen delivery; DO2 may be decreased in nonresponders. The changes of ScvO2 and lactate levels do not track the changes of CO after fl uid challenge. The DO2/VCO2 ratio may be a useful index to identify signifi cant increases of CO after fl uid challenge in cases where CO measurement is not feasible. Results Thirty-one patients were included. Baseline parameters: MAP 70.5 mmHg (SD 13.3) 87% under catecholamine, SV 55.32 ml (SD 20.2), CO 5.2 l (SD 2), SVV 16.8% (SD 12), PPV 19.1% (SD 14), HR 96 bpm (SD 18) and CVP 9.2 mmHg (SD 2.5). In total, 41.9% of patients increased 15% CO after FC (selected as responders), 38.7% after the PLR. Diff erences in responders versus nonresponder patients were: baseline SVV (23.9 vs. 11.6; P = 0.02) and PPV (28.4 vs. 12.4; P = 0.01). Diff erences in SV and CO were not statistically signifi cant. The best parameter to predict positive response to FC was PLR with cutoff 12.6% for CO increase: sensitivity 84.6% (95% CI = 65 to 104), specifi city 94.4% (95% CI = 84 to 105) and AU ROC 0.94 (95% CI = 0.86 to 1.0). ROC was also good for SVV 0.835 (95% CI = 0.66 to 1.0; P = 0.002) and PPV 0.833 (95% CI = 0.681 to 0.985; P = 0.002) in this cutoff value. In SV increase, PLR, SVV and PPV had P <0.05, but with worse ROC. In addition, SVV <13% identifi ed patients who will not increase MAP with FC: sensitivity 91.7% (95% CI = 76 to 107.3%), negative predictive value 93.5 (95% CI = 80.7 to 106). CVP failed to distinguish responders from nonresponders. References volume status. In this way, dynamic echocardiographic parameters have been proposed in mechanically ventilated children [1,2], using the heart–lung interactions. This study aimed to investigate whether respiratory variations of aortic blood fl ow velocity (DELTA Vpeak ao) and inferior vena cava diameter (DELTA IVC) by transthoracic echocardiography (TTE) could accurately predict fl uid responsiveness in ventilated children. volume status. In this way, dynamic echocardiographic parameters have been proposed in mechanically ventilated children [1,2], using the heart–lung interactions. This study aimed to investigate whether respiratory variations of aortic blood fl ow velocity (DELTA Vpeak ao) and inferior vena cava diameter (DELTA IVC) by transthoracic echocardiography (TTE) could accurately predict fl uid responsiveness in ventilated children. 1. Monnet X, et al. Esophageal Doppler monitoring predicts fl uid responsiveness in critically ill ventilated patients. Intensive Care Med. 2005;31:1195-201. 2. Tibby SM, et al. Are transoesophageal Doppler parameters a reliable guide to paediatric haemodynamic status and fl uid management? Intensive Care Med. 2001;27:201-5. 2. Tibby SM, et al. Are transoesophageal Doppler parameters a reliable guide to paediatric haemodynamic status and fl uid management? Intensive Care Med. 2001;27:201-5. Methods A prospective observational and interventional study conducted in a pediatric ICU investigated 40 mechanically ventilated children with preserved left ventricular (LV) function using TTE. Each patient had tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs. Intervention: standardized volume expansion (VE). P183 Fluid management in mechanically ventilated children with acute circulatory failure based on the pleth variability index in a pediatric ICU H Bouguetof, M Negadi, K El Halimi, L Zemour, D Boumendil, Z Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P183 (doi: 10.1186/cc14263) Results The VE-induced increase in LV stroke volume was ≥10% in 28 patients (responders) and <10% in 12 patients (nonresponders). Before VE, the DELTA Vpeak ao and DELTA IVC in responders was respectively higher than that in nonresponders (18.75% (12 to 32) vs. 13.5% (6 to 16) and 31% (18 to 57) vs. 17.5% (14 to 25)). The prediction of fl uid responsiveness was higher with DELTA Vpeak ao (ROC curve area 0.894 (95% CI = 0.756 to 0.969), P = 0.0001) and DELTA IVC (ROC curve area 0.869 (95% CI = 0.717 to 0.957), P = 0.0001). P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Introduction Prediction of fl uid responsiveness is defi ned by an increase in stroke volume (SV) of at least 10% after volume expansion. Dynamic [1] and static [2] esophageal Doppler (OD) parameters have been proposed in mechanically ventilated children to guide fl uid therapy. This study aimed to compare dynamic parameters using the respiratory variation in aortic blood fl ow with static parameters using Doppler corrected fl ow times (FTc) obtained by OD. Introduction The accuracy of predicting fl uid responsiveness (FR) using IVC collapsibility is high in patients on controlled mechanical ventilation, but remains unknown in spontaneously breathing patients with mechanical ventilation. Also, adequate ultrasound images of IVC are diffi cult to obtain in a substantial number of patients. The aim of the current study is to evaluate utility of collapsibility of jugular veins (IJV) and subclavian veins (SCV) in comparison with collapsibility of IVC in patients on pressure support ventilation. ppl y Methods A prospective, observational and interventional study was conducted in our pediatric ICU from March 2012 to September 2014. We investigated 18 mechanically ventilated children with acute circulatory failure (ACF) – tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs – using OD for each patient. Intervention: standardized volume expansion (VE). Results The VE-induced increase in stroke volume was ≥10% in 14 patients (responders) and <10% in four patients (nonresponders). Before VE, the DELTA Vpeak ao in responders was higher than in nonresponders (19.5% (12 to 29) vs. 11.5% (7 to 13)), whereas FTc was lower in responders than in nonresponders (262.5 milliseconds (180 to 340) vs. 285 milliseconds (205 to 300)). The prediction of fl uid responsiveness was higher with DELTA Vpeak ao (ROC curve area 0.964 (95% CI = 0.756 to 1.000); P = 0.0001) than with FTc (ROC curve area 0.562 (95% CI = 0.314 to 0.790); P = 0.7203). pediatric PICU pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Results A total of 211 fl uid challenges were studied in 91 patients. We observed a signifi cant relationship between the GEDV index before volume loading and the percentage increase in GEDV index in the EV1000 group and changes in GEDV index were signifi cantly correlated with changes in stroke volume index (r = 0.75, P <0.001), but an insignifi cant relationship between SVV variation and cardiac index variation (P >0.05) in the Vigileo group. Introduction Volume expansion remains the fi rst treatment step for most children with acute circulatory failure in order to assess blood Introduction Volume expansion remains the fi rst treatment step for most children with acute circulatory failure in order to assess blood S63 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Prediction of fl uid responsiveness in mechanically ventilated children using dynamic and static parameters by esophageal Doppler in a pediatric ICU Prediction of fl uid responsiveness in mechanically ventilated children using dynamic and static parameters by esophageal Doppler in a pediatric ICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P182 (doi: 10.1186/cc14262) Conclusion In this study, PVI seems to predict fl uid responsiveness in ventilated children with ACF. pp p K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P182 (doi: 10.1186/cc14262) References The best cutoff value for DELTA Vpeak ao was 16% with sensitivity and specifi city predictive values of 71.6% and 83.3%, respectively, and DELTA IVC was 20% with sensitivity and specifi city predictive values of 88.5% and 90.9%, respectively. Introduction The pleth variability index (PVI) is a new dynamic index obtained by automatic estimation of respiratory variations in the pulse oximeter waveform amplitude. This noninvasive and continuous hemodynamic monitoring has been recently proposed in mechanically ventilated patients to guide fl uid therapy. We recently acquired a PVI monitor in 2014. PVI is calculated by measuring changes in perfusion index (PI) during the respiratory cycle as follows: PVI = ((PImax – Pimin) / PImax) × 100. This study aimed to investigate whether PVI at baseline can predict fl uid responsiveness. pl p Methods In our pediatric ICU we started a prospective and observational study. Between January and November 2014, nine mechanically ventilated children were investigated using PVI and transthoracic echocardiography for each patient with acute circulatory failure (ACF): tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs. Intervention: standardized volume expansion. Conclusion In this study, DELTA Vpeak and DELTA IVC were appropriate variables to predict fl uid responsiveness by TTE in ventilated children. References 1. Durand P, et al. Respiratory variations in aortic blood fl ow predict fl uid responsiveness in ventilated children. Intensive Care Med. 2008;34:888-94. 1. Durand P, et al. Respiratory variations in aortic blood fl ow predict fl uid responsiveness in ventilated children. Intensive Care Med. 2008;34:888-94.l 2. Choi DY, et al. Respiratory variation in aortic blood fl ow velocity as a predictor of fl uid responsiveness in children after repair of ventricular septal defect. Pediatr Cardiol. 2010;31:1166-70. 2. Choi DY, et al. Respiratory variation in aortic blood fl ow velocity as a predictor of fl uid responsiveness in children after repair of ventricular septal defect. Pediatr Cardiol. 2010;31:1166-70. Results Signifi cant changes in stroke volume were observed after volume loading (VL) ≥10% in eight patients (responders (R)) and <10% in one patient (nonresponder (NR)). Before VL, PVI was signifi cantly higher in R than NR at baseline ((19.75 ± 3.15%) vs. (9% ± 0.00%), P <0.0001), and decreased signifi cantly in R from baseline to after VL ((19.75% ± 3.15) vs. (12.5% ± 2.828), P <0.0001). P187 Assessing fl uid status with the vascular pedicle width: relationship to IVC diameter, IVC variability and lung comets N Salahuddin, I Hussain, Q Shaikh, M Joseph, H Alsaidi, K Maghrabi King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P187 (doi: 10.1186/cc14267) Assessing fl uid status with the vascular pedicle width: relationship to IVC diameter, IVC variability and lung comets Methods Mechanically ventilated patients having cardiac arrhythmia who have been considered for volume expansion were recruited in this prospective study. Each patient was sedated, paralyzed and monitored with a central venous catheter and a thermistor-tipped femoral arterial VolumeView catheter connected to the EV1000 monitor. We assessed hemodynamic changes after PLRT via a pulse wave contour analysis method. Then we compared it with hemodynamic changes after volume expansion (NSS 500 ml in 15 minutes) via the transpulmonary thermodilution (TPTD) method. N Salahuddin, I Hussain, Q Shaikh, M Joseph, H Alsaidi, K Maghrabi King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P187 (doi: 10.1186/cc14267) Introduction This study attempts to determine a vascular pedicle width (VPW) cutoff value that identifi es a fl uid replete state defi ned as an IVC diameter ≥2 cm and ≤15% respiratory variation. p y Methods In a cross-sectional design, consecutive, critically ill patients underwent simultaneous chest radiographs and ultrasounds. The Research Ethics Committee approved the study. Results A total of 17 patients were included in this study. Six patients were volume responders (TPTD cardiac index change ≥15%). A PLRT change cardiac index ≥10% from the pulse wave contour analysis method had predicted VR with a sensitivity of 50%, a specifi city of 72.7% and an area under the ROC curve of 0.591 (P = 0.546). Results Eighty-four data points on 43 patients were collected. VPW correlated with IVC diameter (r = 0.64, P ≤0.001) and IVC variation (r = –0.55, P ≤0.001). No correlation was observed between VPW and number of lung comets (r = 0.12, P = 0.26) or positive fl uid balance (r = 0.3, P = 0.058). On multivariate linear regression, standardized coeffi cients demonstrated that a 1 mm increase in IVC diameter corresponded to a 0.28 mm (Beta) increase in VPW. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Results A total of 204,680 patients met the search criteria, and 76,807 patients developed one or more postsurgical complications (morbidity rate 37.5%). In patients with and without complications, hospital costs (including 30 days readmission costs) were $27,607 ± 32.788 and $15.783 ± 12,282 (P <0.0001), median (interquartile range) hospital lengths of stay (fi rst stay) were 7 (4 to 10) days and 4 (3 to 5) days (P <0.0001), and 30-day readmission rates were 17.2% and 11.9% (P <0.0001), respectively. With PGDT, the morbidity rate was anticipated to decrease from 26.6 to 31.1%, yielding gross cost savings of $153 million to 263 million for the study period, $61 million to 105 million per year, or $754 to 1,286 per patient. used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean difference in total costs volume was 9.8 ml/predicted body weight. The area under the ROC curve of IVC collapsibility was 0.576 (95% confi dence interval (CI): 0.38 to 0.77), while the area under the ROC curves of right IJV, left IJV, right SCV and left SCV collapsibility were 0.870 (95% CI: 074 to 1.0), 0.54 (95% CI: 0.34 to 0.74), 0.62 (95% CI: 0.43 to 0.81) and 0.54 (95% CI: 0.34 to 0.74), respectively. Greater than 11% of right jugular vein collapsibility predicted fl uid responsiveness with a sensitivity of 79% and a specifi city of 94%. volume was 9.8 ml/predicted body weight. The area under the ROC curve of IVC collapsibility was 0.576 (95% confi dence interval (CI): 0.38 to 0.77), while the area under the ROC curves of right IJV, left IJV, right SCV and left SCV collapsibility were 0.870 (95% CI: 074 to 1.0), 0.54 (95% CI: 0.34 to 0.74), 0.62 (95% CI: 0.43 to 0.81) and 0.54 (95% CI: 0.34 to 0.74), respectively. Greater than 11% of right jugular vein collapsibility predicted fl uid responsiveness with a sensitivity of 79% and a specifi city of 94%. pi y Conclusion Our results suggest collapsibility of the right jugular vein can be a useful predictor of fl uid responsiveness in patients on pressure support ventilation, compared with other central large veins. Collapsibility of IVC does not predict FR in those patients. P185 Passive leg raising cannot predict volume responsiveness in septic shock patients having cardiac arrhythmia P Ratanawatkul1, A Wattanathum2 1Srinagarind Hospital, Khon Kaen, Thailand; 2Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P185 (doi: 10.1186/cc14265) Reference 1. Pearse et al. Eff ect of a perioperative, cardiac output-guided hemodynamic therapy algorithm on outcomes following major gastrointestinal surgery: a randomized clinical trial and systematic review. JAMA. 2014;311:2181-90. Introduction The passive leg raising test (PLRT) is a self-volume challenge used in order to predict volume responsiveness (VR) in both spontaneous and mechanically ventilated critically ill patients. However, there were small numbers of arrhythmic patients included in previous studies. Therefore, the accuracy of the PLRT for prediction of VR in arrhythmic patient is still inconclusive. We hypothesized that the PLRT can predict VR in mechanically ventilated patients having cardiac arrhythmia. P187 P187 ROC curve analysis yielded an AUC of 0.843 (95% CI = 0.75 to 0.93), P ≤0.001 and provided the best accuracy with a cutoff VPW value of 64 mm (sensitivity 81%, specifi city 78%, PPV = 88.5%, NPV = 66%, correct classifi cation rate = 79.6%). See Figure 1. Conclusion The PLRT may not be used for prediction of VR in mechanically ventilated patients having cardiac arrhythmia; however, further and larger studies are needed to confi rm this fi nding. References 1. Monnet X, et al. Crit Care Med. 2006;34:1402-7. 1. Monnet X, et al. Crit Care Med. 2006;34:1402-7. 2. Cavallaro F, et al. Intensive Care Med. 2010;36:1475-83. 2. Cavallaro F, et al. Intensive Care Med. 2010;36:1475-83. Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) p y p p Conclusion Postsurgical complications occurred in more than one- third of our study population and had a dramatic impact on hospital costs, length of stay, and readmission rates. Potential cost savings with PGDT were $754 to 1,286 per patient. These projections should help hospitals estimate the return on investment for implementation of PGDT. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) The best cutoff value for DELTA Vpeak ao was 13% with sensitivity and specifi city predictive values of 85.7% and 100%, respectively; and the best cutoff value for FTc was 265 milliseconds with sensitivity and specifi city predictive values of 57.1% and 75%, respectively. Methods Patients on pressure support ventilation were prospectively included when fl uid challenges were clinically indicated. Bilateral IJV were examined at the level of cricoid cartilage. Bilateral SCV were measured where the veins crossed the clavicle. Anteroposterior diameter, cross-sectional area (CSA) of IJV and SCV were measured using frame by frame analysis. IVC was measured 2 cm from the right atrial border in a long axis view. Fluid responsiveness was defi ned as 8% increase of stroke volume calculated by the Vigileo monitor (Vigileo, FloTrac; Edwards Lifesciences) after passive leg raising (started from supine position). Receiver operating characteristic (ROC) curves were generated using EZR. Results Twenty-nine patients (35 measurements) were included. Nineteen measurements had fl uid responsiveness. The mean tidal Conclusion In our study, DELTA Vpeak was the most appropriate variable to predict fl uid responsiveness by OD in ventilated children with ACF. S64 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean diff erence in total costs. Results A total of 204,680 patients met the search criteria, and 76,807 patients developed one or more postsurgical complications (morbidity rate 37.5%). In patients with and without complications, hospital costs (including 30 days readmission costs) were $27,607 ± 32.788 and $15.783 ± 12,282 (P <0.0001), median (interquartile range) hospital lengths of stay (fi rst stay) were 7 (4 to 10) days and 4 (3 to 5) days (P <0.0001), and 30-day readmission rates were 17.2% and 11.9% (P <0.0001), respectively. With PGDT, the morbidity rate was anticipated to decrease from 26.6 to 31.1%, yielding gross cost savings of $153 million to 263 million for the study period, $61 million to 105 million per year, or $754 to 1,286 per patient. used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean diff erence in total costs. Bioimpedance as a measure of fl uid overload in patients recently admitted to intensive care Bioimpedance as a measure of fl uid overload in patients recentl admitted to intensive care M O’Connor, E Galtrey, CJ Kirwan, JR Prowle Barts Health NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) y Barts Health NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) Introduction Fluid overload is associated with adverse outcomes in critical illness; however, better methodology is required for its quantifi cation. Bioelectrical impedance analysis (BIA) represents a non- invasive method for quantifi cation of fl uid overload [1], but has not been widely taken up in the ICU. Results Fifty patients were included, 40.8% of them were responders. The proportion of responders increases with the increase of dose of fl uids (Table 1). The regression equation was: change of Pmsf (%) = 4.4 (dose of fl uids ml/kg, 95% CI 2.3 to 6.5) – 1.6 (95% CI 7.4 to 4.3, R2 = 0.28, F(1.47) = 17.8, P <0.001). The predicted dose of fl uids required to achieve a change in Pmsf of 15% is 3.7 ml/kg crystalloids. y p Methods We assessed changes in fl uid balance and performed daily BIA (using a Maltron BioScan 920-II; Maltron International Ltd, UK) over 3 days in consecutive ICU admissions with LOS >72 hours. Table 1 (abstract P189). Change of Pmsf-arm and CO Table 1 (abstract P189). Change of Pmsf-arm and CO 1 ml/kg 2 ml/kg 3 ml/kg 4 ml/kg (n = 12) (n = 12) (n = 13) (n = 13) P ΔPmsf-arm (%) 0.0 6.5 9.0 18 0.05 (–4 to 9) (3 to 21) (8 to 16) (9 to 21) ΔCO (%) 3.9 6 9.9 12.9 0.1 (0.4 to 10) (2.1 to 9.1) (–1.6 to 14.3) (2.6 to 23.5) Responders (%) 25.0 18.2 46.2 69.2 0.04 Values are median (interquartile range). Results Of 24 patients 71% were male, median age was 65 years and APACHE II score was 15. Eleven patients had a medical diagnosis and 13 a surgical or trauma reason for admission. Seventy-one percent were mechanically ventilated and 67% were on vasopressors or inotropes. Median BIA-estimated extracellular water was 25.2 l (IQR 22 to 28) on day 1, equating to excess fl uid of 7.2 l (IQR 5 to 13.9). Median right body resistance normalized to height at 50 kHz (R50/h) on day 1 was 214 Ω/m (IQR 187 to 256). Return on investment for implementation of perioperative goal-directed therapy in major surgery: a nationwide database study Introduction Preventable postsurgical complications are increasingly recognized as a major healthcare burden. A recent meta-analysis showed a 17 to 29% decrease in complications after major surgery with perioperative goal-directed therapy (PGDT) [1]. We assessed the fi nancial consequences of postsurgical complications in a large population from 541 US hospitals in order to predict potential savings with PGDT. Introduction Preventable postsurgical complications are increasingly recognized as a major healthcare burden. A recent meta-analysis showed a 17 to 29% decrease in complications after major surgery with perioperative goal-directed therapy (PGDT) [1]. We assessed the fi nancial consequences of postsurgical complications in a large population from 541 US hospitals in order to predict potential savings with PGDT. Methods Data from adults who had any one of 10 major noncardiac surgical procedures between January 2011 and June 2013 were selected from the Premier research database. Twenty-six postsurgical complications were tabulated. Hospital costs, length of stay, and readmission rates were compared in patients with and without complications. Risk ratios reported by Pearse’s meta-analysis were Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. S65 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 The objective of this study was to determine the minimum volume of intravenous fl uid required to signifi cantly increase the Pmsf. Conclusion A VPW value of 64 mm accurately identifi es a fl uid replete state. Increased extravascular lung water, however, was not relatable to the VPW measurements. The VPW can be confi dently used to discriminate fl uid repletion from fl uid responsiveness. li Methods Patients following cardiac surgery were randomly allocated to receive 1, 2, 3 or 4 ml/kg (body weight) of crystalloid over 5 minutes using a 60 ml syringe. Pmsf was measured using the arterial pressure after stopping blood fl ow in the arm with a pneumatic tourniquet infl ated for 1 minute. Cardiac output (CO) was also recorded at baseline and immediately after the fl uid infusion. CO was measured with LiDCO or pulmonary artery catheter, and a positive response was considered an increase of 10% from baseline. From previous data, the least signifi cant change for Pmsf was 15%. Return on investment for implementation of perioperative goal-directed therapy in major surgery: a nationwide database study Medians were compared using the independent samples media test, and proportions were compared using a chi-square test. Statistical signifi cance was considered when P <0.05. P188 Bioimpedance as a measure of fl uid overload in patients recently admitted to intensive care Daily change in ECW and R50/h correlated with daily fl uid balance between BIA measurements (R2 = 0.48 and 0.37 respectively) (Figure 1).i y Conclusion BIA suggests many patients already have signifi cant fl uid overload on the fi rst day of ICU admission. Overall, changes in device-specifi c algorithms for ECW estimation and measured resistances correlated with recorded fl uid balance; however, there were inconsistencies in the number of individual patients. Prospective assessment is required to establish whether BIA measurements can be used to assist fl uid management in the ICU. Conclusion The minimum volume required to perform an eff ective fl uid challenge is 4 ml/kg infused in 5 minutes. However, only 30% of the variation of change in Pmsf can be explained by the dose of i.v. fl uid given. The proportion of responders increases with the volume of fl uids. 1. Earthman C, et al. Bioimpedance spectroscopy for clinical assessment of fl uid distribution and body cell mass. Nutr Clin Pract. 2007;22:389. P189 P189 Minimal volume for a fl uid challenge in postoperative patients H Aya, A Rhodes, RM Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P189 (doi: 10.1186/cc14269) Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery 1ICESP, São Paulo, Brazil; 2Heart Institute, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P191 (doi: 10.1186/cc14271) Introduction After cardiac surgery, about 15% of patients develop vasoplegic shock, characterized by systemic vasodilation, increased capillary permeability and edema. We hypothesized that large-volume resuscitation, resulting in positive fl uid balances in the fi rst 24 hours of ICU admission, would be associated with mortality and would not be protective against AKI in this subset of patients. Conclusion Postsurgical complications have a signifi cant impact on hospital margins. Enhanced Recovery Programs have the potential not only to improve quality of care but also to improve hospital margins. References g Methods This is a retrospective analysis of 362 patients submitted to cardiac surgery at the Heart Institute of University of São Paulo in a period of 2 years. Of a total of 2,383 patients, we enrolled 362 patients. Vasoplegic shock was diagnosed if in the 24 hours of ICU admission patients had hypotension, need of vasoactive drugs after fl uid replacement and cardiac index ≥2.2 l/minute/m2. Data were analyzed in logistic regression models for 30-day mortality and acute kidney injury through Acute Kidney Injury Network (AKIN) score as outcomes. Results The mean age of patients was 57 years. Of 362 patients, 53 died at 30 days (14.6%). Nonsurvivors as compared with survivors were slightly older (59 ± 12 vs. 55 ± 13, P = 0.063), had a higher prevalence of AKI through AKIN score ((0) 6.9%, (1) 11.1%, (2) 28.9%, (3) 31.9%, P <0.001), a higher 24-hour fl uid balance (421 ml (–55 to 695) vs. 2,686 ml (1,321 to 2,856), P <0.001), and higher lactate levels at the intraoperative and at 48 hours (5 mmol/l (4.0 to 7.6) vs. 4.4 (3.33 to 6.55), P <0.001; and 8.11 (5.49 to 12.3) vs. 1.5 (1.33 to 1.88), P <0.001). In the multivariate analysis, positive fl uid balance in the fi rst 24 hours (OR = 1.006, 95% CI = 1.003 to 1.008, P <0.001) and higher lactate after 48 hours (OR = 1.204, 95% CI = 1.072 to 1.353, P = 0.002) were predictors of 30-day mortality. Forty-three percent of patients developed AKI during 30 days. P192 Impact of postsurgical complications on hospital costs and margins R Lavender1, M Mythen2, J Bao3, RH Chapman3, F Michard1 1Edwards Lifesciences, Irvine, CA, USA; 2UCLH National Institute of Health Research, London, UK; 3Avalere Health, Washington, DC, USA Critical Care 2015, 19(Suppl 1):P192 (doi: 10.1186/cc14272) y Results Three hundred and thirty-nine patients were included; mean age was 51 ± 20.4 years, 167 (49%) patients were male. Mean APACHE II score was 22 ± 12.8 and SAPS II score was 35.4 ± 18.9. Severe sepsis/ septic shock was the admitting diagnosis in 129 (38%) patients, 108 (32%) patients were postoperative. AKI developed in 148 (44%) patients; Risk 29 (9%); Injury 26 (8%); Failure 89 (26%) by the RIFLE criteria. On univariate regression analysis; positive fl uid balance >2 l on the fi rst ICU admission day, OR 2 (95% CI = 1.3, 3.3, P = 0.002); age, OR 2.7 (95% CI = 1.7, 4.2, P = 0.000); CHF, OR 3.1 (95% CI = 1.2, 7.9, P = 0.013); APACHE II score, OR 1.02 (95% CI = 1.0, 1.04, P = 0.006); SAPS II score, OR 1.04 (95% CI = 1.02, 1.05, P = 0.000); mean MAP on admission, OR 0.98 (95% CI = 0.96, 0.99, P = 0.033); need for vasopressors on admission, OR 2.7 (95% CI = 1.7, 4.2, P <0.001) and for >24 hours, OR 2.7 (95% CI = 1.7, 2.5, P <0.001); and vancomycin use, OR 1.5 (95% CI = 1.02, 2.53, P = 0.04) signifi cantly predicted the development of AKI. On multivariate regression, CHF, OR 3.8 (95% CI = 1.4, 10, P = 0.007); age, OR 1.02 (95% CI = 1.01, 1.03, P = 0.001); vasopressors for >24 hours, OR 2.6 (95% CI = 1.6, 4.2, P <0.001) and a >2 l positive fl uid balance on the fi rst ICU day, OR 1.6 (95% CI = 1.02, 2.7, P = 0.04) remained signifi cant predictors. Introduction The impact of postsurgical complications (PSC) on hospital cost has been studied but the impact on margins remains controversial [1]. We assessed economic consequences of PSC in US Medicare patients, and benefi ts expected from reducing PSC by 14% to 40% with Enhanced Recovery Programs [2]. y Methods Data from patients with ≥1 comorbidity and major cardiac, vascular, gastrointestinal and orthopedic surgeries in 2011 were extracted from Medicare Standard Analytic Files. Hospital margin was calculated as payment minus cost. Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery In the multivariate analysis, positive fl uid balance in the fi rst 24 hours (OR = 1.001, 95% CI = 1.000 to1.001, P <0.001) and higher lactate at 48 hours (OR = 1.011, 95% CI = 1.000 to 1.021, P = 0.0043) were predictors of 30-day AKI. 1. Eappen S, et al. JAMA. 2013;309:1599-606. P192 Patients with and without PSC were compared, and the economic impact of a 14 to 40% relative reduction in PSC was calculated. Results Of 303,432 patients, 37% had ≥1 PSC. Median length of stay was 10 days for patients with ≥1 PSC and 6 days without (P <0.0001 with vs. without PSC), with readmissions for 21% and 16%, respectively (P <0.0001 with vs. without PSC). Average margins for cases with PSC converted into without PSC would be $1,870 higher. A 14 to 40% reduction in patients with PSC (from 37% to 32% to 22%) would result in saving $153 million to $438 million, and increase hospital margins overall by $28 million to $79 million. See Table 1. i Conclusion Fluid overload, defi ned as a >2 l positive fl uid balance on the fi rst day of ICU admission, is an independent risk factor for the development of AKI in the general ICU population. Table 1 (abstract P192) With PSC Without PSC Mean 2011 US$/patient Cost ($) Margin ($) Cost ($) Margin ($) Cardiac 46,535 –2,286 32,887* –778* Gastrointestinal 33,280 –3,088 18,942* –752* Orthopedic 20,798 –3,567 15,194* –1,872* Vascular 31,042 –4,782 17,667* –2,267* P <0.0001 with versus without PSC. Table 1 (abstract P192) P191 Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery A Rezende1, L Camara2, A Leme2, J Ribeiro2, I Bispo2, S Zeferino2, J Jardim2, C Park1, E Osawa2, J Almeida2, A Gerent1, F Galas2, D Fonseca2, J Fukushima1, L Hajjar2 1ICESP, São Paulo, Brazil; 2Heart Institute, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P191 (doi: 10.1186/cc14271) Minimal volume for a fl uid challenge in postoperative patients H A A Rh d RM G d M C i H Aya, A Rhodes, RM Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK g Critical Care 2015, 19(Suppl 1):P189 (doi: 10.1186/cc14269) Introduction In critical illness, fl uid overload may predispose to acute renal dysfunction by a number of mechanisms. Once acute kidney Introduction An eff ective fl uid challenge should increase the mean systemic fi lling pressure (Pmsf) in order to increase the venous return. Figure 1 (abstract P188). Change in BIA-measured ECW (a) or R/h at 50 kHz (b) versus daily fl uid balance. Hz (b) versus daily fl uid balance. Figure 1 (abstract P188). Change in BIA-measured ECW (a) or R/h at 50 kHz (b) versus daily fl uid balance. S66 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Positive fl uid balance after cardiac surgery is an independent risk factor for mortality and for acute kidney injury in patients presenting vasoplegic shock. injury (AKI) develops, positive fl uid balance has been described as a risk factor for overall mortality and delayed renal recovery. We hypothesized that fl uid overload may be an independent risk factor for AKI in the critically ill. injury (AKI) develops, positive fl uid balance has been described as a risk factor for overall mortality and delayed renal recovery. We hypothesized that fl uid overload may be an independent risk factor for AKI in the critically ill. Methods In a cross-sectional design, we collected data on consecutive, critically ill, adult patients admitted over a 5-month period to the medical and surgical ICUs of a single center. AKI was defi ned according to the RIFLE Classifi cation. Logistic regression analysis was performed to determine the predictive ability of variables for AKI. The institutional Research Ethics Committee approved the study. P195 Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Results We included a total of 1,267 patients. The median age was 68 (quartiles: 59, 76), 32% were female, 68% underwent coronary artery bypass grafting and 59% underwent valve surgery. Median length of hospital stay was 6 days (quartiles: 5, 9). Median length of stay in the normal, elevated and high lactate groups were 5 days (quartiles: 4, 7), 6 days (quartiles: 5, 9) and 9 days (quartiles: 6, 17), P <0.001 for comparison. In multivariable analysis, patients with an elevated lactate had a 1.12 times (95% CI: 1.02 to 1.23, P = 0.02) longer length of stay compared with those with normal lactate. Patients with a high lactate had a 1.30 times (95% CI: 1.10 to 1.53, P = 0.002) longer length of stay compared with those with normal lactate. Introduction National University Hospital, Singapore, recently formed a Division of Critical Care – Respiratory Therapy. This service rapidly expanded to provide 24/7 Respiratory Therapy Services in the cardiothoracic intensive care unit (CTICU). One goal of service expansion was a reduction in duration of mechanical ventilation after cardiac surgery. We hypothesized that introduction of a team- based extubation protocol would reduce the duration of mechanical ventilation and ultimately aff ect ICU length of stay. Conclusion Postoperative lactate levels are associated with increased length of hospital stay in patients undergoing major cardiac surgery. Interventions aimed at decreasing postoperative lactate levels may decrease hospital length of stay. f Methods A multidisciplinary group created a team-based extubation protocol. The protocol was applied to all elective postoperative cardiac surgery patients. To assess the protocol’s impact, data were collected in a registry 3 months before and 3 months after protocol initiation. Data collection included cardiopulmonary bypass time, McCormack airway assessment, ICU admission time, initial pH, lactate, inotropes upon arrival at the CTICU, blood gas analysis prior to extubation, time of extubation and length of stay. Patients were excluded from data analysis if they experienced events which contraindicated application of the protocol, such as signifi cant intraoperative or postoperative complications. These events were explicitly stated in the extubation protocol. Lactate levels after major cardiac surgery are associated with hospital length of stay Introduction The objective of the study was to evaluate whether postoperative lactate values are associated with hospital length of stay in patients undergoing major cardiac surgery. Previous studies have shown an association between postoperative lactate levels and increased morbidity and mortality after major cardiac surgery. However, the association between lactate and hospital length of stay has not been adequately characterized. Methods We performed a retrospective analysis of all patients presenting for coronary artery bypass grafting and/or valve surgery between 2002 and 2014 at a tertiary care center in Boston, who had a lactate level measured within 3 hours of skin closure. Lactate values S67 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 were categorized into clinical meaningful categories: 0 to 2 mmol/l (normal), 2 to 4 mmol/l (elevated) and ≥4 mmol/l (high) to allow for nonlinear eff ects. The unadjusted association between lactate group and length of stay was assessed with the Kruskal–Wallis test and post hoc Wilcoxon rank-sum tests. To assess the association between postoperative lactate levels and hospital length of stay we performed multivariable Poisson regression with robust variance estimates. We adjusted for more than 30 variables including patient demographics, comorbidities, cardiac characteristics (for example, New York Heart Association class and ejection fraction), and surgical characteristics (for example, year, status (elective, urgent, emergent), type of procedure, perfusion time, and cross clamp time). severe hypotension (MAP <65 mmHg) and the study was not stopped in any case. The length of the hospital stay was shorter among patients in the intensive group (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). severe hypotension (MAP <65 mmHg) and the study was not stopped in any case. The length of the hospital stay was shorter among patients in the intensive group (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). Conclusion An intensive alveolar recruitment protocol did not result in hemodynamic instability in hypoxemic patients after cardiac surgery (NCT01502332). P195 P195 Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Singapore’s Domain-specifi c review board granted waiver of patient consent to analyze and present these data. Hemodynamic behavior in a randomized trial of intensive alveolar recruitment after cardiac surgery recruitment after cardiac surgery A Leme, M Amato, E Osawa, J Fukushima, M Feltrim, E Nozawa, J Almeida L Hajjar, F Galas Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P194 (doi: 10.1186/cc14274) A Leme, M Amato, E Osawa, J Fukushima, M Feltrim, E Nozawa, J Almeida, L Hajjar, F Galas jj Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P194 (doi: 10.1186/cc14274) Introduction The potential benefi ts of a protocol of intensive alveolar recruitment may be outweighed by its detrimental eff ects in hemodynamic stability after cardiac surgery. The aim of this study was to analyze the hemodynamic behavior of patients included in a trial of intensive alveolar recruitment after cardiac surgery. p y p Results A total of 201 patients undergoing elective open cardiac surgery were included; 99 patients before protocol implementation (pre-protocol) and 102 patients after implementation (post-protocol). There was no signifi cant diff erence in mean age (60 vs. 61 P = 0.823), gender (79.8% vs. 79.4% P = 1.00), EuroSCORE (26 vs. 32 P = 0.576) and proportion receiving bypass surgery (72% vs. 80% P = 0.206) or valve surgery (21% vs. 19% P = 0.722) between the two groups. Median extubation time was reduced by 3.5 hours (620 minutes vs. 408 minutes P <0.001). ICU length of stay was also reduced following introduction of the pre-protocol 48 hours versus 24 hours post protocol (P <0.05).i Methods In this randomized trial, we assigned adult patients with PaO2/ FIO2 <250 at a PEEP of 5 cmH2O to either intensive alveolar recruitment or a standard protocol, both using low-tidal volume ventilation (6 ml/ kg/ibw) after adequate volemia status. Our hypothesis was that an intensive alveolar recruitment protocol with controlled pressure of 15 cmH2O and PEEP of 30 cmH2O during 1 minute, repeated three times at 1-minute intervals between each maneuver, would not cause hemodynamic instability. Conclusion A team-based extubation protocol signifi cantly reduced the duration of mechanical ventilation and this translated to reduced ICU length of stay in patients undergoing elective open-heart surgery. Results In total, 163 patients were included in the standard and 157 in the intensive group. Patients of the intensive group had a signifi cant reduction of the MAP at T1, T2 and T3 (1 hour, 2 hours and 3 hours of the protocol), returning to baseline after T4 (Figure 1). No patients had P196 Impact of patient frailty on outcome in cardiothoracic surgery J Brohan, P Delaney, B O’Brien Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 JL Iribarren, J Jiménez, N Perez, M Brouard, R Perez, E Hurtado, S Diosdado, M Buitrago, A Arbesu, R Martinez, M Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P199 (doi: 10.1186/cc14279) Introduction Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgery. Introduction Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgery. g Results A total of 8,026 were recorded, in 77 of them an IABP was inserted before the surgery. We performed a propensity score analysis by pairing 72 patients with and without BCIAO based on epidemiological factors and type of surgery. In the analysis of all-cause 30-day mortality, 27% of patients in whom IABP was inserted prior surgery died versus 13.1% of patients without IABP preoperative implantation (P = 0.043). A combined endpoint that included need for prolonged mechanical ventilation over 24 hours or reoperation or mediastinitis or stroke after surgery or 30-day mortality was performed and occurred in 58.3% of patients with preoperative IABP versus 41.7% without it (P = 0.046). When stratifi ed by preoperative risk (analyzed with EuroSCORE), no diff erence between groups was observed (P = 0.62, OR 0.75 (0.23 to 2.35)) for mortality rate and (P = 0.11, OR 0.47 (0.19 to 1.18)) for the combined endpoint. The patients with preoperative IABP implantation had a higher ICU length of stay (10.6 ± 7.7 vs. 4.6 ± 6.7, P = 0.046) with no diff erences in terms of overall hospital stay (21.8 ± 18.7 vs. 18.9 ± 22.08, NS). y y Methods We prospectively studied the association between hemodynamic response and polymorphisms of TNF-B and PAI-1 in 563 patients undergoing elective cardiac surgery during the years 2008 to 2011. We tested the Hardy–Weinberg equilibrium in the sample. V18 SPSS was used.if Results We studied 563 patients. We found signifi cant diff erences in TNF-B polymorphisms regarding norepinephrine requirements at 4 hours (F: 15.9; P <0.001), post hoc Scheff é (GG vs. P198 Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery J Muñoz-Bono, MD Delgado-Amaya, E Curiel-Balsera, C Joya-Montosa, G Quesada-García Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P198 (doi: 10.1186/cc14278) Results A total of 120 patients were included in this study, including 100 patients who underwent cardiac surgery and 20 patients who underwent thoracic surgery. Eighty-fi ve patients (70.8%) were male. The mean age was 65.4 years (range 25 to 89 years). The mean baseline frailty score also varied widely within our cohort. Four patients died in the ICU following their surgery (3% ICU mortality rate). Mean length of ICU stay was 2.7 days (range 0 to 20 days), with a mean duration of ventilation of 20 hours (range 0 to 264 hours). Follow-up of these patients at 6 months following their surgery is currently underway. Introduction The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery. Introduction The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery. g y Methods An observational, retrospective, multicenter study of all patients undergoing cardiac surgery included in the ARIAM- ANDALUCIA database of Cardiac Surgery from March 2008 to July 2012. We used the chi-square test and Student t test as needed, establishing the level of statistical signifi cance at 95%. y y y Conclusion Owing to advances in life expectancy, health and perioperative medicine, it has become more diffi cult to determine fi tness for major surgery. Our data suggest that frailty may be a useful prognostic measure to help inform such decisions. R f gi Results Of the 8,026 patients who underwent cardiac surgery during the study period, BCIAO was implemented in 358 (4.5%) of them. In total, 65.4% were male. Surgical times in those patients where IABP was implanted were 146 ± 81 minutes and 90 ± 66 minutes (cardiopulmonary and aortic clamping times, respectively). The in- surgery room mortality was 4.7%, 30-day mortality in these patients was 40.2%. Patients in whom IABP was implanted had a mortality rate eight times higher than those who did not require it during surgery or postoperatively (40.2% vs. 8.4%, P = 0.0001. OR 8.1, 95% CI (6.4 to 10.3)). Impact of patient frailty on outcome in cardiothoracic surgery J Brohan, P Delaney, B O’Brien Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) Figure 1 (abstract P194). J Brohan, P Delaney, B O’Brien , y, Cork University Hospital, Cork, Ireland Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) y p , , Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc142 Introduction Frailty is defi ned as a multidimensional syndrome involving loss of physical and cognitive reserve leading to greater vulnerability to adverse events [1]. Such events include susceptibility to unplanned hospital admissions, and death [1-3]. Frailty is associated with increased ICU and 6-month mortality, and reduced quality of life [4]. The aim of this study is to investigate the impact of baseline frailty on postoperative quality of life indicators and postoperative frailty following cardiothoracic surgery. Methods Adult patients undergoing cardiac surgery or thoracic surgery (involving thoracotomy) were included in this study. Baseline measures of frailty [4] and performance status were prospectively recorded using validated tools. Informed consent was obtained prior to inclusion. Outcome measures of APACHE II scores, duration of ventilation, length S68 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P198 Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery Besides mortality was higher, the later IABP was implanted the higher the mortality rate was (29.6% of the preoperative, 44.2% of surgical and 54.4% of those starting in ICU, P = 0.015). The ICU length of stay was 9 ± 22 days while the hospital length of stay was 21 ± 28 days. In patients who needed IABP, the ICU stay was higher than for those who did not need it (9 ± 22 vs. 5 ± 10 days, P = 0.002) whereas there was no diff erence in hospital stay (21 ± 28 vs. 20 ± 24 days, P = 0.054). 1. Rockwood K, et al. A global clinical measure of fi tness and frailty in elderly people. CMAJ. 2005;173:489-95. 2. Rockwood K, et al. Changes in relative fi tness and frailty across the adult lifespan: evidence from the Canadian National Population Health Survey. CMAJ. 2011;183:E487-94. 3. Makary MA, et al. Frailty as a predictor of surgical outcomes in older patients. J Am Coll Surg. 2010;210:901-8. 4. Le Maguet P, et al. Prevalence and impact of frailty on mortality in elderly ICU patients: a prospective, multicentre observational study. Intensive Care Med. 2014;40:674-82. P198 of ICU stay and mortality were recorded. Follow-up at 6 months was conducted by telephone to assess recovery patterns. of ICU stay and mortality were recorded. Follow-up at 6 months was conducted by telephone to assess recovery patterns. Registry of Cardiac Surgery MD Delgado-Amaya, C Joya-Montosa, J Muñoz-Bono, E Curiel-Balsera Hospital Regional de Málaga, Spain MD Delgado-Amaya, C Joya-Montosa, J Muñoz-Bono, E Curiel-Balsera Hospital Regional de Málaga, Spain f p y y Conclusion The intra-aortic balloon pump was used by 4.5% of surgeries performed during the study period and in patients with an increased risk of perioperative complications, estimated by EuroSCORE. ICU length of stay was higher in patients requiring IABP, with no diff erences in overall hospital stay. Mortality rate was 40% higher, and increases with the delay in the implantation. p g g p Critical Care 2015, 19(Suppl 1):P197 (doi: 10.1186/cc14277) g g Critical Care 2015, 19(Suppl 1):P197 (doi: 10.1186/cc14277) Introduction The aim of our study was to assess whether the preoperative use of IABP is benefi cial in patients undergoing cardiac surgery of any kind. Introduction The aim of our study was to assess whether the preoperative use of IABP is benefi cial in patients undergoing cardiac surgery of any kind. Methods An observational, retrospective, multicenter study of all patients undergoing cardiac surgery and included in the ARIAM- ANDALUCIA Registry of Cardiac Surgery from March 2008 to July 2012. The probability of placing IABP in the preoperative period has been calculated, making a propensity analysis to obtain two homogeneous groups treated with or without the IABP, based on personal history, functional status and type of surgery. Seventy-seven patients with preoperative IABP were matched with 77 patients without BCIAO with the nearest propensity score. We used the chi-square test or Student t test as needed and binary logistic regression for multivariate analysis so we can rule out possible confounding variables. We used the statistical package R v2.12 for MAC. P199 Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 JL Iribarren, J Jiménez, N Perez, M Brouard, R Perez, E Hurtado, S Diosdado, M Buitrago, A Arbesu, R Martinez, M Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P199 (doi: 10.1186/cc14279) P200 Pharyngeal oxygenation during apnoea following conventional pre-oxygenation and high-fl ow nasal oxygenation D Stolady, M Mariyaselvam, H Young, E Fawzy, M Blunt, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P200 (doi: 10.1186/cc14280) y Methods We performed a prospective randomized cross-over study on hypoxemic non-intubated patients (PaO2/FiO2 ≤300 mmHg) admitted to the ICU of the San Gerardo Hospital and prescribed to receive oxygen by facial mask. We delivered the same air/oxygen mix by HFNC (Optifl ow; Fisher & Paykel Healthcare, Auckland, New Zealand) and facial mask (20 minutes per step). Continuous recordings of regional lung volumes by EIT (Pulmovista 500; Drager Medical GmbH, Lubeck, Germany) and of inspiratory eff ort by esophageal pressure (Pes) were obtained and analyzed offl ine by dedicated software. Introduction We hypothesised that pharyngeal oxygen concentrations would be maintained higher and for longer with transnasal humidifi ed rapid insuffl ation ventilatory exchange (THRIVE) than conventional bag-mask pre-oxygenation (CPO). CPO requires the mask to be removed during laryngoscopy; this means that air may enter the mouth so subsequent apnoeic oxygenation will be less eff ective. Oral suctioning could exacerbate this process. However, if high pharyngeal oxygen concentrations and an open airway are maintained, apnoeic oxygenation could be substantially improved. Methods used have included NO-DESAT [1] and recently THRIVE [2], which has been shown to extend apnoea times for up to 1 hour. yfl y Results We enrolled 15 patients (10 male), age 57 ± 16 years. Compared with standard facial mask, HFNC signifi cantly improved PaO2/FiO2 (199 ± 60 vs. 150 ± 46, P <0.001) and end-expiratory lung impedance (corresponding to aeration) (866 ± 568 au vs. baseline, P <0.001). Moreover, HFNC decreased the respiratory rate (22 ± 5 bpm vs. 20 ± 5 bpm, P <0.001), as well as negative Pes swings (ΔPes 8.3 ± 5 mmHg vs. 6.6 ± 1 mmHg, P <0.01) and corrected minute ventilation (that is, actual MV × actual PaCO2 / 40 mmHg) (49,887 ± 16,176 au vs. 41,811 ± 14,042 au, P <0.001). Finally, central venous pressure increased (6 ± 5 mmHg vs. 4 ± 5 mmHg, P <0.01), possibly indicating positive end-expiratory pressure eff ect. Methods A volunteer with a nasopharyngeal sampling catheter underwent simulated emergency airway management (EAM), using both CPO and THRIVE, with and without suction. Reference 1. Sotello D, Rivas M, Mulkey Z, Nugent K. High-fl ow nasal cannula oxygen in adult patients: a narrative review. Am J Med Sci. 2015;349:179-85. Results Pharyngeal oxygen concentrations (mean and SEM) are shown in Figure 1 (all points are signifi cant P <0.05). Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 AA, 0.32 (0.11 to 0.65) vs. 0.06 (0.04 to 0.09) μg/kg/minute, P <0.001; GG vs. AG, 0.32 (0.11 to 0.65) vs. 0.06 (0.03 to 0.08), P <0.001)) and at 24 hours (F: 8; P = 0.005), post hoc Scheff é (GG vs. AA, 0.27 (0.01 to 0.52) vs. 0.10 (0.06 to 0.14), P = 0.019; GG vs. AG, 0.27 (0.01 to 0.52) vs. 0.07 (0.04 to 0.09), P = 0.003)). Unfavorable TNF-B (G homozygous vs. allele A) and PAI-1 unfavorable (4G homozygous vs. allele 5G) were grouped, after adjusting for perioperative signifi cant variables. The homozygous GG and 4G alleles were signifi cant for NA 4 hours (F: 5.5; P = 0.02 and F: 4.1; P = 0.04, respectively) and GG–4G allele interaction (F: 6; P = 0.01) (Figure 1), Conclusion The use of IABP prior to cardiac surgery in patients at high risk does not reduce the mortality rate nor the combined endpoint described above. ICU length of stay was greater in those patients in whom IABP was implanted prior to surgery; there were no diff erences in overall hospital stay. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S69 THRIVE. Assessment of NO-DESAT (15 l/minute) was abandoned due to discomfort. R f Figure 1 (abstract P199). THRIVE. Assessment of NO-DESAT (15 l/minute) was abandoned due to discomfort. Figure 1 (abstract P199). P200 Following 3 minutes of pre-oxygenation with CPO (FiO2 = 1, FEO2 >0.8) or THRIVE (60 l/minute; Optifl ow, Fisher and Paykel), EAM was simulated by voluntary apnoea and pharyngoscopy with the laryngoscope blade tip placed 2 cm from the posterior pharyngeal wall. Capnography at the laryngoscope tip confi rmed apnoea. Pharyngeal gas samples (20 ml) were collected during apnoea, and after 5 seconds of oropharyngeal suctioning. Pre- oxygenation was repeated between sampling. Samples (n = 100) were analysed using calibrated fuel cells. f Conclusion In non-intubated hypoxemic critically ill patients, HFNC improves oxygenation and end-expiratory aeration; moreover, HFNC reduces the inspiratory eff ort and the minute ventilation needed to maintain normal arterial CO2 tension. 0 Eff ects of high-fl ow nasal cannula therapy on oxygenation, lung volumes and CO2 removal in critically ill hypoxemic patients: preliminary results p y T Mauri1, N Eronia2, G Bellani2, G Grasselli2, R Marcolin2, S Marocco Arrigoni2, A Pesenti2 1IRCC Ca’ Granda Maggiore Policlinico Hospital, Milan, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P201 (doi: 10.1186/cc14281) Figure 1 (abstract P199). Figure 1 (abstract P199). while for NA at 24 hours statistics showed GG (F: 3.2; P = 0.07), 4G allele (F: 2; P = 0.15) and interaction (F: 3.6, P = 0.05). Conclusion GG homozygous polymorphism TNF-B is associated with an increased dependence on norepinephrine after cardiopulmonary bypass, showing a synergistic action with the 4G allele of PAI-1. while for NA at 24 hours statistics showed GG (F: 3.2; P = 0.07), 4G allele (F: 2; P = 0.15) and interaction (F: 3.6, P = 0.05). Introduction High-fl ow nasal cannula (HFNC) is increasingly proposed as respiratory support for hypoxemic non-intubated acute respiratory failure patients. Clinically, HFNC therapy decreases dyspnea, improves patient’s comfort, improves oxygenation and enhances clearance of upper airway secretions [1]. We present preliminary results from a clinical study aimed at measuring the eff ects of HFNC on gas exchange, lung volumes and inspiratory eff ort in hypoxemic non-intubated critically ill patients. Conclusion GG homozygous polymorphism TNF-B is associated with an increased dependence on norepinephrine after cardiopulmonary bypass, showing a synergistic action with the 4G allele of PAI-1. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness A similar pattern was seen was seen in the vast majority of ICUs: a single institution reported no capnography available. However, in 141 (66.8%) of the hospitals surveyed, no facility to measure ETCO2 was present on the general wards. Where available, 86.7% used capnography to confi rm ETT placement. Less than 50% used ETCO2 to determine CPR eff ectiveness and 8% to prognosticate.i is located in the same position as the light source on the standard Macintosh blade thus providing a view angle of up to 290° and the USB camera is connected to a laptop. A total of the fi rst 50 patients who presented to the emergency department over a period of 6 months in need of intubation were included in the study and every alternate patient participated in the evaluation of the assembled video laryngoscope (VAL). Information about patient demographics and airway characteristics, Cormack-Lehane (C/L) views and the ease of intubation using the VAL was collected. Failure was defi ned as more than one attempt at intubation. given the option to decline participation. All data were anonymised. Results A total of 211 hospitals met the inclusion criteria. The response rate was 100%. Arrest calls were mainly attended by anaesthesia (47.8%) and ICU doctors (38.3%) with around 2% physicians only. Most were a registrar grade (56.3%). The ability to measure ETCO2 was available in all but four EDs; most used waveform capnography. A similar pattern was seen was seen in the vast majority of ICUs: a single institution reported no capnography available. However, in 141 (66.8%) of the hospitals surveyed, no facility to measure ETCO2 was present on the general wards. Where available, 86.7% used capnography to confi rm ETT placement. Less than 50% used ETCO2 to determine CPR eff ectiveness and 8% to prognosticate. Results Excellent (C/L1) or good (C/L2) laryngeal exposure was obtained in 92% and 8% of patients respectively. In 25 patients in whom VAL was performed, there was a comparable or superior view. Intubation with direct laryngoscopy was successful in 95.2% of patients and VAL was successful in 95.4% of patients. Three patients from the VAL group and four patients from the direct laryngoscopy group were excluded. See Figure 1. Conclusion We believe this is the fi rst study of its kind to follow NAP4 and investigate the availability of capnography throughout for use during cardiac arrest. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness Whilst equipment levels appear adequate (albeit not perfect) in resuscitation areas, there appears a lack of availability of suitable devices on general wards. Figure 1 (abstract P202). New video laryngoscope connected to laptop. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness i Conclusion Pharyngeal oxygen concentration rapidly falls following CPO. This may be detrimental for apnoeic oxygenation during con ven- tional laryngoscopy. Conversely, THRIVE maintains high pharyngeal oxygen concentrations over time. Suction has an immediate negative eff ect on pharyngeal oxygen concentration that is attenuated by f SM Ayyan, Z Ali Figure 1 (abstract P200). Pharyngeal oxygen concentration. Introduction Video laryngoscopes have been introduced in recent years as an alternative choice to facilitate tracheal intubation. Diffi culties with tracheal intubation are mostly caused by diffi cult direct laryngoscopy with impaired view to the vocal cords. Many endoscopic intubation laryngoscopes have been designed to visualize the vocal cords around the corner looking through a proximal viewfi nder. Although they are useful devices, they have limitations for doing direct laryngoscopy and are very expensive, hence they are not used for routine tracheal intubation.i Methods A Macintosh intubating laryngoscope has been modifi ed by attaching a waterproof USB camera with a inbuilt light source, which S70 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 is located in the same position as the light source on the standard Macintosh blade thus providing a view angle of up to 290° and the USB camera is connected to a laptop. A total of the fi rst 50 patients who presented to the emergency department over a period of 6 months in need of intubation were included in the study and every alternate patient participated in the evaluation of the assembled video laryngoscope (VAL). Information about patient demographics and airway characteristics, Cormack-Lehane (C/L) views and the ease of intubation using the VAL was collected. Failure was defi ned as more than one attempt at intubation. examining practice regarding intubation for cardiac arrest and the availability and utilisation of capnography within the ED, ICU and general wards. Questions were directed at the anaesthetist or intensive care doctor ‘responding to cardiac arrest calls’. The respondent was given the option to decline participation. All data were anonymised. Results A total of 211 hospitals met the inclusion criteria. The response rate was 100%. Arrest calls were mainly attended by anaesthesia (47.8%) and ICU doctors (38.3%) with around 2% physicians only. Most were a registrar grade (56.3%). The ability to measure ETCO2 was available in all but four EDs; most used waveform capnography. P203 P203 Availability of appropriate airway monitoring at UK in-hospital cardiac arrest S Turle1, PB Sherren1, T Callaghan1, S Nicholson2, SJ Shepherd1 1Pan-Thames Intensive Care Training Scheme, London, UK; 2KSS Intensive Care Training Scheme, London, UK Critical Care 2015, 19(Suppl 1):P203 (doi: 10.1186/cc14283) Using a laryngoscope and endotracheal tube succeeds in a diffi cult case of nasogastric tube insertion J P k Y L Using a laryngoscope and endotracheal tube succeeds in a diffi cult case of nasogastric tube insertion J Park, Y Lee Ewha Womans Unversity Hospital, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P204 (doi: 10.1186/cc14284) , Ewha Womans Unversity Hospital, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P204 (doi: 10.1186/cc14284) Introduction Nasogastric (NG) tube insertion is necessary in a variety of critically ill patients for intra-abdominal decompression, prevention of aspiration, route of medication administration and nutrition. However, it often fails in patients who showed sedated or comatose mentality with poor cooperation during the procedure. Although there are many reports inserting a NG tube in diffi cult cases, most methods need a special guide wire, tube or nasoendoscope. We report a case of NG tube insertion in a comatose patient using a laryngoscope and endotracheal tube which are easily available. Figure 1 (abstract P202). New video laryngoscope connected to laptop. Conclusion This new assembled VAL is the cheapest video-assisted laryngoscope available costing around $60, which can even be introduced into primary healthcare setup in developing countries. VAL consistently yielded a comparable or superior glottic view compared with direct laryngoscopy despite the limited or lack of prior experience with the device. Because the device can be used for both routine as well diffi cult tracheal intubation, it may be a helpful tool to intubate trauma cases where C-spine immobilization is unavoidable. The presented video-assisted laryngoscope is a useful tool for documentation, teaching and monitoring tracheal intubation. Figure 1 (abstract P204). Insertion of a nasogastric tube through an endotracheal tube in the esophagus. References 1. Grmec S. Intensive Care Med. 2002;28:701-4. 2. Deakin CD, et al. Resuscitation. 2010;81:1305-52. 3. Cook TM, et al. Br J Anaesth. 2011;106:617-31. 1. Grmec S. Intensive Care Med. 2002;28:701-4. Admissions with airway emergencies in the ICU at a tertiary referral centre Results Forty-fi ve patients (31 male) were included with a mean age of 67 ± 15 years. The commonest admission diagnoses were sepsis (10), cardiogenic shock (10), primary respiratory failure (nine) and intracranial haemorrhage (eight). The median transfer delay time was 47 minutes. Only 27 (60%) patients were actually transferred and they were signifi cantly younger than nontransferred patients (62 vs. 73 years, P = 0.02). In-transit mortality was zero. Mean length of stay in the critical care centre was 14.8 ± 16.8 days. Survival to discharge was signifi cantly higher in transferred (14/27) compared with nontransferred (3/18) patients (52% vs. 17%, P = 0.017). Overall mortality rates were 62% and 69% at 1 and 6 months respectively and were signifi cantly lower in the transferred group (P = 0.02). M Gresoiu, M Singer M Gresoiu, M Singer University College London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P205 (doi: 10.1186/cc14285) Introduction As a tertiary referral centre for ENT and maxillo-facial surgery, our ICU receives complex elective and emergency cases. The frequency, aetiology and outcomes of airway emergencies are poorly described. Understanding these factors is key to improving management. g Methods We conducted a retrospective review of the ICU electronic patient database examining unplanned admissions with airway emergencies between December 13 and November 14. Data on demographics, aetiology of airway obstruction (including postprocedural), APACHE II score, therapeutic intervention(s) administered, and outcomes were collected. Conclusion Overall mortality rates of medical patients intubated urgently at BGH were high. Forty per cent of intubated patients were not transferred, indicating signifi cant modifi cation of the protocol over time. Patients transferred to the critical care centre were younger and had signifi cantly better outcomes than patients remaining in BGH, probably due to decisions not to transfer patients with poor prognoses. Most patients who survived to discharge were still alive 6 months later. Reference Results Of 1,516 unplanned admissions, airway emergencies represented 6.3% (96 patients) of whom 40 (41.7%) had malignancy (26 maxillo-facial/trachea, three pulmonary, four haematological, seven other) and 24 infection (abscesses, epiglottitis, Ludwig’s angina). Referring specialties were maxillo-facial surgery (n = 34), internal medicine (n = 25), ENT (n = 21) and other surgical specialties (n = 16). Thirteen patients had complications post bronchoscopy (vocal cord palsy, need for NIV or intubation), one post microlaryngoscopy, and 20 were admitted after diffi cult intubation. Outcomes of medical patients requiring emergency intubation in a rural Irish hospital Outcomes of medical patients requiring emergency intubation in a rural Irish hospital Results A 15-year-old male patient admitted due to abrupt mental change and brain imaging showed severe subdural hemorrhage. NG tube insertion was done for enteral feeding but failed several times though changing position. As we had no guide wire and no nasoendoscope, an endotracheal tube was used as guidance for the NG tube. After making a longitudinal midline cut on the endotracheal tube, it was inserted into the esophagus under a laryngoscope. The NG tube was pushed into the endotracheal tube, and then the endotracheal tube was removed through the cut, reserving the NG tube. We checked the position of the NG tube by air sound and X-ray, and started enteral feeding without complication, such as nasal bleeding, emphysema, and gastric perforation. See Figure 1. p A O’ Connor, MP D’Alton, B Carey Bantry General Hospital, Co. Cork, Ireland Critical Care 2015, 19(Suppl 1):P206 (doi: 10.1186/cc14286) Introduction Bantry General Hospital (BGH) is a small rural hospital serving a large, geographically isolated part of southwest Ireland. Following an infl uential national review of adult critical care services [1], a protocol was introduced in late 2010 mandating the immediate transfer of all medical patients intubated on an emergency basis to a large critical care centre 100 km away. Similar mandatory transfer protocols were introduced at the same time throughout the island of Ireland but few data are available regarding patient outcomes. We designed a study to look at the outcomes of all patients encompassed by the protocol at BGH. Conclusion We report a new method of NG tube insertion using a laryngoscope and endotracheal tube. y Methods We retrospectively reviewed the charts and electronic data of medical patients requiring emergency intubation at BGH from November 2010 to December 2013. We recorded the following data: age, sex, admission diagnosis, comorbidities, time delay to transfer, in- transit mortality, length of stay, survival to discharge and 1-month and 6-month mortality.i p Reference 1. Towards Excellence in Critical Care. Review of adult critical care services in the Republic of Ireland fi nal report. Submitted to the Health Service Executive; September 2009. Admissions with airway emergencies in the ICU at a tertiary referral centre Eighteen were admitted post drainage of abscess (dental, retropharyngeal) and seven for observation for epiglottitis. Thirteen patients had stridor (three tracheal stenosis, one vocal cord cyst, one post CVA, four post vocal cord palsy, one post oesophagoscopy, one post thyroidectomy, two post decannulation). Seven were admitted after emergency tracheostomy, one after blocked tracheostomy, one after emergency laryngectomy, six post bleeding (epistaxis, haemoptysis, bleed form laryngectomy site), and four post evacuation haematoma. Three were admitted following anaphylaxis/ angioedema and one after laryngospasm. Twenty-nine patients required medical management only (for example, steroids, nebulisers, and so forth), 25 were extubated post diffi cult intubation and six needed haemostasis control. Ten (nine surgical) tracheostomies were performed during their ICU stay. Sixteen patients died in hospital, of whom fi ve were in the ICU at the time; 14 of these had an underlying malignancy. Twenty-three patients deteriorated during their ICU stay including HAP (n = 3), bleeding from airway (n = 3), PEA arrest (n = 1), airway swelling (n = 2), blocked laryngectomy (n = 1), and tracheostomy dislodgement (n = 1). 03 Availability of appropriate airway monitoring at UK in-hospital cardiac arrest S Turle1, PB Sherren1, T Callaghan1, S Nicholson2, SJ Shepherd1 1Pan-Thames Intensive Care Training Scheme, London, UK; 2KSS Intensive Care Training Scheme, London, UK Critical Care 2015, 19(Suppl 1):P203 (doi: 10.1186/cc14283) Introduction Airway complications are more common outside the operating theatre and in emergency situations. Capnography remains the gold standard of confi rming correct endotracheal tube (ETT) placement, retaining high sensitivity and specifi city in cardiac arrest [1]. The 2010 European Resuscitation Council guidelines for adult advanced life support recommended waveform capnography in this setting [2]. Failure to use capnography was also identifi ed as a major contributor to airway-related morbidity and mortality in a national UK audit [3]. We sought to investigate current practice relating to the availability and use of capnography equipment cardiac arrest within UK hospitals. Methods Between June and November 2014, a telephone survey was conducted of all UK acute hospitals with adult level 3 ICUs and an emergency department (ED). Hospitals were identifi ed using nationally available data. A standardised telephone questionnaire was developed Figure 1 (abstract P204). Insertion of a nasogastric tube through an endotracheal tube in the esophagus. S71 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 http://ccforum.com/supplements/19/S1 Methods A NG tube was inserted using a laryngoscope and endotracheal tube. Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes Centro Hospitalar Tâmega e Sousa, Penafi el, Portugal Critical Care 2015, 19(Suppl 1):P209 (doi: 10.1186/cc14289) Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes g Centro Hospitalar Tâmega e Sousa, Penafi el, Portugal Critical Care 2015, 19(Suppl 1):P209 (doi: 10.1186/cc14289) g g j p Results A total of 348 patients were included. VL attained better glottic visualization than DL (92.3% vs. 82.6%, respectively: P <0.001). In total, 299 patients with good glottic visualization were included in the analysis. Of these patients, 185 (61.9%) were male, median age and body mass index were 69 (interquartile range (IQR), 51 to 77) and 22 (IQR, 20 to 24) respectively. In univariate analysis, VL group had less respiratory failure (18.3% vs. 46.8%: P <0.001) and included more trauma patients (21.1% vs. 7.9%: P <0.001). The fi rst-attempt success rates were similar between two groups (82.6% vs. 77.4%: P = 0.286). Multivariable logistic regression analysis adjusted for potential confounders showed that the success rate of VL was similar to that of DL (odds ratio, 1.17; 95% confi dence interval, 0.57 to 2.39).i Introduction β-Adrenergic agonists increase the ciliary beat frequency in experimental models, raising the possibility that they may be useful for airway hygiene [1]. Salbutamol increases large airway mucociliary clearance [2], although this may not be true for smaller airways [3]. There are no data from ICU patients, so we decided to test the eff ectiveness of transtracheal instillation of a mixture of ipratropium and salbutamol, assessing the reduction of the number of aspirations and hours of ventilation. Methods An open randomized prospective study was held during 2014. All admitted patients were alternately selected as the study or control group and included if submitted to invasive ventilation for at least for 24 hours. Four patients who had secondary cardiac rhythm eff ects attributable to β-adrenergic agonists were excluded. In the study group, 3 ml of a dilution of 2.5 ml ipratropium (0.52 mg) plus salbutamol (2.5 mg) with 2.5 ml distilled water was instilled after the aspiration of secretions. During the ventilation period we noted for both groups, in addition to the demographic data, the number of tracheal aspirations by day and hours of ventilation. The statistical analysis was done with XLSTAT 2014 and we used the Kolmogorov– Smirnov test to compare the normal distribution of the groups. P210 Simultaneous use of a heat and moisture exchanger and a heated humidifi er causes critical airway occlusion in less than 24 hours M Mariyaselvam, A Doyle, G Wijewardena, N English, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P210 (doi: 10.1186/cc14290) p , g y , Critical Care 2015, 19(Suppl 1):P210 (doi: 10.1186/cc14290) q Results There were 62 RSI intubations using THRIVE (ICU and ED = 30; OR = 33). Diffi cult airway equipment used in 36 cases (videolaryngoscopy in 23). Mean apnoea time was 118 seconds (30 to 480 seconds), with a median SpO2 fall of 1% (0 to 33%). There was no correlation between arterial desaturation and apnoeic time. OR cases had a mean apnoea of 113 seconds with a median SpO2 fall of 0% (0 to 13%). ICU and ED cases had a mean apnoea time of 119 seconds and median SpO2 fall of 1% (0 to 33%). THRIVE was universally readily accepted. Reasons cited included: simplifi cation of pre-oxygenation (hands free) and increased confi dence. Six outlying arterial desaturation events suggested poor airway maintenance at induction or use in particularly high-risk patients. Many anaesthetists reinstituted THRIVE following extubation in selected patients (for example, obesity). No complications occurred during implementation. Introduction We hypothesised that the simultaneous use of a heat and moisture exchanger (HME) and a heated humidifi er (HH) would increase the incidence of airway occlusion over a 24-hour period in comparison with each device in isolation. This bench study compares the incidence of airway occlusion when using (group 1) no airway humidifi cation, (group 2) a HME alone, (group 3) a HH alone and (group 4) both a HME and a HH in combination. Tracheal intubation requires the use of artifi cial humidifi cation systems. HMEs are less effi cient but convenient especially for a short period of intubation and HHs are commonly more expensive. Both devices are often used in close proximity on the ICU depending on the particular clinical scenario and/ or clinical practitioner. Following a critical incident of HME obstruction due to waterlogging on our ICU we realised that HH and HME may be used together inadvertently. This airway obstruction was only resolved by the removal of the HME from the patient’s breathing circuit. Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction M Mariyaselvam, D Stolady, G Wijewardena, M Blunt, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P208 (doi: 10.1186/cc14288) Results These preliminary results included 107 patients. The only normal distribution, according to the Kolmogorov–Smirnov test, was for the number of hours of ventilation: 76.3 ± 100 (24; 478) in the study group versus 111 ± 167 (24; 780) in the control group.i Conclusion The preliminary analysis, referring to the fi rst 6 months of study, showed a tendency in the reduction of both ventilation hours and length of stay in the study group. No signifi cant diff erence was found in the number of aspirations, which may be explained by ICU nursing routines. Further studies will try to fi nd whether signifi cant diff erences in the incidence of VAP exist, allowing this procedure to be implemented in ICU routines. Introduction Rapid sequence induction (RSI) in the ICU, emergency department (ED) and operating room (OR) carries the risk of hypoxemia if laryngoscopy is prolonged especially in high-risk patients. Bag and mask pre-oxygenation is normally used to extend the apnoea time; however, arterial desaturation may still rapidly occur. Transnasal humidifi ed rapid insuffl ation ventilatory exchange (THRIVE) is a new technique that provides modest CPAP during pre-oxygenation and crucially also continuous oxygenation of the pharyngeal space throughout the apnoeic period. In elective surgery, THRIVE provides apnoea times as long as 60 minutes due to apnoeic oxygenation [1]. We report the fi rst implementation of THRIVE with emergency patients into the ICU, ED and OR. References 1. Frohock JI, Wijkstrom-Frei C, Salathe M. Eff ects of albuterolenantiomers on ciliary beat frequency in ovine trachealepithelial cells. J Appl Physiol. 2002;92:2396-402. 1. Frohock JI, Wijkstrom-Frei C, Salathe M. Eff ects of albuterolenantiomers on ciliary beat frequency in ovine trachealepithelial cells. J Appl Physiol. 2002;92:2396-402. 2. Lafortuna CL, Fazio F. Acute eff ect of inhaled salbutamol onmucociliary clearance in health and chronic bronchitis. Respiration. 1984;45:111-23. 2. Lafortuna CL, Fazio F. Acute eff ect of inhaled salbutamol onmucociliary clearance in health and chronic bronchitis. Respiration. 1984;45:111-23. 3. Svartengren K, Philipson K, Svartengren M, Camner P. Eff ect ofadrenergic stimulation on clearance from small ciliatedairways in healthy subjects. Exp Lung Res. 1998;24:149-58. Methods Following training a THRIVE system was installed in each location either as a fi xed system on the anaesthetic machine (OR) or a mobile solution on a wheeled stand (ICU, ER). This was a simplifi ed Optifl ow system (Fisher and Paykel, New Zealand) consisting of a high-fl ow rotameter, a reusable humidifi er, a reusable circuit and a disposable nasal interface. Anaesthetists of all grades were encouraged to use THRIVE (60 l/minute) prior to and during all high-risk intubations. Prospective data of pre and post intubation SpO2 and time to intubate were collected. Anaesthetists were interviewed on acceptability of the technique. 1. Patel A, et al. Anaesthesia. 2014 [Epub ahead of print]. Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes i Conclusion Despite the possible poor alignment of airway, the fi rst- attempt success rate of VL is similar to that of DL among patients with good glottic visualization. P210 Methods A lung simulator underwent pressure controlled ventilation Conclusion We conclude that THRIVE provides a convenient, safe and easy to implement technique for pre-oxygenation and apnoeic oxygenation during laryngoscopy. y p g Methods A lung simulator underwent pressure-controlled ventilation (Pinsp = 25 cmH2O; PEEP = 5 cmH2O; Vt = 500 ml) for 24 hours for seven P209 The primary exposure was use of VL. Potential confounders of success rate examined were age, sex, primary indication of intubation, methods of intubation, and operator level of training and specialty. Among patients with good glottic visualization, we conducted a multivariable logistic regression adjusted for potential confounders. Comparison of video laryngoscopy with direct laryngoscopy in patients with good glottic visualization: an observational study of 348 emergency intubations g y Y Kato, H Okamoto, H Uchino, T Fukuoka Kurashiki Central Hospital, Kurashiki Okayama, Japan Critical Care 2015, 19(Suppl 1):P207 (doi: 10.1186/cc14287) g y Y Kato, H Okamoto, H Uchino, T Fukuoka Introduction Video laryngoscopy (VL) is known to improve glottic visualization and the fi rst-attempt success rate compared with direct laryngoscopy (DL) in emergency tracheal intubations (ETIs). Since VL does not align the oral, pharyngeal, and laryngeal axes of the upper airway, it sometimes leads to failed intubation despite good glottic visualization. We tested the hypothesis that VL has a lower fi rst- attempt success rate of ETI than DL among patients with good glottic visualization. Methods We performed a prospective observational study examining ETIs at our tertiary care institution from July 2012 to June 2014. All consecutive patients who underwent ETIs in the emergency department and ICU were included. Patients under 18 years of age, intubated with VL not using C-MAC, were excluded. After each ETI eff ort, the operator completed a standardized data collection form. We classifi ed glottic visualization as good (C-L grade 1 or 2), and poor (C-L grade 3 or 4). The primary outcome was the fi rst-attempt success rate. Conclusion Marked variation was seen in the type and aetiology of airway emergencies admitted to the ICU. A broad training programme is thus required to off er wide-ranging awareness of potential problems, communication (including an emergency airway plan), and acute management. S72 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P211 P211 Endobronchial streptokinase for airway thrombus: a case series D Lloyd, J Bomford, M Barry, W Berry, N Barrett, L Camporota, N Ioannou, B Lams, C Langrish, C Meadows, A Retter, D Wyncoll, G Glover Guys and St Thomas’ NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P211 (doi: 10.1186/cc14291) Introduction Pulmonary haemorrhage (PH) is common in patients receiving mechanical ventilation and especially during ECMO, due to severe lung pathology and systemic anticoagulation. Whilst PH manifests as worsening ventilation and gas exchange, in ECMO patients who already have low tidal volume and who do not rely on pulmonary gas exchange, deterioration may not be evident until extensive airway thrombus (AT) has developed. Management of AT is challenging, with lavage, suctioning, mechanical disruption and extraction of limited effi cacy in severe cases. Limited reports suggest that topical thrombolytics may have a role in the management of AT [1]. We report the safety and effi cacy of endobronchial streptokinase (EBSK) in patients with extensive AT. Results Control group: 100 patients who received standard intubation and treatment in 2009 to 2010. Of these, four dropped out due screening failure of pneumonia on the day of enrollment. Study group: in 2011 to 2014, 192 patients were screened. Of these, 49 were found eligible and were enrolled. Of these, 14 dropped out (four screening failures with pneumonia on day of enrollment and 10 withdrawals with MV of less than 24 hours). Mean age was 51 (control) and 49 (study). Males were 75% of both groups and mean weight was 81 kg in both. VAP was diagnosed in 26 (27%) of controls and only three (8.5%) of the study group (P = 0.03). Mean time from admission to VAP diagnosis was 4.7 days in controls versus 5.12 in the study group (NS). No serious adverse events occurred. Methods A retrospective case series in a UK ECMO centre. Patients who received EBSK between 2010 and 2014 were identifi ed from pharmacy records. Conclusion Patients connected to the AnapnoGuard system demonstrated a statistically signifi cant lower VAP rate compared with the control group (8.5% vs. 27% respectively, P = 0.03). The estimated relative risk of VAP occurring in the control group was more than three times higher than the study group. Rinsing and aspiration of subglottic secretions combined with cuff pressure and seal management may be an eff ective method to prevent VAP. P211 Results Five patients were identifi ed, 80% were male. Median age was 40 years, APACHE II score 36.5 and Murray score 3.75. Four were on ECMO with systemic heparin. All had ARDS secondary to lung infections (community-acquired pneumonia (two), lung abscess (one), TB (one) and PJP (one)). All had extensive AT, diagnosed on bronchoscopy, causing occlusion of the trachea or major bronchi, refractory to physiotherapy, lavage, suctioning ± rigid bronchoscopy. Patients received up to three administrations of EBSK, 1,000 u/ml in saline 0.9% under bronchoscopic guidance. Dose per administration was 30,000 to 80,000 u and total dose was 30,000 to 150,000 u (375 to 1,500 u/kg), with interval bronchoscopy after several hours for lavage and suctioning of lysed clot. In all cases EBSK was well tolerated with no immediate complications and no clinically signifi cant change in systemic laboratory coagulation parameters at 12 or 24 hours compared with pretreatment baseline. In all cases, signifi cant clearance of airway thrombus was achieved. Median tidal volume increased from 60 ml pre treatment to 170 ml at 24 hours. Median PaO2 during the ‘FiO2 1.0 test’ improved from 9.0 to 17.6 kPa at 24 hours. No major bleeding, intracerebral haemorrhage or ECMO cannulae bleeding was seen up to 7 days post treatment.i P213 Risk factors for bleeding complications after percutaneous dilatational tracheostomy: a 10-year institutional analysis K Pilarczyk1, G Marggraf1, M Dudasova1, E Demircioglu1, D Wendt1, B Huschens2, H Jakob1, F Dusse1 1West German Heart Center Essen, University Hospital Essen, Germany; 2University Hospital Essen, Germany Critical Care 2015, 19(Suppl 1):P213 (doi: 10.1186/cc14293) Introduction Percutaneous dilatational tracheotomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. Bleeding complications after PDT are infrequently observed but have a tremendous impact on further clinical course CFA risk stratifi cation for patients scheduled for PDT. Introduction Percutaneous dilatational tracheotomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. Bleeding complications after PDT are infrequently observed but have a tremendous impact on further clinical course CFA risk stratifi cation for patients scheduled for PDT. Conclusion In this series, the largest reported to date, and the fi rst on ECMO, EBSK was highly eff ective in achieving clearance of AT with subsequent improvements in pulmonary mechanics and gas exchange. No major disturbance of systemic coagulation parameters or major haemorrhagic complications occurred. P212 test periods for each group (n = 24). A HME (Filta-Therm; Intersurgical, Berkshire, UK) was placed between the breathing circuit and catheter mount or the HH (MR850; Fisher & Paykel, Auckland, New Zealand) was used, or both in combination. Circuit manipulation was performed 4-hourly to simulate patient movement. Hourly Vt was recorded to determine airway occlusion. Critical airway occlusion (defi ned as a drop on the TV to <50 ml) was assessed using a Fisher’s exact test. 1. Keane et al. Chest. 1999;115:293-300. P211 The use of EBSK may be considered for refractory AT. Methods We retrospectively reviewed the records of all patients who underwent PDT (using the Ciaglia technique with bronchoscopic guidance) on our cardiothoracic ICU between 2003 and 2013. Patients were stratifi ed into two groups: patients suff ering from acute moderate, severe or major bleeding (Group A) and patients who presented none or only mild bleeding (Group B). y Reference S73 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Reduction of ventilator-associated pneumonia using the AnapnoGuard system Reduction of ventilator-associated pneumonia using the AnapnoGuard system p y Y Bar-Lavie Rambam Medical Center, Haifa, Israel Critical Care 2015, 19(Suppl 1):P212 (doi: 10.1186/cc14292) Introduction Ventilator-associated pneumonia (VAP) is a common complication in mechanically ventilated patients. Frequently the pathogens responsible derive from aspirated secretions of the upper respiratory tract or the stomach. In order to prevent aspiration, two missions should be attained: a good tracheal cuff seal with a well- tolerated pressure, together with continuous evacuation of secretions from the subglottic space. These two goals can be achieved using the AnapnoGuard system and its related endotracheal tube (ETT). Results In all seven of the breathing circuits in group 4 (both a HME and a HH in combination), critical airway occlusion occurred suddenly between 19 and23 hours. No episodes occurred in the other three groups (P <0.0001). g p Conclusion The combination of the use of HME and HH within a single ICU risks inadvertent dual use in a single patient and if uncorrected this is likely to result in a ventilator circuit obstruction. Medical errors can be mitigated by consideration of human factors and system engineering to improve patient safety. A focus on clinical awareness and training may lead to improvements; however, the numbers, experience and turnover of critical care staffi ng would indicate that a systems approach is appropriate and either HME or HH should be used exclusively in an ICU. y Methods A single-center, open-label study in a general ICU. Control group: (retrospective data) mechanically ventilated patients on standard of care regular ETT, manual suction of the trachea and oral– pharyngeal space by nursing staff . Study group: (prospective data) connected at all times to the AnapnoGuard system: an ETT with two above-the-cuff suction ports and a third port and lumen for rinsing and CO2 measurement. A triple lumen harness is connected to a control system designed to measure CO2 levels above the cuff (to identify leaks), infl ate the cuff accordingly, rinse and suction secretions above the cuff . To be included in the study patients had to have no pneumonia on admission and at least 3 days of mechanical ventilation. VAP was diagnosed for a new chest X-ray infi ltrate accompanied by fever, leucocytosis and positive sputum culture. The study was approved by the hospital IRB. Reference Study groups showed signifi cant diff erences in Simplifi ed Acute Physiology Score (SAPS) on the day of PDT (Group A: 47.0 ± 13.1, Group B: 32.9 ± 11.2, P = 0.042), renal replacement therapy on the day of PDT (Group A: 53 (60.2%), Group B: 439 (48.1%), P = 0.026), presence of coagulopathy (Group A: 48 (54.5%), Group B: 393 (43.0%), P = 0.043), platelet count (Group A: 91.6 ± 59.2, Group B: 111.5 ± 79.8 × 1,000/μl, P = 0.037), fi brinogen levels (Group A: 373.6 ± 159.1, Group B: 450.6 ± 259.0 mg/dl, P = 0.012), proportion of PDTs performed by residents (Group A: 72 (81.8%), Group B: 632 (69.2%), P = 0.034) and moderately to very diffi cult PDT (Group A: 31 (35.2%), Group B: 141 (15.4%), P = 0.001). Using logistic regression analyses, diffi cult PDT, low-experienced operator, SAPS >40 and low fi brinogen were independent predictors of relevant bleedings after PDT. Methods The study was conducted in a Danish eight-bed, non- university ICU. Since 2007, all patients admitted to the ICU have been registered on an electronic patient record system, in which daily vital values, diagnoses, procedures and healthcare providers’ notes are entered. When searching for ‘percutaneous dilatation tracheostomy’ in the electronic system, we found all patients who had undergone this specifi c procedure. Afterwards we analyzed each of these patients’ hospital records, looking for any periprocedure or postprocedure complications noted within 7 days. In addition we registered patients’ age, sex, BMI, SOFA score, methods used in procedures and experience of operators. p Results A total of 136 patients admitted to the UCI had undergone a PDT between 2007 and 2014. Of these, two were excluded due to the PDT being performed in another hospital before admission to our ICU. All 134 PDTs were performed with the Ciaglia Blue Rhino Method. No PDTs were performed with bronchoscopic guidance. In 12 cases some kind of complication due to the PDT was registered: six cases with need of surgical hemostasis, three cases of bleeding with need of transfusion of blood products, one case of PDT displacement, one case of ventilation-related problems during procedure and, fi nally, one case of tracheal cartilage fracture. There were no incidents of pneumothorax. No PDTs had a lethal outcome due to the procedure itself. The total complication rate was 9.0%. Reference Results A total of 1,001 patients (46% male, mean age 68.1 years) that underwent PDT were analyzed. In the majority of patients, no or S74 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of PDT are described in the literature, each with disadvantages and benefi ts. The aim of this study was to analyze complications due to PDTs performed without the use of bronchoscopic assistance. only mild bleeding during PDT occurred (none: 425 (42.5%), mild: 488 (48.8%)). In 84 patients (8.4%), bleeding was classifi ed as moderate. Three patients suff ered from severe bleeding, only one major bleeding with need for emergency surgery was observed. Study groups showed signifi cant diff erences in Simplifi ed Acute Physiology Score (SAPS) on the day of PDT (Group A: 47.0 ± 13.1, Group B: 32.9 ± 11.2, P = 0.042), renal replacement therapy on the day of PDT (Group A: 53 (60.2%), Group B: 439 (48.1%), P = 0.026), presence of coagulopathy (Group A: 48 (54.5%), Group B: 393 (43.0%), P = 0.043), platelet count (Group A: 91.6 ± 59.2, Group B: 111.5 ± 79.8 × 1,000/μl, P = 0.037), fi brinogen levels (Group A: 373.6 ± 159.1, Group B: 450.6 ± 259.0 mg/dl, P = 0.012), proportion of PDTs performed by residents (Group A: 72 (81.8%), Group B: 632 (69.2%), P = 0.034) and moderately to very diffi cult PDT (Group A: 31 (35.2%), Group B: 141 (15.4%), P = 0.001). Using logistic regression analyses, diffi cult PDT, low-experienced operator, SAPS >40 and low fi brinogen were independent predictors of relevant bleedings after PDT. only mild bleeding during PDT occurred (none: 425 (42.5%), mild: 488 (48.8%)). In 84 patients (8.4%), bleeding was classifi ed as moderate. Three patients suff ered from severe bleeding, only one major bleeding with need for emergency surgery was observed. Percutaneous dilatational tracheostomy: complications and safety without the use of bronchoscopic guidance Sygehus Lillebælt, Vejle, Denmark Sygehus Lillebælt, Vejle, Denmark yg j Critical Care 2015, 19(Suppl 1):P214 (doi: 10.1186/cc14294) Reference Of the 12 cases, four (33%) complications occurred during the procedure, the rest (66%) occurred after the procedure. The overall periprocedure complication rate was 3%. Conclusion In this study, PDTs without the use of bronchoscopic guidance were performed safely with a low rate of complications. Conclusion Periprocedural bleeding complications during PDT are rare. However, low fi brinogen levels as well as diffi cult PDT, low-experienced operator and SAPS >40 are associated with an increased risk for bleeding complications. Therefore, preprocedural risk evaluation for bleeding complications should include these factors and further studies are necessary to prove whether modifi cation of risk factors – for example, substitution of fi brinogen prior to PDT – is able to reduce incidence of bleeding complications. P215 Introduction Since the introduction and development of percu- taneous dilatational tracheostomy (PDT), this procedure is accepted and incorporated in ICUs worldwide. In spite of obvious benefi ts for the patients, who obtain more comfort and mobility and less use of sedatives, the procedure also implies the risk of several complications, some of which may be lethal. Severe complications include hemorrhage, displacement and pneumothorax. Diff erent methods P216 Impact of antibiotic therapy during a bedside percutaneous tracheotomy procedure in an ICU E Brotfain1, A Borer1, L Koyfman1, A Frenkel1, S Gruenbaum2, A Smolikov1, A Zlotnik1, M Klein1 1Ben Gurion University of The Negev, Soroka Medical Center, Beer Sheva, Israel; 2Yale University School of Medicine, New Haven, CT, USA Critical Care 2015, 19(Suppl 1):P216 (doi: 10.1186/cc14296) Results In total 12,839 records of patients discharged from the ICU were analyzed. Tracheostomy was present in 133 patients. Two groups were defi ned: (1) TCU (n = 56) and (2) GW (n = 77). Patients of the TCU group were older (60.1 ± 13.1 vs. 54.9 ± 15.8 years; P <0.05) with higher APACHE II score (23 (CI: 21.5 to 25.6) vs. 18.5 (CI: 17.1 to 19.9); P <0.001), and had longer stay in the ICU (45.8 (CI: 38.2 to 53.3) vs. 28.4 (CI: 24.2 to 32.6) days; P <0.001) and on the ward (71.1 (CI: 57.4 to 84.8) vs. 46.1 (CI: 33.7 to 58.2) days; P <0.001) than those of the GW group. The GW group had category 1 of Sabadell score more frequently than the TCU group (25.9% vs. 8.9%; P = 0.019). Rates of nosocomial infections were similar in both groups. No signifi cant diff erences on vasoactive use (50% vs. 40.2%), renal failure (23.2% vs. 20.7%), blood transfusions (25% vs. 23.2%), parenteral nutrition (10.7% vs. 12.9%), in-hospital deaths (14.3% vs. 24.6%), decannulation (55% vs. 50%), or discharge to home (53.6% vs. 37.7%) were found between groups 1 and 2, respectively. Introduction Percutaneous bedside tracheostomy (PBT) is a frequently done procedure in the ICU. PBT is a clean-contaminated procedure, and the duration of the procedure is 15 to 20 minutes depending on the physician’s procedural skills. The rate of infectious complications and effi cacy of perioperative therapy in reducing infections after PBT is currently unknown. Currently there have been no defi nitive recommendations for prophylactic antibiotic therapy before PBT in the ICU. Methods All clinical and microbiological data were retrospectively collected and analyzed during the ICU stay before PBT performance and 72 hours after the PBT procedure from 110 patients in our ICU. Controls were defi ned as patients in whom the PBT procedure was performed in the ICU, with antibiotics administered 72 hours prior to and during the procedure (Group 1, n = 82). P216 Cases were defi ned as patients in whom the PBT procedure was performed in the ICU without antibiotics administered 72 hours prior to and during the procedure (Group 2, n = 28). Secondary bacteremia, line sepsis and VAP during the 72 hours after PBT were considered infectious complications. Two- tailed P <0.05 was considered to be signifi cant.f Conclusion In our setting the TCU helps to care more safely for severe tracheostomized patients after ICU discharge, and furthermore facilitates discharge home. References 1. Martinez GH, et al: Respir Care. 2009;54:1644-52. 1. Martinez GH, et al: Respir Care. 2009;54:1644-52. 2. Fernandez R, et al: Crit Care Med. 2011;39:2240-5. 2. Fernandez R, et al: Crit Care Med. 2011;39:2240-5. i Results No diff erences were found in age, gender, admission diagnoses, length of ICU stay and in-hospital mortality rate between the two study groups. Overall Gram-negative, Gram-positive and fungal fl ora were Reference Reference Methods We retrospectively reviewed medical records of ICU adults patients who had TQ when discharged to a new TCU and to a GW. The study was carried out in two tertiary care university hospitals from January 2007 to November 2014. Study variables were age, sex, APACHE II score, principal diagnosis, associated major procedures, length of stay in ICU and out in hospital, TCU and GW, Sabadell score, in-hospital mortality, types of tracheotomy procedure, decision to decannulate and discharge to home or long-care facilities. 1. Kollig E, et al. Injury. 2000;31:663-8. 1. Kollig E, et al. Injury. 2000;31:663-8. 1. Kollig E, et al. Injury. 2000;31:663-8. Decision-making algorithm for TS in the ICU Second University of Naples, Italy Critical Care 2015, 19(Suppl 1):P215 (doi: 10.1186/cc14295) Introduction Nowadays more percutaneous dilatational tracheostomy (PDT) methods are in use, but there is no ideal risk-free technique. We Figure 1 (abstract P215). Figure 1 (abstract P215). Figure 1 (abstract P215). S75 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 similar in both groups before and after PBT. Patients who received antibiotic therapy had a lower incidence of new ventilator-associated pneumonia (VAP) episodes (15/82 (18.2%) in Group 1 vs. 14/28 (50%) in Group 2, P <0.001 (0.23, 0.87 to 0.13)) (Table 1). There were no diff erences in the incidence of bacteremia or line sepsis (Table 1). Conclusion Our fi ndings highlight the importance of conducting a prospective randomized control trial to better understand the role of antibiotic prophylaxis in PBT. have outlined a decisional algorithm to choose the most appropriate technique in each case to reduce the incidence of complications. have outlined a decisional algorithm to choose the most appropriate technique in each case to reduce the incidence of complications. Methods A retrospective review was performed using data from the last 14 years. Two hundred patients were selected. Patients were divided into two groups: one including the fi rst 100 PDTs treated without the algorithm (nA-group) and the other including the last 100 patients treated with the algorithm (A-group). Valuation of clinical and anatomical features of the patients, neck ultrasound and fi brobronchoscopy came before the procedure [1]. The algorithm was formulated by our experience with PDT techniques, comparing the specifi c characteristics of each one with the physiopathological characteristics of each patient. Conclusion Our fi ndings highlight the importance of conducting a prospective randomized control trial to better understand the role of antibiotic prophylaxis in PBT. P217 P217 Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards J Rubio1, JA Rubio2, E Palma2, R Sierra1, F Carmona1, F Fuentes2 1Hospital Universitario Puerta del Mar, Cadiz, Spain; 2Hospital Infanta Cristina, Badajoz, Spain Critical Care 2015, 19(Suppl 1):P217 (doi: 10.1186/cc14297) Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards Results We recorded complications (bleeding, tracheoesophageal fi stula, subglottic stenosis, tracheal rings’ fracture, diffi culty of placement, change of procedure) related to PDTs performed with and without applying the algorithm. We considered complications that occurred in our experience and we changed our modality in technique choice (Figure 1). Compared with the complications reported in the nA-group, use of the algorithm as a guide to choose the kind of PDT technique seems to reduce the incidence of complications (37% vs. 19%; P = 0.001 chi-square test). Introduction Patients with a tracheostomy (TQ) tube in place discharged from the ICU to a general ward (GW) are a fragile group and the TQ may be a risk factor for morbidity and mortality [1,2]. For this reason they need closer monitoring and more airway care. The Transitional Care Unit (TCU) assists patients with serious medical conditions and bridges the gap between the ICU and home or long-term care facilities providing the necessary medical, nursing, psychological and rehabilitative care. The purpose of this study was to evaluate the impact of the TCU on outcomes of tracheostomized patients discharged from the ICU. Conclusion In our experience the application of the proposed algorithm may reduce the incidence of complications related to PDT in the ICU. However, a randomized controlled multicenter study would be necessary in order to confi rm the effi ciency and validity of the proposed algorithm. P218 Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT procedure Infection Group 1 Group 2 P value OR Bacteremia 21/82(25.6%) 11/28(39.3%) 0.149 Colonization 67/82(82.7%) 23/28(82.1%) 0.718 Line sepsis 10/82 (12%) 4/28 (14.3%) 0.39 New VAP (%) 15/82(18.2%) 14/28 (50%) 0.001 Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT procedure Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT d Introduction Incidence of obesity in developed countries is rising. Currently, Europe has a prevalence of 9 to 30% with signifi cant impact on public health systems. Obese patients in the ICU require special management and treatment. Altered anatomy in obese patients complicates procedures such as mechanical ventilation. Obesity aff ects cardiopulmonary physiology and requires elevated ventilation pressures. In our retrospective study, we determined the eff ect of early S76 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefi ts may be underestimated. Conclusion There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefi ts may be underestimated. percutaneous dilatational tracheotomy (PDT) and cessation of sedation on respiratory parameters in severely obese patients. percutaneous dilatational tracheotomy (PDT) and cessation of sedation on respiratory parameters in severely obese patients. p y p y p Methods From June 2010 to July 2014, we included all patients with a body weight of >130 kg, respiratory failure and PDT who were admitted to the ICU of the University Hospital of Muenster. We compared respirator parameters and blood gas analysis before and after PDT. Parameters were recorded on days –1, 0, 1, 3, 5, and 10, with day 0 describing the day of PDT. P220 P220 Is the gastric tube a burden for noninvasive ventilation? I Minev, C Stefanov University Hospital ‘St. Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H K d J N i J S id l Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H Kennedy, J Navein, J Seidel Doncaster Royal Infi rmary, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P219 (doi: 10.1186/cc14299) H Kennedy, J Navein, J Seidelifi y Doncaster Royal Infi rmary, Sheffi eld, UK Methods In this study, six of the COPD patients admitted to our ICU, who required NGT placement, were ventilated with the Draeger Evita 2 dura through a modifi ed reusable silicone face mask (UMDNS code: 12-453 with 22 mm ID connection; sizes 4 and 5) with silicone headgear and a hook ring. All of them had a NGT during their stay in the ICU. We evaluated the effi cacy of our modifi cation comparing the achieved Vt with modifi ed and unmodifi ed face mask, during two periods of 10 minutes. The mode and parameters of ventilation were not changed. We assessed patient comfort with a visual analogue scale. Introduction Respiratory failure is a well-known complication of aortic aneurysm surgery. We describe the impact of a protocol, using CPAP after elective surgery to reduce the need for unplanned invasive ventilation. Methods In 2012 we introduced a CPAP protocol for patients undergoing elective aortic aneurysm surgery, either open (AAA) or as an endovascular repair (EVAR). According to pre-existing risk factors (see Table 1) and arterial blood gas analysis in the anaesthetic room, they were assigned to two alternative options on the ITU: prophylactic CPAP for 9 hours in each of the fi rst two postoperative nights or oxygen via face mask. CPAP was applied at any time in the patients stay, if their P/F ratio dropped below 40. Criteria to stop CPAP were also predefi ned. Previously, CPAP was initiated at the discretion of nursing staff , P/F ratios were not utilised. p g Results The average duration of NIV was 3.5 days (SD = 1.6). We examined two sets of 10 consequent breathing cycles for every patient. The mean Vt was 472 ml (SD = 76 ml) with standard face mask and 460 ml (SD = 86 ml) with the modifi ed one. There was statistically signifi cant correlation between the two datasets (P <0.05). No additional leaks were detected. According to the VAS evaluation, fi ve of the patients (83%) had comfort improvement with the modifi ed mask. Table 1 (abstract P219). Criteria for the use of prophylactic CPAP FEV1: <1.5 l/minute Poor exercise tolerance (<100 yards) due to chest problems Poor exercise tolerance (<100 yards) due to chest problems SpO2 <92% on FiO2 >0.4 in theatre SpO2 <92% on FiO2 >0.4 in theatre Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H K d J N i J S id l Conclusion With this modifi cation of the face mask we achieved adequate drainage of the stomach and/or the enteral nutrition of the patients and improvement in their comfort during NIV, compared with the ventilation with a standard mask, without additional air leaks and at a low cost. Table 1 (abstract P219). Criteria for the use of prophylactic CPAP P218 Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) George’, Plovdiv, Bulgaria Critical Care 2015, 19(Suppl 1):P220 (doi: 10.1186/cc14300) y g y Results Twenty-one patients were included in the study. Mean age was 56 ± 10.3 years and 14 (66.7%) of the patients were male. Body weight was 164.5 ± 39.4 kg, body height accounted for 176.8 ± 8.7 cm (n = 20) and body mass index was 49.7 ± 16.9 kg/m2. Patients stayed in the ICU for 18.4 ± 13.8 days. Mean time of mechanical ventilation by endotracheal tube was 2.4 ± 1.5 days (n = 20) and via tracheostomy 9.8 ± 7.0 days. After PDT, peak inspiratory pressure (P <0.0001), positive end-expiratory pressure (P <0.0001) and insuffl ated oxygen concentration (P <0.0001) could signifi cantly and rapidly be reduced. Respiratory minute volume increased signifi cantly (P = 0.004). PDT was not associated with relevant complications. Introduction The application of noninvasive ventilation (NIV) in ICUs has spread widely during the years. It is used in the treatment of diff erent forms of acute respiratory failure and COPD exacerbations. Although NIV is thought to be more comfortable for patients than invasive mechanical ventilation, its failure rates in the ICUs range between 10 and 40%. Except for the interface-related problems, there are some specifi c considerations for the patient–ventilator interaction and the applied mechanical forces. During NIV there is a predisposition for the stomach to be infl ated with gas, which could cause severe respiratory complications, especially in COPD patients, and thus prolong the mechanical ventilation and the weaning process. This remains one of the major causes for NIV failure. Although a lot of face masks with diff erent interfaces are available on the market, just a few have additional ports for a NGT. They are characterized by higher price and a complex setup. In order to perform NIV in patients, requiring NGT placement, without additional air leaks and to be able to ensure their enteral nutrition and/or stomach drainage, we installed a port for a NGT on a standard face mask. Conclusion Early PDT rapidly improves respiratory distress in severely obese patients due enabling of spontaneous breathing and reduction of dead space ventilation. Lung ultrasound aeration assessment: comparison of two techniques S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) p p Methods The authors prospectively enrolled 17 patients with septic shock who were admitted to the medical ICU, Phramongkutklao Hospital between September 2013 and June 2014. EVLWI was measured by TPTD (VolumeView Set, EV1000; Edwards Lifesciences) method. According to international evidence-based recommendations for point-of-care lung ultrasound 2012, three methods of LUS (LOGIQ e ultrasound; GE Healthcare) were compared to assess EVLW daily in each patient until no indication for invasive blood pressure monitoring [1]. Firstly, B-lines were measured in 28 lung zones. The total numbers of B-lines seen in each patient were counted as total B-line scores (TBS). Secondly, upper and lower BLUE points were anterior two-region scans each side marked by physician hands. Pulmonary edema was diagnosed if three or more B-lines were presented in all regions. Lastly, scanning eight regions, two anterior and two laterals per side, was considered abnormal if more than one scan per side had three or more B-lines.i Introduction Lung ultrasound (LUS) allows semiquantifi cation of lung aeration in PEEP trials [1], pneumonia [2] and weaning [3]. LUS score is based on number/coalescence of vertical artifacts (B-lines) in longitudinal scan (LONG) [4]: the pleura is identifi ed between two ribs and its visualization limited by intercostal space (ICS) width. We hypothesized that a transversal scan (TRANSV) aligned with ICS would visualize longer pleura and a higher number of artifacts, with better assessment of loss of aeration (LoA). Methods LONG and TRANSV were performed in six areas per lung (anterior, lateral and posterior, each divided into superior and inferior). Once LONG was performed, TRANSV was obtained by a probe rotation until the ribs disappeared. We considered pleural length, B-line number/coalescence, and subpleural/lobar consolidations. P221 P221 Lung ultrasonography as a marker of pulmonary edema in cardiac surgery patients: visual versus quantitative evaluation F Corradi1, C Brusasco2, T Manca3, F Nicolini3, F Nicosia1, T Gherli3, V Brusasco2, A Vezzani3 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria di Parma, Italy Critical Care 2015, 19(Suppl 1):P221 (doi: 10.1186/cc14301) Results We compare patient cohorts in the years 2010 and 2011 (pre protocol) with 2013 and 2014 (post protocol). Results are reported as the split between open surgery and endovascular repair. Table 2 presents requirements for invasive ventilation (IPPV) and length of stay (LOS) for both patient groups. Table 2 (abstract P219). IPPV requirements and length of stay data Table 2 (abstract P219). IPPV requirements and length of stay data 2010 to 2011 2013 to 2014 LOS LOS LOS LOS ITU hospital ITU hospital IPPV (days) (days) IPPV (days) (days) EVAR 2/67 (3%) 2 6 1/77 (1.3%) 2 5 AAA 10/46 (21.7%) 5 9 6/37 (16.2%) 5 12 Introduction Lung ultrasonography (LUS) has been used for non- invasive detection of pulmonary edema. LUS visual scores (V-LUS) based on B-lines are poorly correlated with either pulmonary capillary wedge pressure (PCWP) or extravascular lung water (EVLW). A new quantitative LUS analysis (Q-LUS) has been recently proposed [1,2]. The aim of the study was to investigate whether Q-LUS is better correlated with PCWP and EVLW than V-LUS, and to what extent positive end- expiratory pressure (PEEP) aff ects the assessment of pulmonary edema by Q-LUS and V-LUS. S77 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Thirty-nine patients mechanically ventilated with PEEP of 5 cmH2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.i Methods Thirty-nine patients mechanically ventilated with PEEP of 5 cmH2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.i versus 3.9 ± 0.1 cm (range 3.0 to 4.3; variance 0.1) (P <0.0001), B-lines per scan were 1.1 ± 1.6 versus 1.8 ± 2.5 (P <0.0001), coalescent B-lines were detected in 24 versus 30% (P <0.05) and subpleural consolidations in 16 versus 22% (P <0.05), respectively. LUS scores’ prevalence signifi cantly diff ered in LONG versus TRANSV (Figure 1). P221 Results PCWP was signifi cantly and strongly correlated with Q-LUS Grey Unit value (r2 = 0.64) but weakly with V-LUS B-line score (r2 = 0.19). EVLW was signifi cantly and strongly correlated with QLUS Grey Unit mean value (r2 = 0.65) more than with V-LUS B-line score (r2 = 0.42). Q-LUS showed a better diagnostic accuracy than V-LUS for the detection of PCWP >15 mmHg or EVLW >10 ml/kg. With a PEEP of 5 cmH2O, the correlations with PCWP or EVLW were stronger with Q-LUS than V-LUS. With a PEEP of 10 cmH2O, the correlations with PCWP or EVLW were still signifi cant for Q-LUS but insignifi cant for V-LUS. Intraobserver repeatability and interobserver reproducibility were much better for Q-LUS than V-LUS. f Conclusion TRANSV visualizes signifi cantly longer pleura and greater number of artifacts useful for lung disease assessment. References f Conclusion TRANSV visualizes signifi cantly longer pleura and greater number of artifacts useful for lung disease assessment. References 1. Bouhemad B, et al. Am J Resp Crit Care Med. 2011;183:341-7. 2. Bouhemad B, et al. Crit Care Med. 2010;38:84-92. 3. Soummer A. et al. Crit Care Med. 2012;40:264-72. 4. Volpicelli G. et al. Int Care Med. 2012;38:577-91. Lung ultrasound aeration assessment: comparison of two techniques LUS score was assigned: 0 normal lung, 1 moderate LoA (≥3 well-spaced B-lines), 2 severe LoA (coalescent B-lines), 3 complete LoA (tissue-like pattern). Results We enrolled 38 patients (21 males, age 60 ± 16 years, BMI 24.7 ± 4.7 kg/m2) corresponding to 456 ICSs. In 63 ICSs, a tissue-like pattern was visualized in both techniques. In the other 393, LONG versus TRANSV pleural length was 2.0 ± 0.6 cm (range 0.8 to 3.8; variance 0.31) Results A total of 40 comparisons were obtained. Signifi cant positive linear correlations were found between TBS and EVLWI determined by TPTD (r = 0.637, P <0.001). The TBS ≥39 has sensitivity of 91.7% and specifi city of 75.0% to defi ne EVLWI >10 ml/kg. There was low sensitivity (33.3% and 50.0% respectively) but high specifi city (100% and 96.0% respectively) of the positive BLUE points and eight regions to defi ne EVLWI >10 ml/kg. Figure 1 (abstract P222). LUS scores. P <0.01 TRANSV versus LONG. i Conclusion TBS is the best method for assessing EVLW compared with BLUE points and eight regions. These data support the benefi t of LUS with summation of B-line scores of 28 rib interspaces for assessment of the increment of EVLW in septic shock patients. f Reference Reference 1. Volpicelli G, et al. Intensive Care Med. 2012;38:577-91. 1. Volpicelli G, et al. Intensive Care Med. 2012;38:577-91. Ultrasound assessment for extravascular lung water in patients with septic shock Conclusion Both V-LUS and Q-LUS are acceptable indicators of pulmonary edema in patients mechanically ventilated with low PEEP but at high PEEP only Q-LUS provides data that are signifi cantly correlated. Computer-aided Q-LUS has the advantages of being not only independent of operator perception but also of PEEP. References P Pirompanich1, A Wattanathum2 1Thammasat University, Pathumthani, Thailand; 2Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P223 (doi: 10.1186/cc14303) 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. Introduction Extravascular lung water (EVLW) refers to fl uid within the lung but outside the vascular compartment. Increment of EVLW was associated with mortality in critically ill patients. Extravascular lung water index (EVLWI) >10 ml/kg was found in patients with cardiogenic pulmonary edema and correlated with pulmonary capillary wedge pressure >20 mmHg. Measurement of EVLW needs sophisticated tools and an invasive method by transpulmonary thermodilution (TPTD) technique. In contrast, multiple B-lines by lung ultrasound (LUS) have been recently proposed to correlate with increased EVLW in patients with pulmonary edema. This study aims to compare three methods of LUS and EVLWI measured by TPTD to assess pulmonary edema in patients with septic shock. bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) Introduction One of the aims of lung recruitment is to improve oxygenation [1], but it has not yet been investigated in spontaneously breathing patients. Our objective was to evaluate the eff ects of recruitment maneuvers on oxygenation in patients ventilated in CPAP/ pressure support (CPAP/PS) mode. p pp Methods In a prospective, observational study, 30 patients with a Lung Injury Score ≥2 were recruited. Following baseline measurements (t0) PEEP was increased by 5 cmH2O (t1). Recruitment maneuver was applied for 40 seconds with 40 cmH2O PS. Measurements were taken immediately after recruitment (t2) then 15 minutes (t3) and 30 minutes later (t4).f 2 3 4 Results According to the diff erence of PaO2/FiO2 between t2 and t0, three groups were defi ned: nonresponders (NR: diff erence of PaO2/FiO2 ≤0%, n = 8), low responders (LR: diff erence of PaO2/FiO2 = 0 to 50%, n = 11) and high responders (HR: diff erence of PaO2/FiO2 >50%, n = 11). In the NR-group, PaO2/FiO2 decreased signifi cantly: median (interquartile), PaO2/FiO2 = 178 (159 to 240) versus 165 (118 to 210) mmHg; in the LR- group and in the HR-group there was signifi cant improvement: 119 (98 to 164) versus 161 (123 to 182) mmHg and 141 (130 to 183) versus 239 (224 to 369) mmHg, P <0.05, respectively. Dynamic compliance (Cdyn) signifi cantly dropped at t2 as compared with t0 in the NR-group, Cdyn = 62 (48 to 87) versus 53 (43 to 78) ml/cmH2O, while there was no signifi cant change in the LR- and HR-groups, P <0.05. At the same time points the dead space to tidal volume ratio (Vds/Vte) signifi cantly increased in the NR-group, Vds/Vte = 30 (23 to 37) versus 37 (26 to 42)%, but not in the LR- and HR-groups, P <0.05. Figure 1 (abstract P224). Sample ultrasound recording. Lines 1 and 2 are diaphragm thickness measurements. Results We were successfully able to record the diaphragm thickness in all included patients. Median time on the ventilator was 9 days (IQR 4 to 15 days). Mean baseline thickness was 1.9 mm (SD ±0.4 mm), and mean nadir was 1.3 mm (SD ±0.4 mm), corresponding with a mean change in thickness of 32% (SD ±18%). As early as after only 72 hours of MV, we already noted an average drop of diaphragm thickness of 20%, illustrating the rapid progression of the atrophy in VIDD. P226f Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) References 1. Grosu H, et al. Chest. 2012;142:1455-60. Conclusion Recruitment maneuvers improved PaO2/FiO2 in the majority of patients (73%) without aff ecting Cdyn or Vds/Vte; therefore it may be a safe approach to improve oxygenation in patients ventilated in CPAP/PS mode. 1. Grosu H, et al. Chest. 2012;142:1455-60. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) Conclusion On average, diaphragm thickness decreased 32% in our cohort. The decrease occurred rapidly, with two-thirds of the maximal thinning already present after 72 hours of MV. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemithorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography. Methods Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemithorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography. diaphragmatic atrophy can be assessed using ultrasound, the biggest trial in humans published so far included seven patients and only measuring the thickness at two moments during the disease process [1]. We aimed to assess the time course of diaphragm atrophy in a larger cohort of MV patients using ultrasound. y Results Quantitative ultrasonography showed higher sensitivity (93%), specifi city (95%), and diagnostic accuracy (94%) than chest X-ray (64%, 80%, and 69%, respectively), or visual ultrasonography (68%, 95%, and 77%, respectively), or their combination (77%, 75%, and 77%, respectively). Methods A total of 54 patients from an adult ICU were included in this prospective single-centre cohort trial. Patients who needed <72 hours of MV or had been recently admitted to an ICU were excluded. Patients were ventilated in a controlled, assisted, and/or hybrid ventilation mode. The thickness of the diaphragm was assessed daily; the fi rst recording was within 24 hours after the start of mechanical ventilation and we continued the measurements until the patients were extubated or tracheotomised. We measured the diaphragm at the zone of apposition, as described by McCool and colleagues [2] using a linear 13 MHz ultrasound probe. Figure 1 shows a sample measurement. y Conclusion Quantitative lung ultrasonography was considerably more accurate than either chest X-ray or visual ultrasonography in the diagnosis of community-acquired pneumonia and it may represent a useful fi rst-line approach for confi rmation of clinical diagnosis in emergency settings. Quantitative ultrasonography for pneumonia F C di1 C B 2 T M 3 F Ni i 1 A V 1. Lachman B. Intensive Care Med. 1992;18:319-21. y F Corradi1, C Brusasco2, T Manca3, F Nicosia1, A Vezzani3, V Brusasco2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria, Parma, Italy Critical Care 2015, 19(Suppl 1):P225 (doi: 10.1186/cc14305) F Corradi1, C Brusasco2, T Manca3, F Nicosia1, A Vezzani3, V Brusasco2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria, Parma, Italy Critical Care 2015, 19(Suppl 1):P225 (doi: 10.1186/cc14305) References Figure 1 (abstract P224). Sample ultrasound recording. Lines 1 and 2 are diaphragm thickness measurements. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. g p y 2. Quanta Imaging Technology. www.quanta.camelotbio.com. A better way to determine sample size to detect changes in length of mechanical ventilation? A better way to determine sample size to detect changes in length of mechanical ventilation? YS Chiew1, C Pretty1, D Redmond1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P227 (doi: 10.1186/cc14307) Atrophy of diaphragm muscle visualized with ultrasound in mechanically ventilated patients T Schepens, M Mergeay, W Verbrugghe, P Parizel, M Vercauteren, PG Jorens Antwerp University Hospital, Edegem, Belgium p y p , g , g Critical Care 2015, 19(Suppl 1):P224 (doi: 10.1186/cc14304) Introduction Mechanical ventilation (MV) induces diaphragmatic muscle atrophy and contractile fi bre dysfunction, the so-called ventilator-induced diaphragm dysfunction (VIDD). Although Figure 1 (abstract P222). LUS scores. P <0.01 TRANSV versus LONG. S78 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 A better way to determine s of mechanical ventilation? Introduction Chest-X-ray is recommended for routine use in patients with suspected pneumonia, but its use in emergency settings is limited. In this study, the diagnostic performance of a new method for quantitative analysis of lung ultrasonography was compared with bedside chest X-ray and visual lung ultrasonography for detection of community-acquired pneumonia, using thoracic computed tomography as a gold standard. YS Chiew1, C Pretty1, D Redmond1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P227 (doi: 10.1186/cc14307) Introduction Estimation of eff ective sample size (N/arm) is important to ensure power to detect signifi cant treatment eff ects. However, S79 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 monitoring of the eff ect of benzodiazepines on respiratory status in non-intubated patients has been diffi cult, putting patient safety at risk. A non-invasive respiratory volume monitor (RVM) that provides continuous measurement of minute ventilation (MV), tidal volume (TV) and respiratory rate (RR) was used to quantify the eff ects of midazolam on respiratory status in spontaneously breathing patients. traditional parametric sample size estimations depend upon restrictive assumptions that often do not hold in real data. This study estimates N to detect changes in length of mechanical ventilation (LoMV) using Monte-Carlo simulation (MCS) and mechanical ventilation (MV) data to better simulate the cohort. Methods Data from 2,534 MV patients admitted to Christchurch Hospital ICU from 2011 to 2013 were used. N was estimated using MCS to determine a sample size with power of 80%, and compared with the Altman’s nomogram for two patients groups, (1) all patients and (2) targeted patients with 1 <LoMV ≤15 days. MCS allows any range of intervention eff ect to be simulated, where this study tested a 10 and 25% diff erence in LoMV (0.5 to 1.25 days for mean LoMV of 5 days). The simulated LoMV for the intervention group is compared with the LoMV in a control group using the one-sided Wilcoxon rank-sum test, Student t test, and Kolmogorov– Smirnov test to assess central tendency and variation. Methods An impedance-based RVM (ExSpiron; Respiratory Motion Inc., Waltham, MA, USA) was used in 30 patients who received 2 mg midazolam prior to induction of anesthesia and were sedated but spontaneously breathing. A better way to determine s of mechanical ventilation? Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications. Conclusion Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications. Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. A better way to determine s of mechanical ventilation? Eleven of these patients (58 ± 19 years, average BMI 27.7) received midazolam at least 20 minutes prior to induction. Digital RVM data were collected and MV, TV and RR calculated and evaluated from 30-second segments 10 minutes before and after the fi rst dose of midazolam. Ten patients were analyzed as a group and one patient was analyzed separately (due to idiosyncratic reaction). y Results The distribution of LoMV is heavily skewed. Altman’s nomogram assumes a normal distribution and found N >1,000 to detect a 25% LoMV change. Figure 1 panels (1) and (2) show N for 80% power if all patients were included, and panels (3) and (4) for the targeted patient group. Panels (1) and (3) show that it is impossible to achieve 80% power for a 10% intervention eff ect. For 25% eff ect, MSC found N = 400/arm (all patients) and N = 150/arm (targeted cohort). Results Following administration of midazolam, the group MV and TV decreased an average of 19 ± 7% and 16 ± 5%, respectively (mean ± SEM, P <0.01, both) while RR remained essentially unchanged (decrease of 3 ± 8%, P >0.3). In the younger half of the cohort (45 ± 16 years), the decreases in MV and TV were not signifi cant, only 6 ± 3% and 8 ± 5%, respectively. The older half of the cohort (72 ± 8 years) displayed fourfold greater MV and TV decreases (32 ± 11%, P <0.05 and 25 ± 6%, P <0.05), when compared with the younger cohort, P <0.01, Figure 1). Figure 1 (abstract P227). Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. P228 Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G M ll 1 D L dd2 Figure 1 (abstract P227). Figure 1 (abstract P228). Figure 1 (abstract P228). Figure 1 (abstract P227). Conclusion Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. P229 P229 Nebulized heparin for patients under mechanical ventilation: a conventional data meta-analysis GJ Glas1, A Serpa Neto2, J Horn1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Israelita Albert Einstein, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P229 (doi: 10.1186/cc14309) Nebulized heparin for patients under mechanical ventilation: a conventional data meta-analysis y GJ Glas1, A Serpa Neto2, J Horn1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Israelita Albert Einstein, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P229 (doi: 10.1186/cc14309) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 1Vidant Medical Center, Greenville, NC, USA; 2Respiratory Motion, Inc., Waltham, MA, USA Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 1Vidant Medical Center, Greenville, NC, USA; 2Respiratory Motion, Inc., Waltham, MA, USA Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Introduction Mechanical ventilation has the potential to induce pulmonary coagulopathy. Local treatment by nebulization of heparin could be benefi cial in ventilated patients. The aim of this data meta- analysis is to determine the association between nebulization of heparin and outcome of mechanically ventilated critically ill patients. Methods PubMed, Scopus, EMBASE, and Web of Science were searched for relevant articles. Articles were selected if they compared Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Introduction Benzodiazepines are used in many of settings to induce sedation, but can cause a reduction in respiratory drive. Objective Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S80 Figure 1 (abstract P229). Eff ect of heparin nebulization on mortality. Figure 1 (abstract P229). Eff ect of heparin nebulization on mortality. nebulization of heparin with standard care. The primary endpoint was overall mortality. Secondary endpoints included occurrence of pneumonia and number of ventilator-free days and alive at day 28.i pulse oximetry with derived heart rate; snoring and nasal airfl ow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic diffi culties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI). pulse oximetry with derived heart rate; snoring and nasal airfl ow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic diffi culties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI). Results Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/m2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3 –: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Diagnosis of obstructive sleep apnea with respiratory polygraph in hypercapnic ICU patients P231 G Gursel, A Zerman, M Aydogdu, B Basarik Aydogan, K Gonderen, S Memmedova, N Sevimli, I Koroglu, Z Isikdogan, M Badoglu, O Ozdedeoglu Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P230 (doi: 10.1186/cc14310) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Diagnosis of obstructive sleep apnea with respiratory polygraph in hypercapnic ICU patients G Gursel, A Zerman, M Aydogdu, B Basarik Aydogan, K Gonderen, S Memmedova, N Sevimli, I Koroglu, Z Isikdogan, M Badoglu, O Ozdedeoglu Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P230 (doi: 10.1186/cc14310) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identifi ed to have OSA. Conclusion Although it underestimates AHI, RPLG is important and technically feasible in ICU patients in suggesting the presence of OSA and in providing information for appropriate NIV management. Results Six articles were found: fi ve retrospective cohorts with historical controls, one randomized controlled trial, covering 423 patients. Dosages of nebulized heparin varied from 30,000 to 150,000 IU/day. Fifty out of 222 patients (22.5%) receiving nebulized heparin and 48 out of 201 patients (23.9%) receiving standard care died (risk ratio (RR) 0.79 (95% CI 0.47 to 1.35)) (see Figure 1). Occurrence of pneumonia (RR 1.36 (95% CI 0.54 to 3.45); I2 = 59%), and number of ventilator-free days and alive at day 28 (standardized mean diff erence 0.11 (95% CI –0.14 to 35); I2 = 0%), were not diff erent between the two groups. Results Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/m2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3 –: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identifi ed to have OSA. Conclusion Nebulization of heparin is not associated with improved outcome in mechanically ventilated critically ill patients. This meta- analysis is limited by methodological problems in most included studies. Only one randomized controlled trial could be included. Also, most patients in the meta-analyzed studies suff ered from inhalation trauma, and heparin dosages diff ered widely. Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU G Besch1, J Revelly2, P Jolliet2, L Piquilloud-Imboden2 1CHRU Besançon, France; 2CHUV, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P232 (doi: 10.1186/cc14312) G Besch1, J Revelly2, P Jolliet2, L Piquilloud-Imboden2 1CHRU Besançon, France; 2CHUV, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P232 (doi: 10.1186/cc14312) y 2 2 Results The study group was comprised of four men and three women. Median age was 69 (IQR: 61.0 to 72.5) years old. The reason for admission was ARDS (n = 5) and acute exacerbation of idiopathic pulmonary fi brosis (n = 2). The reasons for ARDS are bacterial pneumonia (n = 3), necrotizing fasciitis (n = 1) and extensive burn (n = 1). We identifi ed NETs in the bronchial aspirates of all the patients. NET formation had persisted in four cases during the study period, their P/F ratio did not improve and all patients were dead due to respiratory failure. On the other hand, NETs decreased and vanished in three cases, their P/F ratio improved and all patients recovered from ARDS. Introduction As the objective clinical criteria [1] are imperfect to assess patients before extubation, simple physiological parameters are used to try to improve extubation failure (EF) prediction. The rapid shallow breathing index (RSBI) (respiratory rate (RR) over tidal volume (VT) ratio) recorded during a T-piece spontaneous breathing trial (SBT) is known as the most reliable physiologic predictor. However, RSBI is nowadays usually computed during a pressure support (PS) SBT using the values displayed on the ventilator screen and not based on spirometry measurements without any assist as initially published. The aim of the present study was to prospectively assess the ability of currently measured RSBI to predict EF. Conclusion NET formation was observed in bronchial aspirates of all the patients diagnosed with ARDS or acute exacerbation of idiopathic pulmonary fi brosis. It may be one of the prognostic factors of ARDS or acute exacerbation of idiopathic pulmonary fi brosis. Methods Retrospective analysis of prospectively collected data from patients intubated for more than 48 hours admitted in the medico- surgical ICU of Lausanne, Switzerland, from January 2007 to December 2008. Respiratory muscle training during mechanical ventilation: a systematic reviewfi a systematic review D Brace, M Parrotto, C Urrea, A Goffi , A Murray, E Fan, L Brochard, N Ferguson, E Goligher UHN, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P231 (doi: 10.1186/cc14311) Introduction The most frequent reasons for hypercapnic respiratory failure (HRF) in ICUs are COPD and in recent years obesity hypoventilation syndrome (OHS) and obstructive sleep apnea (OSA). Even 15 to 30% of COPD patients also have accompanying OSA. Due to increased upper airway resistance, those patients require higher expiratory pressures (EPAP) during noninvasive ventilation (NIV). In order to prescribe optimal mode and pressures during the ICU stay and at discharge, the intensivist should diagnose the underlying OSA. Portable recording devices have been developed and they were approved at least for the diagnosis in high pretest probability patients with results equal to in-laboratory polysomnography. The aim of this study is to assess whether respiratory polygraph (RPLG) can be used for obtaining diagnostic information of OSA in hypercapnic ICU patients. Methods Patients, with HRF requiring NIV, were included in the study. RPLG studies were conducted under nasal oxygen before NIV, using the Philips Respironics Alice PDx® device, which provides the records of Introduction Respiratory muscle weakness is common in mechanically ventilated patients and impairs liberation from ventilation. Inspiratory muscle training (IMT) might accelerate liberation from mechanical ventilation. We undertook to summarize previously published IMT protocols and the impact of IMT on respiratory muscle function and clinical outcomes. Methods We searched multiple databases using a sensitive search strategy combining MeSH headings and keywords for studies of IMT during MV. Studies were adjudicated for inclusion and data were abstracted independently and in duplicate. Methodological quality was assessed using the GRADE system. Results Eleven studies met the inclusion criteria; of these, six were randomized controlled trials and fi ve were observational studies. Figure 1 (abstract P231). Eff ect of IMT on the rate of successful weaning from mechanical ventilation. S81 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 A variety of IMT techniques were employed including inspiratory threshold loading (eight studies), biofeedback to increase inspiratory eff ort (one study), chair-sitting (one study) and diaphragmatic breathing pattern training (one study). Threshold loading was achieved by application of an external device (six studies) or increases in the inspiratory pressure trigger setting (two studies). University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) Introduction Acute lung injury (ALI) and its hypoxemic form, acute respiratory distress syndrome (ARDS), has no approved pharma co- logical treatment and is a condition with high mortality. Vascular leakage is one of the early events of ALI/ARDS. CD73 activity (ecto- 5’-nucleotidase) maintains the endothelial barrier function of lung capillaries via its enzymatic endproduct adenosine. Interferon-beta (IFNβ) increases synthesis of CD73 and has been eff ective in a mouse model of ALI [1]. Results A total of 478 extubated patients were included, 25 of whom (5.2%) were reintubated. ICU mortality (ICU-m) and in-hospital mortality (H-m) were higher in reintubated patients: ICU-m = 6 (24) versus 22 (5), P = 0.002 and H-m = 9 (36) versus 63 (15), P = 0.009. The reasons for EF were: acute lung failure (n = 15), congestive heart failure (n = 4) and aspiration/bronchial congestion (n = 6). Demographic data were similar between patients successfully and nonsuccessfully extubated: age: 58 ± 17 versus 58 ± 19 years, P = 0.85; male gender: 15 (60) versus 263 (61), P = 0.99. SAPS II score was higher in the EF group: 30 ± 22 versus 42 ± 27, P = 0.04. RSBI were signifi cantly higher in patients who experienced EF: RSBI=59 ± 44 versus 43 ± 26, P = 0.04. The area under the ROC curve for currently measured RSBI was: 0.617 (95% CI = 0.571 to 0.662), P = 0.035. Methods Therefore we conducted a phase I/II trial [2], in which intravenously administered human recombinant IFNβ1a (FP-1201) was used in the study, which consisted of dose escalation (phase I) and expansion (phase II) parts to test the FP-1201 safety, tolerability and effi cacy in ALI/ARDS patients. CD73, MxA (a marker for IFN β response) and other biomarkers were measured to follow pharmacokinetics/ dynamics of the intravenously administered FP-1201 and therapeutic effi cacy. Conclusion In a cohort of 458 medico-surgical ICU patients, RSBI measured during a pressure support SBT was higher in patients experiencing EF but very imperfect to predict EF. Reference fi y Results The optimal tolerated dose of FP-1201 (10 μg/day for 6 days) resulted in maximal MxA stimulation. Also soluble CD73 values increased, while IL-6 decreased in sera of the FP-1201-treated ALI/ARDS patients. Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU EF was defi ned as the need for reintubation within 48 hours after extubation. Reintubations for a procedure requiring general anesthesia were not considered as EFs. RR and VT during the PS SBT were recorded from the ventilator and RSBI was computed accordingly. Baseline characteristics and currently measured RSBI were compared between patients who experienced EF versus success (t test or chi-square test as appropriate). The ability of currently measured RSBI to predict EF was assessed using ROC curve analysis. P234 1. MacIntyre NR, et al. Chest. 2001;120:375S. Respiratory muscle training during mechanical ventilation: a systematic reviewfi Most studies implemented IMT in the weaning phase (n = 5) or after diffi cult weaning (n = 5); one study implemented IMT within 24 hours of intubation. IMT was associated with greater increases in maximal inspiratory pressure compared with control (six studies, mean diff erence 7.6 cmH2O (95% CI 5.8, 9.3), I2 = 0%). There were no signifi cant diff erences in the duration of MV (six studies, mean diff erence –1.1 days (95% CI –2.5, 0.3), I2 = 71%) or the rate of successful weaning (Figure 1; fi ve studies, risk ratio 1.13 (95% CI 0.92, 1.40), I2 = 58%). The GRADE quality of evidence was low for all these outcomes; risk of bias was high for most studies and summary eff ects were imprecise and inconsistent. No serious adverse events related to IMT were reported. P233 Presence of neutrophil extracellular traps in bronchial aspirate of patients diagnosed with acute respiratory distress syndrome or acute exacerbation of idiopathic pulmonary fi brosis M Ojima1, N Yamamoto1, T Hirose1, S Hamaguchi1, N Matsumoto1, R Takegawa1, M Seki1, O Tasaki2, K Tomono1, T Shimazu1 1Osaka University Graduate School of Medicine, Suita, Japan; 2Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Critical Care 2015, 19(Suppl 1):P233 (doi: 10.1186/cc14313) Introduction Neutrophil extracellular traps (NETs) are fi brous structures that are produced extracellularly from activated neutrophil. They can trap and kill various pathogens, and their release is one of the fi rst lines of immune system. Meanwhile, recently it was reported that NETs also exert adverse eff ects in infl ammatory diseases. Acute respiratory distress syndrome (ARDS) and acute exacerbation of idiopathic pulmonary fi brosis is acute-onset respiratory failure. It is characterized by excessive neutrophil infi ltration into the alveolus, and great amounts of neutrophil elastase are released. The purpose of this study is to evaluate whether NETs are produced in bronchial aspirate of patients with ARDS or acute exacerbation of idiopathic pulmonary fi brosis, and to identify correlations with the respiratory function. p Conclusion IMT in mechanically ventilated patients appears safe and well tolerated and improves respiratory muscle function. IMT was not associated with accelerated liberation from mechanical ventilation. However, because the included studies had important methodological limitations and employed varying methods of IMT, we cannot draw fi rm conclusions about the eff ect of IMT on clinical outcomes. i y y Methods This was a prospective observational study of seven patients admitted to the ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated at the time of admission. The bronchial aspirates were collected serially from the patients by suction through a tracheal tube. To identify NETs, extracellular components including DNA, histone H3, and citrullinated histone H3 were simultaneously detected using immunohistochemistry. The respiratory function was evaluated by PaO2/FiO2 ratio (P/F ratio). New molecules controlling endothelial barrier S Jalkanen New molecules controlling endothelial barrier S Jalkanen New molecules controlling endothelial barrier S Jalkanen University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314 References 1. Crit Care Med. 2006;34:532-7. 2. J Crit Care. 2012;27:522.e11-7. 3. Crit Care. 2013;17:R132. Methods Forty-one CD-1 mice were divided into two groups: an HCl group underwent orotracheal instillation of hydrochloric acid on day 0, and a group control. Mice were evaluated on days 0, 1, 2 and 4 after a 30-minute period of mechanical ventilation. Blood and lung edema fl uid (EF) were sampled. Before initiation of MV, all mice received a tracheal instillation of bovine serum albumin (5%) to detect changes in alveolar protein levels over 30 minutes. Plasma levels of sRAGE and total protein levels were measured. AFC rate values were corrected after measurement of mouse serum albumin in EF. P235 P235 Endocan can be a predictive marker of severity of sepsis but cannot be a marker of acute respiratory distress syndrome in ICU patients M Mizunuma, H Ishikura, Y Nakamura, K Muranishi, S Morimoto, H Kaneyama, Y Izutani, T Nishida, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P235 (doi: 10.1186/cc14315) f References 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. P237 Elevated levels of soluble RAGE predict impaired alveolar fl uid clearance in a translational mouse model of acute respiratory distress syndrome R Blondonnet1, M Jabaudon1, G Clairefond2, J Audard1, D Bouvier2, G Marceau1, P Blanc2, P Dechelotte1, V Sapin2, JM Constantin1 1CHU Clermont-Ferrand, France; 2Laroratoire R2D2-EA7281, Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P237 (doi: 10.1186/cc14317) Results We enrolled 70 ARDS patients during the investigation periods. We met six patients with nonsepsis, 27 with severe sepsis and 37 with septic shock. The serum Endocan levels were signifi cantly higher in patients with septic shock (3.7 to 3.9 ng/ml) than in patients with severe sepsis (1.7 to 2.3 ng/ml, P <0.05), nonsepsis (0.6 to 0.3 ng/ml, P <0.05) and healthy donors (0.4 to 0.1 ng/ml, P <0.05). However, there was no signifi cant correlation between the Endocan level and the severity of ARDS. In addition, signifi cant correlation between the Endocan level and EVLWi and PVPI was not observed. Introduction Receptor for advanced glycation endproducts (RAGE) is a transmembrane receptor expressed in the lung and primarily located on alveolar type I cells. RAGE is implicated in acute respiratory distress syndrome to alveolar infl ammation and, when its soluble form sRAGE is assayed in plasma or pulmonary edema fl uid, as a marker of AT I cell injury. Functional activity of AT 1 cells can be assessed by the measurement of the alveolar fl uid clearance (AFC) rate [1], but the relationship between sRAGE plasma levels of sRAGE and AFC rates has never been investigated. Our objectives were to report plasma levels of sRAGE in a mouse model of direct acid-induced epithelial injury, and to test their correlation with AFC rates. Conclusion These results suggested that there was good relationship between the Endocan level and the severity of sepsis. But unlike the trauma patients, correlation between the severity of ARDS and Endocan value was not recognized. P236 Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Q Chen, A Liu, H Qiu, Y Yang Southeast University, Nanjing, China Critical Care 2015, 19(Suppl 1):P236 (doi: 10.1186/cc14316) Endocan can be a predictive marker of severity of sepsis but cannot be a marker of acute respiratory distress syndrome in ICU patients M Mizunuma, H Ishikura, Y Nakamura, K Muranishi, S Morimoto, H Kaneyama, Y Izutani, T Nishida, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P235 (doi: 10.1186/cc14315) l Results The permeability signifi cantly increased after LPS stimulation in a dose-dependent and time-dependent manner (P <0.01). Mean- while, MSC-EC interaction more signifi cantly decreased endothelial permeability induced by LPS (P <0.05 or P <0.01). Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group (P <0.01). However, neutralizing HGF with anti- HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability (P <0.05). Compared with the MSC group, MSC-EC interaction increased VE-cadherin protein expression (P <0.01), and restored remodeling of F-actin and junctional localization of VE- cadherin. However, the MSC eff ect was signifi cantly blocked by anti- HGF antibody (P <0.05 or P <0.01). Introduction Endocan (endothelial cell specifi c molecule-1), a 50 kDa dermatan sulfate proteoglycan, is expressed by endothelial cells in the lung and kidney. It was reported that the serum Endocan level is related to the severity of sepsis and positive correlation with the mortality rate. On the other hand, it was also reported that lower levels of serum Endocan are associated with subsequent development of chronic kidney disease, and chronic liver disease acute lung injury (ALI) in trauma patients. The aim of this study is confi rmation of the relationship between serum Endocan level and the severity of sepsis, and also the severity of acute respiratory distress syndrome (ARDS) in septic patients. y Conclusion These data suggest that MSC-EC interaction decreased endothelial permeability induced by LPS, which was mainly attributed to HGF secreted by hMSCs. The main mechanisms of HGF restoring the integrity of EC monolayers are remodeling of endothelial intercellular AJs and decreasing caveolin-1 protein expression. p p Methods This study was conducted as a single-center, retrospective, observational study in the emergency department of Fukuoka University Hospital from April 2010 to August 2013. Blood samples were collected within 2 hours when the patients were diagnosed with ARDS. In this time we adopted the Berlin defi nition as the categorized of ARDS severity. Furthermore, we evaluated the extravascular lung water index (EVLWi) and pulmonary vascular permeability index (PVPI) as a condition of ARDS using the transpulmonary thermodilution method. Additionally, 10 healthy donors were entered as a control. The patients were divided into nonsepsis, severe sepsis and septic shock using the ACCP/SCCM guidelines. University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) The overall mortality of the 37 patients treated with FP-1201 S82 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secreted more factors by MSC–endothelial cell (MSC-EC) interaction. We hypothesized that MSC-EC interaction restored endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF. was only 8.1%, fourfold to fi vefold less than the expected rate based on APACHE II score values of 21.9. The control group (n = 59), which was eligible for the trial but not possible to recruit, had mortality of 32.2% (P = 0.01) and APACHE II score 23.0. Conclusion Restriction of vascular leakage with FP-1201 seems to signifi cantly benefi t ALI/ARDS patients. Our results suggest that FP- 1201 could be the fi rst eff ective, mechanistically targeted, disease- specifi c pharmacotherapy for patients with ARDS. However, these fi ndings warrant conduction of a large multicenter study to establish safe and eff ective FP-1201 treatment of ARDS. f p p y p Methods We investigated the endothelial permeability induced by LPS under two co-culture conditions in transwells. HPMECs were added into the upper chambers of cell-culture inserts, while there two diff erent co-culture conditions in the lower side of transwells as follows: MSC-EC interaction group: MSCs and HPMEC contact co- culture in the lower chambers; and MSC groups: MSCs only in the lower chambers. The endothelial permeability in the upper side of transwells was detected. Then the concentration of HGF was measured in the culture medium using an enzyme-linked immunosorbent assay kit, following by neutralizing HGF with anti-HGF antibody in the co-culture medium. In addition, VE-cadherin protein expression were measured under the co-culture conditions by western blot, adherens junctions (AJs) protein including F-actin and VE-cadherin were detected by immunofl uorescence technique as well.i P240 y Results Of those 4,652 patients, there were 2,947 nonsmokers, 1,148 exsmokers, and 557 active smokers. There was no diff erence of APACHE II score between three groups of patients. The active smokers exhibited the highest incidence of ARDS (active smokers 5.4%, exsmokers 4.8%, and nonsmokers 3%, P = 0.003). There was no diff erence of 28-day mortality between the three groups of patients. Active smokers had the highest incidence of SIRS (active smokers 41%, exsmokers 37%, and nonsmokers 34%, P = 0.006). Compared with nonsmokers and exsmokers, active smokers had a longer SICU LOS (P <0.01) and higher total SICU cost (P = 0.02). Patients who smoked more than 15 pack- years were 2.5 times more likely to develop ARDS than patients who smoked ≤15 pack-years (95% CI: 1.65 to 3.66, P <0.001). In multivariate analysis we found that every 1 pack-year of cigarette smoking before References 1. Hoshino Y, et al. Am J Physiol Lung Cell Mol Physiol. 2001;281:L509-16. 2. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 (Suppl 1):S45-54. P238 Smoking increased risk of ARDS in surgical critically ill patients: results from the multicenter THAI-SICU study P Wacharasint1, P Fuengfoo1, N Sukcharoen1, R Rangsin2, THAI-SICU Trial Group3 1Phramongkutklao Hospital, Bangkok, Thailand; 2Phramongkutklao College of Medicine, Bangkok, Thailand; 3THAI-SICU Collaborating Centre, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P238 (doi: 10.1186/cc14318) 2 Results A total of 101 ARDS patients was enrolled; 44 (43.6%) patients were normal weight, 36 (35.6%) overweight and 21 (20.8%) obese. Lung and chest wall elastance were not diff erent between groups, both at PEEP levels of 5 cmH2O and 15 cmH2O (P = 0.580 and P = 0.113, respectively), and the end-inspiratory transpulmonary pressure was also similar. We did not observe any diff erence between groups regarding PaO2/FiO2 ratio (P = 0.178 at PEEP 5 cmH2O; P = 0.073 at PEEP 15 cmH2O) and PaCO2 (P = 0.491 at PEEP 5 cmH2O; P = 0.237 at PEEP 15 cmH2O). Introduction Cigarette smoking slowly and progressively damages the respiratory system [1]. In surgical critically ill patients, whether active cigarette smoking until admission to the surgical intensive care unit (SICU) is associated with increased risk of acute respiratory distress syndrome (ARDS) is not clearly identifi ed. 2 Conclusion In ARDS obese patients the chest wall elastance and the end-inspiratory transpulmonary pressure are not aff ected by the body weight, suggesting that normal weight and obese patients present similar risks for lung stress and VILI onset. The severity of hypoxemia was not diff erent between groups. y yi Methods We conducted a cohort study using the THAI-Surgical Intensive Care Unit (THAI-SICU) study databases [2], which recruited 4,652 Thai patients admitted to the SICUs from nine university-based hospitals in Thailand (April 2011 to November 2012). The enrolled patients were divided into three groups (active smokers, exsmokers, and nonsmokers). Primary outcome was the incidence of patients diagnosed with ARDS and the secondary outcomes included 28-day mortality, incidence of systemic infl ammatory response syndrome (SIRS), SICU length of stay (LOS), and total SICU cost. f References 1. Pelosi et al. Chest. 1996;109:144-51. 1. Pelosi et al. Chest. 1996;109:144-51. . Suratt et al. J Appl Physiol Respir Environ Exerc Physiol. 1984;57:403 2. Suratt et al. J Appl Physiol Respir Environ Exerc Physiol. 1984;57:403-7. P239l P239 Infl uence of body weight on lung mechanics and respiratory function in ARDS patients I Algieri1, D Chiumello2, C Mietto1, E Carlesso1, A Colombo1, G Babini1, M Cressoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS, ‘Ospedale Maggiore Policlinico Mangiagalli Regina Elena’ di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P239 (doi: 10.1186/cc14319) Figure 1 (abstract P237). Relation between AFC rates and sRAGE plasma levels. (P = 0.03) and day 2 (P = 0.02). The rate of AFC was inversely correlated with sRAGE levels in the plasma (Spearman’s ρ = –0.73, P <0.001). See Figure 1. Introduction Traditionally, ARDS obese patients are ventilated with higher tidal volumes and higher PEEP due to expected increased in pleural pressure. However, data from the literature regarding the infl uence of body mass on lung mechanics and, particularly, on chest wall elastance are not univocal [1,2]. Failure to account for how the increase in body weight could aff ect the respiratory function can result in injurious mechanical ventilation and in the onset of VILI. The aim of this study was to evaluate the role of the body weight on respiratory mechanics in ARDS patients. g Conclusion The highest impairment in AFC is reported on day 1. sRAGE levels are also higher in injured mice and may be a good surrogate marker of AT I cell injury. This is a newly described relationship between AFC rates and sRAGE plasma level in a mouse model of direct epithelial injury. Our results support further translational investigation on the role of RAGE in alveolar injury and recovery. 1. Eur Respir J. 2012;39:1162-70. Methods A group of deeply sedated and paralyzed ARDS patients was divided into three classes according to the body mass index: normal weight (between 18.5 and 24.9 kg/m2), overweight (between 25.0 and 29.9 kg/m2) and obese (between 30.0 and 39.9 kg/m2). They were mechanically ventilated in volume-controlled mode with a tidal volume of 6 to 8 ml/kg according to predicted body weight. Respiratory mechanics and gas exchange were assessed at PEEP levels of 5 and 15 cmH2O. Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Q Chen, A Liu, H Qiu, Y Yang Results Basal AFC rate was 35% over 30 minutes in HCl-injured mice, but it was signifi cantly depressed on day 1 (16% over 30 minutes; P = 0.02). Over time, AFC reached basal levels again. Plasma levels of sRAGE were higher in HCl-treated animals than in control animals on day 1 Introduction Mesenchymal stem cells (MSCs) have potent stabilizing eff ects on the vascular endothelium injury, inhibiting endothelial Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S83 Figure 1 (abstract P237). Relation between AFC rates and sRAGE plasma levels. admission to the SICU is associated with increased risk of new ARDS with a hazard ratio of 1.02 (95% CI: 1.01 to 1.02, P = 0.001) after adjustment for APACHE II score, age, gender, and chronic obstructive pulmonary disease. Conclusion In surgical critically ill patients, active smokers are associated with increased risk of new ARDS, longer SICU LOS, and higher total ICU cost, compared with exsmokers and nonsmokers. Our fi ndings emphasize the essential need for a smoking cessation program. f Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Introduction Acute respiratory distress syndrome (ARDS) develops on the basis of nosocomial pneumonia (NP) in 30 to 35% of cases. A complex clinical approach is required for diagnosing it. The aim of this study was to investigate into the role of the PaO2/FiO2 ratio (P/F), extravascular lung water index (EVLWI) and surfactant protein D (SPD) as a complex diagnostic tool for ARDS in NP. S84 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The observational study in ICU ventilated septic patients with peritonitis (70%), pancreonecrosis (25%) and mediastinitis (5%) was done in 2010 and 2014. ARDS was diagnosed and staged according to the V.A. Negovsky Research Institute criteria and the Berlin defi nition. Plasma SPD was measured on ARDS diagnosis (day 0) and days 3 and 5 by the immunoenzyme essay (BioVendor, USA). Patients were treated according to the international guidelines. Data were statistically analyzed by STATISTICA 7.0, ANOVA and presented as median and 25 to 75th percentiles (ng/ml); P <0.05 was considered statistically signifi cant. Areas under the receiver operating curves were calculated. Results Sixty-fi ve patients (out of 450 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned into groups: NP + ARDS (n = 43, 43 ± 4.9 years old, M/F 39/4, mortality 23%) and NP (n = 22, 40 ± 5.1 years old, M/F 20/2, mortality 18%). Groups were comparable in APACHE II and SOFA scores on the baseline. In the NP + ARDS group SPD was higher at all points than in the NP group. Plasma SPD on day 0 >111.2 ng/ml yielded a sensitivity of 68.2% and specifi city of 92.3% (AUC 0.85; 95% CI 0.684 to 0.945; P <0.0001) for diagnosing ARDS in NP. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Results Forty-fi ve oesophageal balloon pressure–volume curves were obtained in 31 patients undergoing controlled mechanical ventilation (PEEP 12 ± 5 cmH2O, FiO2 0.7 ± 0.2, tidal volume/ideal body weight 8.0 ± 1.6 ml/kg). According to the graphically detected minimum slope section of the curve, the minimum and maximum appropriate balloon volumes were 1.5 ± 0.6 ml and 5.3 ± 0.9 ml, respectively. Between these two volumes, the slope of the curve was 1.1 ± 0.5 cmH2O/ml, ranging from 0.3 to 3.1 cmH2O/ml. Eff ects of a recruitment maneuver on plasma soluble rage in patients with diff use ARDS: a prospective randomized crossover study M Jabaudon, N Hamroun, L Roszyk, R Blondonnet, R Guerin, JE Bazin, V Sapin, B Pereira, JM Constantin CHU Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P241 (doi: 10.1186/cc14321) y M Jabaudon, N Hamroun, L Roszyk, R Blondonnet, R Guerin, JE Bazin, V Sapin, B Pereira, JM Constantin CHU Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P241 (doi: 10.1186/cc14321) Introduction The soluble form of the receptor for advanced glycation endproducts (sRAGE) is a promising marker for epithelial dysfunction, but it has not been fully characterized as a biomarker during ARDS. Whether sRAGE could inform on the response to ventilator settings has been poorly investigated, and whether recruitment maneuver (RM) may infl uence plasma sRAGE remains unknown. p p gl Results Forty-fi ve oesophageal balloon pressure–volume curves were obtained in 31 patients undergoing controlled mechanical ventilation (PEEP 12 ± 5 cmH2O, FiO2 0.7 ± 0.2, tidal volume/ideal body weight 8.0 ± 1.6 ml/kg). According to the graphically detected minimum slope section of the curve, the minimum and maximum appropriate balloon volumes were 1.5 ± 0.6 ml and 5.3 ± 0.9 ml, respectively. Between these two volumes, the slope of the curve was 1.1 ± 0.5 cmH2O/ml, ranging from 0.3 to 3.1 cmH2O/ml. l Methods Twenty-four patients with moderate/severe, nonfocal ARDS were enrolled in this prospective monocentric crossover study and randomized into a ‘RM-SHAM’ group when a 6-hour-long RM sequence preceded a 6-hour-long sham evaluation period, or a ‘SHAM-RM’ Figure 1 (abstract P241). Evolution of plasma levels of sRAGE (pg/ml) in both randomization sequences. 2 Conclusion The oesophageal artefact – that is, the reaction of the oesophageal wall to balloon infl ation – may be clinically signifi cant, being on average 1 cmH2O for each millilitre of volume injected in the catheter, but reaching values as high as 3 cmH2O/ml. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Our fi ndings could help to design future studies of sRAGE as a marker of response to therapeutic interventions during ARDS. P242 Oesophageal artefact may signifi cantly aff ect oesophageal pressure measurement in mechanically ventilated patients F Mojoli, F Torriglia, M Pozzi, S Bianzina, G Tavazzi, A Orlando, A Braschi Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy Critical Care 2015, 19(Suppl 1):P242 (doi: 10.1186/cc14322) Introduction Oesophageal pressure is increasingly used to monitor and manage mechanically ventilated patients. Even if the oesophageal balloon catheter is correctly positioned, the measurement can be aff ected by inappropriate balloon fi lling and/or oesophageal reaction to balloon infl ation. We aimed to assess the oesophageal reaction to oesophageal balloon fi lling in mechanically ventilated patients. i Methods An oesophageal balloon catheter (NutriVent; Sidam, Mirandola, Italy) was introduced in mid/distal thoracic position in 31 patients under invasive mechanical ventilation for acute respiratory failure. At ambient pressure, the balloon of the NutriVent catheter can be infl ated up to 6 ml without generation of recoil pressure. The balloon was progressively infl ated in 0.5 ml steps up to 9 ml and end-expiratory values of balloon pressure were used to assemble the balloon pressure–volume curve. The minimum slope section of the curve was graphically detected and infl ation volumes corresponding to this part of the curve were considered appropriate. Overdistension of the balloon being excluded by defi nition in this section of the curve, its slope was attributed to the oesophageal reaction to balloon infl ation.i Methods An oesophageal balloon catheter (NutriVent; Sidam, Mirandola, Italy) was introduced in mid/distal thoracic position in 31 patients under invasive mechanical ventilation for acute respiratory failure. At ambient pressure, the balloon of the NutriVent catheter can be infl ated up to 6 ml without generation of recoil pressure. The balloon was progressively infl ated in 0.5 ml steps up to 9 ml and end-expiratory values of balloon pressure were used to assemble the balloon pressure–volume curve. The minimum slope section of the curve was graphically detected and infl ation volumes corresponding to this part of the curve were considered appropriate. Overdistension of the balloon being excluded by defi nition in this section of the curve, its slope was attributed to the oesophageal reaction to balloon infl ation. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) The pressure generated by the oesophageal reaction leads to overestimation of pleural pressure. Therefore, the oesophageal artefact may signifi cantly aff ect clinical decision-making based on absolute values of oesophageal pressure. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) P/F ratio on day 0 <280 yielded a sensitivity of 94.1% and specifi city of 76.9% (AUC 0.89; 95% CI 0.744 to 0.952; P <0.0001) and EVLWI on day 0 >8.3 ml/kg yielded a sensitivity of 94.1% and specifi city of 92.3% (AUC 0.92; 95% CI 0.810 to 0.982; P <0.0001) for the diagnosis of ARDS in NP. A complex ROC analysis (for SPD in the group of patients with P/F <280 and EVLWI >8.3) yielded a much better diagnostic accuracy of SPD: cutoff >93.7 ng/ml, sensitivity 81.0%, specifi city 100.0% (AUC 0.96; 95% CI 0.817 to 0.998; P <0.0001). Conclusion A complex approach (P/F <280, EVLWI >8.3, SPD >93.7) presents as a sensitive and highly specifi c method for diagnosing ARDS in NP patients. group (inverted sequences). Protective ventilation was applied, and RM consisted of the application of 40 cmH2O airway pressure for 40 seconds. Arterial blood was sampled for gas analyses and sRAGE measurements, 5 minutes pre RM (or 40-second-long sham period), 5 minutes, 30 minutes, 1 hour, 4 hours and 6 hours after the RM (or 40-second-long sham period). g p ) Results Mean PaO2/FiO2, tidal volume, PEEP and plateau pressure were 125 mmHg, 6.8 ml/kg (ideal body weight), 13 and 26 cmH2O, respectively. Median baseline plasma sRAGE levels were 3,232 pg/ml. RM induced a signifi cant decrease in sRAGE (–1,598 ± 859 pg/ml) in 1 hour (P = 0.043). At 4 and 6 hours post RM, sRAGE levels increased back toward baseline values. Pre-RM sRAGE was associated with RM- induced oxygenation improvement (AUC 0.87). See Figure 1. Conclusion We report the fi rst kinetics study of plasma sRAGE after RM in ARDS. Our fi ndings could help to design future studies of sRAGE as a marker of response to therapeutic interventions during ARDS. Results Mean PaO2/FiO2, tidal volume, PEEP and plateau pressure were 125 mmHg, 6.8 ml/kg (ideal body weight), 13 and 26 cmH2O, respectively. Median baseline plasma sRAGE levels were 3,232 pg/ml. RM induced a signifi cant decrease in sRAGE (–1,598 ± 859 pg/ml) in 1 hour (P = 0.043). At 4 and 6 hours post RM, sRAGE levels increased back toward baseline values. Pre-RM sRAGE was associated with RM- induced oxygenation improvement (AUC 0.87). See Figure 1. Conclusion We report the fi rst kinetics study of plasma sRAGE after RM in ARDS. P244 P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation Regional distribution of excess tissue mass in ARDS lung I Algieri1, D Massari1, A Colombo1, G Babini1, F Crimella1, M Brioni1, A Cammaroto1, K Nikolla1, C Montaruli1, M Guanziroli1, M Gotti1, C Chiurazzi1, M Amini1, M Chiodi1, M Cressoni1, D Chiumello2, L Gattinoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS, ‘Ospedale Maggiore Policlinico Mangiagalli Regina Elena’ di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P244 (doi: 10.1186/cc14324) Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation y p g y T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) Introduction ARDS has a wide variability of lung morphological charac- teristics. Both alveolar collapse and airway narrowing or closing are present, often heterogeneously. Despite advances in ARDS imaging, we have thus far been unable to distinguish regional airway opening from airway dilatation in PEEP-induced lung recruitment. We demonstrate the technique of functional respiratory imaging (FRI) to diff erentiate these two entities. Introduction ARDS is characterized by edema diff use to all lung fi elds. Distribution of excess tissue mass had been studied with CT scan in a few patients on a single slice, comparing with data obtained in healthy controls. Methods ARDS patients underwent CT scan imaging during their ICU stay at 45 cmH2O end-inspiratory pressure. After hospital discharge, patients underwent a follow-up CT scan performed at end inspiration. Each lung was divided into three sections along the apex–base axis and into three sections along the sternum–vertebral axis (nine regions per lung). Excess tissue mass in each lung region was defi ned as the diff erence in lung tissue (grams) between the CT scan performed during ARDS course and the follow-up CT scan. Results are presented as mean ± SD. Methods Six patients with early-stage ARDS were included in this prospective single-centre cohort trial. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) Figure 1 (abstract P244). Figure 1 (abstract P244). demonstrated recently its eff ectiveness on prognosis. Extrapulmonary etiologies of ARDS include abdominal emergencies. In cases of severe hypoxemia in the early postoperative period, intensivists discuss prone positioning based on the risk/benefi t ratio. demonstrated recently its eff ectiveness on prognosis. Extrapulmonary etiologies of ARDS include abdominal emergencies. In cases of severe hypoxemia in the early postoperative period, intensivists discuss prone positioning based on the risk/benefi t ratio. p gi Methods We conducted a retrospective two-center study of 5 years. The aim was to compare the prevalence of surgical complication potentially related to prone positioning between patients who had at least one prone positioning session and patients that remained in a supine position after abdominal surgery. Patients with ARDS in a context of recent (<7 days) abdominal surgery (except laparoscopy) were included. The primary outcome was the number of patients who had at least one surgical complication potentially related to prone positioning. We defi ned a priori these complications: scar dehiscence, abdominal compartment syndrome, stoma leakage, stoma necrosis, scar necrosis, wound infection, displacing of a drainage system, removal of gastro- or jejunostomy feeding, digestive fi stula, evisceration.i g j j y g gi Results We identifi ed 43 patients with postoperative ARDS (62 ± 8 years, SAPS II 50 ± 13), among whom 34 (79%) had emergent surgery. Fifteen patients had at least one stoma after surgery. Nineteen patients (44%) had at least one prone positioning session (number of sessions: 2 (1 to 3)). At baseline, prone group patients had minimum PaO2/FiO2 ratio lower than the supine group (77 ± 23 vs. 110 ± 46 mmHg, P = 0.005). Plateau pressure was higher in the prone group (28 ± 4 vs. 23 ± 5 cmH2O, P = 0.002). The fi rst prone positioning session signifi cantly increased the PaO2/FiO2 ratio: 106 ± 52 vs. 192 ± 90 mmHg (P = 0.001). Mean duration of the fi rst prone positioning session was 20 ± 10 hours. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 In the prone group, 11 patients (58%) had at least one surgical complication, in comparison with nine (38%) in the supine group (P = 0.2). These complications resulted in revision surgery for two (10%) patients in the prone group and two (8%) in the supine group (P = 0.8). Mortality in the ICU was respectively 42% and 38% in prone group and supine group (P = 0.8).if Conclusion These preliminary results confi rm the eff ectiveness of prone positioning in terms of oxygenation in ARDS after abdominal surgery without signifi cant increase in surgical complications and no eff ect on the need for surgical revisions. Hence, if necessary, clinicians should not refrain from proning patients with postabdominal surgery ARDS. P243 P243 Prone positioning in acute respiratory distress syndrome after abdominal surgery S Gaudry1, S Tuff et1, AC Anne-Claire2, N Zucman1, S Msika1, M Pocard2, D Payen2, D Dreyfuss1, JD Ricard1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Lariboisière, Paris, France Critical Care 2015, 19(Suppl 1):P243 (doi: 10.1186/cc14323) Introduction Prone positioning has been used for many years as an alveolar recruitment strategy in acute respiratory distress syndrome (ARDS). Prone positioning in ARDS improves oxygenation and Introduction Prone positioning has been used for many years as an alveolar recruitment strategy in acute respiratory distress syndrome (ARDS). Prone positioning in ARDS improves oxygenation and Figure 1 (abstract P241). Evolution of plasma levels of sRAGE (pg/ml) in both randomization sequences. S85 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Effi cacy and safety of open lung ventilation in patients with impaired peripheral chemorefl ex sensitivity N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Kuban State Medical University, Krasnodar, Russia Critical Care 2015, 19(Suppl 1):P246 (doi: 10.1186/cc14326) g Results Piglets ventilated with higher energy loads (RR 15 and 12) developed ventilator-induced lung injury (VILI), while piglets ventilated with lower energy loads (RR 9, 6 and 3) did not. The energy dissipated in the lung parenchyma at each breath was 0.56 J (0.52 to 0.62). Dissipated energy increased with time in piglets that developed VILI, while it remained near-constant in all the other piglets. Recruitability was 6% (3 to 8) of lung parenchyma, strain was 3.1 (2.6 to 3.5) and lung inhomogeneity extent (that is, the percentage of lung parenchyma that is inhomogeneous) was 10% (9 to 11). The energy dissipated in the lung parenchyma was well related to lung recruitability (r2 = 0.50, P <0.0001), strain (r2 = 0.57, P <0.0001) and lung inhomogeneity extent (r2 = 0.42, P <0.0001). Multiple linear regression showed that dissipated energy was independently related to all of the three determinants of energy dissipation: lung opening and closing (P = 0.025), strain (P <0.0001) and lung inhomogeneities (P <0.01). Introduction Mechanical ventilation during anesthesia leads to the development of atelectasis, poor oxygenation and postoperative pulmonary complications. Application of PEEP and recruitment maneuver (RM) can signifi cantly reduce the severity of atelectasis and improve lung function. But the application of this strategy often leads to hemodynamic instability, which may be associated with impaired reactivity of the cardiovascular system. The purpose of this study was to evaluate the effi cacy and safety of RM in patients with increased sensitivity of peripheral chemoreceptors (SPCR), which refl ects the decreasing reactivity of the cardiovascular system. g y y Methods We conducted a prospective study in 116 patients with high SPCR, evaluated using the breath-holding test. The test was performed by measuring of voluntary breath-holding duration (BHD) after two- thirds of maximal inspiration. The end of breath-hold was determined by a palpation of contraction of the diaphragm. BHD <38 seconds was the marker of high SPCR [1]. All patients received a major abdominal surgery and were randomized into an open lung ventilation group or a PEEP group. P244 The lower infl iction point on a pressure–volume curve was considered as the clinically acceptable minimal PEEP value. Subsequently, four distinct PEEP levels were chosen to perform CT scans: at 20 cmH2O; median value between 1 and 3; clinically acceptable minimal; and 0 cmH2O. FRI methods as described by De Backer and colleagues [1] were used to evaluate airway opening and airway dilatation. Results We studied eight ARDS patients (55 ± 18 years) with a BMI of 27 ± 6 kg/m2. At ICU admission, patients had the following clinical parameters: PaO2/FiO2 106 ± 33 with PEEP 15 ± 5 cmH2O; PaCO2 43 ± 10 mmHg; pH 7.35 ± 0.05. The average increase in lung weight during ARDS compared with follow-up CT scan was 68 ± 40% (680 ± 320 g). Figure 1 presents the tissue volume during ARDS (white bars) and after ARDS resolution (black bars) and compares the ratio between the two (P <0.01 vs. dependent region). Results Airway stretching (that is, bronchodilatation) could be quantifi ed and distinguished from airway recruitment with this technique. Higher PEEP pressures not only recruit, but also expand the bronchi. The ratio of dilation/recruitment of bronchi was higher in the upper lobes than in the lower lobes, as illustrated in Figure 1. We were able to phenotype each patient, allowing a prediction on when an increase in PEEP further recruits atelectasis/bronchi or distends certain airway regions. Conclusion The excess tissue mass was not diff erent between apex, hilum and base, but was increased in the dependent lung regions at apex and hilum, being uniformly distributed at the lung base. y g Conclusion The novel technique of FRI can be used to visualise the airway structures in ARDS and distinguish airway stretching from Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S86 more pronounced. So CI on average decreased by 34% (from 3.7 to 2.5 l/minute/m2) compared with 10% with no RM (P <0.05), and SVR decreased by 19% (from 1,310 to 1,150 dyn × sec–1 × cm–5, P <0.05), while in the PEEP group it did not change. No signifi cant diff erences between groups in the incidence of complications, length of stay in the ICU and in the hospital were noted. Figure 1 (abstract P245). Bronchodilation and bronchial recruitment split per lobe for one patient. Effi cacy and safety of open lung ventilation in patients with impaired peripheral chemorefl ex sensitivity The concept of open lung ventilation was performed as follows: PEEP was increased from 4 to 10 cmH2O for three breaths, from 10 to 15 cmH2O for three breaths, and from 15 to 20 cmH2O for 10 breaths [2]. Then PEEP was reduced to 12 cmH2O. This RM was repeated every hour. In the PEEP group PEEP was maintained at 12 cmH2O during the whole anesthesia. Hemodynamics, blood gases and dynamic compliance were evaluated. Conclusion Energy dissipation was largely dependent on strain, obtained with very high tidal volumes, but also lung inhomogeneities and lung opening and closing played a signifi cant role. Reference 1. Cressoni M. et al. Am J Respir Crit Care Med. 2014;189:149-58. P244 Conclusion RM patients with high SPCR and with reduced reactivity of the cardiovascular system improve lung function, but this is associated with the risk of hemodynamic instability. References 1. Zabolotskikh I, et al. Eur J Anesth. 2014;31:62. 2. Weingarten TN, et al. Br J Anesth. 2010;104:16-22. Determinants of energy dissipation in the respiratory system during mechanical ventilation Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P247 (doi: 10.1186/cc14327) Introduction Mechanical ventilation performs at each breath mechanical work on the lung parenchyma. Part of this energy is recovered and part is dissipated into the respiratory system. The purpose of this study is to assess the respective role of three known determinants of energy dissipation: lung opening and closing, strain and lung inhomogeneities [1]. Figure 1 (abstract P245). Bronchodilation and bronchial recruitment split per lobe for one patient. g g Methods Thirteen female piglets (21 ± 2 kg) were ventilated with a strain (VT/FRC) greater than 2.5 (VT ~ 38 ± 3 ml/kg) for 54 hours or until massive lung edema developed. Piglets were divided into fi ve groups characterized by diff erent energy loads obtained varying the respiratory rate: 15 breaths/minute (n = 3), 12 (n = 3), 9 (n = 3), 6 (n = 2) and 3 (n = 2). Every 6 hours two CT scans were performed (end-expiration and end- inspiration) and a static pressure–volume curve was obtained. A total of 51 CT scan couples and 51 corresponding pressure–volume curves was analyzed. The energy dissipated in the lung parenchyma at each breath was determined as the hysteresis area of the pressure–volume curve. CT scans were quantitatively analyzed with dedicated software. Data are presented as median (interquartile range). airway recruitment. This pilot study shows that, in ARDS, the upper lung regions are subject to airway dilation, whereas the lower (atelectatic) lung lobes have more airway opening with higher PEEP levels. Reference 1. De Backer et al. Radiology. 2010;257:854-62. P246fi P248 P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Results RM improved oxygenation compared with the PEEP group. The mean increase in the oxygenation index at the end of surgery was 31% (from 340 to 445 mmHg, P <0.05), in the PEEP group the increase was less signifi cant and amounted to 12% (from 330 to 370 mmHg, P <0.05). Dynamic compliance increased by 35% in the RM group and did not change in the PEEP group. Hemodynamic changes at RM were Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) Introduction Mechanical ventilation and hyperoxia independently promote pulmonary injury and infl ammation. However, the time course of the immune response following concurrent exposure is unclear. The Introduction Mechanical ventilation and hyperoxia independently promote pulmonary injury and infl ammation. However, the time course of the immune response following concurrent exposure is unclear. The Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S87 lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy. lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy. Figure 1 (abstract P248). Infl ammatory mediators independent of tidal volumes after 12 hours of MV. Methods In 40 patients we performed EIT measurements at fi ve points of time (Table 1) with the Swisstom BB2 prototype. Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) p g g Methods Healthy male C57Bl/6J mice, aged 9 to 10 weeks, were randomly assigned to experimental groups (n = 8), in which the applied fractions of oxygen (FiO2) were 30%, 50% or 90% and tidal volumes were either 7.5 or 15 ml/kg. Anesthetized mice were tracheotomized and ventilated for 8 or 12 hours. Infl ammatory cells and mediators were measured in bronchoalveolar lavage fl uid (BALf).i Introduction The still unclear mechanisms causing ventilator- induced diaphragm dysfunction (VIDD) are considered intrinsic to the diaphragm muscle fi bers. VIDD delays and complicates weaning from mechanical ventilation (MV) and accordingly contributes to prolonged ICU stay by 50%, with older patients being more aff ected than the young. The main aim of this study was to measure the eff ects of aging and 5 days of MV on rat diaphragm muscle fi ber structure and function. We also aimed to investigate the biological age of the old rats to obtain data useful to design future experimental studies focusing on the eff ects of age in an ICU setting. Results Mice exposed to higher FiO2 had signifi cantly higher PaO2 levels at the end of the experiment. The total number of infl ammatory cell in the BALf was not signifi cantly diff erent between the experimental groups (P = 0.28), yet an increasing trend in the percentage of neutrophils was observed with increasing FiO2 (P = 0.03). Cytokine and chemokine levels did not diff er between FiO2 groups at 8 hours of ventilation. In mice ventilated for 12 hours, a signifi cantly increasing trend in IFNγ, IL-1β, IL- 10, MCP-1 and TNFα (Fig. 1, P <0.01) was measured with increasing FiO2, whereas IL-6, KC, MIP-2, GM-CSF and VEGF remained virtually unchanged. Diff erences between the tidal volume groups were small and did not markedly infl uence the eff ects of hyperoxia. See Figure 1.f Methods We used a unique ICU rat model, which allows us to maintain the vital parameters stable under deep sedation and MV for long durations (several weeks). Diaphragm fi ber cross-sectional area (CSA) and force-generating capacity (specifi c force = absolute force / CSA) were measured in young (6 months) and old (28 to 32 months) F344/ BN hybrid rats in response to 5 days of deep sedation and volume- controlled MV. P250 Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) To investigate the biological age of the old rats, we performed a second set of experiments, comparing muscle fi ber CSA and specifi c force in fast and slow-twitch distal hind limb muscles in three diff erent age groups: young adults (6 months), middle aged (18 months) and old rats (28 months). ylf yp g Conclusion Hyperoxia induced a time-dependent and diff erentiated immune response that was independent of tidal volumes in a model of mechanically ventilated mice. The presence of cytokines and chemokines in the pulmonary compartment was more pronounced with prolonged and severe hyperoxia. P248 Thirty-two electrodes were used to apply weak alternating currents to the thorax and to measure the resulting voltages, from which tomographic images of the changes in regional impedance caused by ventilation were created. We describe the ventilation distribution using a novel EIT lung function parameter called Silent Spaces that provides information about areas that do not receive much or any air during tidal breathing and are divided into nondependent (NSS) and dependent Silent Spaces (DSS) using a reference line that runs perpendicular to the gravity vector right through the centre of ventilation. NSS and DSS are expressed as a percentage of the total lung area. Results Perioperative changes of NSS and DSS are shown in Table 1 as mean ± SD. Statistically signifi cant diff erences marked by § when compared with 1 or by * when compared with 3 (P <0.05). Conclusion We describe for the fi rst time the mapping of Silent Spaces during spontaneous breathing and changing ventilation conditions and body positions in patients with healthy lungs using EIT. This mapping of Silent Spaces might prove useful for developing perioperative protective ventilation strategies. Figure 1 (abstract P248). Infl ammatory mediators independent of tidal volumes after 12 hours of MV. aim of this preclinical study was to study both time-dependent and dose-dependent eff ects of supplemental oxygen during prolonged ventilatory support on pulmonary infl ammation in a well-established murine model of ventilation comparing low and high tidal volumes. aim of this preclinical study was to study both time-dependent and dose-dependent eff ects of supplemental oxygen during prolonged ventilatory support on pulmonary infl ammation in a well-established murine model of ventilation comparing low and high tidal volumes. Methods Healthy male C57Bl/6J mice, aged 9 to 10 weeks, were randomly assigned to experimental groups (n = 8), in which the applied fractions of oxygen (FiO2) were 30%, 50% or 90% and tidal volumes were either 7.5 or 15 ml/kg. Anesthetized mice were tracheotomized and ventilated for 8 or 12 hours. Infl ammatory cells and mediators were measured in bronchoalveolar lavage fl uid (BALf).i Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods All 89 inpatients on the CCU who received mechanical ventilation continuously for 7 days or more between October 2012 and December 2013 were initially included. Forty patients who were intubated prior to arrival at RCHT, had incomplete notes, or were extubated during end-of-life care were excluded. Patients were divided into groups by fi rst airway intervention; 31 TOE, 18 TR. fi bers from young, middle aged and old animals confi rmed the 28 to 32 month rats to be senescent from a skeletal muscle point of view. p Conclusion These results demonstrate intrinsic changes in diaphragm muscle fi bers of signifi cant importance for the prolonged and complicated weaning from MV. Moreover, the increased number of frail diaphragm muscle fi bers observed after MV in old age, both controls and mechanically ventilated, off ers a further age-related possible mechanism which may be of signifi cant clinical importance. These results also provided useful information to design future experimental studies focused on the eff ect of age in an ICU setting, pharmacological intervention strategies as well as mechanisms underlying rat strain diff erences. g p yi y ; , Results A total 52% (16/31) of patients had TOE, required no other airway intervention and survived to discharge from hospital, compared with 72% (13/18) of TR patients. Four patients from each group failed the fi rst intervention and died prior to a second intervention. In total, 8/11 patients who had a second intervention after failed TOE survived to discharge from hospital. One patient had a second TR but died before discharge. This gave an in-hospital mortality rate of 19% for the TOE group and 28% for the TR group. TOE was performed earlier, all 31 on days 7 to 15. TR was performed later; 14/18 on days 7 to 15, and 4/18 on days 17 to 23. Early TR was more successful; 11/11 survived to discharge without a second intervention who had TR on days 7 to 12, compared with 29% (2/7) after day 12. TOE was more successful when performed later; 64% (7/11) survived to discharge without a second airway intervention when TOE was after day 10, 45% (9/20) between days 7 and 10. Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Results Twenty-eight patients were included in G1 and 22 patients in G2. There was no signifi cant diff erence between the patients’ admission parameters and daily NIV usage times. PaCO2 decreased >5 mmHg in 93% of G1 patients and in 60% of G2 patients in the fi rst 6 hours (P = 0.044). A 10 mmHg reduction in PaCO2 occurred in more patients (93% vs. 60%, P = 0.004) and in a shorter time (1.8 ± 1.2 vs. 3 ± 3 days, P = 0.044) in G1. At the time of discharge, PaCO2 levels were <50 mmHg in 79% of G1 and 41% of G2 patients (P = 0.006). Both groups showed similar and signifi cant improvements in PaO2, PaCO2 and HCO3 – levels within the fi rst 4 days but only in G1 patients were HCO3 – levels decreased more rapidly than G2 patients (P = 0.007). Duration of NIV (6 ± 2 vs. 8 ± 3 days, P = 0.002) and the number of mode and pressures changes (0.3 ± 1.8 vs. 2 ± 2 times, P >0.0001) were signifi cantly less in G1. While mean IPAP was similar in both groups, maximum and minimum EPAP titrated automatically in G1 were signifi cantly diff erent from G2. Mean tidal volume and amount of leakage were also signifi cantly higher in G1. fi g pfi Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) fi g pfi Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Introduction Bilevel non-invasive ventilation (NIV) is an established therapy in chronic obstructive pulmonary disease (COPD) but confl icting evidence exists for its use in patients with pneumonia. Initial arterial pH <7.25 is used as a marker of severity and need for admission to critical care (CC) [1]. We examined the impact of pH and condition on outcome in patients with acute respiratory failure (ARF) of mixed aetiology treated with NIV. Methods Data were collected retrospectively for a 5-year period from 2008 to 2013 using the Metavision electronic patient record. We identifi ed all patients admitted with ARF treated with bilevel NIV. Patients who received continuous positive airway pressure or had a primary surgical problem were excluded. Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) We recorded primary cause of respiratory failure, arterial blood gas values and mortality.i g gi y g Conclusion These results suggest that the AVAPS-AE mode may provide some advantages in hypercapnic ICU patients such as rapid PaCO2 reduction, less NIV duration and workload. Results A total of 145 patients were identifi ed. Mean age was 64 and 51% were male. The primary diagnosis was pneumonia in 69 patients and exacerbation of COPD in 57. The overall mortality was 19% on CC and 39% at 1 year. In patients with COPD, infective exacerbations had a higher CC mortality (17%) compared with non-infective (0%). However, by 1 year the mortality was 28% in infective and 29% in non-infective. Patients with pneumonia had a higher mortality on CC (25%) and at 1 year (48%). Patients with an initial pH <7.25 were less likely to survive. The mortality at discharge from CC was 16% (pH ≥7.25) and 26% (pH <7.25) but narrowed to 38% and 39% by 1 year. When subdivided, it was found that patients with infective COPD and pH <7.25 had the lowest 1-year mortality (17%) while those with pneumonia and pH <7.25 had the highest mortality (67%). P251 P251 Does it make a diff erence to add automatic EPAP titration to the volume-targeted pressure support mode in noninvasive ventilation of hypercapnic ICU patients? G Gursel, A Zerman, B Basarik, K Gonderen, M Aydogdu, S Memmedova Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P251 (doi: 10.1186/cc14331) Introduction Obese patients are increasing in number in ICUs and more than 90% of them also have sleep apnea syndrome. Variability in upper airway resistance during sleep and awakening periods makes it diffi cult to set EPAP in these patients. A new mode that automatically titrates EPAP according to upper airway resistance and IPAP according to target tidal volume may be more eff ective. The aim of this study is to evaluate whether adding automatic EPAP titration to the volume-targeted pressure support mode will provide any therapeutic advantages in hypercapnic ICU patients. Conclusion TOE is more common and is associated with shorter time spent ventilated, in the CCU and in hospital than TR. It is also associated with a lower in-hospital mortality rate. TOE is more successful when performed after day 10; TR is more successful when performed before day 13. After failed TOE, a second TOE is associated with longer time in hospital but a better mortality rate than secondary tracheostomy. y Methods The hypercapnic patients treated with average volume- assured pressure support-automatic EPAP (AVAPS-AE) mode (Group1 (G1)) were compared with those treated with AVAPS mode (Group 2 (G2)). G2 was recruited retrospectively and matched with G1 according to diagnoses, demographic characteristics, arterial blood gas values and daily noninvasive ventilation (NIV) usage times. Trilogy 100® devices and their software Directview® (Philips Respironics) were used to reveal the respiratory data such as pressures, volumes, and daily usage times. For statistical analyses, t test, chi-square test and repeated measures of ANOVA were used. P253 Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) P249 Perioperative assessment of regional ventilation during changing body positions and ventilation conditions by electrical impedance tomography with increased spatial resolution and signal quality A März1, A Ukere1, K Wodack1, C Trepte1, A Waldmann2, S Böhm2, D Reuter1 1University Medical Center Hamburg-Eppendor, University Hamburg, Germany; 2Swisstom AG, Landquart, Switzerland Critical Care 2015, 19(Suppl 1):P249 (doi: 10.1186/cc14329) Perioperative assessment of regional ventilation during changing body positions and ventilation conditions by electrical impedance tomography with increased spatial resolution and signal quality A März1, A Ukere1, K Wodack1, C Trepte1, A Waldmann2, S Böhm2, D Reuter1 1University Medical Center Hamburg-Eppendor, University Hamburg, Germany; 2Swisstom AG, Landquart, Switzerland Critical Care 2015, 19(Suppl 1):P249 (doi: 10.1186/cc14329) Results This study demonstrated an unexpected increase in CSA (P <0.001) of the diaphragm fi bers in response to 5 days of MV in both young and old animals. Maximum force decreased 39.8 to 45.2% (P <0.001) in both young and old animals compared with controls, resulting in a dramatic loss of specifi c force. This increase in CSA and the concomitant decrease in specifi c force observed in both young and old diaphragm fi bers are compatible with an ineff ective compensatory hypertrophy in response to the MV. The comparison of the limb muscles Introduction Electrical impedance tomography (EIT) is a functional imaging technology allowing one to regionally monitor aeration of the Table 1 (abstract P249) 1 2 3 4 5 (sitting, (supine, (ventilated, (ventilated, (ventilated, spontaneous breathing) spontaneous breathing) supine position) 30° head down position) supine position) NSS (%) 5.25 ± 2.9 4.12 ± 1.89§ 3.05 ± 1.9§ 2.8 ± 2.7§ 2.67 ± 1.9§ DSS (%) 0.07 ± 0.3 2.29 ± 2.3§ 9.23 ± 6.3§ 11.5 ± 8.9§* 8.5 ± 5.8§ Table 1 (abstract P249) 1 2 3 4 5 (sitting, (supine, (ventilated, (ventilated, (ventilated, spontaneous breathing) spontaneous breathing) supine position) 30° head down position) supine position) NSS (%) 5.25 ± 2.9 4.12 ± 1.89§ 3.05 ± 1.9§ 2.8 ± 2.7§ 2.67 ± 1.9§ DSS (%) 0.07 ± 0.3 2.29 ± 2.3§ 9.23 ± 6.3§ 11.5 ± 8.9§* 8.5 ± 5.8§ S88 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 After fi rst failed TOE, four patients had a successful second TOE; all four survived to discharge resulting in a median CCU stay of 29 days and median hospital stay of 39 days (excluding prior to CCU admission). Seven patients had TR after the fi rst failed TOE, fi ve survived to discharge from the CCU and four to discharge from the hospital. This group had shorter median stays in both the CCU (27 days) and hospital (32 days). Overall, the median duration of time ventilated, in the CCU, and in hospital was shorter for the TOE group; 13, 17 and 24 days respectively, compared with 22, 27.5 and 34 days for the TR group. P252 Retrospective study of patients receiving long-term mechanical ventilation I D d B H d R Si l i I Dunwoody, B Hopwood, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2015, 19(Suppl 1):P252 (doi: 10.1186/cc14332) Introduction This study analysed the practice of clinicians managing patients requiring long-term mechanical ventilation in the critical care unit (CCU) of the Royal Cornwall Hospital, Truro (RCHT), comparing outcomes of primary tracheostomy (TR) with trial of extubation (TOE). Conclusion NIV is used in our unit with comparable success rates to published series [2,3]. COPD patients responded well to NIV, while Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S89 patients with pneumonia treated with NIV have the highest mortality. A low presenting pH is associated with a higher mortality in patients with pneumonia treated with NIV. However, in COPD patients, pH <7.25 is not associated with higher mortality in CC or at 1 year. Further work defi ning the precise role of pH as a prognostic indicator is warranted. References patients with pneumonia treated with NIV have the highest mortality. A low presenting pH is associated with a higher mortality in patients with pneumonia treated with NIV. However, in COPD patients, pH <7.25 is not associated with higher mortality in CC or at 1 year. Further work defi ning the precise role of pH as a prognostic indicator is warranted. References care units within the NoECCN. Patient data collected included patient demographics, admission diagnosis and speciality, hospital length of stay (LOS) pre and post critical care admission, severity of illness scores, critical care LOS and status at hospital discharge. p g Results During the study period 134 patients met the criteria for PMV representing 1% of annual admissions and 6.9% NoECCN bed- days. The majority of patients receiving PMV were medical (50.7%), followed by emergency surgery (20.1%), elective surgery (16.4%) and specialist services such as spinal cord injury (8.2%) and cardiothoracic transplant (4.5%). The commonest admission diagnosis in the medical population was pulmonary infection followed by acute neurological disorders, while 89.4% of surgical patients were admitted to critical care during the perioperative period. At the end of the study period the highest hospital mortality was observed in the nonspecialist surgical population (26.5%). In contrast, the medical population had one of the lowest hospital mortality rates (11.8%), lower than predicted using the intensive care national audit research network illness severity score. P254 Lung protective ventilation with lower tidal volumes and development of pulmonary complications in critically ill patients without ARDS FD Simonis1, A Serpa Neto2, M Gama de Abreu3, P Pelosi4, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Isrealita Albert Einstein, São Paulo, Brazil; 3University Hospital Carl Gustav Carus, Dresden, Germany; 4IRCCS San Martino IST, Genoa, Italy Critical Care 2015, 19(Suppl 1):P254 (doi: 10.1186/cc14334) Reference 1. Pilcher DV, et al. Outcomes, cost and long term survival of patients referred to a regional weaning unit. Thorax. 2005;60:187-92. Methods An individual patient data meta-analysis of studies of ventilation in ICU patients without ARDS. Corresponding authors of retrieved studies provided individual patient data. The primary outcome, pulmonary complications, was a composite of development of ARDS or pneumonia during hospital stay. Secondary outcomes included ICU and hospital length of stay, and in-hospital mortality. Patients were assigned to three groups based on tidal volume size (≤7 ml/kg predicted body weight (PBW), 7 to 10 ml/kg PBW, or ≥10 ml/ kg PBW). P252 Retrospective study of patients receiving long-term mechanical ventilation I D d B H d R Si l i Comparable rates of hospital discharge were found in both medical (85%) and nonspecialist surgical patients (88.9%). 1. 2013 BTS NIV audit. https://www.brit-thoracic.org.uk/document-library/ audit-and-quality-improvement/audit-reports/ bts-adult-niv-audit-report-2013/ 2. Carillo et al. ICM. 2012;38:458-66. 3. Lightowler et al. BMJ. 2003;326:185-9. Lung protective ventilation with lower tidal volumes and development of pulmonary complications in critically ill patients without ARDS FD Simonis1, A Serpa Neto2, M Gama de Abreu3, P Pelosi4, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Isrealita Albert Einstein, São Paulo, Brazil; 3University Hospital Carl Gustav Carus, Dresden, Germany; 4IRCCS San Martino IST, Genoa, Italy Critical Care 2015, 19(Suppl 1):P254 (doi: 10.1186/cc14334) g Conclusion The results of this study highlight an expanding proportion of NoECCN critical care bed-days occupied by stable patients undergoing PMV. In keeping with published UK data, elevated hospital mortality was observed in the nonspecialist surgical subpopulation. Although the literature suggests the medical cohort of patients has poorer prognosis, within our region all were liberated from mechanical ventilation and over 80% were discharged from hospital. R f Introduction A large meta-analysis suggests that use of low tidal volumes benefi ts patients without ARDS [1] but most studies in this meta-analysis included patients receiving ventilation during general anesthesia for surgery. The aim of the present meta-analysis is to determine the association between tidal volume size and development of pulmonary complications in ICU patients. Eff ects of positive end-expiratory pressure on lung ventilation/ perfusion matching: a clinical study N Eronia1, T Mauri2, G Bellani1, R Marcolin1, S Marocco Arrigoni1, G Grasselli1, A Pesenti1 , 1San Gerardo Hospital, Monza, Italy; 2IRCC Ca’ Granda Maggiore Policlinico Hospital, Milan, Italy Critical Care 2015, 19(Suppl 1):P256 (doi: 10.1186/cc14336) Results Seven investigations (2,184 patients) were meta-analyzed. Pulmonary complications occurred in 23%, 28% and 31% respectively in the ≤7 ml/kg PBW, 7 to 10 ml/kg PBW and ≥10 ml/kg PBW group (adjusted RR, 0.72; 95% CI, 0.52 to 0.98; P = 0.042). Occurrence of pulmonary complications was associated with a lower number of ICU- free days and alive at day 28, a lower number of hospital-free days and alive at day 28 and increased in-hospital mortality. Introduction Positive end-expiratory pressure (PEEP) exerts multiple protective eff ects in hypoxemic critically ill patients: PEEP can increase end-expiratory lung volume (EELV) and induce recruitment, thus reducing lung strain and opening and closing of alveoli and potentially improving the ventilation/perfusion matching. In particular, estimation of PEEP-induced ventilation/perfusion matching might help identify the optimal PEEP setting, but bedside non-invasive methods are few and complex to be applied in daily clinical practice. Electrical impedance tomography (EIT) is a non-invasive bedside technique that claims to track global and regional changes in perfusion and ventilation over time. In the present study we aimed at verifying the eff ects of PEEP on ventilation/perfusion matching, as assessed by EIT, in acute respiratory failure patients. Conclusion Ventilation with low tidal volumes is associated with a lower risk of development of pulmonary complications. Occurrence of pulmonary complications is associated with an increased ICU and hospital length of stay and in-hospital mortality in ICU patients without ARDS. Reference Reference 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. Reference 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. p y p Methods We enrolled 20 intubated critically ill patients undergoing controlled mechanical ventilation, sedated, paralyzed and with PaO2/FiO2 ≤300 at PEEP ≥5 cmH2O. We started EIT monitoring (Pulmovista500®; Dräger Medical GmbH, Lübeck, Germany) and applied two PEEP levels (clinical and clinical + 5 cmH2O) for 20 minutes each. We collected ventilatory and EIT parameters and, by offl ine analysis, we calculated the increase of EELV at higher PEEP and the EIT- based indexes of ventilation heterogeneity (VtHetend-insp) and of the regional homogeneity of ventilation/perfusion matching (HA/P). P255 Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 1Sunderland Royal Hospital, Sunderland, UK; 2James Cook University Hospital, Middleborough, UK Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 1Sunderland Royal Hospital, Sunderland, UK; 2James Cook University Hospital, Middleborough, UK Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) Pressure reconstruction method for spontaneous breathing eff ort monitoring Pressure reconstruction method for spontaneous breathing eff ort monitoring N Damanhuri1, YS Chiew1, NA Othman1, PD Docherty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P259 (doi: 10.1186/cc14339) N Damanhuri1, YS Chiew1, NA Othman1, PD Docherty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P259 (doi: 10.1186/cc14339) Introduction Estimating respiratory mechanics of mechanically venti lated patients is unreliable when patients exhibit spontaneous breathing (SB) eff orts on top of ventilator support. This reverse triggering eff ect [1] results in an M-wave shaped pressure wave. A model-based method to reconstruct the aff ected airway pressure curve is presented to enable estimation of the true underlying respiratory mechanics of these patients. Table 1 (abstract P257) Ki <27.8 ml/ Ki ≥27.8 ml/ minute/ml minute/ml × 104 (n = 7) × 104 (n = 8) P value PaO2/FiO2 280 (261 to 346) 239 (212 to 271) 0.31 pH 7.46 (7.43 to 7.48) 7.41 (7.39 to 7.44) 0.15 PaCO2 (mmHg) 37 (34 to 44) 50 (46 to 53) 0.01 WBCs (103 cell/mm3) 8 (8 to 9) 9 (9 to 16) 0.34 Total lung volume (ml) 1,298 (1,092 to 1,494) 1,516 (1,408 to 1,665) 0.18 Total lung weight (g) 592 (488 to 741) 732 (597 to 774) 0.39 Total lung gas (ml) 706 (551 to 843) 804 (755 to 1,080) 0.15 Not-infl ated lung tissue (%) 25 (12 to 30) 23 (17 to 30) 0.95 Poorly infl ated lung tissue (%) 34 (42 to 42) 31 (29 to 34) 0.39 Well-infl ated lung tissue (%) 36 (27 to 47) 47 (35 to 52) 0.53 Conclusion Patients clinically defi ned as having primary graft dysfunction did not have an increased rate of 18-FDG uptake. 18-FDG uptake was not diff erent in single-lung versus bilateral transplantation and, in single-lung procedures, the native lung showed elevated infl ammatory activity. Methods Airway pressure and fl ow data from 72 breaths of a pneu- monia patient were used for proof of concept. A pressure wave reconstruction method fi lls parts of the missing area caused by SB eff orts and reverse triggering by connecting the peak pressure and end-inspiration slope (Figure 1). A time-varying elastance model [2] was then used to identify underlying respiratory elastance (AUCEdrs). P258 4.8) vs. 1.9 (1.5 to 2.1), P <0.01) and lower compliance of dependent lung regions (Crsdep, 13 ± 3 ml/cmH2O vs. 18 ± 6 ml/cmH2O, P <0.05), as compared with patients with smaller improvement. 4.8) vs. 1.9 (1.5 to 2.1), P <0.01) and lower compliance of dependent lung regions (Crsdep, 13 ± 3 ml/cmH2O vs. 18 ± 6 ml/cmH2O, P <0.05), as compared with patients with smaller improvement. P257 18-FDG PET in lung transplantation I Al 1 F V l 1 M G l 1 B S f P257 18-FDG PET in lung transplantation I Al 1 F V l 1 M G l 1 B S f 18-FDG PET in lung transplantation I Algieri1, F Valenza1, M Guanziroli1, B Safaee Fakhr1, M Cressoni1, M Brioni1, A Colombo1, G Babini1, F Crimella1, D Massari1, K Nikolla1, G Crisafulli1, S Paladini1, L Rosso2, A Palleschi2, F Zito2, D Chiumello1, L Gattinoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda- Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2015, 19(Suppl 1):P257 (doi: 10.1186/cc14337) p g p g p g p g p Results The median value of LU comets was signifi cantly lower in Group A compared with Group B. There was a signifi cant increase in hypoxic index in Group A compared with Group B. There was no signifi cant diff erence between both groups as regards PIP, while Pplat showed a signifi cant increase in Group B compared with Group A and Cs showed a signifi cant decrease in Group B. Introduction Lung transplantation is associated with an infl ammatory reaction known as primary graft dysfunction. This clinical syndrome occurs within the fi rst 72 hours after transplantation and is characterized by hypoxemia (PaO2/FiO2 <300) and bilateral infi ltrates not secondary to cardiac dysfunction, viral or bacterial pneumonia and venous anastomotic obstruction. gi p Conclusion LU is a useful to quantify EVLW; it is a good predictor of weaning. References Methods 18-FDG PET scan was used to study 15 lung transplantation patients. The rate of 18-FDG uptake (Ki) was computed voxel by voxel with the Patlak method. Patients were divided according to the median Ki (27.8 (20.3 to 34.6) ml/minute/ml × 104). Data are reported as median and interquartile range. 1. Nektaria X, Eumorfi a K, George P, et al. Impact of lung ultrasound on clinical decision making in critically ill patients. Intensive Care Med. 2014;40:57-65. 2. Jambrik Z, Monti S, Coppola V, et al. Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water. Am J Cardiol. 2004;93:1265-70. 2. Jambrik Z, Monti S, Coppola V, et al. Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water. Am J Cardiol. 2004;93:1265-70. Results Five patients developed primary graft dysfunction; median Ki in these patients was not diff erent from patients who did not (24.5 (18.2 to 33.6) ml/minute/ml × 104 vs. 29.1 (23 to 35.4) ml/minute/ ml × 104 respectively, P = 0.64). Bilateral lung transplantation patients were characterized by a median Ki of 30.5 (22.9 to 34.5) ml/minute/ ml × 104, while patients undergoing single-lung transplantation presented a median Ki of 24.4 (21 to 34.1) ml/minute/ml × 104 (P = 0.61). Considering single-lung transplantation, graft and native lung had similar Ki: 24.4 (21 to 34.1) ml/minute/ml × 104 versus 24.2 (17.7 to 30.1) ml/minute/ml × 104 respectively (P = 0.64). When patients were divided according to the median Ki value, higher Ki was associated with higher PaCO2 values (50 (46 to 53) mmHg vs. 37 (34 to 44) mmHg, P = 0.01). See Table 1. Usefulness of extravascular lung water assessment as a predictor of weaning from mechanical ventilation Usefulness of extravascular lung water assessment as a predictor of weaning from mechanical ventilation Conclusion EIT might represent a feasible, bedside method to estimate PEEP-induced improvement in ventilation/perfusion matching. Assessing regional ventilation and mechanical lung properties might help identify patients who would benefi t more from higher PEEP. g Alexandria University Hospital, Alexandria, Egypt Critical Care 2015, 19(Suppl 1):P258 (doi: 10.1186/cc14338) Introduction Quantitative measurement of extravascular lung water (EVLW) would be extremely useful for clinical management, both as an index of severity and to guide treatment lung ultrasonography (LU) as a tool to quantify EVLW. q y Methods Forty mechanically ventilated patients were examined for 5 successive days after being eligible for weaning; counting B-lines (comets) in both lung fi elds by LU, peak airway pressure, plateau airway pressure, static compliance and ABG analysis every 6 hours. Patients were divided into two groups according to success of the weaning process: group A (weaned group), and group B (nonweaned group). Results The median value of LU comets was signifi cantly lower in Group A compared with Group B. There was a signifi cant increase in hypoxic index in Group A compared with Group B. There was no signifi cant diff erence between both groups as regards PIP, while Pplat showed a signifi cant increase in Group B compared with Group A and Cs showed a signifi cant decrease in Group B. Pressure reconstruction method for spontaneous breathing eff ort monitoring The area of the unreconstructed M-wave has less pressure, resulting in a lower overall AUCEdrs without reconstruction. The missing area of the airway pressure or AUCEdrs is hypothesized to be a surrogate of patient- specifi c inspiratory to assess the strength of SB eff orts. AUCEdrs and missing area A2 are compared with/without reconstruction.if 2 Results Median AUCEdrs and breath-specifi c eff ort using reconstruction were 24.99 (IQR: 22.90 to 25.98) cmH2O/l and 3.64 (IQR: 0.00 to 3.87)% versus AUCEdrs of 20.87 (IQR: 15.24 to 27.48) cmH2O/l for unreconstructed M-wave data, indicating signifi cant patient and breath-specifi c SB eff ort, and the expected higher elastance (P <0.05). Conclusion A simple reconstruction method enables the real-time measurement of respiratory system properties of a SB patient and measures the surrogate of the SB eff ort, that latter of which has clinical use in deciding whether to extubate or re-sedate the patient. Conclusion Patients clinically defi ned as having primary graft dysfunction did not have an increased rate of 18-FDG uptake. 18-FDG uptake was not diff erent in single-lung versus bilateral transplantation and, in single-lung procedures, the native lung showed elevated infl ammatory activity. Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) g g y p g Results Patients were 62 ± 12 years old, mean PaO2/FiO2 was 197 ± 52, lower PEEP level was 7 (7 to 9) cmH2O, while higher PEEP was 12 (12 to 14) cmH2O (P <0.001). At higher PEEP, EELV increased (391 (334 to 555) ml vs. PEEP low, considered as baseline, P <0.001); VtHetend- insp was reduced (1.8 (1.5 to 2.4) vs. 2.2 (1.8 to 2.6), P <0.001) and HA/P increased (0.29 ± 0.19 vs. 0.2 ± 0.15, P <0.05). Interestingly, the increase of HA/P was signifi cantly correlated with the decrease of VtHetend- insp (r = –0.48, P <0.05). Moreover, patients with higher potential for improvement of ventilation/perfusion matching (that is, patients with increase of HA/P >16%) had higher baseline VtHetend-insp (2.6 (2.3 to Introduction The combination of a global demographic shift and increased survival following critical illness has led to an increasing number of patients requiring prolonged mechanical ventilation (PMV) and longer critical care stay. This is a prospective observational study evaluating the characteristics and speciality-based outcome of critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network (NoECCN). Methods A weekly survey was conducted over a 1-year period screening patients older than 16 years of age requiring PMV in all 18 adult critical S90 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P257 q y Methods Forty mechanically ventilated patients were examined for 5 successive days after being eligible for weaning; counting B-lines (comets) in both lung fi elds by LU, peak airway pressure, plateau airway pressure, static compliance and ABG analysis every 6 hours. Patients were divided into two groups according to success of the weaning process: group A (weaned group), and group B (nonweaned group).i Breath-to-breath respiratory mechanics variation: how much variation should we expect? Each patient was sedated to prevent spontaneous breathing eff ort, and ventilated using the volume control mode with a square fl ow profi le. Varied PEEP levels were maintained for 30 minutes before 1 minute of data were collected for analysis. This dataset provides a wide range of respiratory mechanics values, and the clinical protocol detail is in [1]. A clinically proven, single compartment model respiratory system elastance (Ers) is identifi ed from data for every breathing cycle at each PEEP level using a linear regression method. The dynamic elastance (Edynamic = (peak airway pressure – positive end-expiratory pressure) / tidal volume) of the corresponding breathing cycle is calculated for comparison.fii p g g y p Results The coeffi cient of variation (CV) of identifi ed Ers across all patients was low (<0.005), as expected, as the 30-minute period allows time-dependent alveolar recruitment to fully occur and stabilise. However, even with substantial stabilisation periods, there remains a diff erence between the minimum and maximum estimated Ers within each 1-minute period of analysed data. The diff erences were median 2.8% (interquartile range (IQR): 1.5 to 4.6%, 90% range (90R): 0.8 to 13.4) for Ers and 3.6% (IQR: 2.3 to 5.2, 90R: 0.9 to 10.8) for Edynamic, and in extreme cases, can be up to 16%. Results Over 275 paired samples were analysed over the following concentration ranges: pH 7.249 to 7.545; pCO2 3.32 to 11.01 kPa; pO2 5.59 to 21.80 kPa; Hct 23.6 to 41.4%; K+ 3.3 to 4.79 mM. The imprecision for each sensor was calculated to be: pH –0.00 ± 0.03; pCO2 –0.48 ± 0.34 kPa; pO2 –0.85 ± 0.96 kPa; Hct –4.49 ± 3.42%; K+ 0.08 ± 0.15 mM, respectively. The data collected on the new analyser for Hct showed relative imprecision of 3.42%. The pooled SD was calculated to be 1.21% and the mean SD of each of the novel devices used was 1.14%. This indicates that scatter observed on the Hct sensor was largely due to inter-device rather than intra-device factors. Conclusion This study quantifi ed the variability (over short periods) of identifi ed and estimated respiratory mechanics properties used to (potentially) guide ventilation care in sedated patients. It is also important to note that this minimum level of variability occurs even when stabilisation is achieved. Breath-to-breath respiratory mechanics variation: how much variation should we expect? 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) p KT Kim1, YS Chiew1, C Pretty1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P260 (doi: 10.1186/cc14340) KT Kim1, YS Chiew1, C Pretty1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P260 (doi: 10.1186/cc14340) Introduction Patient-dedicated arterial blood analysers off er the potential to allow close monitoring of critically ill patients without leaving the patient’s bedside or net loss of blood for the patient. A new patient-attached blood gas analyser was evaluated in a clinical environment to compare the results against the reference bench-top analyser. Introduction Model-based respiratory mechanics can be used to guide mechanical ventilation therapy. However, identifi ed mechanical properties vary breath to breath, leading to potential treatment errors when using model-based care that requires accuracy. This study investigates and quantifi es this variability to improve its application in guiding clinical interventions. Methods Twenty ICU patients with a range of clinical conditions, including trauma, head injury, post-surgical recovery and sepsis, were included in the study. The new analyser was operated by clinical staff at the Queen Elizabeth Hospital, Birmingham, who each underwent a 90-minute training programme. Patients were monitored using the patient-dedicated analyser (Proxima; Sphere Medical) for up to 3 days. Each time a sample was tested on the patient-dedicated analyser, a concurrent sample was drawn and tested on the hospital’s bench- top analyser (Cobas b221; Roche Diagnostics). Samples were assessed using methods described by Bland and Altman for repeated measures. Performance of the novel device was analysed by calculating bias as the mean diff erence between the new analyser and the comparator device, imprecision as ±1 standard deviation (SD) from the mean and limits of agreement as ±1.96 SD from the mean. Percentage values for bias and precision were calculated from analysis of the percentage diff erence between the two methods of analysis. Intra-device imprecision for the haematocrit (Hct) sensor was calculated using a pooled variance calculation. Methods Retrospective data from 12 acute respiratory distress syndrome (ARDS) patients were used [1]. 1. Bersten. Eur Respir J. 1998;12:526-32. References 1. Akoumianaki E, et al. Chest. 2013;143:927-38. 2. Chiew YS, et al. Biomed Eng Online. 2011;10:111. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 S91 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P259). Edrs for M-wave and reconstructed airway pressure at PEEP = 15 cmH2O. t PEEP = 15 cmH O Figure 1 (abstract P259). Edrs for M-wave and reconstructed airway pressure at PEEP = 15 cmH2O. P261 P261 Evaluation of a patient-dedicated blood gas analyser JJ Fox1, TH Clutton-Brock2 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) Breath-to-breath respiratory mechanics variation: how much variation should we expect? Thus, clinically, if this information was to be used to guide ventilation in real time, such as titrating PEEP to minimal elastance, larger errors, at least up to 15% variation in Ers, could be expected, which could well aff ect care. Such levels thus also begin to defi ne the minimum levels of change necessary to be larger than natural variation. Conclusion The patient-dedicated blood gas analysers demonstrated excellent agreement with the bench-top analyser for pH, pCO2, pO2 and K+, while Hct provides a reasonable trend monitor with variable bias. Proxima is well suited to enable staff to more closely manage unstable and critically ill patients. This device may be of signifi cant benefi t to patients at risk of iatrogenic anaemia or those being treated in side rooms/isolation rooms. Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) Introduction Maintaining the appropriate tidal volume (VT) is important for success of lung protective ventilation. However, in neonates, the presence of airway leaks may increase the errors in the delivery of tiny VT, which raises a concern of ventilator-induced lung injury. This study is to investigate the accuracy of VT delivery during non-invasive ventilation (NIV) with modern neonatal ventilators. Results There was a signifi cant correlation between the indexed EE and indexed VE in both groups (R = 0.51, P <0.0001 in the control group; R = 0.63, P <0.0001 in the MV group). The VR was signifi cantly suppressed in the MV group compared with the control group (0.041 ± 0.003/minute/ BEE vs. 0.069 ± 0.003/minute/BEE, P = 0.012; respectively). Although the indexed VE was comparable in the MV and control groups (0.19 ± 0.07 l/ kg vs. 0.17 ± 0.04 l/kg, P = 0.23; respectively), the indexed EE was shifted to a higher range in the MV group than in the control group (maximal indexed EE; 2.26 ± 0.68 vs. 1.74 ± 0.20, P = 0.008; respectively). The TV was smaller (maximal TV; 985 ± 592 ml vs. 1,410 ± 299 ml, P = 0.018; respectively) and the RR was more frequent (maximal RR; 30 ± 8/minute vs. 16 ± 4/minute, P <0.0001; respectively) in the MV group than in the control group. Methods Using a lung simulator for a patient body weight of 3 kg, we measured the actual delivered VT in the lung and compared it with the value displayed on the ventilator in six ventilators. We tested 18 conditions with various combinations of respiratory mechanics (normal, restrictive, obstructive), leak levels (0, 1.0, 1.5 l/minute), and PEEP settings (5, 10 cmH2O). All conditions were tested in NIV mode. The pressure level was set to achieve VT to the lung at 6 to 7 ml/kg. All other settings were: FIO2 0.21, Itime 0.6 seconds, f 25/minute, and default rise time. We calculated the mean errors of the ventilator-displayed VT at various levels of airway leak. Eff ective capnography training in the ICU using the ‘hats and caps’ training tool Eff ective capnography training in the ICU using the ‘hats and caps training tool CA Lobo, FE Kelly, S Steynberg, G Thomas, C Pope, M Eveleigh, M Charlton, TM Cook Royal United Hospital, Bath, UK Critical Care 2015, 19(Suppl 1):P264 (doi: 10.1186/cc14344) Reference Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S92 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P262 The VE was indexed by body weight and the EE was also indexed by the basal energy expenditure (BEE) estimated by the Harris–Benedict formula. VR was defi ned as the slope in the indexed VE–indexed EE plot with an assumption of those relationship in the linear manner. We examined the correlation between indexed EE and indexed VE in both groups, and the diff erences of the maximal indexed EE, the maximal indexed MV, and the others between both groups were investigated using the unpaired t test. For all the data, signifi cance was accepted at values of P <0.05. P262 Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) Table 1 (abstract P262) Leak amount Hamilton Hamilton (l/minute) C3 G5 Servo i PB840 PB980 VN500 0 26 (15) 4 (6) 3 (2) 2 (7) –4 (2) 2 (2) 1.0 –9 (5) 9 (5) –2 (2) –5 (4) –2 (4) 20 (5) 1.5 16 (8) –14 (8) –2 (2) –6 (4) –2 (3) 33 (6)a Data presented as mean (SD), %. aUnable to measure in one case. Introduction Failure to use or correctly interpret capnography in patients dependent on an artifi cial airway in ICUs was thought to have contributed to 74% of ICU airway-related deaths in the NAP4 study [1]. However, capnography is only of value if those using it can interpret it correctly, with recommendations for training all ICU staff in capnography [1,2]. A recent UK survey identifi ed that only 48% of ICUs have trained all staff in capnography interpretation (TM Cook, personal communication). In this study, we used a capnography teaching aid (‘hats and caps’) to educate all ICU staff during a 1-month period, and evaluated its eff ectiveness. Conclusion Tidal volume accuracy during neonatal NIV varies greatly among diff erent ventilators and leak conditions. This must be considered in neonatal ventilation management to avoid over- ventilation or underventilation. Figure 1 (abstract P264). ‘Hats and caps’ capnography training guide. Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators Conclusion The VR to external stress with mechanical ventilation is more suppressed than in healthy volunteers. The VE in the mechanical ventilation was earned by a higher RR rather than by increased TV. Careful monitoring of VE or RR would be benefi cial in early rehabilitation with mechanical ventilation. y Results The VT mean error values are presented in Table 1. When no leak existed, the mean error was less than 5% in all ventilators except one (C3) which showed a mean error of 26%. As the leak level increased, three ventilators (C3, G5, and VN500) showed marked diff erences between the delivered and displayed VT. In particular, the VN500 could not operate in the large leak condition. The other three ventilators (PB840, PB980, Servo i) showed acceptable VT accuracy across all conditions tested. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods ‘Hats and caps’ was devised on our ICU [3] and used for the training: this teaches that capnography traces on the left signify the airway is functional, in contrast to the traces on the right which indicate immediate attention is required (Figure 1). This was presented to staff working on the ICU in individual bedside teaching sessions with feedback obtained and evaluated. percentage variation –1.4040 ± 4.73 (–2.15 to –0.67); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.82 ± 3.85 (3.04 to 4.69); fl ow percentage variation 9.76 ± 8.08 (8.11 to 11.41); r = 0.969 and r2 = 0.939 (P <0.000). (3) FiO2 variation –0.005 ± 0.26 (–0001 to 0009); FiO2 percentage variation –0.72 ± 5.2 (–1.5 to 0.1); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.91 ± 1.26 (3.69 to 4.13); fl ow percentage variation 12.77 ± 5.33 (11.84 to 13.7); r = 0.996 and r2 = 0.992 (P <0.000). See Figure 1. percentage variation –1.4040 ± 4.73 (–2.15 to –0.67); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.82 ± 3.85 (3.04 to 4.69); fl ow percentage variation 9.76 ± 8.08 (8.11 to 11.41); r = 0.969 and r2 = 0.939 (P <0.000). (3) FiO2 variation –0.005 ± 0.26 (–0001 to 0009); FiO2 percentage variation –0.72 ± 5.2 (–1.5 to 0.1); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.91 ± 1.26 (3.69 to 4.13); fl ow percentage variation 12.77 ± 5.33 (11.84 to 13.7); r = 0.996 and r2 = 0.992 (P <0.000). See Figure 1. Results We delivered teaching sessions to 100% (9/9) of junior doctors, 100% (71/71) of nursing staff and other health professionals. We obtained feedback from 90% (76/84), showing an improvement in understanding of capnography from 73% of respondents to 100%, with 87% reporting that the teaching aid made capnography interpretation much easier. All felt the training would improve patient safety, and 97% felt it would be worthwhile training in other ICUs. g Conclusion The FiO2 percentage variation in the Airvo® is higher than the other two devices, with no clinical relevance. The fl ow percentage variation of Evita XL® is superior to the other two devices; this may have some clinical relevance. Comparison of three methods of applying high fl ow nasal oxygen: in vitro study yl y in vitro study M Muñoz Garach1, O Olga1, ME Yuste Osorio1, R Fernandez Fernandez2, R Ramirez Puerta1, F Acosta Díaz1, AM Perez Bailón1, S Narbona Galdó1, L Peñas Maldonado1 1Hospital Universitario San Cecilio, Granada, Spain; 2Hospital Nuestra Señora del Prado, Talavera de la Reina, Spain Critical Care 2015, 19(Suppl 1):P265 (doi: 10.1186/cc14345) Methods Twenty-one adult and clinically stable patients undergoing assisted mechanical ventilation for more than 48 hours were investigated during pressure support (baseline) and during a 2-hour CPAP trial. sEMG of diaphragm (costmar), intercostal and sternocleidal (accessory muscles) was recorded with a dedicated device (Dipha16; Inbiolab, Groningen, the Netherlands) simultaneously with airway waveforms and expressed as the ratio of the signal during baseline. Diaphragmatic electrical activity from a nasogastric tube (EAdi) of 14 of those patients was also measured. 1Hospital Universitario San Cecilio, Granada, Spain; 2Hospital Nuestra Señora del Prado, Talavera de la Reina, Spain Critical Care 2015, 19(Suppl 1):P265 (doi: 10.1186/cc14345) Introduction High fl ow nasal (HNF) requires precise control of the fraction of inspired oxygen (FiO2) and fl ow contributed as well as an adequate adjustment of temperature and humidity of the gas provided. There are several equipments for HNF. We evaluated the FiO2 and fl ow supplied with three diff erent systems Results The rapid shallow breathing index was lower than 105 in all patients and only one patient failed the trial. We observed that the mean inspiratory value of costmar increased immediately after switch to CPAP but did not signifi cantly vary during the CPAP trial (ANOVA, P = 0.7). On the other hand, the activation of accessory muscles increased signifi cantly during the same period (P = 0.01) and was strongly correlated with respiratory rate (r = 0.41, P <0.001) and inversely with f y Methods There have been analyzed: (1) ‘Oxygen Therapy’ from Dräger Evita-XL®; (2) Fisher & Paykel Airvo® option; and (3) pack of fl owmeters Debson®. Measurements were made in the distal part of the circuit that is used in clinical practice. Variables: programmed and measured FiO2, programmed and measured fl ow. We used the Oxygen Monitor Ohmeda 5120® and Flow Meter® Fisher-Porter. Before each measurement we checked and/or calibrated each of them. All measurements were performed at room temperature in the ICU of our hospital (23 to 26º).The data were processed using SPSS v.15.0.1, accepting a signifi cance level of 95%. Figure 1 (abstract P266). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Use of ‘hats and caps’ enabled delivery of short bedside teaching sessions to clinical staff in ICU during everyday work. Feedback shows a marked improvement in confi dence around capnography interpretation. It may have value in other ICUs to improve staff understanding of capnography and improve patient safety. References P266 Surface electromyography of respiratory muscles during a CPAP trial for weaning g S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) 1. Cook TM, et al. BJA. 2011;106:617-31. 2. McGrath BA. BJA. 2014;113:521. 3. Cook TM, et al. Anaesthesia. 2013;68:421. Introduction Weaning from mechanical ventilation is an important concern in ICU clinical practice. Surface electromyography (sEMG) [1] is a non-invasive tool to assess activity of diff erent muscles. We describe sEMG patterns of respiratory muscles during a CPAP trial [2] in patients undergoing pressure support ventilation. Surface electromyography of respiratory muscles during a CPAP trial for weaning S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) Surface electromyography of respiratory muscles during a CPAP trial for weaning S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) Ventilatory response during intentional early rehabilitation in patients with mechanical ventilation K Obata1, N Shiba2, T Takahashi3, S Ichiba2, Y Ujike2 1 d C Ok l Ok , , , , j 1Japanese Red Cross Okayama Hospital, Okayama, Japan; 2Okayama University Graduate School of Medicine, Okayama, Japan; 3Tokyo University of Technology, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P263 (doi: 10.1186/cc14343) Introduction Intentional early rehabilitation with mechanical ventilation in ICUs is performed in clinical settings. However, strict indexes for safe rehabilitation have not been fully elucidated. The purpose of this study is to analyze ventilator response (VR) between healthy volunteers and patients with mechanical ventilation. Methods Sixteen healthy volunteers (Control group) and 13 patients on mechanical ventilation (MV group) were enrolled in this study. Both groups were positioned in a variety of postures (baseline in a supine position, settled back 30°, settled back 60°, and sitting position) and measured with an indirect calorimeter. The instantaneous energy expenditure (EE), tidal volume (TV), respiratory rate (RR) and minute expiratory volume (VE) were non-invasively measured in each posture. Figure 1 (abstract P264). ‘Hats and caps’ capnography training guide. S93 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Systematic procedures including non-invasive ventilation improve morbidity in sleeve gastrectomy I Auriant, N Devos, S Rossi I Auriant, N Devos, S Rossi Critical Care 2015, 19(Suppl 1):P269 (doi: 10.1186/cc14349) Table 1 (abstract P267). Mean ventilator-days/reintubation rates Table 1 (abstract P267). Mean ventilator-days/reintubation rates 2009 2010 2011 2012 2013 2014 MICU mean ventilator-days 7.71 6.81 5.90 5.88 5.50 5.50* MICU reintubation rates 14.5% 9.8% 9.7% 9.8% 13.9% 8.3% SICU mean ventilator-days 5.91 5.93 5.68 5.93 4.93** 5.20* SICU reintubation rates 5.2% 3.0% 4.1% 4.3% 4.7% 7.5% , Signifi cance as described in Results. Introduction Postoperative morbidity after sleeve gastrectomy is decreasing, but remains signifi cant. Bleeding and surgical fi stules remain the leading causes of morbidity and mortality. In several studies in postoperative care of obese patients, non-invasive positive pressure ventilation (NPPV) reduced the risk of lower respiratory tract infection and pneumonia [1], thereby reducing in-hospital morbidity. The aim of study was to describe whether systematic use of NPPV improves morbidity in the postoperative care of sleeve gastrectomy. Methods A 4-year before–after study was conducted in a 19-bed intermediate care unit of a private hospital. Before period: standard treatment – all patients received oxygen supplementation to achieve SaO2 above 90%. After period: standard treatment plus NPPV – all patients were submitted to a systematic postoperative protocol: NPPV was provided using an oxygen CIPAP system with 5 cmH2O. Statistical analysis: complication rates were compared using the chi-square test. P <0.05 was considered statistically signifi cant. Conclusion Initiatives to reduce ventilator-days per patient realized signifi cant reductions from 2009 to 2014 while reintubation rates were unaff ected. One component of the bundle, early mobilization, introduced in the SICU in 2013 was associated with an additional reduction in mean ventilator-days. y gi Results A total of 857 patients were included. Inclusion characteristics were similar in the two groups: Before group – noNPPV: 352 patients, 2010 to 2011. Age: 40.58 ± 10.94, BMI: 42.79 ± 5.51, sex ratio F/M: 0.81. After group – NPPV: 504 patients, 2012 to 2013. Age: 40.81 ± 11.24, BMI: 42.92 ± 5.09, sex ratio F/M: 0.77. There is a signifi cant between- group diff erence in the complication rate: Before group – noNPPV: 10 surgical fi stula (2.84%) and six postoperative bleeding (1.70%); After group – NPPV: seven surgical fi stula (1.39%) and three postoperative bleeding (0.6%). The overall complication rate fell from 4.54% to 1.98%. The chi-square statistic = 4.58. The number of degrees of freedom is 1. References 1. Chittawatanarat K, Thongchai C. Spontaneous breathing trial with low pressure support protocol for weaning respirator in surgical ICU. J Med Assoc Thai. 2009;92:1306-12. 1. Chittawatanarat K, Thongchai C. Spontaneous breathing trial with low pressure support protocol for weaning respirator in surgical ICU. J Med Assoc Thai. 2009;92:1306-12. Methods Ventilator-day data were compiled from 2009 to 2014 for MICU and SICU in our facility. Reintubation rates were calculated when intubations were required >1 day after an extubation. 2. Esteban A, Alia I, Gordo F, et al. Extubation outcome after spontaneous breathing trials with T-tube or pressure support ventilation. The Spanish Lung Failure Collaborative Group. Am J Respir Crit Care Med. 1997;156:459-65. 2. Esteban A, Alia I, Gordo F, et al. Extubation outcome after spontaneous breathing trials with T-tube or pressure support ventilation. The Spanish Lung Failure Collaborative Group. Am J Respir Crit Care Med. 1997;156:459-65. Results Ventilator volume ranged from 639 to 766 distinct patients/ year in the MICU and from 555 to 687 for the SICU. Ventilator-day reduction was signifi cant (P <0.01) for the MICU (7.7 to 5.5, –29%) and the SICU* (5.91 to 5.20, –12%). Reduction patterns diff ered between the units as the SICU had a distinct reduction (*P = 0.007) between 2012 and 2013 coinciding with implementation of an early mobilization program. Reintubation rates diff ered between the units and rates did not increase with decreasing mean patient ventilator- days. See Table 1. Comparison of three methods of applying high fl ow nasal oxygen: in vitro study p g gi Results (1) FiO2 variation –0.001 ± 0.09 (–0.01 to 0.002); FiO2 percentage variation –0.012 ± 1.88 (–0.27 to 0.25); r2 = 0.999 and r = 0.998 (P <0.000). Flow variation (l/minute) 5.45 ± 3.23 (4.94 to 5.96); fl ow percentage variation 19.59 ± 11.63 (17.75 to 21.43); r = 0. 997 and r2 = 0.994 (P <0.000). (2) FiO2 variation –0.007 ± 0.26 (–0.011/–0.003); FiO2 Figure 1 (abstract P265). Programmed and measured fl ow. Figure 1 (abstract P265) Programmed and measured flow Figure 1 (abstract P266). Figure 1 (abstract P265). Programmed and measured fl ow. S94 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 before discontinue mechanical ventilation and extubation in the surgical intensive care unit (SICU). tidal volume (r = –0.16, P = 0.02). In patients with EAdi we confi rmed a tight correlation between costmar and EAdi (r = 0.62, P <0.001). See Figure 1. tidal volume (r = –0.16, P = 0.02). In patients with EAdi we confi rmed a tight correlation between costmar and EAdi (r = 0.62, P <0.001). See Figure 1. Methods We performed a prospective open-label randomized control study (non-inferiority trial) in SICU patients who were intubated and used mechanical ventilation, and appropriated discontinuation of the ventilator between June 2011 and November 2013. All patients underwent the same weaning protocol. The appropriated patients for discontinuation of MV were randomized into low pressure support up to 7 cmH2O and T-piece method. Reintubation within 72 hours, pneumonia after extubation, and hospital mortality were recorded. The statistical signifi cant diff erence was considered when P <0.05. Conclusion sEMG indicated that while diaphragm activation remains constant during the CPAP period, accessory muscles were progressively recruited and particularly in the conditions of increased respiratory rate and lower tidal volumes. Ventilator-day reductions not associated with reintubations and further reduced by an early mobilization program Ventilator-day reductions not associated with reintubations and further reduced by an early mobilization program M Palmquist-Tess, A Adams, J Mangan, D Owen, M LeClaire HCMC, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P267 (doi: 10.1186/cc14347) y y p g M Palmquist-Tess, A Adams, J Mangan, D Owen, M LeClaire HCMC, Minneapolis, MN, USA Introduction Mechanical ventilation is associated with increased risk of pneumonia, barotrauma, VILI, VAP, ARDS and mortality. From 2009 to 2014 in the MICU/SICU of our facility, eff orts to reduce ventilator-days included: noninvasive ventilation, sedation reduction, daily sedation vacations and weaning protocols. In 2013, an early mobilization of ventilated patients in the SICU was initiated. Aggressive ventilator-day reduction eff orts may be expected to lead to premature extubations and reintubations. Conclusion The outcomes after discontinuation of the mechanical ventilator between low pressure support and T-piece was not diff erence in term of reintubation, pneumonia after extubation and hospital mortality. Systematic procedures including non-invasive ventilation improve morbidity in sleeve gastrectomy The value returned from the chi-square statistic is P <5%.i References 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. gif Results A total of 520 patients were randomized into two groups: low pressure support group (260 patients) and T-piece group (260 patients). There was no diff erence in age, gender, body mass index, comorbidity, site of surgery, Charlson Comorbidity Index and Acute Physiologic and Chronic Health II score between groups (P >0.05). Regarding the intention to treat analysis, there were no diff erences between groups in reintubation rate (PSV 10% vs. T 14.6%; P = 0.109), pneumonia after extubation (PSV 14.2% vs. T 11.9%; P = 0.435) and hospital mortality rate (PSV 3.1% vs. T 3.5%; P = 0.805). Chiumello D, et al. Non-invasive ventilation in postoperative patients: a sytematic review. Intensive Care Med. 2011;37:918-29. P271 Systematic alveolar recruitment after cardiac surgery AL Lafarge, CK Kerneis, F Scalbert, LL Larnier, AB Brusset, PE Estagnasie, PS Squara Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P271 (doi: 10.1186/cc14351) Introduction The aim of our study was to investigate the value of C-reactive protein (CRP) and procalcitonin (PCT) in identifi cation of the systemic infl ammatory response syndrome (SIRS) and other complications in the early postoperative period after cardiac surgery with cardiopulmonary bypass (CPB) [1]. Methods In 93 patients undergoing coronary artery bypass graft surgery with CPB, after Ethical Committee approval in a prospective study, serum PCT and CRP values were collected before operation and daily until postoperative day 5. All patients were divided post hoc into patients with SIRS (n = 42) and patients without SIRS (n = 51). Student’s t test, the Mann–Whitney U test and receiver operating characteristic (ROC) curves were used. Introduction We designed a pilot study to evaluate the interest of an early systematic acute recruitment maneuver (ARM) in postcardiac surgery hypoxemic patients in order to properly design a larger trial. Methods This randomized controlled trial included consecutive patients operated on in our institution. Three hours after surgery, hypoxemic patients (PaO2 <300 mmHg, FIO2 = 1) were randomly assigned to ARM or control (H0). ARM was performed by applying once a positive end-expiratory pressure of 35 cmH2O during 45 seconds. Blood gases and hemodynamic variables were collected at H1, H8, H24 and H48. The primary endpoint was the duration of mechanical ventilation (MV). Secondary endpoints were survival rate, ICU length of stay and the occurrence of pneumonia. Results The comparison of serum CRP values in patients with or without SIRS on postoperative day 1 until postoperative day 5 demonstrated an increase in both groups without signifi cant diff erences (P >0.05). The PCT levels increased more signifi cantly in SIRS patients (5.78 ± 3.21 ng/ml vs. 1.23 ± 0.31 ng/ml) compared with patients without SIRS (P = 0.0001) on postoperative day 1. In patients with postoperative complications (21/93, 22%) (circulatory failure = 10, pneumonia = 2, respiratory insuffi ciency = 9, sepsis = 0), PCT levels remained elevated until postoperative day 5 (6.11 ± 2.87 ng/ml) but diminished in patients with SIRS (0.96 ± 0.23 ng/ml) (P <0.0001). Early postoperative pulmonary complications following heart transplantation A Camkiran Firat, Ö Kömürcü, P Zeyneloglu, M Türker, A Sezgin, A Pirat Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P270 (doi: 10.1186/cc14350) Introduction The aim of this study was to determine the types, incidence, and risk factors for early postoperative pulmonary complications in heart transplantation recipients. Methods We retrospectively collected data from the records of consecutive heart transplantations from January 2003 to December 2013. A total of 83 patients underwent heart transplantation. Those patients younger than 10 years (n = 9) and the patients who died intraoperatively (n = 1) or during the fi rst postoperative day (n = 1) were not included in the analyses. The data collected for each case were demographic features, duration of mechanical ventilation, respiratory problems that developed during the ICU stay, and early postoperative mortality (<30 days). The respiratory complications that we sought were pleural eff usion, pneumonia, pulmonary atelectasis, pulmonary edema, pneumothorax, and acute respiratory failure. Figure 1 (abstract P271). p p y Results Of the 72 patients considered, 52 (72.2%) were male. The mean age at the time of transplantation was 32.1 ± 16.6 years. The mean duration of postoperative mechanical ventilation was 71.8 ± 126.6 hours. The mean length of ICU stay was 13.5 ± 18.0 days. Two patients (2.8%) and one patient (1.4%) required extracorporeal membrane oxygenation support and intra-aortic balloon pump support, respectively, due to low cardiac output or primary graft failure postoperatively. Twenty-fi ve patients (34.7%) developed early postoperative respiratory complications. The most frequent problem was pleural eff usion (n = 19, 26.4%) followed by atelectasis (n = 6, 8.3%), acute respiratory distress syndrome (n = 5, 6.9%), pulmonary edema (n = 4, 5.6%), and pneumonia (n = 3, 4.2%). Postoperative duration of mechanical ventilation (44.2 ± 59.2 hours vs. 123.8 ± 190.8 hours, P = 0.005) and the length of ICU stay postoperatively (10.1 ± 5.8 hours vs. 19.8 ± 28.9 hours, P = 0.03) were longer among patients who had respiratory problems. Postoperative length of stay in the hospital (22.3 ± 12.5 days vs. 30.3 ± 38.3 days, P = 0.75) was similar in the two groups. The overall mortality rate was 12.5% (n = 9 patients). The patients who had respiratory problems did not show higher mortality than those who did not have respiratory problems (16.0% vs. 10.6%, P = 0.71). the two groups. Early postoperative pulmonary complications following heart transplantation At H1, PaO2/FIO2 was 367 ± 15 in the recruited group versus 299 ± 15 mmHg in the control group, P = 0.002. At H8 and 24 the diff erence was not signifi cant. At H48, the PaO2/FIO2 was lower in the recruited group (296 ± 10 vs. 343 ± 11 mmHg, P = 0.003) (Figure 1). The duration of mechanical ventilation (invasive + non-invasive) was lower in the recruited group (total 6.4 ± 1.4 vs. 8.4 ± 1.4 hours, P = 0.02). The survival rate, the length of stay in the ICU and the occurrence of pneumonia were similar in the two groups (P >0.2). Conclusion We can speculate that the inverse evolution of the blood oxygenation between the ARM group versus control may be due to: barotraumatism of normal alveoli during the ARM and/or a higher de- recruitment rate after ARM due to the shorter mechanical ventilation support. This pilot study shows that a unique ARM decreased the duration of MV in cardiac surgery patients but this may have subsequent detrimental eff ects on blood oxygenation. P272 Open-label randomized control trial between low pressure support and T-piece method for discontinuation from mechanical ventilator and extubation in general surgical ICUs g g K Chittawatanarat, S Orrapin, S Orrapin Chiang Mai University, Chiang Mai, Thailand Chiang Mai University, Chiang Mai, Thailand g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) Introduction In routine practice for most surgical patients in Thailand, all appropriated patients with planned discontinuation of mechanical ventilator (MV) are routinely changed to T-piece before extubation. However, this method needs to alter the instrument for testing tolerability of the patient. The objective of this study was to compare continuous low pressure support (PSV) and the T-piece method (T) Conclusion Systematic use of NPPV signifi cantly improves morbidity in the postoperative care of sleeve gastrectomy. Reference Conclusion Systematic use of NPPV signifi cantly improves morbidity in the postoperative care of sleeve gastrectomy. Chiumello D, et al. Non-invasive ventilation in postoperative patients: a sytematic review. Intensive Care Med. 2011;37:918-29. S95 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P271). P270 Figure 1 (abstract P271). P270 Early postoperative pulmonary complications following heart transplantation A Camkiran Firat, Ö Kömürcü, P Zeyneloglu, M Türker, A Sezgin, A Pirat Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P270 (doi: 10.1186/cc14350) Is procalcitonin a valuable marker for identifi cation of postoperative complications after coronary artery bypass graft surgery with cardiopulmonary bypass? y y A Baysal1, M Dogukan2, H Toman3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Adiyaman University Research and Training Hospital, Adiyaman, Turkey; 3Canakkale 18 Mart University Hospital, Canakkale, Turkey Critical Care 2015, 19(Suppl 1):P272 (doi: 10.1186/cc14352) A Baysal1, M Dogukan2, H Toman3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Adiyaman University Research and Training Hospital, Adiyaman, Turkey; 3Canakkale 18 Mart University Hospital, Canakkale, Turkey Critical Care 2015, 19(Suppl 1):P272 (doi: 10.1186/cc14352) Conclusion Respiratory complications were relatively common in our cohort of heart transplant recipients. However, these complications were mostly self-limiting and did not result in increased mortality. Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery A Baysal1, I Ozkaynak2, M Dogukan3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Kahramanmaraþ Sütçü Ýmam University Hospital, Kahramanmaraþ, Turkey; 3Adiyaman University Research and Training Hospital, Adiyaman, Turkey Critical Care 2015, 19(Suppl 1):P273 (doi: 10.1186/cc14353) A Baysal1, I Ozkaynak2, M Dogukan3 A Baysal , I Ozkaynak , M Dogukan 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Kahramanmaraþ Sütçü Ýmam University Hospital, Kahramanmaraþ, Turkey; 3Adiyaman University Research and Training Hospital, Adiyaman, Turkey Critical Care 2015, 19(Suppl 1):P273 (doi: 10.1186/cc14353) Conclusion Increasing sweep gas fl ow results in eff ective CO2 removal which can be further reinforced by raising blood fl ow. The clinically relevant oxygenation eff ect even in this setting of low invasivity could broaden the range of indications towards hypercapnic lung failure with mild to moderate hypoxia. Figure 1 (abstract P274). PaO2 with increasing blood fl ow (P <0.0001). Introduction Our goal was to investigate the incidence of postoperative jaundice in open heart surgery patients and to determine the risk factors associated with hepatic dysfunction. Methods A total of 292 patients were included in a prospective study design. Patients undergoing on-pump coronary artery bypass graft surgery (CABG) (n = 154) and valve repair surgery (mitral, mitral and aortic valve and/or tricuspid valve) (n = 138) were included. Postoperative hyperbilirubinemia was defi ned as occurrence of a plasma total bilirubin concentration of more than 34 μmol/l (2 mg/ dl) in any measurement during the postoperative period. All patients were divided into groups with or without hyperbilirubinemia. Liver enzymes were collected on postoperative days 1, 7, 14 and 30. The risk factors including age, cardiopulmonary bypass time, number of blood transfusions, inotropic support, use of intra-aortic balloon pump and ICU stay were evaluated with logistic regression. Figure 1 (abstract P274). PaO2 with increasing blood fl ow (P <0.0001). y g g Results Postoperative hyperbilirubinemia was observed in 27 of 292 patients (9.3%). The numbers of valves replaced, preoperative total bilirubin concentration, increased cardiopulmonary bypass time, higher number of inotropic support agents, and use of intra-aortic balloon pump correlate with hyperbilirubinemia on postoperative day 7 (P <0.05). P271 A PCT threshold value of 2.79 ng/ml was able to discriminate between postoperative complications in patients with or without SIRS with a sensitivity of y Results We included 124 patients, age 67.5 ± 10.6 years, M/F sex ratio 95/29, left ventricle ejection fraction 58.8 ± 10.6%, forced expiratory volume 94 ± 23% of the predicted value, bypass/valve ratio 82/53. The preoperative and postoperative PaO2/FIO2 were 401 ± 66 and 204 ± 66 mmHg, respectively (P <0.0001). The hemodynamic and ventilation status as well as the fl uid and inotrope supports were comparable in S96 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 developed miniaturized device consisting of a centrifugal pump and a membrane ventilator (iLA Activve®; Novalung, Germany) allows eff ective decarboxylation via a jugular double lumen cannula. So far no data on gas exchange in this setting exist to date. 82.5% and a specifi city of 70% (area under the curve: 0.76 ± 0.05; P <0.01) on postoperative day 1. Conclusion After cardiac surgery with CPB, PCT values increased signifi cantly in the SIRS group of patients compared with patients without SIRS on postoperative day 1 and remained elevated until postoperative day 5. In the early postoperative period, early rise of PCT values may help to discriminate the development of postoperative complications in patients with or without SIRS. Methods We included 10 patients receiving iLA Activve® due to hypercapnic respiratory failure as bridge-to-transplant or obstructive lung disease. Sweep gas fl ow was increased in steps from 1 to 14 l/ minute at constant blood fl ow (phase 1). Similarly, blood fl ow was gradually increased at constant sweep gas fl ow (phase 2). At each step, gas transfer via the membrane as well as arterial blood gas samples were obtained. 1. Delannoy B, Guye ML, Slaiman DH, Lehot JJ, Cannesson M. Eff ect of cardiopulmonary bypass on activated partial thromboplastin time waveform analysis, serum procalcitonin and C-reactive protein concentrations. Crit Care. 2009;13:R180. 1. Delannoy B, Guye ML, Slaiman DH, Lehot JJ, Cannesson M. Eff ect of cardiopulmonary bypass on activated partial thromboplastin time waveform analysis, serum procalcitonin and C-reactive protein concentrations. Crit Care. 2009;13:R180. P275 P275 Safety and effi cacy of extracorporeal CO2 removal combined with continuous renal replacement therapy in patients presenting both acute respiratory distress syndrome and acute kidney injury J Allardet-Servent, M Castanier, T Signouret, A Lepidi, R Soundaravelou, J Seghboyan Hopital Européen Marseille, France Critical Care 2015, 19(Suppl 1):P275 (doi: 10.1186/cc14355) P271 Results During phase 1, we observed a signifi cant increase in CO2 transfer together with a decrease in PaCO2 levels from a median of 66 mmHg (range 46 to 85) to 49 (31 to 65) mmHg from 1 to 14 l/ minute sweep gas fl ow, while arterial oxygenation deteriorated with high sweep gas fl ow rates. During phase 2, oxygen transfer signifi cantly increased leading to an increase in PaO2 from 67 (49 to 87) at 0.5 l/ minute to 117 (66 to 305) mmHg at 2.0 l/minute. Higher blood fl ow rates also signifi cantly enhanced decarboxylation. Increasing blood fl ow to 2.0 l/minute led to negative suction pressures of more than –100 mmHg and signs of hemolysis. See Figure 1. Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery Independent risk factors of early postoperative jaundice are; multiple valve replacement surgery, ejection fraction and use of intraaortic balloon pump (R = 0.58, R2 = 0.33, F = 26.44, P <0.001). The ICU stay was signifi cantly longer in group 2 (11.52 ± 3.76 days) as compared with group 1 (2.79 ± 1.36 days) (P <0.001). g y Conclusion Patients undergoing multiple valve replacement procedures are at greater risk for the development of postoperative hyperbilirubinemia and an association with prolonged ICU stay was observed. Other risk factors including ejection fraction, increased cardiopulmonary bypass time and use of intra-aortic balloon pump are also important as they have been reported to increase postoperative complications [1]. Conclusion Increasing sweep gas fl ow results in eff ective CO2 removal which can be further reinforced by raising blood fl ow. The clinically relevant oxygenation eff ect even in this setting of low invasivity could broaden the range of indications towards hypercapnic lung failure with mild to moderate hypoxia. p Reference 1. Nishi H, Sakaguchi T, Miyagawa S, et al. Frequency, risk factors and prognosis of postoperative hyperbilirubinemia after heart valve surgery. Cardiology. 2012;122:12-9. Interhospital transfer of patients in extracorporeal membrane oxygenation F Socci, S Di Valvasone, M Ciapetti, A Franci, M Bonizzoli, G Cianchi, S Batacchi, A Peris Careggi Hospital, Firenze, Italy Critical Care 2015 19(Suppl 1):P276 (doi: 10 1186/cc14356) gg y Critical Care 2015, 19(Suppl 1):P276 (doi: 10.1186/cc14356) gg y Critical Care 2015, 19(Suppl 1):P276 (doi: 10.1186/cc14356) Introduction The transfer of patients in extracorporeal membrane oxygenation (ECMO) from a peripheral hospital to a tertiary center represents a high-risk situation of adverse events [1]. The aim of this retrospective study is to determine the feasibility and safety of interhospital transfer for critically ill patients with ECMO support. Methods We collected data for the ECMO Regional Reference Centre Careggi Hospital activity from September 2009 to June 2014. In this study, 57 transfers were examined. The ECMO service is activated by a telephone call from a peripheral hospital. The team is represented by an intensivist, a heart surgeon, a cardiologist, a perfusionist and an intensive care nurse, all previously trained in the management of patients with ECMO. Medical personnel and the necessary equipment are transported by an ambulance and a van, specially designed and equipped for the transfer of patients with ECMO. Methods Retrospectively, we examined 20 patients with cerebral MRI (including SWI) who had suff ered from severe ARDS and received ECMO therapy. The MRI slides were anonymized and analyzed by two experienced neuroradiologists. Based on the distribution pattern and characteristic, a modifi ed HACE score (mHCS) was surveyed [2]. i y Results Six of 20 patients (30%) showed multiple MH with emphasis in the splenium of the corpus callosum. Eight patients had sporadic MH in the parenchyma of the brain but not in the corpus callosum. The remaining six patients had no intracerebral alterations. The distribution of MH with involvement of the splenium resembled that seen in HACE survivors. Results In this study, 57 patients transferred from the peripheral hospital to the ECMO center were examined; in all cases the ECMO system was implanted in the peripheral hospital (54 venovenous ECMO and three venoarterious ECMO). On average, trails were 271 km ± 304 (round trip) (minimum 14 km to maximum 939 km). The activation time from the call to the ambulance departure from our hospital was an average of 2 hours 27 minutes 13 seconds ± 1 hour 25 minutes 35 seconds. Determinants of gas exchange during extracorporeal CO2 removal using a novel pump-driven venovenous gas exchange system in a minimally invasive setting y g A Hermann, K Riss, P Schellongowski, A Bojic, P Wohlfarth, O Robak, W Sperr, T Staudinger Medical University of Vienna, Austria Critical Care 2015, 19(Suppl 1):P274 (doi: 10.1186/cc14354) A Hermann, K Riss, P Schellongowski, A Bojic, P Wohlfarth, O Robak, W Sperr, T Staudinger Medical University of Vienna, Austria Critical Care 2015, 19(Suppl 1):P274 (doi: 10.1186/cc14354) Introduction Pump-driven venovenous extracorporeal CO2 removal (ECCO2-R) increasingly takes root in hypercapnic lung failure to minimize ventilation invasiveness or to avoid intubation. A recently Introduction Pulmonary overdistension has been observed in 33% of patients with acute respiratory distress syndrome (ARDS) despite low tidal volume (6 ml/kg ideal body weight) ventilation [1]. Tidal volume S97 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (VT) reduction from 6 to 4 ml/kg attenuates overdistension but is associated with hypercarbia [2]. We thought to combine extracorporeal CO2 removal (ECCO2R) with continuous renal replacement therapy (CRRT) through the insertion of an oxygenator membrane within the hemofi ltration circuit in patients presenting both ARDS and acute kidney injury (AKI).i the devices during transport were not recorded; in some cases it was necessary to manage minor complications (circuit cavitation, minor vascula accesses bleeding). Conclusion Some studies have found several complications during transfer of patients in ECMO [2]. In our experience, there were no complications during the transfer of ECMO patients, even for longer trips. A wide and thorough clinical evaluation and multidisciplinary ECMO team allowed the optimization of clinical parameters before transport and a safely transfer. The start of ECMO treatment at peripheral hospitals and the transfer of patients in ECMO may be a viable option compared with conventional ventilation. Our data suggest that ECMO can be set up safely in peripheral hospitals by a multidisciplinary highly specialized ECMO team [3]. y j y Methods A fi rst set of measurement was performed at 6 ml/kg before and after ECCO2R. Twenty minutes later, VT was reduced to 4 ml/kg for the remainder of the study period (72 hours). Ventilator settings were those of the ARMA trial. The CRRT mode was hemofi ltration with 33% of predilution. Ultrafi ltration was adjusted to achieve a fi ltration fraction of 15%. Sweep gas fl ow was constant at 8 l/minute. The primary endpoint was a 20% reduction of PaCO2 at 20 minutes after initiation of ECCO2R. P276 h Introduction Cerebral microhemorrhages (MH) are diminutive focal bleedings which can be detected best by MRI using susceptibility- weighted imaging sequences (SWI). They can be found in a variety of neurologic diseases. The pattern of distribution can lead to the underlying pathomechanism [1]. Survivors of high-altitude cerebral edema (HACE) showed multiple MH, predominantly in the splenium of the corpus callosum. Mountaineers with a lack of acclimatization to high altitudes tend to suff er from HACE. Hypoxemia in great heights is discussed to be the main trigger of HACE [2]. Acute respiratory distress syndrome (ADRS) is characterized by oxygenation failure in mechanically ventilated patients. The severity is classifi ed by the ratio of arterial oxygen tension to fraction of inspired oxygen [3]. In some patients suff ering from severe ARDS, refractory to conventional therapy, venovenous extracorporeal membrane oxygenation therapy is the therapeutic option to ensure oxygenation. References 2 2 Results Eight patients were studied. Age was 69 ± 11 years, SAPS II was 68 ± 9 and SOFA score was 13 ± 4 at inclusion. Blood fl ow, at the inlet of the oxygenator membrane, was 400 ± 4 ml/minute. CO2 removal rate was 84 ± 4 ml/minute. Initiating ECCO2R, at 6 ml/kg, induced a mean PaCO2 reduction of 17% (41 ± 5.5 to 33.9 ± 5.6 mmHg, P <0.001). Then, lowering the VT to 4 ml/kg induced a mean PaCO2 increase of 25% (33.9 ± 5.6 to 42.6 ± 8 mmHg) and a mean PaO2/FIO2 ratio increase of 8% (176 ± 63 to 190 ± 61). Minute ventilation decrease from 7.4 ± 1.6 to 5 ± 1.2 l/minute. Respiratory system compliance did not vary. No major complications were observed. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 2. Haneya A, Philipp A, Foltan M, Mueller T, Camboni D, Rupprecht L, et al. Extracorporeal circulatory systems in the interhospital transfer of critically ill patients: experience of a single institution. Ann Saudi Med. 2009;29:110-4. 3. Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 2. Haneya A, Philipp A, Foltan M, Mueller T, Camboni D, Rupprecht L, et al. Extracorporeal circulatory systems in the interhospital transfer of critically ill patients: experience of a single institution. Ann Saudi Med. 2009;29:110-4. p p g 3. Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. p p g 3. Determinants of gas exchange during extracorporeal CO2 removal using a novel pump-driven venovenous gas exchange system in a minimally invasive setting Results Eight patients were studied. Age was 69 ± 11 years, SAPS II was 68 ± 9 and SOFA score was 13 ± 4 at inclusion. Blood fl ow, at the inlet of the oxygenator membrane, was 400 ± 4 ml/minute. CO2 removal rate was 84 ± 4 ml/minute. Initiating ECCO2R, at 6 ml/kg, induced a mean PaCO2 reduction of 17% (41 ± 5.5 to 33.9 ± 5.6 mmHg, P <0.001). Then, lowering the VT to 4 ml/kg induced a mean PaCO2 increase of 25% (33.9 ± 5.6 to 42.6 ± 8 mmHg) and a mean PaO2/FIO2 ratio increase of 8% (176 ± 63 to 190 ± 61). Minute ventilation decrease from 7.4 ± 1.6 to 5 ± 1.2 l/minute. Respiratory system compliance did not vary. No major complications were observed. y j y Methods A fi rst set of measurement was performed at 6 ml/kg before and after ECCO2R. Twenty minutes later, VT was reduced to 4 ml/kg for the remainder of the study period (72 hours). Ventilator settings were those of the ARMA trial. The CRRT mode was hemofi ltration with 33% of predilution. Ultrafi ltration was adjusted to achieve a fi ltration fraction of 15%. Sweep gas fl ow was constant at 8 l/minute. The primary endpoint was a 20% reduction of PaCO2 at 20 minutes after initiation of ECCO2R. Interhospital transfer of patients in extracorporeal membrane oxygenation Transfer duration (from activation to return to the ECMO center) was an average of 8 hours 25 minutes 6 seconds ± 3 hours 27 minutes 58 seconds (minimum 3 hours to maximum 16 hours 55 minutes). The stop time (necessary for evaluation of the patient and for placement of the ECMO system) was an average of 3 hours 53 minutes 40 seconds ± 1 hour 6 minutes 35 seconds (minimum 2 hours 5 minutes to maximum 7 hours 30 minutes). Major complications related to malfunctions of Conclusion Based on these results, we postulate that hypoxemia is one of the main players in the development of splenium-associated MH, not only in HACE but also in severe ARDS and other diseases accompanied with severe hypoxemia. Further investigations have to examine potential triggers and special circumstances concerning ARDS treatment which lead to MH in this distinctive pattern. 1. Renard D, et al. J Neurol Neurosurg Psychiatry. 2014;85:1041-8. 2. Schommer K, et al. Neurology. 2013;81:1776-9. 3. Force ADT, et al. JAMA. 2012;307:2526-33. References Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. Conclusion Combining ECCO2R and CRRT in patients with ARDS and AKI is safe and feasible through the insertion of an oxygenator membrane within a RRT circuit. References 1. Terragni PP, Rosboch G, Tealdi A, et al. Tidal hyperinfl ation during low tidal volume ventilation in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2007;175:160-6. P277 Microhemorrhages in the corpus callosum after treatment with extracorporeal membrane oxygenation S Riech, P Hellen, O Moerer, K Kallenberg, M Müller, M Quintel, M Knauth University Medical Center Goettingen, Germany Critical Care 2015, 19(Suppl 1):P277 (doi: 10.1186/cc14357) 2. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal CO2 removal. Anesthesiology. 2009;111:826-35. 2. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal CO2 removal. Anesthesiology. 2009;111:826-35. P279 Introduction Diff erential hypoxia is a pivotal problem in cardio- pulmonary failure patients with femoral venoarterial extracorporeal membrane oxygenation (VA ECMO) support. Although there was some attempt to deliver more oxygenated blood to the upper body, the mechanism of diff erential hypoxia has not been well investigated. Successful approach for emergent consent for ECMO research J Board1, S Vallance1, C Aubron2, D Pilcher1, V Pellegrino1, DJ Cooper1 1The Alfred Hospital, Melbourne, Australia; 2Monash University, Melbourne, Australia Critical Care 2015, 19(Suppl 1):P279 (doi: 10.1186/cc14359) f y Methods We used a sheep model of acute respiratory failure that was supported with femoral VA ECMO (from inferior vena cava to femoral artery (IVC-FA)), ECMO from superior vena cava to FA (SVC-FA), ECMO from IVC to carotid artery (IVC-CA) and ECMO adding an additional return cannula to internal jugular vein based on femoral VA ECMO (FA- IJV). Angiography and blood gas analysis were performed. Introduction The HELP-ECMO pilot study (Heparin low dose protocol versus standard care in critically ill patients undergoing ECMO; ACTRN12613001324707) is a randomised controlled phase II study evaluating two levels of heparin anticoagulation in patients with no requirement for full anticoagulation. This work is a substudy of the HELP-ECMO trial and describes the consent process of the parent study. At our site, consent for research is often obtained by the research coordinator with little involvement from investigators. However, the nature of the ECMO population required a modifi ed consent approach to be implemented given that ECMO is often inserted emergently. It required a model that would be successful out of hours and could be delivered by any member of the treating team. g g p y g y p Results Blood oxygen saturation (SO2) of IVC (83.6 ± 0.8%) was higher than that of SVC (40.3 ± 1.0%) in sheep with IVC-FA. Oxygen-rich blood was drained back to the ECMO circuit and poorly oxygenated blood in the SVC entered the right atrium (RA). SVC-FA achieved the oxygen-rich blood return from IVC to RA without shifting the arterial cannulation. SO2 of SVC and pulmonary artery increased (70.4 ± 1.0% and 73.4 ± 1.1%, respectively) subsequently. Compared with IVC-FA, Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. e 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. References 1. Renard D, et al. J Neurol Neurosurg Psychiatry. 2014;85:1041-8. 2. Schommer K, et al. Neurology. 2013;81:1776-9. 3. Force ADT, et al. JAMA. 2012;307:2526-33. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S98 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P278f less diff erences of venous oxygen return and attenuated diff erential hypoxia were also observed in IVC-CA and FA-IJV. See Figure 1.f P278 Diff erential venous oxygen return: a key factor of diff erential hypoxia in venoarterial extracorporeal membrane oxygenation X Hou1, X Yang1, Z Du1, J Xing1, C Jiang1, J Wang1, Z Xing1, H Wang1, H Zeng2 1Beijing Anzhen Hospital, Beijing, China; 2Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China Critical Care 2015, 19(Suppl 1):P278 (doi: 10.1186/cc14358) Conclusion Diff erential venous oxygen return is a key factor of diff erential hypoxia in VA ECMO. We can take advantage of the notion of diff erential venous oxygen return to choose better cannula confi guration in clinical practice. In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock M Bottiroli, T Maraffi , D Decaria, S Nonini, E Montrasio, K Gervasio, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P281 (doi: 10.1186/cc14361) y g M Bottiroli, T Maraffi , D Decaria, S Nonini, E Montrasio, K Gervasio, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P281 (doi: 10.1186/cc14361) Introduction Venoarterial (VA) ECMO is used for mechanical support in patients with cardiogenic shock (CS) unresponsive to medical therapy. Long-term survival and quality of life after hospital discharge have not yet been well analyzed. Methods We performed a retrospective observational study of patients admitted to the ICU for refractory CS from January 2010 to November 2014. Patients with postcardiotomy and/or post-transplant CS were excluded. Demographic, clinical and biochemical variables were collected. Continuous variables are presented as mean (standard deviation) and categorical variables as percentage. Long-term outcome and quality of life were assessed during scheduled follow-up evaluations or telephonic interviews. g p Results From April to December 2014, 30 patients were screened. Fourteen met the eligibility criteria and were approached for consent. Twelve patients were enrolled and randomised to receive either standard anticoagulation or low-dose heparin as per the study protocol. Consent was provided by the person responsible for 10 patients. One patient was competent to consent for themselves and one was enrolled under legislation allowing enrolment into research in the absence of a person responsible. There were two refusals. Seventy- one per cent of participants were approached out of hours. Eighty-six per cent were consented by clinicians. Twenty-one per cent of patients were consented by a non-investigator. Results We analyzed 23 consecutive patients undergoing VA ECMO for refractory CS. Etiologies of cardiac collapse were: 11 acute myocarditis, fi ve acute myocardial infarctions and seven acute decompensation of chronic cardiomyopathies (CCM). Thirteen patients died during the hospital stay and 10 survived. The main cause of ICU death was progressive multiple organ dysfunction (12/13). Baseline variables are presented in Table 1. All patients discharged from the hospital are still alive at follow-up (median 27 months, range 4 to 56) with a median NYHA class of 1 (range 1 to 2). All patients except one returned to an active style of life. Extracorporeal membrane oxygenator and ventricular assist device activity of a tertiary cardiothoracic centre: survival rates and length of ITU stay y D Sarridou1, CP Walker1, N Rashid1, D Heaton1, I McGovern1, N Marczin2, J Mitchell1 1The Royal Brompton & Harefi eld NHS Foundation Trust, London, UK; 2Imperial College, London, UK Critical Care 2015, 19(Suppl 1):P280 (doi: 10.1186/cc14360) Introduction Harefi eld Hospital accepts patients as a tertiary centre for end-stage heart and respiratory failure for the south of England. Interventions include VA-ECMO, VV-ECMO as a bridge to lung transplantation, left ventricular assist devices (LVAD) as a bridge for heart transplantation, right ventricular assist devices (RVAD) and BIVADs. The aim of this review was to identify the total number of such patients, analyse the individual length of ITU stay and calculate survival and mortality rates for each intervention. Conclusion VA-ECMO is an eff ective treatment tool for refractory CS in patients with acute life-threatening heart failure. Patients aff ected by acute decompensation of CCM had poorer outcomes characterized by multiple organ dysfunction, as already known in the literature. y Methods Patients consisted of six groups: Group 1: VA ECMO, Group 2: VV-ECMO, Group 3: LVAD, Group 4: RVAD, Group 5: BIVAD, Group 6: combination of devices. Data were extracted from the Perfusion Department records and the intensive care dataset from 2011 to 2013. Data included length of ITU stay, outcome, indication for device insertion and device-related major complications.i P280 P280 Extracorporeal membrane oxygenator and ventricular assist device activity of a tertiary cardiothoracic centre: survival rates and length of ITU stay D Sarridou1, CP Walker1, N Rashid1, D Heaton1, I McGovern1, N Marczin2, J Mitchell1 1The Royal Brompton & Harefi eld NHS Foundation Trust, London, UK; 2Imperial College, London, UK Critical Care 2015, 19(Suppl 1):P280 (doi: 10.1186/cc14360) P282 h P282 Characteristics of trauma patients with creatine kinase elevation N Assanangkornchai, O Akaraborworn, C Kongkamol, K Kaewsaengrueang Prince of Songkla University, Hatyai, Thailand Critical Care 2015, 19(Suppl 1):P282 (doi: 10.1186/cc14362) j p Results Forty patients were identifi ed. Twenty-nine were male and 11 female. Group 1 included 22 patients, Group 2: two patients, Group 3: four patients, Group 4: four patients, Group 5: zero, Group 6: eight patients treated with various combinations of ECMO or ventricular assist devices. ITU stay varied from 1 day to a maximum of 6 months intermittently for one patient. Duration of ITU stay for all 40 patients was 1,052 days with an average of 26.3 days per patient. Sixteen patients survived and were discharged to the Transplant Unit. Twenty- four patients died, putting the survival rate at 40% for this group. Conclusion This review demonstrates that the majority of these patients occupied intensive care beds for a prolonged period of time and despite the use of advanced support devices survival rates were signifi cantly lower than mortality rates. Results Forty patients were identifi ed. Twenty-nine were male and 11 female. Group 1 included 22 patients, Group 2: two patients, Group 3: four patients, Group 4: four patients, Group 5: zero, Group 6: eight patients treated with various combinations of ECMO or ventricular assist devices. ITU stay varied from 1 day to a maximum of 6 months intermittently for one patient. Duration of ITU stay for all 40 patients was 1,052 days with an average of 26.3 days per patient. Sixteen patients survived and were discharged to the Transplant Unit. Twenty- four patients died, putting the survival rate at 40% for this group. Introduction Rhabdomyolysis is a condition that results in the release of mainly creatine kinase (CK) and myoglobin from the breakdown of myocytes. Myoglobin has been known to cause renal failure (RF) and the CK level is routinely used as an indicator. A CK level >5,000 U/l was found to be associated with the risk of RF [1]. However, data are lacking on the level of CK to predict RF, especially in general trauma patients. The purpose of this study was to determine the initial CK level that predicts markedly elevated CK and the characteristics of trauma patients with elevated CK. Introduction Rhabdomyolysis is a condition that results in the release of mainly creatine kinase (CK) and myoglobin from the breakdown of myocytes. P279 Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. S99 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The HELP-ECMO pilot study is enrolling patients admitted to a large metropolitan ICU who require ECMO. Education on eligibility criteria and study processes was given to all ICU senior medical staff . Consent must be performed prior to the commencement of anticoagulation, often only a short time after ECMO cannulation. To facilitate recruitment at all hours, a HELP-ECMO study box is located in the ICU. Within the box is an instruction page outlining the screening, consent and randomisation process, as well as administrative tasks. Plain language statements, randomisation envelopes and consent documentation stickers are provided. A consent script is provided to ensure consistency across consenting personnel. The process is reviewed by the research coordinator the following day to confi rm local governance compliance. In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock Multivariate analysis (Cox) revealed pre-ECMO SOFA score (HR = 2.18, 95% CI = 1.016 to 4.6) and history of CCM (HR = 19, 95% CI = 2 to 178) and pre-ECMO lactate (HR = 1.2, 95% CI = 1.02 to 1.4) as independent risk factors for hospital mortality. Conclusion The model of consent described has proven to be successful in this challenging patient population. The ability of all staff to perform consent for the study has been a signifi cant factor in the success of the pilot. The review of study processes by research coordinators has supported this model. Table 1 (abstract P281) Alive Dead P value Age (years) 30 ± 18 43 ± 14 0.012 CCM 0 (0%) 7 (100%) 0.014 SOFA 8 ± 1.4 10 ± 1.9 0.002 Creatinine (mg/dl) 1.1 ± 0.3 2.2 ± 0.7 0.001 Bilirubin (mg/dl) 1 ± 0.4 2 ± 1.8 0.09 Lactate (mmol/l) 5 ± 2.1 8 ± 5.3 0.021 Platelets (103/μl) 257 ± 79 162 ± 99 0.03 Conclusion VA-ECMO is an eff ective treatment tool for refractory CS in patients with acute life-threatening heart failure. Patients aff ected by acute decompensation of CCM had poorer outcomes characterized by multiple organ dysfunction as already known in the literature References 1. Cartin Ceba R, et al. Risk factors for development of acute kidney injury in critically ill patients: a systematic review and meta-analysis of observational studies. Crit Care Res Pract. 2012:691013. Methods A retrospective observational study of adults with confi rmed rhabdomyolysis admitted to the Neurosciences Critical Care Unit between 2002 and 2012. Data collection included APACHE score, daily CK (with PEAK CK defi ned as the maximum CK recorded throughout ICU stay), creatinine, calcium, phosphate and bicarbonate levels, AKI, RRT, ICU length of stay and mortality. 1. Cartin Ceba R, et al. Risk factors for development of acute kidney injury in critically ill patients: a systematic review and meta-analysis of observational studies. Crit Care Res Pract. 2012:691013. 2. Suh SH, et al. Acute kidney injury in patients with sepsis and septic shock: risk factors and clinical outcomes. Yonsei Med J. 2013;54:965-72. 2. Suh SH, et al. Acute kidney injury in patients with sepsis and septic shock: risk factors and clinical outcomes. Yonsei Med J. 2013;54:965-72. g y y Results A total of 232 patients met the inclusion criteria. Rhabdo- myolysis was associated with trauma (76%), medical (15%) and surgical (9%) admission diagnoses. Forty-fi ve (19%) patients developed AKI, with 29 (12.5%) requiring RRT. Mortality was signifi cantly higher in patients who developed AKI (62% vs. 18%, P <0.001). Average CK on admission was 5,009 IU/l (SD 12,403 IU/l). CK values remained greater than 2,000 IU/l for an average of 3.3 days (range 1 to 10 days). Although PEAK CK was greater in patients requiring RRT compared with those that did not (PEAK CK: 32,354 IU/l vs. 7,353 IU/l, P = 0.001), receiver operator characteristic curves revealed that a threshold for PEAK CK >5,000 IU/l is only 55% specifi c and 83% sensitive for the prediction of need for RRT. CK peaks on the day of admission in 46% of patients, on day 2 in 37%, and on day 3 or later in 17% of cases. A McMahon Score >6 calculated on admission is 68% specifi c and 86% sensitive for RRT. P284 Methods Data from the Songklanagarind Hospital trauma registry were reviewed over 1 year (January 2013 to December 2013). Patients with at least two records of CK and creatinine (Cr) levels were included. Creatine kinase levels were analyzed during the fi rst 3 days of hospital admission. RF was defi ned as a Cr increment >0.3 mg/dl within 48 hours. Results Of the 1,491 patients admitted to the trauma service, 47 patients had CK levels drawn twice. These patients had a mean age of 32 years and a median Injury Severity Score (ISS) of 14. The predominant mechanism of injury was motorcycle crash. Only three patients developed RF. The median CK during the fi rst 3 days after admission was 3,088 (IQR 1,327, 6,072) U/l. The CK peaked at 11 hours after admission at a mean value of 16,114.167 (SD 34,010.80) U/l. There were no signifi cant diff erences in demographic data, ISS scores and fl uid balance between the groups of CK level over or below 5,000 U/l. A mean positive fl uid balance observed; however, initial CK was signifi cantly diff erent between the two groups. None of the patients with initial CK of <900 U/l had a peak of CK >5,000 U/l. Risk factors for acute kidney injury in patients with complicated intra-abdominal infection A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) P283 Rhabdomyolysis: early prognostication of renal failure and other adverse outcomes J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) Rhabdomyolysis: early prognostication of renal failure and other adverse outcomes J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) Introduction The clinical diagnosis of rhabdomyolysis is confi rmed by creatine kinase (CK) levels >1,000 IU/l [1]. A local therapeutic protocol triggers aggressive renoprotective treatment in all patients with CK >2,000 IU/l. To evaluate local practice and refi ne CK thresholds for the instigation of renoprotective treatment, we studied the correlation between CK time trends and adverse outcomes such as acute kidney injury (AKI), the need for emergency renal replacement therapy (RRT) and mortality. We also evaluated the McMahon Score, a risk prediction model based on demographic, clinical, and laboratory variables available on admission [2].i p y Conclusion Severe AKI with RRT need is highly associated with previous HTN. The number of previous medications is related to severe AKI too. HTN has been described as a risk factor for developing AKI in critically ill patients [1]. ACEI and ARB use has been associated with AKI development in septic patients [2]. To our knowledge, this is the fi rst study that investigates risk factors associated with AKI in surgical septic patients with CIAI. 2. McMahon GM, et al. JAMA Intern Med. 2013;73:1821-8. 1. Khan FY. Neth J Med. 2009;67:272-83. Risk factors for acute kidney injury in patients with complicated intra-abdominal infection Risk factors for acute kidney injury in patients with complicated intra-abdominal infection A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) Introduction AKI has been poorly studied in surgical septic patients. The aim of our study was to determine the factors related to AKI in surgical septic patients with complicated intra-abdominal infection (CIAI) and mortality associated with AKI in this setting. Methods An observational study was performed of all adult patients with CIAI requiring surgery and ICU admission from June 2011 to June 2013. We recorded demographic data, SAPS II, SOFA score at admission, presence of septic shock, history of pre-existing comorbidities, angiotensin-converting enzyme inhibitor (ACEI), angiotensin receptor blocker (ARB), NSAIDs, statins or diuretics consumption, baseline creatinine and at admission, and standard biomarkers. Factors associated with developing AKI and renal replacement therapy (RRT) were studied using a multivariate analysis. Association between mortality and AKI and RRT need was also analyzed. Conclusion Trauma patients had varying levels of elevated CK. Initial CK shows a promising result as a predictor of high peak CK levels. A larger sample size is needed to demonstrate the predictors of RF in trauma patients with elevated CK levels. Reference 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. Results A total of 114 patients were included, with a mean SAPS II of 42.14. Sixty-seven patients (58.8%) developed AKI and 33 (28.9%) required RRT. Development of AKI (R2 = 0.498; P <0.0001; AUC = 0.926) was independently associated with SOFA (OR = 1.57; 95% CI = 1.29, 2.02) and creatinine at admission (OR for 0.1 units = 1.56; 95% CI = 1.30, 1.99). RRT need (R2 = 0.382; P <0.0001; AUC = 0.892) was independently associated with arterial hypertension (HTN) (OR = 4.90; 95% CI = 1.50, 15.97) and SOFA score (OR = 1.71). In another model with more predictive capacity (R2 = 0.433; P <0.0001; AUC = 0.918) the number of previous medications (OR = 3.73; 95% CI = 1.92, 8.38) and SOFA score (OR = 1.86; 95% CI = 1.47, 2.54) were related to RRT need. Both AKI and RRT need were related to ICU (RR = 8.41, 95% CI = 1.14, 62.5; and RR = 8, 95% CI = 2.40, 27.85 respectively) and 28-day mortality (RR = 2.8, 95% CI = 1.00, 7.86; and RR = 4.65, 95% CI =1.99, 10.40 respectively). P282 h Myoglobin has been known to cause renal failure (RF) and the CK level is routinely used as an indicator. A CK level >5,000 U/l was found to be associated with the risk of RF [1]. However, data are lacking on the level of CK to predict RF, especially in general trauma patients. The purpose of this study was to determine the initial CK level that predicts markedly elevated CK and the characteristics of trauma patients with elevated CK. Conclusion This review demonstrates that the majority of these patients occupied intensive care beds for a prolonged period of time and despite the use of advanced support devices survival rates were signifi cantly lower than mortality rates. S100 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Introduction Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients [1]. Early detection and treatment may improve outcome. Introduction Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients [1]. Early detection and treatment may improve outcome. y Methods A retrospective analysis of prospectively collected data from 2,158 patients without end-stage renal disease from the EPaNIC trial [2]. For early detection of AKI, defi ned according to the creatinine-based KDIGO guidelines [3], three multivariate logistic regression models (LR) were developed using data available at baseline (LR_B), upon ICU admission (LR_BA), and at the end of the fi rst day in the ICU (LR_BAD1). In a subpopulation (n = 580) where plasma neutrophil gelatinase- associated lipocalin (pNGAL), an early biomarker of AKI, was measured at ICU admission, the value of adding pNGAL to LR_BA and LR_BAD1 was assessed. The models were evaluated via bootstrapping, by comparing receiver operator characteristic (ROC) and decision curves. pi Conclusion Although higher CK levels are associated with adverse outcomes, instigation of renoprotective treatment should not be based solely on CK levels. A McMahon Score >6 on admission allows for a more sensitive, specifi c and timely identifi cation of patients at risk of renal failure requiring RRT. S101 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P285). Area under ROC curves for the diff erent models LR_BA + pNGAL on LR_BA on pNGAL on subpopulation subpopulation subpopulation LR_B LR_BA LR_BAD1 with pNGAL with pNGAL with pNGAL (n = 2,158) (n = 2,067) (n = 1,808) (n = 528) (n = 528) (n = 528) 0.73 0.76 0.82 0.64 0.70 0.76 with the AMPK activator AICAR (100 mg/kg intraperitoneal, 24 hours before CLP), and sacrifi ced 24 hours after CLP. Blood and tissue samples were collected for all animals. AMPK activation (pThr172), B-ENaC, and mitophagy (LC3 II/I) were examined by western blot of kidney lysates. Plasma creatinine (Scr) was assessed using ELISA. Results Table 1 presents the performance of the models and admission pNGAL. References 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. P286 Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? K Jin, H Li, J Volpe, D Emlet, N Pastor-Soler, MR Pinsky, BS Zuckerbraun, K Hallows, JA Kellum, H Gomez University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P286 (doi: 10.1186/cc14366) Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Performance improved when predictions were made at a later time point, and was highest for LR_BAD1. Similar results were obtained in subgroups of septic and cardiac surgery patients. As an independent predictor, pNGAL alone did not perform better than a model using routine clinical data available upon admission. However, when combining pNGAL with LR_BA, predictive performance improved. The performance of LR_BAD1 was not improved by including pNGAL. with the AMPK activator AICAR (100 mg/kg intraperitoneal, 24 hours before CLP), and sacrifi ced 24 hours after CLP. Blood and tissue samples were collected for all animals. AMPK activation (pThr172), B-ENaC, and mitophagy (LC3 II/I) were examined by western blot of kidney lysates. Plasma creatinine (Scr) was assessed using ELISA. g Results The acute response to sepsis was characterized by early activation of AMPK which increased from 6 to 18 hours, peaked at 24 hours, and decreased by 48 hours (Figure 1A). This activation was associated with a consistent decrease in B-ENaC expression. In AICAR pretreated animals, AMPK was only activated in WT mice, which was associated with a decrease in the expression of B-ENaC as compared with AMPK KO mice (Figure 1B). p p y g p Conclusion This study shows the potential of data-driven models based on routinely collected patient information for early detection of AKI during the fi rst week of ICU stay. Although adding admission pNGAL to admission data improved early detection of AKI, this added value is lost upon inclusion of data from the fi rst day of ICU. R f Conclusion AMPK was activated early after induction of sepsis, and was associated with a consistent decrease in Beta-ENaC expression in the apical membrane of tubular epithelial cells. In addition, absence of AMPK activation in KO animals was associated with increased expression of Beta-ENaC at 24 hours after CLP. These data support the hypothesis that early activation of AMPK decreases energy consumption through ion channel downregulation. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? S102 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 AKI as per the AKIN KDIGO defi nition, as well as their worst level of TIMP2IGFBP7 during their fi rst 2-day stay. Values of mean and 25th to 75th percentile for the worst value of TIMP2IGFBP7 were 0.24 (0.11 to 0.46), 0.50 (0.28 to 1.24), 0.94 (0.34 to 3.28) and 3.34 (1.47 to 6.22) for no AKI, AKIN I, II and II respectively (P <0.0001). The worst values for no AKI/no sepsis, no AKI/sepsis, AKI/no sepsis and AKI/sepsis were 0.21 (0.10 to 0.4), 0.32 (0.15 to 0.63), 1.05 (0.41 to 2.31) and 0.98 (0.36 to 3.94) respectively, with P <0.05 for AKI and P = NS for sepsis. The AUC ROC curve for prediction of AKI of the worst value was 0.80 with sensitivity of 73.5% and specifi city of 71.4% (P <0.0001). In contrast to the Sapphire study, in our population cutoff values of 0.4 and 0.8 (ng/ml)2/1,000 predict AKI and AKIN ≥II respectively, regardless of the presence of sepsis. See Figure 1. Results There were 101 patients without AKI, 95 patients with KDIGO 1 AKI, and 42 patients with KDIGO 2 to 3 AKI within 48 hours of the start of surgery. In patients without AKI, median TIMP-2·IGFBP7 values were less than 0.3 (ng/ml)2/1,000 (dashed line in Figure 1) for all time points. In patients with KDIGO 1 AKI, median [TIMP-2·IGFBP7] signifi cantly exceeded this cutoff at 24 and 36 hours following the start of surgery (one-sided P <0.025). Median [TIMP-2·IGFBP7] increased earlier in KDIGO 2 to 3 AKI patients, remaining signifi cantly elevated relative to the cutoff from 12 to 60 hours after the start of surgery. The highest median [TIMP-2·IGFBP7] was observed at 24 hours for KDIGO 2 to 3 AKI patients and was nearly fi ve times the 0.3 (ng/ml)2/1,000 cutoff .i p yi gf Conclusion Urinary [TIMP-2·IGFBP7] was signifi cantly elevated as early as 12 to 24 hours from the start of surgery in patients who developed AKI within 48 hours. Monitoring of these biomarkers in the immediate postsurgical period might enable improved management of patients at risk for AKI. Conclusion TIMP-2 and IGFBP-7 can predict AKI in both septic and nonseptic critically ill patients. Further pragmatic randomised controlled trials are needed to prove their role on clinical basis. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? P287 Urinary TIMP-2 and IGFBP7 elevate early in critically ill postoperative patients that develop AKI P Honore1, LS Chauwla2, A Bihorac3, AD Shaw4, J Shi5, JA Kellum6 1VUB, Brussels, Belgium; 2VAMC, Washington, DC, USA; 3UF, Gainesville, FL, USA; 4VUSM, Nashville, TN, USA; 5WB, Carlsbad, CA, USA; 6PiU, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P287 (doi: 10.1186/cc14367) Introduction This study tested the hypothesis that the cellular response in the kidney to sepsis is characterized by early activation of AMP activated protein kinase (AMPK), and that such activation is temporally associated with downregulation of the epithelial sodium channel (B-ENaC). Introduction Little is known about temporal changes in [TIMP- 2·IGFBP7] relative to injury in patients who develop AKI. In this analysis, we examined [TIMP-2·IGFBP7] in serial urine collections from the subset of Sapphire patients who were admitted to the ICU after major surgery. Methods We stratifi ed 238 Sapphire patients into three groups by their maximum AKI stage within 48 hours of the start of surgery using KDIGO criteria (No AKI, KDIGO 1, and KDIGO 2 to 3). Median TIMP-2·IGFBP7 values were calculated from all samples collected at 12 (±6)-hour intervals for 4 days following the start of surgery. Methods Fifteen C57BL/6 wildtype (WT) mice were subjected to cecal ligation and puncture (CLP), and sacrifi ced at 2, 6, 18, 24, or 48 hours. In addition, we pretreated three WT and three AMPK Beta 1 knockout mice Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. intervals for 4 days following the start of surgery. Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Figure 1 (abstract P287). Figure 1 (abstract P287). Figure 1 (abstract P287). Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? Reference 1. Kashani et al. Crit Care. 2013;17:R25. 1. Kashani et al. Crit Care. 2013;17:R25. Single point measurement of cystatin C has similar performance as serum creatinine for assessment of kidney function in critically ill patients J Houthoofd1, M Carlier2, J De Waele2, R Vanholder2, J Delanghe2, J Decruyenaere2, E Hoste2 Introduction Sepsis and acute kidney injury (AKI) have a high prevalence in the ICU population. The aim of this study is to describe the composite of tissue inhibitor of metalloproteinases-2 (TIMP2) and insulin-like growth factor-binding protein-7 (IGFBP7) as novel urinary renal biomarkers in both septic and nonseptic patients. Introduction The gold standard for routine evaluation of kidney function is measurement of serum creatinine concentration (Scr). In ICU patients, muscle loss and dilution leads to decreased Scr. Scr is distributed in total body water, resulting in delay of Scr changes when the glomerular fi ltration rate (GFR) changes (lag time). Cystatin C (CysC) is a protein produced by all cells with a nucleus and therefore less aff ected by muscle mass. Also, the lag time may be shorter as the volume of distribution is only extracellular fl uid. The objective of this study was to evaluate whether single point measurement of CysC is more adequate than Scr for monitoring of kidney function in ICU patients. Methods We conducted a prospective, observational study in two university hospitals. Patients were admitted in ICU either from the emergency department or after undergoing an acute surgery at hospital admission. Two months prior to the admission, recruited patients had not been admitted to hospital. We collected epidemiological, clinical and laboratory data at admission, 24 and 48 hours. TIMP2IGFBP7 was analysed in urine samples by a point-of-care device (Nephrocheck®; Astute Medical). Results The sample included 98 patients (65 men) with mean age 55 ± 17.3 years, length of ICU stay 11.1 ± 14.6 days. In total, 41.4% had sepsis at ICU admission; 59.2% were diagnosed of sepsis within the fi rst 48 hours of stay. We stratifi ed patients based on the presence of Methods Data were collected in two prospective single-center studies on a convenience sample of ICU patients. During the 24-hour study period, we measured CysC, Scr, and urinary inulin clearance (Cinu) as a Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2][IGFBP7] value within the fi rst 48 hours of ICU admission to predict (a) AKI and (b) AKIN KDIGO ≥2. Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2][IGFBP7] value within the fi rst 48 h predict (a) AKI and (b) AKIN KDIGO ≥2. P288 Urinary TIMP2 and IGFBP7 as early biomarkers of acute kidney injury in septic and nonseptic critically ill patients M Cuartero 1, A Betbesé 1, J Sabater2, J Ballús2, J Ordóñez1 1Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Spain; 2Hospital Universitari Bellvitge, Hospitalet Llobregat, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P288 (doi: 10.1186/cc14368) P291 gold standard for assessment of GFR. We compared Scr and CysC, with Cinu. Also, we assessed Cinu in patients who had CysC and Scr within the normal sex and age corrected limits. Finally, we determined the ability of CysC and Scr to detect normal range and decreased GFR (80 to 120 ml/minute/1.73m2 resp. <60). Association between urinary TIMP-2 and IGFBP7 as early biomarkers of AKI and oliguria during liver surgery: a prospective pilot study F Desmet1, M D’Hondt1, H Pottel2, S Carlier1, E Hoste3, J Kellum4, W De Corte1 Association between urinary TIMP-2 and IGFBP7 as early biomarkers of AKI and oliguria during liver surgery: a prospective pilot study F Desmet1, M D’Hondt1, H Pottel2, S Carlier1, E Hoste3, J Kellum4, W De Corte1 1AZ Groeninge, Kortrijk, Belgium; 2Catholic University Leuven, Kortrijk, Belgium; 3Ghent University Hospital, Ghent University, Ghent, Belgium; 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P291 (doi: 10.1186/cc14371) 1AZ Groeninge, Kortrijk, Belgium; 2Catholic University Leuven, Kortrijk, Belgium; 3Ghent University Hospital, Ghent University, Ghent, Belgium; 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P291 (doi: 10.1186/cc14371) p Results We included 68 patients, with median age 58 years (IQR 29 years), length of stay in the hospital before study 11 days (IQR 16), and APACHE II score 19 (IQR 9). Scr was 1.12 (IQR 1.55), CysC 0.64 (IQR 0.73), and Cinu 80 ml/minute/1.73 m2 (IQR 82). Cinu was markedly decreased in patients with Scr in normal range (n = 12) compared with patients with CysC in normal range (n = 22) (55 ml/minute/1.73 m2 (IQR 57.4) vs. 100 (IQR 42.2), P <0.001). Patients with normal range Scr had similar proportion of patients with Cinu in the normal range compared with normal range CysC patients (33.3% vs. 45.5%, P = 0.23). ROC analysis showed that Scr and CysC had similar performance for detection of normal range Cinu (AUC: 0.66; 95% CI = 0.53 to 0.77 vs. 0.77; 95% CI = 0.65 to 0.87; P = 0.118), and decreased Cinu (AUC: 0.86; 95% CI = 0.76 to 0.97 vs. 0.94; 95% CI = 0.88 to 1; P = 0.113). Introduction Patients undergoing elective liver surgery have an increased risk for developing AKI [1]. This study was intended to assess [TIMP-2][IGFBP7] and its possible association with urine output (UO) in this population. References 1. Slankamenac et al. Development and validation of a prediction score for postoperative ARF following liver resection. Ann Surg. 2009;250:720-8. 2. Hoste et al. Derivation and validation cutoff s for clinical use of cell cycle arrest biomarkers. Nephrol Dial Transplant. 2014;29:2054-61. 2. Hoste et al. Derivation and validation cutoff s for clinical use of cell cycle arrest biomarkers. Nephrol Dial Transplant. 2014;29:2054-61. g Results Mean age was 81 ± 5.6 years (16 male, 40.0%). Thirty-fi ve patients underwent TA-TAVI and fi ve patients TAo-TAVI. AKI developed in 17 patients (42.5%); seven patients (17.5%) suff ered from AKI 3 and required renal replacement therapy (RRT). Mean maximum value of UTI within 24 hours after TAVI was signifi cantly higher in patients with AKI compared with patients without renal impairment (2.19 ± 3.11 vs. 0.67 ± 0.816, P = 0.037) and higher in patients with AKI 3 compared with patients with AKI 2 (4.73 ± 3.58 vs. 0.59 ± 0.71, P = 0.022). In contrast, preoperative creatinine (AKI (mg/dl) 1.22 ± 0.41 vs. no AKI 1.30 ± 0.59; AKI 3 1.32 ± 0.49 vs. AKI 2 1.26 ± 0.53, P = NS) and early postoperative serum creatinine levels (maximum within 24 hours after TAVI: AKI 1.41 ± 0.50 vs. no AKI 1.34 ± 0.60; AKI 3 1.69 ± 0.56 vs. AKI 2 1.32 ± 0.54, P = NS) did not show any association with the development of AKI. ROC analyses revealed a very good predictive value of early UTI levels for the development of AKI 3 within the next 72 hours after TAVI with a sensitivity of 100% and a specifi city of 80% for a cutoff value of 0.815 (AUC = 0.919, 95% CI = 0.824 to 1.0, SE 0.048, P = 0.001). Single point measurement of cystatin C has similar performance as serum creatinine for assessment of kidney function in critically ill patients Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2][IGFBP7] value within the fi rst 48 hours of ICU admission to predict (a) AKI and (b) AKIN KDIGO ≥2. S103 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P290 Early prediction of acute kidney injury after transcatheter aortic valve implantation with urinary G1 cell cycle arrest biomarkers F Dusse, M Dudásová, D Wendt, M Thielmann, E Demircioglu, HG Jakob, K Pilarczyk University Hospital Essen, Germany Critical Care 2015, 19(Suppl 1):P290 (doi: 10.1186/cc14370) Introduction Acute kidney injury (AKI) is a common complication following transcatheter aortic valve implantation (TAVI) and has been shown to increase mortality. The concentration of the G1 cell cycle arrest proteins TIMP-2 and IGFBP7 in the urine have recently been suggested as sensitive biomarkers for early detection of AKI in critically ill patients. Whether postoperatively elevated levels of urinary [TIMP-2][IGFBP7] (UTI) predict the development of an AKI in patients undergoing TAVI is currently unknown. Conclusion This pilot study demonstrated the association between [TIMP-2][IGFBP7] increase and oliguria and may therefore indicate kidney damage during liver surgery. As Scr could not diff erentiate for these changes, patients did not meet the classical biomarker criteria for AKI. y Methods In a prospective cohort study, 40 patients undergoing TAVI, either trans-apical (TA) or trans-aortic (TAo), were enrolled. Serial measurements of UTI were performed every 12 hours in the postoperative course. Results were calculated for their multiplication and presented as arbitrary values. Urinary output and serum creatinine were recorded simultaneously. The primary clinical endpoint was the occurrence of AKI according to the AKI Network. P291 Secondly we sought to compare [TIMP-2][IGFBP7] with serum creatinine concentration (Scr). Methods A prospective single-center pilot study performed on 12 patients undergoing elective liver surgery. Serial urine samples were analyzed for [TIMP-2][IGFBP7] measured with the Nephrocheck device (Astute Medical, San Diego, CA, USA). Serial Scr was analyzed, UO, blood losses, and mean arterial pressure (MAP) were recorded. Fluid management was standardized, oliguria defi ned as a UO <0.5 ml/kg/ hour. [TIMP-2][IGFBP7] values of >0.3 identify patients at high risk and >2 at highest risk for AKI [2]. Conclusion Single point measurement of Scr and CysC has similar performance for detection of normal and decreased GFR in this cohort of ICU patients. Performance was weak for detection of normal GFR, but both biomarkers had moderate good performance for detection of decreased GFR. g [ ] Results Males comprised 66.7%, median age was 72 years. Median surgical time was 195 minutes. Peroperative median MAP was 71 mmHg (IQR 69; 77). Baseline median GFR was normal in eight patients and decreased in four patients (eGFR >90 and 66.5 ml/ minute/1.73 m2 respectively). Median baseline Scr was 0.75 mg/dl (IQR 0.61; 1.10), 0.74 mg/dl (IQR 0.64; 1.04) at ICU admission and 0.74 mg/ dl (0.64; 1.04) on day 1 postoperatively. No diff erence in Scr and eGFR was seen between these time points (P = 0.457 and P = 0.517 respectively; repeated-measures ANOVA). Median peroperative and postoperative UO was 0.18 ml/kg/hour (IQR 0.13; 0.23) and 0.93 ml/kg/ hour (IQR 0.79; 1.49) respectively. Median baseline [TIMP-2][IGFBP7] was 0.10 (IQR 0.04; 0.34), 2.02 (1.44; 6.23) during surgery, 0.61 (IQR 0.27; 1.22) at ICU admission and 0.74 (0.67; 0.97) on day 1 postoperatively. [TIMP-2][IGFBP7] diff ered at these time points (P <0.0001; repeated- measures ANOVA). Peroperative oliguria was associated with increased [TIMP-2][IGFBP7] (P = 0.018, chi-squared test). Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data y J Dixon1, K Lane1, R Dalton2, I MacPhee1, B Philips1 J Dixon1, K Lane1, R Dalton2, I MacPhee1, B Philips1 p 1St George’s Hospital and University of London, UK; 2King’s College, London, UK Critical Care 2015, 19(Suppl 1):P292 (doi: 10.1186/cc14372) 1St George’s Hospital and University of London, UK; 2King’s College, London, UK Critical Care 2015, 19(Suppl 1):P292 (doi: 10.1186/cc14372) Introduction There is currently no accurate method of measuring the glomerular fi ltration rate (GFR) during acute kidney injury (AKI). Four- hour creatinine clearance (4-CrCl) is often used. We have previously validated a method of measuring the GFR using a continuous infusion of low-dose iohexol (CILDI) in patients with stable renal function (GFR from normal to <30 ml/minute/1.73 m2). Steady state was achieved in <10 hours in all subjects and we calculate that variations >10.3% suggest an AKI. In this study we compare GFR measured by CILDI with 4-CrCl and 1-hour creatinine clearance (1-CrCl). Conclusion Early elevation of urinary [TIMP-2][IGFBP7] after TAVI is associated with the development of postoperative AKI. These biomarkers have an excellent diagnostic accuracy in the prediction of severe AKI requiring RRT that is superior to that of serum creatinine. Further studies are necessary to prove whether UTI-guided therapy of patients with AKI can reduce morbidity and mortality. Methods Critically ill patients with evolving AKI and patients following nephrectomy were recruited. Demographics were compared using the t test. CIDLI was connected for up to 72 hours. Plasma and renal iohexol and creatinine concentrations were measured by tandem mass spectrometry four times daily. Iohexol renal clearance (IRC) and S104 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1-CrCl and 4-CrCl were calculated and compared using Bland–Altman analysis. P294 Impact of kidney injury on fl uid overload and impaired oxygenation A Akcan Arikan, LL Loftis, MA Arnold, CE Kennedy Baylor College of Medicine, Houston, TX, USA Critical Care 2015, 19(Suppl 1):P294 (doi: 10.1186/cc14374) y Results Baseline estimated GFR was similar in the postnephrectomy (88 ± 28) to the evolving AKI group (92 ± 23), P = 0.70. The evolving AKI group had a higher APACHE score (17.8 ± 5.1 vs. 10.6 ± 3.9; P <0.001). Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data When 1-CrCl was compared with IRC, a bias of 0.8% (SD 26%, limits of agreement –52 to 50%; Pearson’s r = 0.90) was observed in the evolving AKI group, whereas bias was –3.3% (SD 16, limits of agreement –35 to 29%; Pearson’s r = 0.95) in the postnephrectomy group. When 4-CrCl was compared with IRC, bias was 5.1% (SD 54, limits of agreement –102 to 112%, Pearson’s r = 0.45) in the established AKI group and bias was –4.5% (SD 38, limits of agreement –79 to 70%; Pearson’s r = 0.78) in the postnephrectomy group. Introduction Severity of acute kidney injury (AKI) and fl uid overload (FO) are not incorporated into current severity of illness measures and are invisible to the practitioner. The causal relationship and timing between AKI and FO and oxygenation is not clear. The Fluid Overload Kidney Injury Score (FOKIS) is a daily score incorporating subscores for AKI (pRIFLE (creatinine (Cr) and urine output (UOP))), FO (total fl uid (in – out) / ICU admission weight) >15% in fi ve percentile increments, and exposure to nephrotoxic medications. We previously reported that FOKIS outperforms PRISM in mortality prediction in our pediatric intensive care unit (PICU). We hypothesized that patients with AKI on admission to the PICU developed worse fl uid overload and in turn worse oxygenation. y g Conclusion Our data suggest that 4-CrCl is not as accurate and precise as 1-CrCl in patients with AKI and following nephrectomy. IRC appears to be more accurate and precise in patients with a predicted AKI risk and outcome (post nephrectomy) than in patients with evolving AKI. We hypothesise that IRC will be useful alternative to creatinine-based measures of AKI. Methods We prospectively calculated daily FOKIS scores and subscores (Cr, UOP, FO) in PICU patients. We excluded patients with <7 day stays in order to properly explore the association between timing of AKI and FO and oxygenation by oxygenation index (OI). y y y Results Over 18 months, there were 2,830 patients, 436 patients with PICU stay >7 days, 361 patients had complete data for all 7 days. Mortality was 4.5% overall and 11% cohort. A total of 246 patients (68%) had AKI (by FOKISCr or FOKISuop); 205 patients (57%) on day 1, 85 patients (24%) on day 3. Admission or day 3 AKI by either FOKIS subscore (FOKISCr or FOKISuop) did not predict maxFO or mortality. P295 P295 Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs M Ostermann1, L Forni2, K Kashani3, M Joannidis4, A Shaw5, M Chawla6, JA Kellum7, on Behalf of Sapphire Investigators7 1Guy’s & St Thomas Foundation Hospital, London, UK; 2Royal Surrey County Hospital, Guilford, UK; 3Mayo Clinic, Rochester, MN, USA; 4University Hospital Innsbruck, Austria; 5Vanderbilt University Hospital, Nashville, TN, USA; 6George Washington University Medical Center, Washington, DC, USA; 7University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P295 (doi: 10.1186/cc14375) FOKIS f Methods Retrospective data from all nondialysed patients undergoing isolated CABG at our institution were collected for the year 2013. Propensity scoring using the software platform MatchIt® was used to match subjects from ONCAB and OPCAB groups with regard to preoperative variables: logistic EuroSCORE, creatinine clearance (CrCl), gender and operative urgency. Postoperative AKI was defi ned as a rise of 50% or more in baseline serum creatinine [2]. Chi-square analysis was used to determine statistically signifi cant diff erences. Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. yif Results From 500 cases (369 OPCAB, 131 ONCAB), 262 subjects were included in the fi nal analysis, with 131 in each group. There was a higher incidence of AKI and renal replacement therapy (RRT) in the ONCAB group, although this was not signifi cantly greater than in the OPCAB group (Table 1). The mortality rate was identical with three deaths in each group. The average length of ICU stay for the OPCAB group was 1.96 days versus 2.49 days for the ONCAB group. 2. Acute Kidney Injury Network criterion. http://www.akinet.org. 1. Lamy A, et al. N Engl J Med. 2012;366:1489-97. Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data Increasing total FOKIS score was associated with increasing mortality and increasing OI (Table 1). FOKIS, controlled for PRISM, was an independent predictor of OI (P = 0.03). Does cardiopulmonary bypass increase the risk of postoperative acute kidney injury after coronary artery bypass grafting? A Karmali, C Walker, L Kuppurao Harefi eld Hospital, London, UK Critical Care 2015, 19(Suppl 1):P293 (doi: 10.1186/cc14373) Does cardiopulmonary bypass increase the risk of postoperative acute kidney injury after coronary artery bypass grafting? A Karmali, C Walker, L Kuppurao Harefi eld Hospital, London, UK Critical Care 2015, 19(Suppl 1):P293 (doi: 10.1186/cc14373) Introduction Acute kidney injury (AKI) following coronary artery bypass grafting (CABG) results in signifi cant morbidity, mortality and prolonged stay on the cardiothoracic ICU. We sought to determine the incidence of AKI in our cardiothoracic centre, hypothesizing a higher occurrence in patients undergoing CABG using cardiopulmonary bypass (ONCAB) compared with off -pump (OPCAB) surgery [1]. Table 1 (abstract P294) P FOKIS 0 <4 4 to 7 >7 value maxOI, median (IQR) 7.4 11.1 16.4 14.2 0.03 (5.9 to 16.4) (6.2 to 23.6) (7.3 to 29.6) (10 to 38.7) Mortality, % 3.6 7.7 13 38 <0.001 Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. Table 1 (abstract P294) P FOKIS 0 <4 4 to 7 >7 value maxOI, median (IQR) 7.4 11.1 16.4 14.2 0.03 (5.9 to 16.4) (6.2 to 23.6) (7.3 to 29.6) (10 to 38.7) Mortality, % 3.6 7.7 13 38 <0.001 Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. Table 1 (abstract P294) Table 1 (abstract P294) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK g Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Introduction This study investigated renal outcomes following radio- contrast (RC) administration in patients from two intensive care units (ITUs), where one gave RC-induced nephropathy (RCIN) prophylaxis, while the other did not. Acute kidney injury (AKI) during critical illness increases morbidity and mortality. ITU patients, who already suff er a variety of renal insults, often require RC, increasing their risk of developing AKI, and requiring renal replacement therapy (RRT). Evidence suggests that hydration alone is inadequate for the prevention of RCIN in ITU patients, and is contraindicated in some disease states [1]. The European Society of Intensive Care Medicine (ESICM) provides recommendations for prophylaxis [2]. The current study aimed to establish the effi cacy of the ESICM recommended prophylaxis. p p y Conclusion Both SOFA score and BD may be used to predict AKI in surgical oncology patients at ICU admission. These variables allow physicians to recognize early patients who might be under risk, and anticipate measures to avoid further renal impairment. References 1. Rocktaeschel J, Morimatsu H, Uchino S, Goldsmith D, Poustie S, Story D, et al. Acid–base status of critically ill patients with acute renal failure: analysis based on Stewart–Figge methodology. Crit Care. 2003;7:R60. 1. Rocktaeschel J, Morimatsu H, Uchino S, Goldsmith D, Poustie S, Story D, et al. Acid–base status of critically ill patients with acute renal failure: analysis based on Stewart–Figge methodology. Crit Care. 2003;7:R60. gg gy 2. Smith I, Kumar P, Molloy S, Rhodes A, Newman PJ, Grounds RM, et al. Base excess and lactate as prognostic indicators for patients admitted to intensive care. Intensive Care Med. 2001;27:74-83. fi Methods Retrospective data from 140 Maidstone (M) ITU patients (men 101, women 39, mean age 63.5, mean APACHE 15.3) and 73 Tunbridge Wells (T) ITU patients (men 41, women 32, mean age 60.2, mean APACHE 20.2) admitted between 22 September 2011 and 22 September 2013, who underwent RC-enhanced CT, were collected. Patients on MITU received ESICM-recommended RCIN prophylaxis: 200 mg aminophylline i.v. over 30 minutes prior to RC, 1.26% sodium bicarbonate 3 ml/kg/hour for 1 hour prior to RC and 1 ml/kg/hour for 6 hours post RC. Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs 1 2 h 3 d 4 h 5 h l 6 Table 1 (abstract P293) OPCAB ONCAB P value AKI 14 (10.69%) 19 (14.50%) 0.35 RRT 6 (4.58%) 9 (6.87%) 0.43 Table 1 (abstract P293) g Critical Care 2015, 19(Suppl 1):P295 (doi: 10.1186/cc14375) Conclusion Our study is limited by its size and the use of logistic EuroSCORE as a composite measure of risk factors. However, it has demonstrated that in our predominantly off -pump cardiac unit, OPCAB conferred no statistically signifi cant advantage over ONCAB with regard to postoperative AKI. Studies to date have standardised patient-specifi c variables, but not the conduct of the procedure itself; for example, minimising renal hypoperfusion during OPCAB. This may be a reason for the lack of clear superiority in the continuing debate over choice of CABG procedure and reducing the risk of AKI. References Introduction Tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) are specifi c urinary biomarkers which can predict acute kidney injury (AKI) in critically ill patients within 12 hours [1]. A [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 is associated with seven times the risk of AKI compared with a test result ≤0.3 [2]. The aim of our study was to explore the use of potentially nephrotoxic medications within the window between a positive biomarker test and the diagnosis of AKI stage 2 or 3.i Introduction Tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) are specifi c urinary biomarkers which can predict acute kidney injury (AKI) in critically ill patients within 12 hours [1]. A [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 is associated with seven times the risk of AKI compared with a test result ≤0.3 [2]. The aim of our study was to explore the use of potentially nephrotoxic medications within the window between a positive biomarker test and the diagnosis of AKI stage 2 or 3. Methods We identifi ed all patients enrolled into the Sapphire study [1] who received at least one potentially nephrotoxic drug on the day of AKI (defi ned by stage 2 or 3 as per KDIGO classifi cation). We subsequently Methods We identifi ed all patients enrolled into the Sapphire study [1] who received at least one potentially nephrotoxic drug on the day of AKI (defi ned by stage 2 or 3 as per KDIGO classifi cation). We subsequently 2. Acute Kidney Injury Network criterion. http://www.akinet.org. pp References 1. Kashani K, et al. Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care. 2013;17:R25. 2. Bihorac A, et al. Validation of cell-cycle arrest biomarkers for acute kidney injury: using clinical adjudication. Am J Resp Crit Care Med. 2014;189:932-9. 2. Bihorac A, et al. Validation of cell-cycle arrest biomarkers for acute kidney injury: using clinical adjudication. Am J Resp Crit Care Med. 2014;189:932-9. Results There were 76 (26.7%) patients who developed AKI within the fi rst 48 hours after ICU admission. In a univariate analysis, patients with AKI were more likely to be male, had higher Sequential Organ Failure Assessment (SOFA) score, higher baseline serum creatinine and urea levels, higher serum lactate levels and had more metabolic academia at admission. These patients also had a higher 24-hour Simplifi ed Acute Physiology III score and higher length of mechanical ventilation as compared with non-AKI patients. There were no diff erences between patients regarding intraoperative vasopressors, type and amount of fl uids, diuresis and blood transfusion. In a multivariate analysis we identifi ed admission base defi cit (BD) (OR = 1.13, 95% CI = 1.02 to 1.24, P = 0.017) and SOFA score (OR = 1.35, 95% CI = 1.2 to 1.51, P <0.001) as independent predictive factors of early AKI. 2. Joannidis M, et al. Intensive Care Med. 2010;36:392-411. 1. Huber W, et al. Radiology. 2006;239:793-804. Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs 1 2 h 3 d 4 h 5 h l 6 S105 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 determined the proportion of patients who had a [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 before meeting the criteria for AKI. determined the proportion of patients who had a [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 before meeting the criteria for AKI. Predictors of renal recovery in critically ill patients with AKI: observations from the ongoing FBI clinical trial f S Robinson, U Larsen, A Zincuk, S Zwisler, P Toft , , , , Odense University Hospital, Odense, Denmark , , , , Odense University Hospital, Odense, Denmark y p Critical Care 2015, 19(Suppl 1):P298 (doi: 10.1186/cc14378) y Critical Care 2015, 19(Suppl 1):P298 (doi: 10.1186/cc14378) Introduction The predictive value of NGAL for renal recovery is not established. Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients Results Of 184 patients who developed AKI, 58% received one or more potentially nephrotoxic drug on the day of AKI. Eighty-nine percent of these patients had a positive biomarker test ≥12 hours earlier. In 41% of patients receiving one or more nephrotoxic drug on the day of AKI, at least one nephrotoxic medication was stopped within 1 day of AKI, and in 24% of patients all nephrotoxic drugs were stopped within 1 day of AKI, which implies that these medications were not absolutely necessary. Introduction Patients who undergo major oncology surgery are under high risk to develop postoperative acute kidney injury (AKI), mainly due to infl ammatory and ischemic insults. This complication results in worse outcomes. The aim of this study is to identify predictive factors of AKI in this population. Conclusion Nephrotoxic medications are commonly used in patients who develop moderate or severe AKI. The [TIMP-2]·[IGFBP7] test could have identifi ed many of these patients earlier and would have off ered an opportunity to reduce exposure to non-essential nephrotoxic drugs. References Methods We performed an observational study in 285 consecutive patients admitted to a surgical ICU after major abdominal oncology surgery. Baseline characteristics, laboratorial, clinical and intraoperative data, such as type of fl uids, blood transfusion, bleeding and use of vasopressor, were collected at ICU admission. Early acute kidney injury was defi ned according to the Acute Kidney Injury Network classifi cation at 48 hours of ICU admission. Logistic regression model was performed using AKI as the outcome. P297 Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients A Gerent, J Almeida, E Almeida, A Lousada, C Park, J Ribeiro, J Fukushima, A Leme, E Osawa, A Rezende, I Bispo, F Galas, L Hajjar Instituto do Cancer do Estado de São Paulo – ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P297 (doi: 10.1186/cc14377) Introduction The predictive value of NGAL for renal recovery is not established. Methods Data from the fi rst 19 patients were assessed during a multicentre low molecular weight heparin trial (FBI, EudraCT Number: 2012-004368-23). Critically ill patients with AKI are randomly assigned into either a treatment arm (1 mg/kg enoxaparin) or a control arm (40 mg enoxaparin) upon commencement of CRRT. The primary outcome is the occurrence of venous thromboembolism. NGAL was measured at baseline and during CRRT-free intervals. Results The total number of patients developing renal injury falling into any RIFLE category for MITU at 24, 28 and 72 hours was eight (0.06%), 12 (0.09%) and 14 (0.1%), while for TITU it was fi ve (0.07%), six (0.08%), and four (0.05%) respectively. A repeated-measures ANOVA revealed no signifi cant diff erences in outcomes between the two groups overall (F = 2.35, P = 0.127) or at each time point (F = 1.93, P = 0.123). Results Patients were comparable at baseline with respects to demographics, APACHE II, creatinine, NGAL, start of dialysis, and the duration of dialysis. The main cause of AKI was sepsis (42%). In 63% of the patients, the reason for starting dialysis was a combination of anuria and electrolyte disturbances. Twenty-six percent of patients were dialysis dependent after the fi rst CRRT-free interval. Plasma NGAL levels were higher in nonrenal recovery patients (1,074 (±694) ng/ml) compared with renal recovery patients (296 (±197) ng/ml; P = 0.01). Urine NGAL levels were higher in nonrenal recovery patients (3,885 Conclusion While RCIN is a recognised problem within the critical care population, there is little clear evidence for any prophylactic strategy to reduce this risk. This study suggests that a RCIN prophylaxis protocol based on the ESICM recommendations has no eff ect on the incidence of RCIN. However, further studies are needed. Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) TITU patients received standard critical care alone. Exclusion criteria were: those undergoing RRT prior to CT, >1 CT in 48 hours, no creatinine (Cr) data available post scan. The Cr prior to CT (baseline), at 24, 48 and 72 hours post CT scan were identifi ed. The RIFLE criteria was used to classify the changes of Cr from baseline into low risk (% change >1.25), risk (% change >1.5), injury (% change >2) or failure (% change >3, or Cr>355 and increase of >44). P299 Comparison of two strategies for initiating renal replacement therapy in the ICU: study protocol plan for a multicenter, randomized, controlled trial from the AKIKI research group S Gaudry1, D Hajage2, F Schortgen3, L Martin-Lefevre4, J Ricard1, D Dreyfuss1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Bichat, Paris, France; 3Hôpital Henri Mondor, Créteil, France; 4CHD La Roche sur Yon, France Critical Care 2015, 19(Suppl 1):P299 (doi: 10.1186/cc14379) Results The total baseline plasma concentration of all standard amino acids was similar between IHD versus SLEDf groups (1,812 ± 517 vs. 2,675 ± 527 μmol/l, respectively) but were higher in the CVVH group (3,194 ± 564 μmol/l). RRT reduced the plasma concentration of amino acids in the SLEDf group (to 1,732 ± 529 μmol/l; P = 0.02), but had no eff ect in the IHD or CVVH groups (IHD; 1,853 ± 523, CVVH; 2,845 ± 512 μmol/l). The average, unadjusted loss of amino acids was signifi cantly infl uenced by mode of RRT (IHD, 5.13 ± 3.1 vs. SLEDf, 8.21 ± 4.07 vs. CVVH, 18.69 ± 3.04 g; P <0.01). The total baseline plasma concentration of trace elements was similar in the IHD, SLEDf and CVVH groups (3,797 ± 827, 3,667 ± 791, 3,642 ± 481 μg/l, respectively). By the end of the RRT session, the plasma concentration of trace elements had reduced (IHD, to 3,103 ± 827; SLEDf, to 2,805 ± 797; CVVH, to 3,433 ± 481 μg/l; P = 0.01). By the end of each RRT session, total losses of trace elements were estimated at IHD, 5,051 ± 2,312; SLEDf, 8,751 ± 2,421; CVVH, 11,258 ± 2,547 μg/l; P = 0.02 for treatment. Introduction There is currently no validated strategy for the timing of renal replacement therapy (RRT) for acute kidney injury (AKI) in the ICU when short-term life-threatening metabolic abnormalities are absent. No adequately powered prospective randomized study has to date addressed this issue. As a result, signifi cant practice heterogeneity exists and may expose patients either to unnecessary hazardous procedures or to undue delay in RRT. Methods This is a multicenter, prospective, randomized, open- label parallel-group clinical trial that compares the eff ect of two RRT initiation strategies on overall survival of critically ill patients receiving intravenous catecholamines and/or invasive mechanical ventilation and presenting with RIFLE F stage of AKI. In the early strategy, RRT is initiated immediately. References S106 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (±2,722) ng/ml) compared with renal recovery patients (597 (±565) ng/ml; P = 0.006). See Figures 1 and 2. Infl ammatory parameters (WBCs, CRP, and procalcitonin) did not diff er signifi cantly between the groups. Conclusion NGAL may be able to predict renal recovery, and allow proper utilization of resources. Figure 2 (abstract P298). Urine neutrophil gelatinase-associated lipocalin concentration by recovery. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. more than 72 hours after randomization, serum urea concentration >40 mmol/l, serum potassium concentration >6 mmol/l, serum potassium concentration >5.5 mmol/l persisting despite medical treatment, arterial blood pH <7.15 in a context of pure metabolic acidosis (PaCO2 <35 mmHg) or in a context of mixed acidosis with a PaCO2 >50 mmHg without possibility of increasing alveolar ventilation, acute pulmonary edema due to fl uid overload despite diuretic therapy leading to severe hypoxemia requiring oxygen fl ow rate >5 l/minute to maintain SpO2 >95% or FiO2 >50% under invasive or non-invasive mechanical ventilation. The primary endpoint is overall survival, measured from randomization (D0) until death, regardless of the cause. The minimum follow-up duration for each patient will be 60 days. To demonstrate a 14% decrease in mortality, a total of 546 subjects (273 per group) should be randomized.i p g p Results Enrollment is ongoing. After the fi rst interim analysis, the DSMB recommended to continue the study. On 5 December 2014, 318 patients were included in the trial. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. Conclusion The AKIKI study will be one of the very few large randomized controlled trials evaluating mortality according to the timing of RRT in critically ill patients with RIFLE F stage of AKI. Results should help clinicians better decide when to initiate RRT. Figure 2 (abstract P298). Urine neutrophil gelatinase-associated lipocalin concentration by recovery. P300 Micronutrient loss in renal replacement therapy for acute kidney injury W Oh1, M Devonald1, D Gardner2, R Mahajan3, D Harvey3, A Sharman3, B Mafrici1, M Rigby1, S Welham2 1Nottingham University Hospitals NHS Trust, Nottingham, UK; 2University of Nottingham, Sutton Bonington, UK; 3University of Nottingham, UK Critical Care 2015, 19(Suppl 1):P300 (doi: 10.1186/cc14380) Micronutrient loss in renal replacement therapy for acute kidney injury j y W Oh1, M Devonald1, D Gardner2, R Mahajan3, D Harvey3, A Sharman3, B Mafrici1, M Rigby1, S Welham2 1Nottingham University Hospitals NHS Trust, Nottingham, UK; 2University of Nottingham, Sutton Bonington, UK; 3University of Nottingham, UK Critical Care 2015, 19(Suppl 1):P300 (doi: 10.1186/cc14380) Introduction The prevalence of malnutrition in acute kidney injury (AKI) is high. Patients with AKI may require renal replacement therapy (RRT), which could result in loss of water-soluble micronutrients. Little is known about these losses in RRT and whether they diff er between types of RRT. This study aims to quantify micronutrient losses during RRT in patients with AKI and to compare them in three diff erent RRT modalities: continuous venovenous haemofi ltration (CVVH), intermittent haemodialysis (IHD) and sustained low-effi ciency diafi ltration (SLEDf). (±2,722) ng/ml) compared with renal recovery patients (597 (±565) ng/ml; P = 0.006). See Figures 1 and 2. Infl ammatory parameters (WBCs, CRP, and procalcitonin) did not diff er signifi cantly between the groups. Conclusion NGAL may be able to predict renal recovery, and allow proper utilization of resources. Methods A prospective observational study is being conducted at NUH. Thirty-three adult patients with AKI requiring RRT (13 IHD, 10 SLEDf, 10 CVVH) have been recruited. Samples of blood and RRT effl uent were obtained at baseline, mid and end-session from each participant during their fi rst RRT treatment. Samples were processed and stored at –80°C for subsequent analysis of amino acids by high- performance liquid chromatography and trace elements by inductively coupled mass spectrometry after derivatization from physiological fl uids. Micronutrient losses were calculated by multiplying mass- corrected concentrations by total volume of RRT effl uent, adjusted for baseline plasma concentrations and RRT dose. Data were analysed by restricted maximum likelihood estimating equations. P299 In the delayed strategy, clinical and metabolic conditions are closely monitored and RRT is initiated only when one or more events (severity criteria) occur, including: oliguria or anuria for Conclusion Micronutrients are lost during RRT in AKI. The degree of micronutrient loss is infl uenced by the type of RRT used. S107 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P301 Methods A comparison of patients receiving CVVHDF on the 11-bed critical care unit at Conquest Hospital, Hastings was undertaken, before and after the implementation of new CVVHDF protocols. All patients receiving CVVHDF were identifi ed from the electronic patient record system between March 2012 to 2013 and September 2013 to 2014. Patient demographics, the duration of CVVHDF and sodium bicarbonate supplementation were analysed between the groups to assess the impact of the new protocols. P301 Super high-fl ux CVVHD using regional citrate anticoagulation: long-term stability of middle molecule clearance M Siebeck, D Kindgen-Milles University Hospital, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P301 (doi: 10.1186/cc14381) Introduction Conventional membranes used for CRRT have a limited middle molecule clearance. New membranes called super high-fl ux (SHF) or high cutoff membranes have been investigated. The loss of albumin with hemofi ltration is a major drawback, but these membranes can be used in CVVHD with regional citrate anticoagulation (Ci-Ca® CVVHD), which may limit albumin loss, and contribute to a prolonged fi lter patency and an improved and stable middle molecule clearance. We evaluated saturation coeffi cients (SC), plasma clearances (PCL) and serum levels of eight small and middle molecules during 72 hours of Ci-Ca® CVVHD with a SHF membrane (Ultrafl ux®EMiC®2). p p Results Sixty-four patients received CVVHDF in 2012 to 2013, 61 receiving citrate and three receiving unfractionated heparin due to fulminant liver failure. Forty-seven patients received CVVHDF in 2013 to 2014, two receiving no anticoagulation due to severe coagulopathy and one receiving unfractionated heparin. The two patient cohorts assessed were similar in age (median 65.5 for March 2012 to 2013 cohort vs. 66 for September 2013 to 2014 cohort), gender mix (64% male vs. 57% male) and severity of illness as assessed by APACHE II score (23 vs. 24). Mean duration of CVVHDF was also similar (71.5 hours vs. 75 hours). P299 A total 30/64 of 2012 to 2013 patients did not require a fi lter change prior to completion of RRT, compared with 23/47 of 2013 to 2014 patients. Sodium bicarbonate was added to the dialysate fl uid in 29/64 2012 to 2013 patients, compared with just 2/47 2013 to 2014 patients.i l Methods After approval of the local committee of medical ethics and written informed consent we enrolled patients on a surgical ICU with AKI RIFLE-F who were treated with a Ci-Ca® CVVHD with a SHF membrane for 72 hours. We measured urea (0.006 kDa), creatinine (0.113 kDa), osteocalcin (5.8 kDa), B2MG (12 kDa), myoglobin (17.2 kDa), FreeLightChains (FLC) kappa (25 kDa) and lambda (50 kDa) and albumin (66 kDa) at 0 hours, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours. PCL, SC and serum levels during 72 hours were compared, using the Wilcoxon signed-rank test with P <0.05. Conclusion Changing protocols resulted in a signifi cant reduction in off -license addition of sodium bicarbonate to dialysate bags without impacting on fi lter life, thus reducing nursing workload and removing a potential source of adverse events in this high-risk group of patients. Results Four females and 10 males (mean age 68.1 ±15.1 years; mean APACHE II score 13.7 ± 14.7; mean SAPS II 38.7 ± 12.7) were included. The SC and the PCL (ml/minute) of small solutes like creatinine at 1 hour (1.0 ± 0.0/23.72 ± 1.04) and 72 hours (0.95 ± 0.16/22.19 ± 3.99) were not statistically signifi cantly diff erent (P = 0.5/P = 0.42), the PCL was slightly reduced by 6%. The creatinine serum level was reduced by 42%. The SC and PCL of B2MG from 1 hour (0.61 ± 0.09/14.49 ± 2.5) to 72 hours (0.48 ± 0.13/11.6 ± 2.96) were signifi cantly decreased (P = 0.0024/P = 0.0061). The reduction was 23% only; the overall clearance still was high. There was almost no reduction in SC or PCL for FLC kappa from 1 hour (0.176 ± 0.047/4.14 ± 1.08) to 72 hours (0.164 ± 0.078/3.854 ± 1.87), not reaching statistical signifi cance (P = 0.94/P = 0.81). The serum levels of B2MG and FLC kappa were decreased by 39% and 23%. The SC of albumin was low (1 hour: 0.0009 ± 0.0004) and clearance decreased rapidly within the fi rst 6 hours from 0.021 ± 0.01 to 0.011 ± 0.009. Descriptive study of the haematological management of adult patients with severe respiratory failure receiving venovenous extracorporeal membrane oxygenation p yg O Tavabie, R Pocock, N Barrett, A Retter Introduction Venovenous extracorporeal membrane oxygenation (VV-ECMO) is a novel therapy for severe respiratory failure (SRF). Its introduction has reduced mortality; however, patients require substantial blood product support and between 10 and 20% of cases develop a life-threatening haemorrhage. p g g Methods We contacted 336 practitioners at 135 centres, examining their haematological management. Conclusion This study shows high middle molecular clearances using a SHF membrane with Ci-Ca® CVVHD for 72 hours with no loss of albumin. This set-up may improve blood purifi cation in critically ill patients with acute kidney injury. Results In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Results In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Scenario 1 was based on a patient with H1N1. Practitioners targeted a haemoglobin (Hb) of 80 to 100 g/l; however, 20% targeted a Hb outside this range; 38% favouring a transfusion threshold of <80 g/l when the patient was improving compared with 32% when the patient had just started on ECMO. Seventeen per cent of practitioners transfused platelets when the count was <30 × 109/l whilst 21% maintained the platelet count >100 × 109/l. Scenario 2 described a patient with SRF secondary to a hospital-acquired pneumonia. The patient developed a haemothorax, with persistent blood loss of 200 ml/hour. Descriptive study of the haematological management of adult patients with severe respiratory failure receiving venovenous extracorporeal membrane oxygenation Practitioners targeted a higher haemoglobin concentration of 100 g/l and targeted a higher platelet count of >100 × 109/l when compared with the patient in scenario 1, neither of these diff erences was statistically signifi cant. Seventy-one per cent stated they would manage the patient off anticoagulation. There was no agreement as to the length of time off anticoagulation; 26% restarted anticoagulation in <12 hours, compared with 22% who advised no anticoagulation for >5 days. Scenario 3 examined the management of an incidental intracranial haemorrhage. There was a lack of consensus regarding the duration off anticoagulation; 14% of responders held anticoagulation for less than P299 Serum levels of albumin did not decrease (1 hour: 2.64 ± 0.51; 72 hours: 2.63 ± 0.25). P302 P302 Citrate anticoagulation for continuous venovenous haemodiafi ltration: the impact of a novel protocol on patients receiving therapy in one regional hospital J Highgate1, G Escott2, A Lowe2, F Stedman2, N McNeillis2 1Worthing Hospital, Worthing, UK; 2Conquest Hospital, Hastings, UK Critical Care 2015, 19(Suppl 1):P302 (doi: 10.1186/cc14382) Introduction Citrate has been used to anticoagulate extracorporeal haemofi ltration circuits since the 1960s, and has been used as the fi rst-line anticoagulant for continuous venovenous haemodiafi ltration (CVVHDF) at Conquest Hospital since 2009. Benefi ts of citrate demonstrated in clinical trials include increased fi lter life and increased bicarbonate formation from metabolism of the citrate complex; citrate also lacks the increased bleeding risk associated with unfractionated heparin use. One of the main issues with new renal replacement therapies is the development of ideal dialysate fl uids. During the initial period of citrate use at Conquest, hyponatraemia was identifi ed as an issue, with off -license supplementation of dialysate fl uid with sodium bicarbonate often necessary to prevent this. New protocols were therefore developed, designed to maximise the fi ltration dose and maintain normal electrolyte balance. S108 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Data were collected from our computerised information system on all AKI patients receiving RRT on our ICU, between October 2008 and October 2013. This included demographics, APACHE II and SOFA scores, modality and dose of RRT and ICU length of stay (LOS). Renal and patient survival at ICU discharge was collected, in addition to outcome data at 28 and 90 days and 12 months. Data were examined 12 hours whilst 37% held anticoagulation for >5 days and tranexamic was considered useful by 25%. y Conclusion There was wide variation in the use of blood products and the intensity of anticoagulation. This is not surprising given the current lack of evidence. Further work is required to provide a standardised approach. using Cox proportional hazard multivariate analysis, with Stata 10.1. Results A total of 620 patients with AKI received RRT on our ICU between October 2008 and October 2013. Sixty-one per cent were males. Median age was 65 years (IQR 54 to 74). Median APACHE II score was 23 (IQR 18 to 27). Median SOFA score was 11 (IQR 8 to 13). Fifty-fi ve per cent were mechanically ventilated. Comparison between nafamostat mesilate and unfractionated heparin as anticoagulant during continuous renal replacement therapy Comparison between nafamostat mesilate and unfractionated heparin as anticoagulant during continuous renal replacement therapy S Makino, H Kita, Y Miyatake, T Yokoyama, K Kubota, N Obata, M Egi, T Misumi, S Izuta, S Mizobuchi Kobe University Hospital, Kobe, Japan Critical Care 2015, 19(Suppl 1):P304 (doi: 10.1186/cc14384) Introduction For continuous renal replacement therapy (CRRT), continuous administration of anticoagulant would be necessary to prevent the circuit clotting. Nafamostat mesilate (NM) is commonly used as its anticoagulant in Japan, although unfractionated heparin (UFH) is the most frequently used anticoagulant internationally. There is little study to compare the risk and benefi t of NM with UFH as an anticoagulant during CRRT. Methods We conducted a single-center retrospective observational study to compare NM with UFH as anticoagulant during CRRT. We screened subsequent critically ill patients requiring CRRT in our ICU from January 2011 to December 2013. We excluded patients who required any other extracorporeal circuit including extracorporeal membrane oxygenation, who used both NM and UFH simultaneously, or who were administered any other anticoagulant including gabexate mesilate or urokinase. The primary outcome of this analysis was fi lter life, and the secondary outcome was the incidence of bleeding complications during CRRT. As an initial dose, NM and UFH were given pre fi lter at 15 to 25 mg/hour and 1,500 to 3,000 IU/hour, respectively. The dose of both drugs was adjusted to maintain activated clotting time at post fi lter between 150 and 200 seconds. Filter life was assessed using the Kaplan–Meier method and the incident of bleeding complications was compared using the chi-square test. P <0.05 was considered to be statically signifi cant. Conclusion Results from our cohort suggest that, in patients with AKI presenting to ICU for RRT, long-term patient survival is signifi cantly impaired. Renal outcomes are poor with 35% being either dialysis dependent or having severe chronic kidney disease (eGFR <15 ml/ minute), at 1 year from ICU discharge. Our data do not suggest a benefi t of using HVHF in AKI patients presenting to ICU for RRT. P306 Plasma antioxidant capacity in critical traumatized patients: severity and anatomical location y G Papakitsos1, A Kapsali1, T Papakitsou2, A Roimba3 1GHA, Arta, Greece; 2GHM, Messologi, Greece; 3General Practitioner, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P306 (doi: 10.1186/cc14386) i Results We included 101 patients in this study. Among them, 76 patients were with NM and 25 patients were with UFH. They used 239 fi lters in total; 173 with NM, 66 with UFH. There were signifi cantly more post- surgical patients in the NM group (P = 0.001). There was no diff erence in age, APACHE II score, days from ICU admission to commencement of CRRT, length of ICU stay and mortality between two groups. There was no diff erence in median number of fi lters used by one patient (NM vs. heparin; median of 1.5 (IQR) vs. 2 (IQR), P = 0.27). Filter life in the UFH group was signifi cantly longer than those in the NM group (NM vs. UFH; median of 24 hours vs. 36 hours; P = 0.01). The incidence of bleeding complications was not signifi cantly diff erent between two groups (P = 0.15). Introduction Oxidative stress (OS) has been invoked as a relevant factor in the evolution and outcome of critical care patients. Indeed, antioxidant therapies have been used in critical care patients, but with controversial results. This may be explained by assuming OS as a homeostatically regulated parameter and both its excess and its defi cit infl uencing severity progression. Nonetheless, antioxidant agents could mask an OS signaling role, blocking otherwise physiological responses aimed at recovery of homeostasis. We have evaluated plasma total antioxidant capacity (TAC) in traumatized patients in the emergency department (ED) and we determined its potential relationship with severity and trauma location. Conclusion In our retrospective analysis with 101 patients, fi lter life with UFH was signifi cantly longer than those with NM. The incidence of bleeding complications was not signifi cantly diff ered between patients with NM and UFH. y Methods In a prospective observational study of ED polytraumatized patients (n = 23, mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score of 11 ± 6) we measured (in the fi rst 24 hours) plasma TAC by the ferric reducing activity/antioxidant power (FRAP). For control subjects, we used age-matched and gender-matched volunteers (n = 32). We also evaluated the contribution of antioxidant molecules (uric acid, bilirubin, and albumin) to these values. P302 A total of 96.7% received CVVH as the principal RRT modality. Twenty-one per cent received a period of high-volume haemofi ltration (HVHF) (80 ml/kg/hour), median LOS was 6 days (IQR 3 to 14). In total, 331 (53.4%) patients recovered their renal function at ICU discharge, whilst 237 (38.2%), 220 (35.4%), and 220 (35.4%) patients did not at 28 and 90 days and 12 months respectively. A total of 414 (66.7%) patients survived to ICU discharge, with 368 (59.3%), 341 (55%) and 308 (49.6%) patients being alive at 28 and 90 days and 12 months respectively. Overall patient survival at the end of follow-up was 43%. Adjusting for age and sex; APACHE II score, SOFA score and use of HVHF were associated with worse patient survival at ICU discharge (HR: 1.07, 95% CI: 1.03 to 1.11, P <0.001, HR: 1.11, 95% CI: 1.03 to 1.19, P = 0.006 and HR: 2.27, 95% CI: 1.4 to 3.66, P = 0.001, respectively). Adjusting for age and sex; APACHE II score and use of HVHF were associated with worse renal recovery at ICU discharge (HR: 1.06, 95% CI: 1.03 to 1.09, P <0.001 and HR: 1.55, 95% CI: 1.03 to 2.3, P = 0.032 respectively). SOFA score did not appear to signifi cantly impact renal recovery (HR: 0.99, 95% CI: 0.94 to 1.04, P = 0.81). Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, China Critical Care 2015, 19(Suppl 1):P309 (doi: 10.1186/cc14389) Introduction The aim was to analyze the injury features and treatment strategies of surviving casualties in the explosion accident on 2 August 2014 in Kunshan city (Kunshan ‘August 2’ Explosion Accident). Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. Introduction The aim was to analyze the injury features and treatment strategies of surviving casualties in the explosion accident on 2 August 2014 in Kunshan city (Kunshan ‘August 2’ Explosion Accident). Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. p p p p Results In total, 722 trauma patients were included, of which 300 patients were hypothermic. The mortality in the hypothermia group was signifi cantly higher than in normotherm patients (OR = 3.73, 95% CI = 2.02 to 7.13, P <0.001). A cutoff point of 36°C was observed as the best threshold for hypothermia (sensitivity 74%, specifi city 56%). Besides hypothermia, other predictors found for 28-day mortality were APACHE II score corrected for temperature, minimum thrombocytes in fi rst 24 hours and urea and included in the fi nal model with an AUC of 0.89 (95% CI = 0.85 to 0.92). External validation of the model was associated with a predicted probability of an AUC of 0.64 (95% CI = 0.51 to 0.77).i Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. Results A total of 40 cases were admitted to our hospital within 6.3 ± 0.8 hours after the explosion accident on 2 August 2014 in Kunshan city, including 28 males and 12 females. The major injury types included burn injury, inhalation injury, blast injury (lung, eye, eardrum, and so forth), traumatic brain injury and bone fractures. All cases suff ered from burn injury caused by the explosion. The mean burned area in the surviving casualties accounted for 92 ± 14% total body surface area (TBSA) and those with third-degree burns for 77 ± 19% TBSA. Additionally, incidence rate of inhalation injuries was 97.5%. There were 34 cases complicated by multiple organ dysfunction syndrome, which accounted for 85.0%. Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu The common organ dysfunction of surviving casualties included the lungs, circulation, liver, gastrointestinal tract, kidney, brain, and coagulation. During hospitalization, the most common infectious site in surviving casualties was a burn wound, followed by blood and lung. The most common infectious strain of bacteria was Gram- negative bacteria, which accounted for 91.3%. Further analysis showed that Proteus mirabilis ranked fi rst in occurrence, followed by Acinetobacter baumannii and Pesudomonas aeruginosa, and Klebsiella pneumonia and Enterobacter cloacae ranked fourth and fi fth. After intensive treatment, the mean 28-day mortality was 20.0% and 90- day mortality was 62.5%, mainly due to septic shock and multiple organ dysfunction syndrome. Conclusion Hypothermia, defi ned as <36°C, is associated with an increased 28-day mortality. The discriminative ability of the developed model for predicting mortality in a new patient population is moderate. P309 Methods A retrospective cohort study was performed in adult trauma patients admitted to a level 1 trauma center and who were transferred to the ICU between 2007 and 2012. Diff erent cutoff points for hypothermia were compared to fi nd the best defi nition for hypothermia. Logistic regression analysis was performed to quantify the net eff ect of hypothermia on admission to the ICU on 28-day mortality and to develop a model with predictors. The developed model was externally validated in data from another level 1 trauma center with a comparable patient population. Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, China Critical Care 2015, 19(Suppl 1):P309 (doi: 10.1186/cc14389) P305 Long-term renal and survival outcomes in acute kidney injury patients receiving renal replacement therapy in intensive care I Elsayed1, N Pawley1, J Rosser1, MJ Heap1, GH Mills1, A Tridente2, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P305 (doi: 10.1186/cc14385) Results Polytraumatized patients show diff erences in TAC with reference to control subjects. ED polytraumatized patients show high FRAP values. We found that FRAP values were inversely correlated with APACHE II score (r = –0.266, P <0.01) suggesting that, in trauma patients, increased antioxidant response, as measured by FRAP assay, could be a pathophysiological response to stress. Albumin and uric acid concentrations reproduced the FRAP trend with severity. Introduction Acute kidney injury (AKI) aff ects 40% of critically ill patients, with UK data reporting 5% needing renal replacement therapy (RRT). Hospital mortality is reported as being up to 60%. We sought to evaluate renal and long-term patient survival outcomes in AKI patients receiving RRT on our ICU. Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, S109 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion An increase in StO2 and lower tissue oxygen extraction rates were associated with H-survival in polytrauma patients. Figure 1 (abstract P308). Figure 1 (abstract P308). P <0.002) and head trauma (β = –0.219, P <0.045). These results accentuate the infl uence of trauma location and severity in TAC changes. TAC response in ED patients reinforces the need for an adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. P <0.002) and head trauma (β = –0.219, P <0.045). These results accentuate the infl uence of trauma location and severity in TAC changes. TAC response in ED patients reinforces the need for an adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. P307 Hypothermia as a predictor for mortality in trauma patients K Balvers1, JM Binnekade1, C Boer2, JC Goslings1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU University Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P307 (doi: 10.1186/cc14387) Introduction Previous studies reported hypothermia as an independent predictor for mortality. However, diff erent cutoff points were used in these studies and external validation has never been applied. The aim of this study was to quantify the net eff ect of hypothermia on admission to the ICU on the 28-day mortality and to test the predictors from the developed model in another level 1 trauma center with a comparable patient population to validate the model. Figure 1 (abstract P308). Conclusion An increase in StO2 and lower tissue oxygen extraction rates were associated with H-survival in polytrauma patients. Near-infrared spectroscopy to assess tissue oxygenation in patients with polytrauma: relationship with outcome A Donati, E Damiani, R Domizi, S Pierantozzi, S Calcinaro, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) Università Politecnica delle Marche, Ancona, Italy , , y Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) y Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) Introduction We evaluated tissue oxygenation by means of near- infrared spectroscopy (NIRS) and explored its relationship with outcome in polytrauma patients. Methods A prospective observational study; 37 polytrauma patients underwent NIRS monitoring (thenar eminence) every day during their stay in the ICU. A VOT was performed with a 40% tissue oxygen saturation (StO2) target. Healthy volunteers (n = 27) were studied as controls. y y Conclusion During the Kunshan ‘August 2’ Explosion Accident, burn injury was the leading cause of injuries. Most surviving casualties are accompanied by multiple organ dysfunction syndrome and infection. Results StO2 increased over the fi rst 7 days only in hospital survivors (n = 29), who showed higher values as compared with healthy volunteers at days 5 and 7 (Figure 1). StO2 downslope and upslope tended to be lower in H-nonsurvivors (n = 8) (P <0.05 at days 2 and 4) as compared with H-survivors. Tissue hemoglobin index was lower in H-no survivors over the fi rst 7 days and tended to normalize only in H-survivors (P >0.05 vs. healthy at day 7). Five patients were discharged from the ICU but did not survive until H-discharge. At discharge from the ICU, these patients were similar to H-survivors in SOFA score, heart rate, mean arterial pressure and lactate, but showed lower StO2 downslope (–13 (–16.5, –11.7)%/minute vs. –8.6 (–11.7, –6.5)%/minute, P = 0.01). P310 In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users A Franci1, S Piantini2, M Pierini2, A Peris1, M Mangini1 1A.O.U. Careggi, Firenze, Italy; 2University of Florence, Italy Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) Introduction The purpose of this study is to analyze anatomic distributions, diagnostic methods, and prognosis of missed fractures in patients with severe injury. Introduction The purpose of this study is to analyze anatomic distributions, diagnostic methods, and prognosis of missed fractures in patients with severe injury. gg y y y Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) Methods A review of single-institutional medical records between January 2001 and May 2012 identifi ed 58 patients with 62 delayed diagnoses of fractures among 4,643 severely injured patients older than 20 years with Injury Severity Scores higher than 16. We evaluated combined injuries, location of fractures, diagnostic methods, and reasons for missed diagnosis at initial examination. Introduction Road accidents are the leading cause of death for young people, 50% being represented by vulnerable road users (VRU) (pedestrians, cyclists). In-depth accident studies assess the consequences of lack of use of safety devices and the need to develop new ones. Since 2009 a permanent team (physicians and engineers) has performed in-depth studies on road trauma admitted to our ICU [1]. Results Among 62 missed fractures, there were eight cases of spine fracture, 10 cases of peri-shoulder joint fracture, eight cases of upper extremity fracture, 10 cases of pelvis of acetabulum fracture, and 26 cases of lower extremity fracture. Head injury was the most common concomitant injury (23 cases). Initially missed fractures were most commonly discovered by offi cial reading by radiologists. The most common reasons for misdiagnosis were the use of improper radiologic study and missed-reading of proper radiologic studies. Methods The team studied 52 VRU crashes that occurred in an urban area. The clinical data included an injury assessment using total body CT scan, Injury Severity Score (ISS), Abbreviated Injury Score (AIS), ICU and hospital length of stay and outcome score. Engineers collect data onsite with the partnership of the police, and assess the dynamics of the vehicles with the most advanced reconstruction techniques. Medical and engineering data were cross-matched during the correlation process. Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) I Turriziani, A Cecchi, A Giugni, L Copertino Introduction In the literature, duration of mechanical ventilation (DMV) is often considered an important risk factor (RF) [1] for VAP [2] in critical patients; generally the whole duration of MV is taken into S110 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P311). Frequency of lesions. account, including days before and after infection onset. We tried to assess whether, counting only MV days prior to VAP development (MVp), something would change. Methods We considered, in a 10-year period, data prospectively collected in our database (4D solution, V11) on trauma patients admitted to the ICU directly from the emergency department. Inclusion criteria were: age ≥16 years, ICU length of stay (ICUlos) ≥4 days, DMV ≥48 hours; we excluded patients who received antibiotics before VAP (or during the whole stay, for patients without VAP) and with incomplete data. Data were: age, sex, prehospital GCS <9, prehospital intubation (preHTI), admission base excess (BE), Injury Severity Score (ISS), surgery, massive transfusion, feeding, antacids, nursing, DMV, ICUlos and MVp. MVp was calculated as the diff erence between the fi rst day of VAP and the fi rst day of MV in patients who developed VAP (vapY) and whole DMV in patients that did not (vapN). We only considered the fi rst infectious episode. The outcome was VAP onset. Group comparison was made with Fisher’s exact test and Student’s t test. Signifi cant variables were evaluated in a logistic regression (LR) model; the Hosmer–Lemeshow test (HL) was used as the post-estimation test. Odds ratio (OR) and 95% confi dence interval (95% CI) were calculated. Statistical signifi cance for P <0.05. We used Stata/IC 10.1 for analysis. Figure 1 (abstract P311). Frequency of lesions. Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Dynamic N injured Speed (SD) Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Dynamic N injured Speed (SD) Car vs. pedestrian 29 41.1 (13.9) Motorcycle vs. pedestrian 7 40.6 (4.3) Car vs. ferences Richmond J, Egol KA, Koval KJ. Management of orthopaedic injuries in polytrauma patients. Bull Hosp Jt Dis. 2001-2002;60:162-7. Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) cyclist 10 40.7 (15.9) Other 6 49.0 (5.4) y Results A total of 541 patients met the inclusion criteria, 378 (69.9%) developed VAP. MVp does not seem to be a RF for VAP because they are longer in vapN than in vapY (mean MVp 5.5 vs. 4.41, P = 0.001). PreHTI (vapY/N: 49.74%/38.65%; OR: 1.57; 95% CI: 1.08 to 2.28), ISS (mean vapY/N: 28.4/25.55; P = 0.0018), BE (mean vapY/N: –3.76/–3.04; P = 0.03) were signifi cantly diff erent between the two groups. In LR only preHTI (OR: 1.47; 95% CI: 1.01 to 2.15) and ISS (OR: 1.03; 95% CI: 1.01 to 1.05) are RF for VAP (HL: P = 0.133). Conclusion In our study MVp are not a RF for VAP in trauma patients, although the whole DMV is longer in patients with VAP (mean DMV vapY/N: 13.57/6.09; P = 0.0001). Further studies could confi rm whether the whole DMV in trauma patients with VAP is a consequence of infection. Conclusion The head is still the most frequently and severely injured region. The severity of injuries increases in the most rigid part of the car. Improving VRUs’ safety devices (active and passive) to reduce impact speed and severity of the primary impact has to be the next step. Reference References 1. Charles MP, et al. Ventilator-associated pneumonia. Australas Med J. 1. Charles MP, et al. Ventilator-associated pneumonia. Australas Med J. 2014;7:334-44. 1. Charles MP, et al. Ventilator associated pneumonia. Australas Med J. 2014;7:334-44. 1. Piantini et al. BMC Emerg Med. 2013;13:3. 2. CDC ventilator-associated event (January 2014). www.cdc.gov. 2. CDC ventilator-associated event (January 2014). www.cdc.gov. P311 Dankook University Hospital, Cheonan City, South Korea Critical Care 2015, 19(Suppl 1):P312 (doi: 10.1186/cc14392) Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) Introduction Severe trauma aff ects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. Figure 1 (abstract P314). sRAGE kinetics (days). on days 1, 3 and 5. sRAGE was measured by ELISA and cfDNA was measured by UV absorbance after plasma isolation. g p g Methods A prospective, observational substudy of the Acute Coagulopathy and Infl ammation in Trauma (ACIT) II study was performed at our academic level 1 trauma center. Adult multiple trauma patients with an Injury Severity Score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. Results Median ISS was 39 and mortality was 21% (8/38). During the fi rst 5 days after trauma, the median concentration of sRAGE (Figure 1) decreased signifi cantly over time (P <0.0001) while median levels of DNA did not (P = 0.73), and remained elevated compared with normal control. No correlation was found with ISS. Patients initially in shock had lower levels of sRAGE or cfDNA (P <0.05) and had received more fl uid (10.6 l vs. 5.25 l) or blood (6 l vs. 0.5 l). Day 3 and day 5 sRAGE levels were inversely correlated with PRBC received. Medians of sRAGE on days 0 (1,301 vs. 730 pg/ml) and day 1 (925 vs. 760 pg/ml) were signifi cantly higher in nonsurvivors (P <0.01). Finally, day 0 sRAGE was correlated with the maximal (r = 0.44; P = 0.007) and the cumulative renal failure component of the MODS, over the 10 days (r = 0.48; P = 0.005).f Results Ten trauma patients and 10 healthy individuals were included. Trauma patients were signifi cantly injured with a median ISS of 19 (17 to 45). On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with healthy individuals, as refl ected by decreased production of IL-6, IL-10 and TNFα (P <0.001). In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users Injuries suff ered by each person were related to specifi c impact objects. y g p p g Conclusion In order to prevent misdiagnosis of fractures in patients with severe injury, meticulous physical examination with suspicion of fractures should come fi rst. In addition, obtaining proper radiologic study and thorough evaluation of radiologic images are important to decreasing the rates of missed fracture diagnoses. In addition, thorough surveillance for ipsilateral fractures is important in extremities with identifi ed fractures. i Results The average ISS is 21.5 (SD 10.9). Cars are the most involved in serious urban VRU crashes. Car-to-pedestrian crashes are the most frequent (50%). The impact speed is always over 40 km/hour (Table 1). The head and face are the most frequently injured part (48% of the 571 injuries collected), followed by lower extremities (15%). In terms of maximum AIS (MAIS), the head is the most severely injured region with 42% of MAIS 3+ (Figure 1). i References S111 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P314). sRAGE kinetics (days). 2. Houshian S, Larsen MS, Holm C. Missed injuries in a level I trauma center. J Trauma. 2002;52:715-9. 3. Janjua KJ, Sugrue M, Deane SA. Prospective evaluation of early missed injuries and the role of tertiary trauma Survey. J Trauma. 1998;44:1000-6; discussion 1006-7. 3. Janjua KJ, Sugrue M, Deane SA. Prospective evaluation of early missed injuries and the role of tertiary trauma Survey. J Trauma. 1998;44:1000-6; discussion 1006-7. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients In trauma patients, MP-positive plasma was associated with a signifi cantly higher synthesis of IL-6 and TNFα compared with plasma depleted from MPs (P = 0.047 and 0.002 respectively). Compared with healthy individuals the number of circulating MPs was signifi cantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in healthy controls. Conclusion DNA and sRAGE kinetics diff er following trauma. Early elevation of sRAGE predicts mortality in univariate analysis and correlates with subsequent renal failure. P313 P313 Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients N Curry1, K Balvers2, DJ Kleinveld2, AN Boïng2, R Nieuwland2, JC Goslings2, NP Juff ermans2 1John Radcliff e Hospital, Oxford, UK; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) P313 Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients N Curry1, K Balvers2, DJ Kleinveld2, AN Boïng2, R Nieuwland2, JC Goslings2, NP Juff ermans2 1John Radcliff e Hospital, Oxford, UK; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) References 1. Cohen MJ. J Trauma. 2010;68:1273-8. 1. Cohen MJ. J Trauma. 2010;68:1273-8. . Cohen MJ. J Trauma. 2010;68:1273-8. 1. Cohen MJ. J Trauma. 2010;68:1273 8. 2. Simmons JD. Ann Surg. 2013;258:591-6. 2. Simmons JD. Ann Surg. 2013;258:591-6. 2. Simmons JD. Ann Surg. 2013;258:591-6. Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 1National Center for Global Health and Medicine Hospital, Tokyo, Japan; 2Seirei Hamamatsu General Hospital, Shizuoka, Japan Critical Care 2015, 19(Suppl 1):P315 (doi: 10.1186/cc14395) Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis p g y T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 Conclusion On admission, trauma patients have a reduced immune response towards endotoxin challenge which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an infl ammatory host response post injury. g y T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 1National Center for Global Health and Medicine Hospital, Tokyo, Japan; 2Seirei Hamamatsu General Hospital, Shizuoka, Japan Critical Care 2015, 19(Suppl 1):P315 (doi: 10.1186/cc14395) Introduction Cervical spinal cord injury (CCI) without bony injury (CCIWOBI) is more frequent among Asian than among Caucasian populations and shows various extents of severity. Cervical magnetic resonance imaging (MRI) is useful for detecting intramedullary lesions, ligament injuries and intervertebral disk hernias, but some patients with mild CCIWOBI do not show clinically signifi cant abnormalities on MRI. To date, the cost–benefi t ratio of performing MRI in addition to computed tomography (CT) is unclear. We have developed a clinical decision rule for cervical MRI (MR-CDR), indicating MRI for patients >70 years old with ossifi cation of the posterior longitudinal ligament on CT or injury in a ground-level fall or a fall down stairs. The objective of the present study was to prospectively validate this MR-CDR for cervical MRI in patients with suspected mild CCIWOBI. P314 DNA and sRAGE circulation in the early phase after polytrauma P Joly1, C Massé2, D Dwivedi2, P Liaw2, J Marshall3, Y Berthiaume2, E Charbonney4 1University of Montreal, QC, Canada; 2McMaster University, Hamilton, ON, Canada; 3University of Toronto, ON, Canada; 4CRHSCM-University of Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P314 (doi: 10.1186/cc14394) Introduction Various DAMPS, alarmins are released after trauma. The soluble receptor for advanced glycation endproducts (sRAGE) was reported to be associated with acute renal failure and duration of ventilation [1]. Cell-free DNA (cfDNA) has been associated with prognosis in trauma patients [2]. We studied the kinetics of these two biomarkers over the fi rst 5 days, in a cohort of severely ill trauma patients. Methods We have been conducting a prospective observational study in two institutions in Japan since September 2012, enrolling patients with CCIWOBI among head or neck trauma patients >16 years old brought in by ambulance. We collect data about patient characteristics, injury profi les, neurological fi ndings, results of radiological examinations, and medical courses. We then analyze the sensitivity and specifi city of MR-CDR for detecting intramedullary lesions on MRI and conduct further analysis. Methods Patients who had sustained serious traumatic injury, within 24 hours of trauma, were recruited in a level I trauma center. We collected ISS, baseline demographic characteristics, resuscitation information and daily organ dysfunction (MOD) scores, over 10 days. Blood samples were collected within 24 hours of trauma (day 0) and S112 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results During the study period, 63 patients were brought in with CCIWOBI. Mean age was 60.6 years (standard deviation, 17.9 years) and 76% were male. Forty-fi ve patients presented with mild symptoms (Frankel Grade D). Cervical MRI was performed for 23 patients. Sensitivity and specifi city of MR-CDR in detecting intramedullary lesions on T2- weighted imaging among cases of suspected mild CCIWOBI were 85.7% (95% confi dence interval (CI), 60.1 to 96.0%) and 33.3% (95% CI, 12.1 to 35.4%). Further analysis showed a signifi cant diff erence in minimal spinal canal diameter as measured on sagittal T2-weighted imaging between the MR-CDR-positive and MR-CDR-negative groups (5.0 mm vs. 8.3 mm, P = 0.0003). One patient underwent surgery during hospitalization and no patients experienced exacerbated neurological fi ndings. P316 Accuracy of targeted wire-guided tube thoracostomy in comparison with classical surgical chest tube placement: a clinical study A Protic, I Barkovic, A Sustic University Hospital Rijeka, Croatia Critical Care 2015, 19(Suppl 1):P316 (doi: 10.1186/cc14396) Introduction Chest tube malfunction, after the tube thoracostomy, is often the result of an inappropriate chest tube tip position. The aim of this study was to analyze the precision of chest tube placement using the targeted wire guide technique (TWG technique) with a curve dilator and to compare it with the classical surgical technique (CS technique). Methods In this clinical study 80 patients with an indication for thoracic drainage due to pneumothorax or pleural eff usion were included. The experimental group contained 39 patients whose chest tube was placed using the TWG technique. The control group contained 41 patients whose chest tube was placed using the CS technique. Introduction Chest tube malfunction, after the tube thoracostomy, is often the result of an inappropriate chest tube tip position. The aim of this study was to analyze the precision of chest tube placement using the targeted wire guide technique (TWG technique) with a curve dilator and to compare it with the classical surgical technique (CS technique). Results There is positive signifi cant correlation between T day and LOS of injured patients (P <0.001, Spearman coeffi cient = 0.1672). Statistical analysis by Mann–Whitney test, between groups A and B, showed signifi cant diff erences in ICU LOS (P <0.001); no signifi cant diff erences (P <0.05) were found for age, ISS and outcome (Table 1). p g q q Methods In this clinical study 80 patients with an indication for thoracic drainage due to pneumothorax or pleural eff usion were included. The experimental group contained 39 patients whose chest tube was placed using the TWG technique. The control group contained 41 patients whose chest tube was placed using the CS technique. Conclusion The optimum and early time point of tracheostomy seems to be directly related with LOS in the ICU, independently of the rate of ISS, patient’s age and outcome. These results could account for ICU cost-eff ectiveness, as diminished LOS decreased the overall cost. Reference Results The comparison of the outcomes of the two techniques applied suggests that the TWG technique was signifi cantly more successful, irrespective of patient diagnosis (TWG vs. CS in all patients, 78.4% vs. 36.6%, P <0.001). See Table 1. 1. Alali et al. P317 Evaluating trauma care: comparison of early versus late tracheostomy ICU data outcome on injured patients V Kaldis1, N Mourelatos1, D Markopoulou1, K Venetsanou2, E Diogou1, E Papadaki1, D Chroni1, I Alamanos1 1General Hospital KAT-EKA, Kifi sia Athens, Greece; 2ICU Research Unit KAT, Kifi ssia Athens, Greece Critical Care 2015, 19(Suppl 1):P317 (doi: 10.1186/cc14397) Introduction In the surgical ICU, bedside tracheostomy (T) is one of the most frequently applied surgical techniques for multi-injured patients mainly with TBI [1]. The optimum surgical time decision for T still remains a contradiction in trauma. This retrospective study was designed to register all trauma patients who underwent T, during 60 months of observation (2009 to 2013), in order to identify factors associated with their ICU outcome on the basis of the T day (A <10th day >B) after tracheal intubation. g Conclusion MR-CDR was not validated for predicting the existence of intramedullary lesions on cervical MRI. MR-CDR is useful in predicting the severity of cervical stenosis. Methods Seventy-eight injured patients in the SICU underwent T, from a total of 403 issues; 58 male and 20 female, with mean age 59.3 and 74.7 years respectively. The total length of ICU stay recorded was 2,098 days, nursing time 26.55 (4/93), whereas the T time was adjusted between the 6th and 16th day (mean 11th). Mean ISS score was 22.59 (9 to 50). Classifi cation according to trauma type was TBI (n = 44) followed by thoracic trauma. Thirty-one male survivors were discharged from the ICU, to the ward. The mortality rate amounts to 47 cases due to infectious/non-infectious nosocomial complications and multiorgan dysfunction syndrome. Clinical ISS, the type of injury, ICU length of stay (LOS), T day, demographic (gender, age) data and ICU outcome were registered. Statistical analysis was performed with GraphPad 5.0.i Factors related to sepsis and outcome in multiple trauma patients H Pavlou, E Pappa, M Eforakopoulou KAT-EKA General Hospital, Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) pi Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) Introduction The outcome of multiple trauma patients is related to a number of diagnostic and therapeutic interventions during hospitali- zation. ICU patients with severe trauma are susceptible to sepsis leading to poor outcome. Factors associated with the occurrence of sepsis and the outcome of these patients were investigated. Conclusion Using a curved dilator and the TWG technique for the thoracic drainage procedure we found statistically signifi cant advantage to the TWG technique in comparison with the CS technique regarding precise chest tube placement within the pleural cavity. References Methods We studied retrospectively all trauma patients admitted to the A’ ICU of KAT General Hospital in Athens during the last 3 years and were treated for more than 5 days. Age, gender, the type of injury, the severity of injury (Injury Severity Score), the length of ICU stay, severe sepsis, coexisting diseases, the outcome and the cause of death were recorded. Logistic regression and chi-square tests were used for statistical analysis. 1. Chen F, Yamada T, Aoyama A, et al. Position of a chest tube at video-assisted thoracoscopic surgery for spontaneous pneumothorax. Respiration. 2006;73:329-33. 2. Protic A, Barkovic I, Bralic M, et al. Targeted wire-guided chest tube placement: a cadaver study. Eur J Emerg Med. 2010;17:146-9. P314 No signifi cant diff erences were evident between groups in discharge status, duration of hospitalization, or neurological fi ndings at discharge. P316 Tracheostomy timing in traumatic brain injury: a propensity- matched cohort study. J Trauma Acute Care Surg. 2014;76:70-8. Table 1 (abstract P316) CS technique TWG technique Diagnosis N (% of success) (% of success) P value Pleural eff usion 47 37.5 78.2 0.005 Pneumothorax 31 35.3 78.6 0.029 Total 78 36.6 78.4 <0.001 P318 Factors related to sepsis and outcome in multiple trauma patients H Pavlou, E Pappa, M Eforakopoulou KAT-EKA General Hospital, Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) Ventilator-associated pneumonia in a trauma ICU M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399 Figure 1 (abstract P320). M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Royal London Hospital, London, UK normoxia will create a condition of relative hypoxia, which acts in turn as a stimulus for erythropoietin (EPO) production [1]. Variations in GSH and oxygen free radical (ROS) levels may be involved in this process. We tested the NOP in critically ill patients. y p Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399) Introduction Ventilator-associated pneumonia (VAP) is associated with increased length of ventilation, ICU stay, mortality, cost and antibiotic burden [1]. There is a large variation in reported rates of VAP, partly as a result of inconsistencies in defi nition [2]. We explored a more pragmatic defi nition to describe the VAP rate, antibiotic burden and outcome of VAP in a 44-bed adult critical care unit in a level 1 trauma centre. Methods A prospective observational study on 38 mechanically ventilated (FiO2 <50%) patients with no active bleeding, no blood transfusion needed, and no kidney failure. Eighteen patients underwent a 2-hour period of normobaric hyperoxia (FiO2 = 100%), 20 patients were evaluated as controls (no FiO2 variations). EPO was assayed at baseline (t0), 24 hours (D1) and 48 hours (D2). Serum GSH and ROS were assayed at t0 (baseline), t1 (2-hour FiO2 100%) and t2 (2- hour return to normoxia) in 12 patients in the hyperoxia group. Methods A retrospective review of all adult patients admitted to the ICU at The Royal London Hospital over a 6-month period (February to August 2014). The diagnosis of VAP was based on the Clinical Pulmonary Infection Score. Patients were identifi ed with VAP if they were started on antibiotics for chest sepsis 48 hours after start of mechanical ventilation. Demographic, clinical, microbiological and radiological data were collected to identify risk factors, and compare VAP and non- VAP groups. Chi-squared and ANOVA tests were performed using the SOFA statistics package. Results EPO tended to increase in the hyperoxia group over time (P = 0.05), while it remained stable in the control group (P = 0.53) (Figure 1). ROS levels increased at t1 and decreased at t2, GSH tended to decrease at t1 and increased at t2 in the hyperoxia group. Conclusion Relative hypoxia after a transient period of normobaric hyperoxia induces an increase in GSH levels, thus enhancing ROS scavenging. This may act as a stimulus for EPO production. Table 1 (abstract P317). SICU data Table 1 (abstract P317). SICU data Table 1 (abstract P317). SICU data Outcome ICU data ISS Age ICU LOS (days) Tracheostomy day Survival Mortality A <10 16 ± 87 (16/45) 66 ± 94 (28/87) 602 17.2 ± 21.5 (4/47) 5.5 ± 6.5 (1/10) n = 13 (43.3%) n = 17 (57.7%) B >10 16 ± 25 (9/50) 64 ± 87 (23/93) 1,496 25 ± 34 (9/95) 15 ± 26 (11/23) n = 18 (40%) n = 27 (60%) t test NS NS <0.001 <0.001 NS NS Data presented as median ± IQR S113 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results A total of 106 multiple trauma patients, 85 men and 21 women, met the inclusion criteria. Depending on their age, patients were divided into two groups: <60 years old and >60 years old. In both groups, gender, the type and severity of injuries and the length of ICU stay were not associated with outcome. The length of ICU stay was correlated with severe sepsis and coexisting diseases (P <0.01) in both groups. Mortality was not diff erent in the two groups. The presence of at least one coexisting disease was signifi cantly associated with mortality (P <0.007). Sepsis was signifi cant cause of death in trauma patients >60 years (P <0.05). Figure 1 (abstract P320). Figure 1 (abstract P320). y Conclusion In multiple trauma patients, the length of ICU stay and comorbidities infl uence the occurrence of severe sepsis, comorbidities increase mortality, and sepsis is the leading cause of death in trauma patients >60 years old. Ventilator-associated pneumonia in a trauma ICU M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399 Reference Results A total of 535 mechanically ventilated patients were admitted in the study period, with 281 ventilated for more than 48 hours. The incidence of VAP was 11% in all ventilated patients and 19.6% in those ventilated more than 48 hours. VAP rates were 31% in polytrauma, 25% in neurotrauma and 18% in the neuromedical/surgical cohort. Early and late onset VAP were equal in number. Patients with VAP spent longer on mechanical ventilation (9 ± 9 in no VAP vs. 18 ± 18 days in VAP patients; P <0.001), and had longer ICU and hospital LOS (11 ± 10 vs. 22 ± 20 days; P <0.001). However, APACHE II scores and hospital mortality were unaff ected (VAP 33.3% vs. no VAP 37.6%; P = 0.173). Despite rising infl ammatory markers and secretion load, many patients did not exhibit oxygenation defi cits. Sputum microbiology showed S. aureus, H. Infl uenza, Klebsiella and Enterobacter as predominant pathogens with low rates of Pseudomonas, Acinectobacter and other resistant organisms. Average length of antibiotic use was 6 (3 to 18) days. Conclusion Chest sepsis after 48 hours of mechanical ventilation commonly complicates neurocritical illness and polytrauma requiring signifi cant ICU resources and antibiotic burden. However, it does not aff ect mortality. Further research should focus on pathophysiology and new preventative measures to reduce VAP in the at-risk population. R f 1. Balestra et al. J Appl Physiol. 2006;100:512-8. p References 1. Taylor MD, et al. J Trauma. 2002;53:407-14. 2. Osborn TM, et al. Crit Care Med. 2004;32:2234-40. References References 1. Safdar N, et al. Crit Care Med. 2005;33:2184-93. 2. Nair GB, et al. Intensive Care Med. 2014 [Epub ahead of print]. 1. Safdar N, et al. Crit Care Med. 2005;33:2184-93. 2. Nair GB, et al. Intensive Care Med. 2014 [Epub ahead of print]. Results Mean D-dimer concentration of the control group and of the patient group with PE was 0.28 (95% CI: 0.25 to 0.31) mg/l and 1.45 (95% CI: 1.23 to 1.72) mg/l, respectively. Receiver operator characteristics analysis revealed an optimized cutoff value of 0.466 mg/l for the PATHFAST D-dimer assay (AUC = 0.975 (95% CI: 0.938 to 0.993); sensitivity: 95% (95% CI: 86 to 99%); specifi city: 89% (95% CI: 82 to 95%)). Therefore we used a rounded up cutoff value of 0.5 mg/l to examine the diagnostic accuracy of PATHFAST D-dimer to exclude PE. The correlation between PATHFAST and VIDAS results was particularly close for concentrations at or around the critical cutoff value of P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, R Castagnani, N Mininno, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P320 (doi: 10.1186/cc14400) P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, R Castagnani, N Mininno, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P320 (doi: 10.1186/cc14400) P321 Comparison of the PATHFAST D-dimer assay with two POC D-dimer assays E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) Comparison of the PATHFAST D-dimer assay with two POC D-dimer assays E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Methods A total of 272 patients with symptoms of PE and VTE were included. The diagnoses of VTE and PE were established by duplex ultrasound, venography and spiral CT. D-dimer values were determined in the patients and in plasma samples obtained from 102 healthy individuals who served as the control group. Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Methods A total of 272 patients with symptoms of PE and VTE were included. The diagnoses of VTE and PE were established by duplex ultrasound, venography and spiral CT. D-dimer values were determined in the patients and in plasma samples obtained from 102 healthy individuals who served as the control group. Impact of introducing guidelines for thrombolysis of submassive pulmonary embolism at a large UK teaching hospital GP Misselbrook g p p Results The peak action of enoxaparin and rivaroxaban was observed at 4 hours post administration. LPTEG indicators that determine the coagulation state after 4 hours in the fi rst group: CTA was decreased by 72.12% (P <0.05), ICD was decreased by 68.44% (P <0.05), GP was increased by 17.9%; in the second group: CTA was decreased by 76.24% (P <0.05), ICD was decreased by 74.52% (P <0.05), GP was increased by 23.34%. After 12 hours, CTA in the fi rst group decreased by 22.41%, ICD decreased by 5.3%, GP increased by 8.12%, indicating reduction of hypocoagulation eff ect; in the second group, CTA decreased by 39.35% (P <0.05), ICD decreased by 40.24% (P <0.05), GP increased by 18.25%. After 24 hours in the fi rst group LPTEG indicators returned to the original value, and in the second group of patients CTA was decreased by 15.14%, ICD was decreased by 6.62%, GP increased by 14.22%.f University Hospitals Southampton NHS Foundation Trust, Southampton, UK Critical Care 2015, 19(Suppl 1):P324 (doi: 10.1186/cc14404) Introduction Pulmonary embolism (PE) is a signifi cant cause of death with 10% of patients dying within 3 months [1]. Multiple studies now advocate the use of thrombolysis (TPA) in both massive and submassive PE [1,2]. This audit assessed the impact of introducing a guideline allowing for thrombolysis of submassive and massive PE at a large UK teaching hospital. g p Methods Retrospective data collection using notes and imaging to risk-stratify patients. First audit ran from January to June 2012. New guidance was introduced in March 2013 (Figure 1) after which a second cycle ran for a further 6 months. y y y Conclusion Using LPTEG showed the hypocoagulation eff ect of continuous rivaroxaban 24 hours after oral administration compared with enoxaparin, which retains less hypocoagulation eff ect 12 hours after administration. LPTEG indicators in the second group were bigger than in the fi rst group after 12 hours: CTA 43.07%, ICD 69.72%, GP 54.12%. Results Re-audit revealed 46 patients with radiological evidence of massive or submassive PE on CTPA (32% of all PEs). Ten patients had clinical features of submassive PE and nine presented as massive PE. Previous guidelines suggested consideration of TPA in only seven patients in 6 months. TPA was given to two patients; however, six patients had no contraindications to treatment (Table 1). P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study Introduction The normobaric oxygen paradox (NOP) postulates that a period of normobaric hyperoxia followed by a rapid return to S114 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 0.5 mg/l. The correlation between PATHFAST and STRATUS results was particularly close in the patient group with VTE (r = 0.9694), whereas slightly lower results were obtained with STRATUS in the control group. With the widely used cutoff value 0.5 mg/l, PATHFAST demonstrated suitable sensitivity but not STRATUS. ROC analysis indicated that optimal cutoff values could be set at either 0.5 or 0.6 mg/l and at 0.3 or 0.4 mg/l for PATHFAST and STRATUS, respectively. clinical guidelines recommend the administration of pharmacological thromboprophylaxis (PTP) to reduce the risk of developing TEC [2]. However, it is unknown whether delayed PTP initiation increases risk of TEC. We hypothesize that delayed PTP initiation is associated with increased TEC rates. Methods A retrospective chart review (2010 to 2013) was conducted on adult trauma patients that were admitted into a level 1 trauma centre in Toronto. Demographics, date of PTP initiation, date of TEC diagnosis (CT-PE/US Doppler), injury type and severity were collected. A comparison between early and late PTP initiation has been made with regards to TEC development. Student’s t test, univariate and multivariate logistic regression analyses were performed. y Conclusion By use of the PATHFAST D-dimer assay only six of diagnoses were missed at the time of fi rst presentation compared with 10 diagnoses missed by the VIDAS D-dimer Exclusion assay, yielding higher sensitivity of the PATHFAST D-dimer assay compared with the VIDAS assay (90% vs. 83%). The STRATUS assays showed comparable performance and appeared to be suitable for the exclusion of VTE in the emergency room setting, whereas PATHFAST demonstrated superior sensitivity. Moreover, the PATHFAST analyzer allows simultaneous determination of D-dimer and cardiac troponin I within 16 minutes from whole blood samples. Therefore, this method might be useful at the point of care for early diagnostic assessment of patients with symptoms of PE or chest pain admitted to the ER or to the chest pain unit. g g y p Results A total of 1,312 patients received PTP, 821 (62.5%) initiated early PTP (within 48 hours) while 491 (37.5%) initiated after 48 hours. The group that initiated early prophylaxis was younger (mean: 46 vs. Using rivaroxoban in patients with venous thromboembolism I Tyutrin, O Tarabrin, B Todurov, S Shcherbakov, D Gavrychenko, G Mazurenko Odessa National Medical University, Odessa, Ukraine Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Conclusion Mortality rates in patients with delayed PTP are higher. Our study shows LOS as the only independent predictor for TEC. However, this might not necessarily refl ect causation. Delayed PTP appears not to be an independent predictor to TEC events in trauma patients, which favours current clinical trends when it comes to contraindicating early PTP initiation. Introduction A prospective study was conducted in patients for treatment of venous thromboembolism (VTE) to compare the eff ect of enoxaparin and rivaroxaban using the method of low-frequency piezoelectric thromboelastography (LPTEG) for checking coagulation activation markers. P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study 55, P <0.0005), had lower ISS (mean: 17 vs. 24, P <0.0005), shorter length of stay (LOS) (mean: 11 vs. 23, P <0.0005), more pelvic fractures (19% vs. 13%, P = 0.0058), more head injury (AIS Head ≥3, P <0.0005), less blunt trauma (85% vs. 95%, P <0.0005), lower incidence of TEC (5.3% (44) vs. 8.5% (42), P = 0.023), and lower mortality rate (1.5% vs. 7.5%). Univariate analysis showed LOS (P <0.0005), ISS (P <0.0005), time to PTP initiation (P = 0.0018) and blunt MOI (P = 0.0099) signifi cantly associated with TEC events. Multivariate analysis, however, showed TEC events correlated only to LOS (P = 0.0001). Stepwise multiple logistic regression confi rmed LOS as independently associated with TEC events (95% CI = 0.003, 0.006, P <0.0005). P323 P323 Is delaying pharmacological thromboprophylaxis associated with thromboembolic complications? P Padim1, A Alshafai2, S Canestrini3, S Rizoli2, J De Rezende Neto2, A McFarlan2 1Universidade de São Paulo, Ribeirao Preto, Brazil; 2St Michael’s Hospital, Toronto, ON, Canada; 3St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P323 (doi: 10.1186/cc14403) Is delaying pharmacological thromboprophylaxis associated with thromboembolic complications? P Padim1, A Alshafai2, S Canestrini3, S Rizoli2, J De Rezende Neto2, A McFarlan2 1Universidade de São Paulo, Ribeirao Preto, Brazil; 2St Michael’s Hospital, Toronto, ON, Canada; 3St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P323 (doi: 10.1186/cc14403) q g g g Conclusion Delivering a service that off ers TPA to patients with submassive PE signifi cantly increases the need to consider this therapy. Introducing this service is only eff ective if doctors initially assessing these patients are aware of recent changes to guidelines for PE. R f References Methods A total of 60 patients entered the Odessa Clinical Regional Hospital for treatment venous thromboembolism. Patients were divided into two groups. The fi rst group (n = 30) were receiving enoxaparin in dosage 1.5 mg/kg subcutaneously per day. The second group (n = 30) were receiving rivaroxaban orally 15 mg/day. For checking the coagulation state we were using such indicators of LPTEG as constant thrombin activity (CTA), intensity of coagulation drive (ICD) and gel point (GP). We performed LPTEG three times per day: 4, 12 and 24 hours after taking the drug to check for changes in the coagulation state in both groups of patients. 1. Geerts W, Code K, Jay R, Chen E, Szalai J. A prospective study of venous thromboembolism after major trauma. N Engl J Med. 1994. 331;1601-6. 1. Geerts W, Code K, Jay R, Chen E, Szalai J. A prospective study of venous thromboembolism after major trauma. N Engl J Med. 1994. 331;1601-6. 2. Rogers FB, Cipolle MD, Velmahos G, et al. Practice management guidelines for the prevention of venous thromboembolism: the EAST Practice Management Guidelines Work Group. J Trauma. 2002;53:142-64. Impact of introducing guidelines for thrombolysis of submassive pulmonary embolism at a large UK teaching hospital GP Misselbrook Limitations to TPA administration were late recognition of submassive PE and inadequate knowledge of changes to guidelines.f p References Introduction Thromboembolic complications (TEC) are very common and lethal in patients suff ering from traumatic injury [1]. The trauma Introduction Thromboembolic complications (TEC) are very common and lethal in patients suff ering from traumatic injury [1]. The trauma Meyer G, et al. N Engl J Med. 2014;370:1402-11. Kearon C, et al. Chest. 2012;141:419-96S. S115 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (P <0.001). The type of thrombosis was directly associated with poor outcome, especially one that resulted from central catheters (P <0.001) and pulmonary embolism (P <0.001). However, no correlations were found with temperature, white blood cells and platelet counts on admission (P > 0.05). (P <0.001). The type of thrombosis was directly associated with poor outcome, especially one that resulted from central catheters (P <0.001) and pulmonary embolism (P <0.001). However, no correlations were found with temperature, white blood cells and platelet counts on admission (P > 0.05). Figure 1 (abstract P324). New local guidelines. Conclusion Thrombosis aff ects the ICU patient’s fi nal outcome. The type of thrombosis contributes to a poor outcome and mainly the occurrence of pulmonary embolism signifi cantly increases the mortality rate. P325 Results A total of 51 patients were included. The median age and BMI were 73 years and 23 kg/m2, respectively; 31% were female and 69% were surgical critical care patients. The median APACHE II and SOFA scores were 20 and 8, respectively. Risk factors associated with DVT were presence of central venous catheter 63%, malignancy 9% and hemodialysis 14%. The rate of DVT was 18.6% and the rate of CRT was 13.7%. All of these were asymptomatic and seen in neck and upper extremities. There was no DVT-associated adverse event (pulmonary embolism, bleeding) during hospital stay. The 28-day all-cause mortality rate was 3.4%. Introduction Despite preventive anticoagulation therapy measures, venous thromboembolic disease is a major cause of morbidity and mortality among patients hospitalized in ICUs. In fact, pulmonary embolism is not only the most serious manifestation of the disease, but also one of the primary causes of sudden death. The aim of this study is to investigate the frequency of thromboembolism and pulmonary embolism in ICU hospitalized trauma and neurosurgical patients. Conclusion While incidence of asymptomatic DVT is relatively high in adult critically ill patients, they were found only in the neck and upper extremities without any adverse event. Further research is needed to evaluate the clinical signifi cance of this type of DVT. Methods One hundred ICU patients, 51 postoperative neurosurgical and 49 trauma, were included in the study. Patients’ demographic data as well as medical history, temperature, white blood cells and platelets counts were recorded on admission, the day of thrombosis diagnosis and the fi nal outcome of their treatment. Statistics were performed with SPSS-19. P <0.05 was considered signifi cant. P326 P326 Incidence and outcome of asymptomatic deep vein thrombosis in critically ill patients: a prospective cohort study R Echigoya, H Okamoto, H Uchino, A Kuriyama, N Tamura, K Sato, T Fukuoka Kurashiki Central Hospital, Okayama, Japan Critical Care 2015, 19(Suppl 1):P326 (doi: 10.1186/cc14406) Figure 1 (abstract P324). New local guidelines. Figure 1 (abstract P324). New local guidelines. Introduction Asymptomatic deep vein thrombosis (DVT) including catheter-related thrombosis (CRT) is an increasingly recognized disease entity in critically ill patients. However, the reported rate and outcome of DVT vary widely depending on study design, patient background and detecting method. The objective of this study is to evaluate the incidence and outcome of DVT in adult critically ill patients. Table 1 (abstract P324). TPA decisions High Intermediate Total risk risk Considered TPA and given 2 2 0 Considered TPA but contraindications 1 1 0 Considered TPA and not given on balance 3 0 3 Considered TPA and not given but fi t 3 2 1 criteria Not considered TPA but contraindicated 4 1 3 anyway Not considered TPA and on balance 5 1 4 would not be given Not considered TPA but fi t criteria 1 0 1 Table 1 (abstract P324). TPA decisions y p Methods This study is a prospective cohort study of patients admitted to a medical and surgical ICU from 1 July 2014 to 15 October 2014. All consecutive patients over 18 years of age and with expected ICU stay over 72 hours were included. Patients who had previous history of DVTs were excluded. We examined internal jugular vein, subclavian vein, axillary vein, brachial vein, femoral vein, superfi cial femoral vein, and popliteal vein, on ICU admission and within 48 hours after ICU discharge. The DVT was diagnosed using compression ultrasonography with color Doppler. Images were interpreted by two independent investigators trained in ultrasonography. All patients received intermittent pneumatic compression and unfractionated heparin twice daily during their IUC stay. Once the DVT was detected, therapeutic anticoagulation was initiated. Contrast-enhanced CT was performed when the patients were suspected to have pulmonary embolism. The primary outcome was the incidence of DVT during the ICU stay. Patients were followed until their hospital discharge. P327 P327 Computed tomographic pulmonary angiographic fi ndings to predict adverse outcomes in acute pulmonary embolism P Tajarernmuang, J Euathrongchit, C Liwsrisakun, A Deesomchok, T Theerakittikul, C Bumroongkit, C Pothirat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2015, 19(Suppl 1):P327 (doi: 10.1186/cc14407) gi Results Thirty-eight out of 100 patients presented thrombosis, 14 trauma and 24 neurosurgical. We examined the correlation of thrombosis development during hospitalization with diagnosis, treatment allocated time and overall patient outcome. It was found that neurosurgical patients developed thrombosis more frequently than trauma patients (P <0.05). In relation to diagnosis, thrombosis was prevalent among patients with brain lesions (P = 0.018). Regarding the type of thrombosis, pulmonary embolism was also commonly apparent among individuals with brain lesion (P = 0.020). In addition, there was a statistically signifi cant correlation in thrombosis occurrence between hospitalization day (P <0.01) and patients’ outcome on discharge Introduction Computed tomographic pulmonary angiography (CTPA) has been used as a standard tool for diagnosing an acute pulmonary embolism (APE). The right ventricular (RV) strain signs may be used to predict adverse outcomes. However, the results are still controversial. The primary objective of our study was to evaluate the relationship between the RV strain signs and respiratory failure requiring mechanical ventilation or death in APE. The secondary objective was to identify clinical factors which related to those outcomes. Introduction Computed tomographic pulmonary angiography (CTPA) has been used as a standard tool for diagnosing an acute pulmonary embolism (APE). The right ventricular (RV) strain signs may be used to predict adverse outcomes. However, the results are still controversial. The primary objective of our study was to evaluate the relationship between the RV strain signs and respiratory failure requiring mechanical ventilation or death in APE. The secondary objective was to identify clinical factors which related to those outcomes. S116 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P329). Kaplan–Meier analysis of ESRD progression for SC and Ecu treatment for all patients. Methods CTPA and the medical records of patients with suspected APE on admission from June 2011 to March 2013 were reviewed. Mean platelet volume and mean platelet volume/platelet count ratio in risk stratifi cation of pulmonary embolism T Yardan1, M Meric1, C Kati1, Y Celenk2, A Atici1 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) T Yardan , M Meric , C Kati , Y Celenk , A Atici 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) Introduction Recently, mean platelet volume (MPV) was reported to predict early death in acute pulmonary embolism (PE). The aim of this study was to investigate the role of MPV and MPV/platelet count ratio (MPV/P) in risk stratifi cation of patients with acute PE. Conclusion Ecu treatment reduces the number of ESRD events and the rate of progression to ESRD; thus initiation of Ecu early after aHUS diagnosis may prevent cumulative kidney damage and progression to ESRD. i p Methods We retrospectively reviewed the medical records of patients with acute PE admitted to the emergency department. In addition to the clinical evaluation, platelet count and MPV were measured on admission. P328 Mean platelet volume and mean platelet volume/platelet count ratio in risk stratifi cation of pulmonary embolism T Yardan1, M Meric1, C Kati1, Y Celenk2, A Atici1 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) p Reference 1. Ghuysen A, Ghaye B, Willems V, Lambermont B, Gerard P, Dondelinger RF, et al. Computed tomographic pulmonary angiography and prognostic signifi cance in patients with acute pulmonary embolism. Thorax. 2005;60:956-61. p p Results The SC and Ecu treatment phases included 32 and 33 patients, respectively. With SC, during a median (range) of 211 (7 to 745) days, 13 (41%) patients had a total of 16 ESRD events. On Ecu treatment, during a median (range) of 924 (73 to 1,254) days, three (9%) patients had a total of fi ve ESRD events. The ESRD event rate was 92% lower during Ecu treatment versus the SC phase (0.36 vs. 0.07; P = 0.001); the incidence rate ratio was 0.08 (95% CI = 0.02 to 0.37; P = 0.001). HR for progression to ESRD for patients on Ecu versus SC was 0.03 (95% CI <0.01 to 0.34), a 97% reduction (Figure 1). Stratifi cation by baseline CKD stage showed no patients with CKD stage 2 or 3 at baseline progressed to ESRD over 3 years of Ecu treatment. Early initiation of eculizumab treatment in patients with atypical haemolytic uraemic syndrome improves long-term outcomes: a pooled analysis of clinical trials p y J Vande Walle1, Y Delmas2, G Ardissino J Vande Walle1, Y Delmas2, G Ardissino3, J Wang4, J Kincaid4, H Haller5 1University Hospital Ghent, Belgium; 2Centre Hospitalier Universitaire de Bordeaux, France; 3Ospedale Maggiore Policlinico, Milan, Italy; 4Alexion Pharmaceuticals, Cheshire, CT, USA; 5Medical School Hannover, Germany Critical Care 2015, 19(Suppl 1):P330 (doi: 10.1186/cc14410) Introduction Atypical haemolytic uraemic syndrome (aHUS) is a severe, life-threatening disease requiring rapid treatment to inhibit complement-mediated thrombotic microangiopathy (TMA) and avoid irreversible organ damage. Four prospective clinical trials have reported the safety and effi cacy of eculizumab (Ecu) in the treatment of aHUS [1,2]. We report data from a pooled analysis of these trials on renal function in patients starting Ecu within ≤7 days or >7 days after the current aHUS manifestation. Introduction Atypical haemolytic uraemic syndrome (aHUS) is a severe, life-threatening disease requiring rapid treatment to inhibit complement-mediated thrombotic microangiopathy (TMA) and avoid irreversible organ damage. Four prospective clinical trials have reported the safety and effi cacy of eculizumab (Ecu) in the treatment of aHUS [1,2]. We report data from a pooled analysis of these trials on renal function in patients starting Ecu within ≤7 days or >7 days after the current aHUS manifestation. P327 RV dysfunction signs included right ventricular to left ventricular (RV/LV) diameter ratio, interventricular septal shift, main pulmonary artery to ascending aorta (mPA/AA) diameter ratio, IVC contrast refl ux, SVC diameter, IVC diameter, PA diameter and azygos vein diameter. Clinical factors included cardiovascular, respiratory parameter and also time to diagnosis and treatment. g Results There were total of 36 cases with suspected APE on admission. Ten patients required mechanical ventilation (27.8%) and seven patients died (19.4%). Interventricular septal (IVS) shift was a signifi cant risk factor of in-hospital death (85.7% vs. 27.6%, P = 0.008) and respiratory failure (70% vs. 26.9%, P = 0.026). The sensitivity, specifi city, positive predictive and negative predictive values of IVS shift to predict in-hospital death were 85.7%, 70%, 42.8% and 95.5%, respectively. The sensitivity, specifi city, positive predictive and negative predictive values of IVS shift to predict respiratory failure were 70%, 73.1%, 50% and 86.4%, respectively. The ratios of RV to LV diameter and the ratio of main pulmonary artery to ascending aorta diameter tended to be higher in the nonsurvivor group. The clinical factor that predicted mortality was the PaO2 to FiO2 ratio (P/F ratio). Mean P/F ratio in survivor and nonsurvivor groups was 246.1 ± 94.1 vs. 132.2 ± 78.1, respectively (P = 0.011). P/F ratio ≤150 was the best predictor of mortality (66.7% vs. 8.7%, P = 0.008). Figure 1 (abstract P329). Kaplan–Meier analysis of ESRD progression for SC and Ecu treatment for all patients. with aHUS progress to end-stage renal disease (ESRD) after the fi rst episode [1]. Two prospective clinical trials have assessed the effi cacy of eculizumab (Ecu) in patients with aHUS [2]. We now evaluate data on progression to ESRD before and during Ecu treatment. Methods Patients with chronic kidney disease (CKD) stage 1 to 4 were analysed for progression to an ESRD event (two consecutive glomerular fi ltration rate measurements <15 ml/minute/1.73m2 (CKD stage 5)). ESRD incidence rate ratios during supportive care (SC) and Ecu treatment phases were calculated using a negative binomial regression analysis. Kaplan–Meier analyses were calculated for all patients and stratifi ed by CKD stages 2 to 4 at baseline. Hazard ratios (HR) were calculated from Cox proportional hazard models. Conclusion The IVS shifting from CTPA and P/F ratio ≤150 helps predict poor outcomes in APE. References 1. Fremeaux-Bacchi V, et al. CJASN. 2013;8:554-62. Results One hundred and fi fty-two patients were included. Patients with right ventricular (RV) dysfunction had signifi cantly higher MPV levels and MPV/P than patients without RV dysfunction. Receiver operating characteristic analysis revealed that a MPV cutoff of 7.85 fl provided 53.3% sensitivity and 68.5% specifi city, and a MPV/P cutoff of 0.0339 fl /(109/l) provided 69.6% sensitivity and 65% specifi city for prediction of RV dysfunction. There was a positive correlation between MPV and systolic pulmonary artery pressure (SPAP) and between MPV and RV diameter. There was a positive correlation between MPV/P and SPAP and between MPV/P and RV diameter. The low-risk PE group had lower MPV and MPV/P than the massive PE and submassive PE groups. Conclusion MPV and MPV/P are associated with RV dysfunction and clinical severity in acute PE. Low MPV and MPV/P levels may be an indicator of low risk in patients with acute PE. 2. Legendre C, et al. N Engl J Med. 2013;368:2169-81. Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemiai Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemia A Taha, A Shafi e, M Mostafa, N Syed, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P331 (doi: 10.1186/cc14411) Results We identifi ed 21 eligible studies (16,951 patients). After pooling data from the 20 included cohort studies (16,884 patients), at least around 33% (95% CI: 27 to 39; I2: 97.8%) of patients with TBI in published reports received transfusions at some point during their hospital stay. In a post hoc analysis of one RCT comparing transfusion strategies, 82% of patients were transfused RBCs. Thresholds for transfusion were rarely available and varied from 6 to 10 g/dl. From raw data, Glasgow Coma Scale scores were lower in patients who were transfused than those who were not (three cohort studies; n = 1,371; mean diff erence of 1.38 points (95% CI: 0.86 to 1.89); I2 = 12%). Mortality was not signifi cantly diff erent among transfused and nontransfused patients both in univariate and multivariate analyses. Hospital length of stay was longer among patients who were transfused (three studies; n = 455; mean diff erence 9.58 days (95% CI: 3.94 to 15.22); I2 = 74%). Due to the observational nature of included studies, results should be considered cautiously due to the high risk of confounding. i A Taha, A Shafi e, M Mostafa, N Syed, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P331 (doi: 10.1186/cc14411) Introduction Hemoglobin as the main trigger parameter for blood transfusion usually gives diminutive information about oxygen delivery and consumption. Although central venous oxygen saturation (ScvO2) is an alternative parameter, its changes are unable to detect regional hypoxia. Our aim was to evaluate the quotient of the central venous-to- arterial carbon dioxide gradient (δPCO2) and the arteriovenous oxygen content diff erence (Ca-cvO2) as a valid transfusion trigger parameter in hemodynamically stable anemic patients to reduce the amount of potentially counterproductive erythrocyte transfusions [1].i Methods Forty-fi ve postoperative patients admitted to our cardiac ICU were enrolled between January 2013 and September 2014. Three groups were defi ned according to the trend of blood loss over the surgical drains in the fi rst 24 postoperative hours. Red blood cell transfusion in patients with traumatic brain injury: a systematic review Red blood cell transfusion in patients with traumatic brain injury: a systematic review A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada Critical Care 2015, 19(Suppl 1):P332 (doi: 10.1186/cc14412) minute/1.73 m2 were included. Changes from baseline in eGFR were analysed at study visits using a one-sample t test. y A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada Critical Care 2015 19(Suppl 1):P332 (doi: 10 1186/cc14412) Results Data from 97 patients were analysed: median (range) age at enrolment was 29 (0 to 80) years; 62% of patients were females; median (range) duration of current manifestation to start of Ecu treatment was 23 (1 to 1,447) days; median (range) baseline eGFR was 15.9 (5.6 to 76.1) ml/minute/1.73 m2. Ecu treatment was started in 21 patients in ≤7 days and 76 patients in >7 days after presentation with TMA. Median eGFR was 11 ml/minute/1.73 m2 for the patients started within 7 days and 16 ml/minute/1.73 m2 for those initiating >7 days. The mean change from baseline in eGFR for patients starting Ecu in ≤7 days and in >7 days after presentation with TMA were 57 and 23 ml/minute/1.73 m2 at 1 year, respectively (Figure 1). Critical Care 2015, 19(Suppl 1):P332 (doi: 10.1186/cc14412) Introduction We aimed to evaluate the frequency of red blood cell (RBC) transfusion in patients with traumatic brain injury (TBI) as well as determinants and outcomes associated with RBC transfusion in this population. Methods We conducted a systematic review of cohort studies and trials of patients with TBI. Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemiai Mild blood loss was defi ned as 500 to 1,000 ml/24 hours, moderate (1,000 to 1,500/24 hours) and severe (>1,500 ml/24 hours). In addition to the δPCO2 the following parameters were monitored: CI, CO, SVR, serum lactate, ScvO2 and hemoglobin. Ca-cvO2 was calculated and the δPCO2/ Ca-cvO2 quotient was assessed for a total of 400 paired blood samples. All enrolled patients were hemodynamically stable. A retrospective analysis of this data was performed.i Conclusion RBC transfusion is frequent in patients with TBI, but practices varied widely in cohort studies in this population. The paucity of data precludes defi nitive conclusions and highlights the lack of clinical evidence guiding transfusion strategies in TBI. P329 Progression to end-stage renal disease is reduced with eculizumab in patients with atypical haemolytic uraemic syndrome J Vande Walle1, S Johnson2, E Harvey3, J Kincaid3 1University Hospital Ghent, Belgium; 2Medicus Economics, LLC, Boston, MA, USA; 3Alexion Pharmaceuticals, Cheshire, CT, USA Critical Care 2015, 19(Suppl 1):P329 (doi: 10.1186/cc14409) Methods Data from four phase 2, open-label, single-arm trials including both paediatric and adult patients with aHUS were pooled. Patients with a documented date of onset of current TMA manifestation and a baseline estimated glomerular fi ltration rate (eGFR) of <90 ml/ Introduction Atypical haemolytic uraemic syndrome (aHUS) is associated with severe kidney damage; almost one-half of adults S117 Critical Care 2015, Volume 19 Suppl 1 http://ccforum com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P330). Mean change in eGFR from baseline over 1 year (standard error). hemoglobin capacity decline and signifi cantly improved after erythrocyte transfusions (P <0.005). Figure 1 (abstract P330). Mean change in eGFR from baseline over 1 year (standard error). Conclusion Blood transfusions carry risks of adverse eff ects and should be carried out responsibly. Our fi ndings suggest an additive and easy detectable transfusion trigger parameter (δPCO2/Ca-cvO2) providing physiological information on anemia-related altered oxygen extraction conditions and hence the indication for erythrocyte transfusions. However, additional studies are warranted to confi rm these fi ndings. Reference 1. Mekontso-Dessap A, Castelain V, Anguel N, Bahloul M, Schauvliege F, et al. Combination of venoarterial PCO2 diff erence with arteriovenous O2 content diff erence to detect anaerobic metabolism in patients. Intensive Care Med. 2002;28:272-7. 1. Mekontso-Dessap A, Castelain V, Anguel N, Bahloul M, Schauvliege F, et al. Combination of venoarterial PCO2 diff erence with arteriovenous O2 content diff erence to detect anaerobic metabolism in patients. Intensive Care Med. 2002;28:272-7. y References 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. Red blood cell transfusion in patients with traumatic brain injury: a systematic review We searched Medline, Embase, The Cochrane Library and BIOSIS databases from their inception up to 30 June 2014. We selected cohort studies and RCTs of adult patients with TBI reporting data on RBC transfusions. We extracted data related to demographics, baseline characteristics, blood product use and any relevant clinical patient-oriented outcome. Cumulative incidences of transfusion were pooled through random eff ect models with a DerSimonian approach, after a Freeman–Tukey transformation to stabilize variances. To evaluate the association between RBC transfusion and potential determinants as well as outcomes, we pooled risk ratios or mean diff erences with random eff ect models and the Mantel–Haenszel method. Sensitivity and subgroup analysis were planned a priori.i y p y g Conclusion This pooled analysis indicates that patients treated with Ecu within 7 days of a TMA manifestation had a greater improvement in eGFR over time than patients in whom treatment was delayed. These data show the importance of rapid diagnosis and treatment of aHUS for recovery of renal function. Red blood cell transfusion is associated with an increased mortality in critically ill surgical patients y A Piriyapatsom, O Chaiwat, S Kongsayreepong Siriraj Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P334 (doi: 10.1186/cc14414) Results δPCO2/Ca-cvO2 showed signifi cant correlation with the moderate and severe blood loss groups (P <0.01), while no signifi cant correlation was detected in the mild blood loss group. The abnormality of the δPCO2/Ca-cvO2 was easy detectable and refl ected intracapillary Introduction The aim of this study is to explore the association between red blood cell transfusion (RBCT) and mortality in Thai critically ill surgical patients. Introduction The aim of this study is to explore the association between red blood cell transfusion (RBCT) and mortality in Thai critically ill surgical patients. S118 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods This study was a part of the multicenter, prospective, observational study performed in nine surgical intensive care units (SICUs) across the nation between April 2011 and November 2012 [1]. This study included adult patients admitted to the SICUs after surgery. Patients were categorized into transfusion and no transfusion groups according to whether or not they received RBCT at any time during SICU stay. Demographic data, clinical outcomes as well as SICU and hospital length of stay (LOS) and SICU and hospital mortality were collected. Patients were followed for up to 28 days or until discharge from the SICUs. The primary endpoint was hospital mortality. Data were compared between groups and logistic regression analysis was performed to determine whether RBCT was an independent risk factor of hospital mortality. In addition, patients were matched between groups based on the propensity score of the requirement of RBCT and were then compared. confounders, a Hb level of ≤9 g/dl was associated with a poor neurologic outcome (OR = 2.572, 95% CI = 1.058 to 6.250), whereas an Hb level of ≤10 g/dl was not. A Hb level of ≤10.3 g/dl had a sensitivity of 82% and a specifi city of 62% to predict poor neurologic outcome in TBI patients. Conclusion In TBI patients a Hb level of ≤9 g/dl is associated with poor neurologic outcome. Red blood cell transfusion is associated with an increased mortality in critically ill surgical patients A transfusion threshold of ≤10.3 g/dl may be a reasonable target to be tested in future transfusion trials aimed at improving neurologic outcome of TBI patients.fi Acknowledgements All of the author institutes are affi liated members of the International Trauma Research Network (INTRN) and as such this work represents a combined output resulting from this Network. Microparticles from red blood cell transfusion products induce a strong infl ammatory host response y p q g Results Supernatant from blood bags containing MPs strongly induced production of all cytokines compared with supernatant without MPs, a reaction which equaled that of LPS stimulation. MPs from stored RBC bags induced higher production of TNF (868 (263 to 1,625) vs. 2,596 (407 to 3,040) pg/ml, P = 0.049), IL-6 (1,088 (234 to 3,716) vs. 6,952 (1,507 to 21,990), P = 0.042) and IL-8 (1,333 (535 to 3,569) vs. 5,562 (833 to 13,904), P = 0.081) compared with MPs from fresh RBC bags. There was no diff erence in IL-10 responses between groups (8.0 (3.9 to 32.1) vs. 3.9 (3.9 to 22.2), P = 0.390). The host response was dose dependent both for fresh and stored MPs. In addition, the same amount of older MPs induced a stronger host response compared with fresh MPs. Conclusion MPs from RBC transfusion bags induce a strong proinfl ammatory response, which is largely negated when MPs are removed. This MP-mediated response depends both on the amount of MPs as well as on alterations in MPs as a result of storage. Reference Reference Reference 1. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 Suppl 1:S45-54. 1. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 Suppl 1:S45-54. Microparticles from red blood cell transfusion products induce a strong infl ammatory host response Results Overall, 968 of 2,374 (40.8%) patients received RBCT. Trans- fused patients, when compared with those without RBCT, had more frequency of admission after emergency surgery, higher APACHE II score, higher SOFA score, higher number of organ dysfunctions and lower hemoglobin level at admission. When compared with patients without RBCT, those with RBCT had more frequency of all adverse events including infection, AKI, ALI/ARDS and MI, and longer SICU and hospital LOS. Both SICU and hospital mortality were also higher in the transfusion group compared with the no transfusion group (9.4% vs. 1.6% and 13.7% vs. 3.6%, both P <0.001, respectively). The logistic regression analysis showed that RBCT was an independent risk factor of hospital mortality with odds ratio of 1.60 (95% CI 1.05 to 2.45). In the propensity-score matched cohort of 852 patients, when compared with patients without RBCT, transfused patients had more frequency of adverse events including infection and AKI, longer SICU and hospital LOS and higher hospital mortality (7.5% vs. 4.0%, P = 0.027). l M Straat1, M Van Hezel1, A Boing1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 f 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin Blood Cell Research, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P336 (doi: 10.1186/cc14416) Introduction Red blood cell (RBC) transfusion is associated with increased morbidity and mortality in the critically ill. Adverse eff ects of transfusion may be mediated by red blood cell storage lesion. In this study, we hypothesized that MPs from stored RBC bags would induce a more pronounced host response than MPs from fresh RBC bags and that this response is dose dependent. p p Methods MPs were isolated by high-speed centrifugation from red blood cell transfusion bags stored for 2 to 7 (fresh) or 25 to 35 (stored) days. Whole blood from healthy volunteers was incubated with supernatant from the bags either containing MPs or depleted from MPs (n = 12 bags per group). Controls were incubated with PBS as a negative and LPS (10 ng/ml) as a positive control. Cytokines in supernatant were measured by ELISA. Data are expressed as medians and interquartile ranges. g y Conclusion This study showed that RBCT was associated with increased morbidity and mortality in critically ill surgical patients. These results supported the restrictive strategy of RBCT suggested by more recent studies. Eff ect of the haemoglobin level on neurologic outcome in patients with severe traumatic brain injury Eff ect of the haemoglobin level on neurologic outcome in patients with severe traumatic brain injury K Balvers1, MR Wirtz1, C Rourke2, S Eaglestone2, K Brohi2, S Stanworth3, C Gaarder4, JC Goslings1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Blizard Institute, Queen Mary University of London, UK; 3John Radcliff e Hospital, Oxford, UK; 4Oslo University Hospital, Oslo, Norway Critical Care 2015, 19(Suppl 1):P335 (doi: 10.1186/cc14415) Introduction Anaemia in patients with severe traumatic brain injury (TBI) may worsen neurologic outcome. The aim of this study was to determine the association of haemoglobin level (Hb) with neurologic outcome and to determine a transfusion threshold which may be used in future transfusion trials aimed at improving neurologic outcome in TBI patients. Value of thromboelastography in managing hypercoagulopathy in intensive care Methods A retrospective audit over 2 months of all blood transfusion forms at St Francis Hospital, Eastern Province, Zambia. Respective patients’ notes were reviewed for: record of observations during transfusion; patient demographics; and length of stay. We surveyed nurses’ attitudes, confi dence and knowledge in relation to blood transfusion standards. J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P340 (doi: 10.1186/cc14420) J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK g p , , Critical Care 2015, 19(Suppl 1):P340 (doi: 10.1186/cc14420 Introduction This aim of this analysis is to explore the use of thromboelastography (TEG) in the management of hypercoagulation in the ICU. TEG allows the assessment of whole blood coagulation and fi brinolysis and hence can identify patients who are hypercoagulable. Methods A prospective audit of TEG tests performed on patients being treated on a general surgical and medical ICU was conducted over a 2-month period. Results In May and June 2014, 457 requests were made for blood, of which 157 (34%) received blood transfusion, of which 108 (69%) had records of observations available. The audit demonstrated that requests were mostly complete (90%), but urgency was indicated in only 32%. The matching of blood to patient by more than one nurse was recorded amongst 66% of cases. Only 2% of transfusions met minimal requirements for transfusion reaction monitoring. Of nurses surveyed (n = 20), most were experienced in their post (mean 7.3 years, range 2 weeks to 20 years). Nurses rated themselves as highly confi dent in handling blood transfusions and identifying and dealing with transfusion reactions. However, 90% believed they could identify all transfusion reactions by measuring temperature alone, and 25% would measure temperature only as a parameter to monitor the transfusion, even in ideal settings. Most knew to check observations before, 15 minutes after the start of transfusion and then hourly thereafter (88%); but only 10% would check at the start, at completion and 4 hours after completing transfusion. The most frequently reported reason for not doing observations was time pressure on the ward (85%). Results Twenty-one out of 78 patients (26.9%) had one or more TEG criteria consistent with hypercoagulopathy. Admission diagnoses included trauma (37%), haemorrhage (23%), postoperative (23%) and sepsis (14.3%). Sixty-two per cent of patients with a primary diagnosis of trauma were in a hypercoaguable state. P337 Improving blood transfusion safety in a low-resource setting: an audit of 1,163 transfusion requests S Kudsk-Iversen1, R Colhoun1, D Chama2, J Mulenga2, MD Bould1, J Kinnear1, D Snell1 1Zambia Anaesthesia Development Project, Lusaka, Zambia; 2Zambia National Blood Transfusion Service, Lusaka, Zambia Critical Care 2015, 19(Suppl 1):P337 (doi: 10.1186/cc14417) Methods A substudy of the prospective multicentre Activation of Coagulation and Infl ammation in Trauma (ACIT) II study was performed on subjects recruited between January 2008 and December 2014. All adult trauma patients admitted to a level 1 trauma centre with severe traumatic brain injury (AIS head ≥3), ICU admission and available Hb levels on admission were selected for analysis. The primary outcome was the cognitive functioning of patients as determined by an estimated Glasgow Outcome Scale (GOS) on discharge. Anaemia was defi ned as a Hb level of ≤9 g/dl (severe anaemia) or ≤10 g/dl (moderate anaemia) within the fi rst 24 hours post injury. Multivariate logistic regression models were used to determine the association between anaemia and neurologic outcome. The receiver operating characteristic curve and the Youden Index were used to determine an optimal transfusion threshold. Introduction Sub-Saharan Africa suff ers from more acute life- threatening indications for blood transfusion compared with high- income countries [1]. The commonest ‘systems failure’ contributing to perioperative death in low-resource settings is the timely availability of correctly cross-matched blood products [2]. Often this is not the result of an absolute shortage of blood products, but failure in the chain of supply and distribution. We audited an early step in this chain, the quality of blood requests, at the University Teaching Hospital (UTH) in Lusaka, Zambia. UTH does not have a formal blood request form, and only the cancer diseases hospital (CDH) has a blood request form developed by the blood bank. Results Of a total of 261 TBI patients, 61 patients (23%) fell below the threshold for severe anaemia and 101 patients (39%) had moderate anaemia within the fi rst 24 hours. In a model adjusted for all relevant S119 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We performed a 1-day retrospective review in June of blood request forms submitted to the cross-match laboratory, followed by a 14-day prospective review in September 2014. Group and save requests were excluded. Inadequate monitoring risks safety of blood transfusion in rural Zambia Results The mean age was 43.5 ± 5.7 years. Of 58 patients (38 male/20 female), 25 (43.1%) had iron defi ciency with outcomes of blood samples used at ICU admission. The overall transfusion rate was 32.8%, being higher in iron-defi ciency patients than in normal iron profi le patients (42.3 vs. 14.9%, P = 0.001). After adjusting for severity of illness and hemoglobin level, iron-defi ciency patients remained signifi cantly associated with transfusion, with a hazard ratio of 4.2 (95% CI, 1.3 to 12.9; P = 0.001). Zambia O Todd1, K Sikwewa1, J Kamp1, I Hodt Rasmussen1, K Mortensen1, S Kudsk-Iversen2 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) O Todd1, K Sikwewa1, J Kamp1, I Hodt Rasmussen1, K Mortensen1, S Kudsk-Iversen2 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) Introduction In Zambia, supply of blood is insuffi cient to meet clinical need, on a national level. Paradoxically, blood is also more often transfused unnecessarily in this setting. The Zambian National Blood Transfusion Service is currently scaling up voluntary blood donation and supply systems, and requires hospitals to improve blood transfusion safety. At a rural district hospital in Zambia, we audited practice and surveyed knowledge amongst staff using standards established in national guidelines. Conclusion Iron defi ciency is common at ICU admission and is associated with higher transfusion requirements. These fi ndings have important implications for transfusion practices in ICU patients. P340 P339 Eff ects of iron defi ciency on transfusion requirements in critically ill patients: a preliminary observational study M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) P339 Eff ects of iron defi ciency on transfusion requirements in critically ill patients: a preliminary observational study M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) Introduction Critically ill patients often need blood transfusion, but no reliable predictors of transfusion requirements are available at ICU admission. We hypothesized that ICU patients admitted with iron defi ciency may be at higher risk for developing anemia, requiring blood transfusion. The aims of this study were to determine the frequency of iron defi ciency in ICU patients at admission and to investigate its relationship with transfusion requirements in ICU patients. p Conclusion The audit revealed an important system failure impacting on effi cacy and safety of transfusion practice at UTH. Full patient identifi ers, as well as vital information such as the indication and urgency, were rarely fi lled in, which are crucial for the blood bank to prioritise the release of blood products. The audit shows that practice may be signifi cantly improved by a cheap intervention such as a standardised blood request form meeting international standards. Acknowledgements UK aid, THET. References 1 L d l T f A h S i 2013 49 416 21 Methods Eighty-fi ve patients admitted to the general ICU were enrolled in the prospective observational study. We studied 58 patients, after excluding those transfused on or before ICU admission. The patients’ age, gender, APACHE II score, diagnosis, severity score, presence of sepsis, ICU complications, ICU treatments, and transfusion-free interval were recorded. Iron defi ciency was assessed on the basis of several parameters, including hemoglobin, hematocrit, levels of serum iron, iron-binding capacity, transferrin saturation, levels of ferritin, soluble transferrin receptor, hepcidin, C-reactive protein, and peripheral blood smear. 1. Lund et al. Transfus Apher Sci. 2013;49:416-21. 2. Ohanaka et al. Turk J Med Sci. 2007;37:219-22. P337 Each form was audited against the American Association of Blood Banks (AABB) minimum standards for content of a blood request form. Analysis was performed with Fisher’s exact test for nominal data and t test for continuous data. Conclusion In this setting, current practice is evidently inadequate to identify and prevent blood transfusion reactions. The survey revealed high confi dence but patchy knowledge amongst nurses of the requirements for safe blood transfusion. Better timing to transfuse at times when nursing staff numbers are higher, alongside compulsory training, may together represent potential low-cost interventions to improve blood transfusion safety. Results A total of 1,163 blood requests were reviewed, 51 from CDH and 1,112 from other wards. Eighteen forms from CDH (35%) and 22 from other wards (2%) met all minimum AABB standards (P <0.0001). The mean number of standards met on the requests from CDH and the rest were 11.25 (SD 0.93) and 8.87 (SD 1.75) respectively (P <0.0001). Considering all blood requests, the standards met in order from least to most were: signature of requesting doctor (36%), urgency of request (43%), hospital number (59%), indication for transfusion (62%), type of product requested (72%), requesting doctor’s name (78%), age or date of birth of patient (84%), gender of patient (89%), quantity of products requested (90%), date form was completed (90%), patient’s ward (95%), and patient’s full name (100%). P339f P342 P342 In trauma, when used in the emergency department, do viscoelastic hemostatic tests decrease mortality? A systematic review J Cousineau, R Daoust, K Doyon, M Marquis, É Piette, JM Chauny, M Clar, D Rose, É Notebaert Hôpital du Sacré Coeur de Montréal, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P342 (doi: 10.1186/cc14422) Introduction This systematic review done in March and updated in November 2014 has been conducted in accordance with the STARD, PRISMA and STROBE recommendations. Retrospective and prospective studies with a comparison group published in English were kept. Methods Two reviewers (JC and ÉN) and two librarians (MC and DR) independently conducted a systematic review and identifi ed abstracts. Full texts were read by two authors (ÉN and ÉP), and data were extracted. The following databases were searched: Cochrane CENTRAL, Medline, Embase, LILACS, Web of Science, Science.gov, SciFinder Scholar, WorldCat, the Transf Evid Lib Database, and proceedings of the congresses of the International Society on Thrombosis and Haemostosis and the American Society of Hematology. Figure 1 (abstract P340). Coagulation modifi cation prior to TEG analysis in hypercoagulable patients. as a result of performing TEG was documented in 14 of these 21 patients. No further blood products were administered in all cases and anticoagulation was commenced or increased in four cases. Conclusion Hypercoagulopathy was present in 27% of patients. One- third of these patients had recently received prothrombotic therapy indicating a possible iatrogenic aetiology. TEG analysis resulted in cessation of prothrombotic drug and blood product administration in all cases. Further research is required to determine whether titrated anticoagulation treatment to normalise the TEG profi le in these patients would be benefi cial. f y y Results We initially kept 2,870 references. In total, 453 articles had mortality in their keywords. After reading the abstracts, 37 papers were analysed, and three articles evaluating mortality in two groups of patients (using and not using a VHT) were identifi ed. Among these three studies, only one had raw data available. We did not succeed in getting this information from the two other authors. We asked the main authors of the 37 selected papers, and renowned authors in the fi eld, if they had studies with new data that could be included in our review. The answers were negative. Thromboelastography may detect hypercoagulation in early sepsis and improve anticoagulation during extracorporeal treatments F T S B R B h AB B ll S M M F l Aurelia and European Hospital, Rome, Italy p p y Critical Care 2015, 19(Suppl 1):P341 (doi: 10.1186/cc14421) Introduction During early sepsis, activation of the infl ammatory response and coagulation occurs. Extracorporeal therapies are used to adsorb mediators, but the coagulation of fi lters is a drawback [1,2]. The aim of this study is to evaluate whether thromboelastography (TEG) may detect hypercoagulation and may improve anticoagulation during extracorporeal treatments. Conclusion With the studies available as of the end of 2014, it is impossible to conclude whether the use of a VHT in the emergency department decreases mortality. Other studies are needed. References 1. Johansson PI, Stensballe J. Eff ect of haemostatic control resuscitation on mortality in massively bleeding patients: a before and after study. Vox Sang. 2009;96:111-8. p Methods Twenty-four patients with early severe sepsis had a TEG monitoring at basal time (T0) and during three diff erent extracorporeal treatments (T1): coupled plasma fi ltration (CPFA) with heparin infusion (Group A), CPFA with citrate infusion (Group B) and RRT with oXiris fi lter – heparin coated – and no heparin infusion (Group C). ANOVA test was used for the statistical analysis. 2. Messenger BM, et al. TEG-guided massive transfusion in trauma patients. Anesth Analg. 2011;112:S-9. 3. Kashuk K, et al. Initial experiences with POC rapid TEG for management of life-threatening postinjuty coagulopathy.Transfusion. 2012;52:23-33. 3. Kashuk K, et al. Initial experiences with POC rapid TEG for management of life-threatening postinjuty coagulopathy.Transfusion. 2012;52:23-33. y Results Table 1 presents the TEG values in early septic patients at T0. At T1, angle and MA decreased and r increased in Group A at diff erence with Group B and Group C (P <0.01). In group C, LY 30 was higher than in Group A and B (P <0.01). Reference 1. Kashuk JL, et al. r-TEG identifi es hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146:764-72. 1. Kashuk JL, et al. r-TEG identifi es hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146:764-72. P341 Thromboelastography may detect hypercoagulation in early sepsis and improve anticoagulation during extracorporeal treatments F Turani, S Busatti, R Barchetta, AB Belli, S Martini, M Falco Aurelia and European Hospital, Rome, Italy Critical Care 2015, 19(Suppl 1):P341 (doi: 10.1186/cc14421) References Figure 1 (abstract P340). Coagulation modifi cation prior to TEG analysis in hypercoagulable patients. 1. Livigni S, Bertolini G, Rossi C, et al. BMJ Open. 2014;4:e003536. 2. Ronco C, et al. Crit Care. 2014, 18:309. Value of thromboelastography in managing hypercoagulopathy in intensive care Hypercoagulopathy was suggested by an abnormally short R time in 16 patients (76%), an abnormal alpha angle in 17 cases (81%), a maximum amplitude >74 mm in nine cases (43%) and a high LY30 in one case. Procoagulant treatment was given to seven patients and fi ve patients had received no coagulation modifi cation prior to testing (Figure 1). Eight patients were receiving prophylactic anticoagulation and only one was receiving treatment-dose anticoagulation. A change in management S120 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P342 The three studies were: Johansson and Stensballe, total 832 cases, 121 traumas, raw data unavailable for trauma [1]; Messenger and colleagues, prospective study, 50 cases, mortality similar, raw data unavailable [2]; and Kashuk and colleagues, prospective study, 68 cases, mortality 59% control group, 28% VHT group [3]. Role of thromboelastography in the management of haemorrhage: an observational analysis y C Ward, J Aron, A Gibbon, J Ball C Ward, J Aron, A Gibbon, J Ball Table 1 (abstract P341) Control Sepsis r 8 ± 2 6 ± 3 k 4 ± 2 1.7 ± 0.5* Angle 47 ± 15 67 ± 7* MA 55 ± 8 72 ± 5* LYS 30 1.8 ± 1 0.95 ± 0.9 P <0.01. St George’s Hospital, London, UK g p Critical Care 2015, 19(Suppl 1):P343 (doi: 10.1186/cc14423) Introduction The aim of this analysis is to explore and evaluate the role of thromboelastography (TEG) in aiding the management of patients admitted with haemorrhage to the ICU. TEG has been shown to rationalise and reduce transfusion requirements [1] but the precise role of TEG in the assessment and management of haemostasis in the bleeding patient is uncertain and has not been previously demonstrated. Methods A prospective audit of TEG analyses performed on patients in the general medical and surgical ICU was recorded over a 2-month period. Operators documented the reason for admission, demographic data, indication for TEG, laboratory results, blood gas analysis, TEG results, diagnosis and subsequent action from the TEG result. Only patients who had been admitted with haemorrhage were included in this analysis. Conclusion In early sepsis, TEG monitoring may detect hyper coagu la- bility. CPFA with heparin, but not CPFA with citrate and oXiris, is able to reverse hypercoagulability. OXiris may induce fi brinolysis. TEG detects alterations of coagulation during early sepsis and extracorporeal treatments. S121 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 TEG analysis Both TEG normal/ Standard normal/ abnormal standard abnormal TEG abnormal Clot factor defi cit 9 (11.5) 20 (25.6) 3 (3.8) Platelet defi cit 11 (14.1) 13 (16.6) 2 (2.6) Fibrinogen defi cit 5 (6.4) 4 (5.1) 5 (6.4) Data presented as n (%). Results Seventy-eight audit sheets were completed, of which 31 identifi ed haemorrhage as the reason for admission. The mean age was 59.3 (range 21 to 90) and the mean APACHE II score was 18.23 (range 11 to 37). The main indications for TEG analysis included coagulopathy (64%) and ongoing haemorrhage (45%). As a result of performing TEG analysis, 23 (74%) patients had a documented change in their management. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) M Popescu, D Tomescu M Popescu, D Tomescu Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P345 (doi: 10.1186/cc14425) Introduction Our aim was to assess hemostasis, using ROTEM, in patients with end-stage liver disease (ESLD) undergoing liver transplantation (LT) and to develop a predictive model for patients prone to high intraoperative blood loss. Introduction This review aims to assess the value of thrombo- elastography (TEG) on the general ICU, which has not been previously demonstrated. TEG is a near-patient assessment of whole blood coagulation and fi brinolysis, which reduces transfusion requirements during cardiothoracic surgery and liver transplantation. Methods We retrospectively analyzed 122 patients who underwent LT between January and December 2013 in a single national center. Patients with acute liver failure or incomplete data were excluded. Demographic data, severity of liver disease assessed by MELD score (model for ESLD), presence of portal vein thrombosis, and laboratory data were recorded preoperatively. We performed concomitant ROTEM assay and standard coagulation tests (prothrombin time (PT), International Normalized Ratio (INR), fi brinogen) 1 hour before surgery and 15 minutes after the neohepatic phase. Intraoperative blood loss was recorded. High blood loss was defi ned as loss of one blood volume during surgery. Correlation between recorded data standard ROTEM parameters and derived thrombodynamic ROTEM parameters (potential index (TPI), maximum velocity of clot formation (MaxV), time to MaxV (MaxVt), AUC) were analyzed using SPSS 19.0. Methods A prospective audit of TEG tests performed on patients being treated on a general ICU was conducted over 2 months. Results A total of 332 TEG tests were performed with a failure rate of 29.8%. Seventy-eight audit sheets were collected. Mean patient age was 68.9 years and mean APACHE II score was 18.1. Admissions included trauma (33.0%), perioperative (43.6%), haemorrhage (42.3%) and sepsis (21%). Standard tests of coagulation demonstrated 22 defi cits in coagulation which were not identifi ed as functionally signifi cant with TEG. Of these, 20 had abnormal clotting factor activity as measured by the INR/APTTr and 13 patients were thrombocytopenic. In total, 52.6% documented that the TEG result changed the management of the patient. In 46.8% of these cases no further blood products were required. In 41% there was no documentation. Role of thromboelastography in the management of haemorrhage: an observational analysis Ten patients did not require any further administration of blood products, which they would have received based on conventional laboratory results. The information gained from TEG also resulted in the omission of anticoagulation in three patients, and with a further two patients anticoagulation increased. Conclusion TEG analysis suggested that 22 patients who were identifi ed as coagulopathic with traditional measures of coagulation did not have a functional defi ciency. Over one-half of TEG studies resulted in a change in management and in 46.8% no further transfusions were required. There was a high technical failure rate and a low audit return rate, which may indicate the need for further training. Reference p g Conclusion TEG aids prompt rationalisation of blood products and titration of anticoagulation in the bleeding patient. TEG identifi es a number of patients who required administration of platelets and other procoagulants which would not have been identifi ed by conventional methods. Several patients would have also received inappropriate transfusions which has both cost and resource implications, alongside the potential adverse eff ects on patients. We recognise that further research is needed to clarify the overall effi cacy of TEG in the bleeding patient. f 1. Da Luz et al. Eff ect of TEG and ROTEM on diagnosis of coagulopathy, transfusion guidance and mortality in trauma: descriptive systematic review. Crit Care. 2014;18:518. 1. Da Luz et al. Eff ect of TEG and ROTEM on diagnosis of coagulopathy, transfusion guidance and mortality in trauma: descriptive systematic review. Crit Care. 2014;18:518. Decreased coagulation kinetics is associated with high blood loss in patients with end-stage liver disease undergoing liver transplantation Decreased coagulation kinetics is associated with high blood loss in patients with end-stage liver disease undergoing liver transplantation M Popescu, D Tomescu Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P345 (doi: 10.1186/cc14425) Reference 1. Bollinger D, et al. Principles and practice of thromboelastography in clinical coagulation management and transfusion practice. Transfusion Med Rev. 2012;26:1-13. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) See Table 1 and Figure 1. Table 1 (abstract P344). Summary of concordance with standard tests versus y g Results After applying exclusion criteria, 72 patients were analyzed with mean age of 54.5 years (SD 11.6) and a median MELD score of 17.4 (7 to 34). Preoperative MCE correlated with age (P = 0.044, 95% CI (–7.50, –0.12)) and MELD score (P = 0.009, 95% CI (–37.21, –6.69)), but not with PT (P = 0.557) or INR (P = 0.623). MaxV correlated with fi brinogen level (P = 0.005, 95% CI (0.01, 0.05)) and AUC correlated with age (P = 0.034, 95% CI (–257.74, –11.91)) and MELD score (P = 0.01, 95% CI (–1,233.14, –215.33)). Patients with portal vein thrombosis had an increase in InTEM CFT (P = 0.002, 95% CI (77.98, 317.97)) and MaxVt (P = 0.03, 95% CI (5.53, 105.63)). No correlation was found between preoperative ROTEM parameters and intraoperative blood loss. We calculated ΔMaxV, ΔMaxVt and ΔAUC as the mathematical diff erence between preoperative and intraoperative MaxV, MaxVt and AUC. High blood loss correlated with ΔAUC (P = 0.005, 95% CI (15.69, 61.03)), ΔMaxV (P = 9=0.002, 95% CI (–20,413, 6,392)) and ΔMaxVt (P = 0.008, 95% CI (15.69, 61.07)). Figure 1 (abstract P344). Change in management due to TEG results. Conclusion MELD score correlated with a decrease in MaxV and AUC on preoperative ROTEM but not with INR. Patients with portal vein thrombosis have increased InTEM CFT and MaxVt. High blood loss was associated with a decrease in thrombodynamic parameters, but no correlations were found between blood loss and standard ROTEM parameters. Figure 1 (abstract P344). Change in management due to TEG results. S122 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P346 time (aPTT), and prothrombin time (PT). In addition, the plasma levels of vitamin K-dependent coagulation factors were determined. P346 Evaluation of fi xed dose four-factor prothrombin complex concentrate for warfarin reversal at a level 1 trauma center H Drone, J Jancik, J Gorlin, M McCarthy Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P346 (doi: 10.1186/cc14426) p g Results Acute coumarin anticoagulation of rats induced a rise in median bleeding time by ≥2-fold from an average of 823 to 1,800 seconds (maximum observation period) compared with untreated animals. Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation E Herzog, F Kaspereit, W Krege, P Niebl, G Dickneite g p g CSL Behring GmbH, Marburg, Germany g g y Critical Care 2015, 19(Suppl 1):P347 (doi: 10.1186/cc14427) Introduction The study was conducted as a head-to-head comparison of a four-factor prothrombin complex concentrate (4F-PCC) and two diff erent three-factor PCCs (3F-PCC) for eff ective reversal of vitamin K antagonist (VKA)-induced anticoagulation using an established rat model of acute bleeding [1]. The 4F-PCC (containing the human coagulation factors II, VII, IX and X) is indicated for the urgent reversal of acquired coagulation factor defi ciency induced by VKA therapy in adult patients with acute major bleeding. In contrast, the 3F-PCCs (containing factors II, IX, X and only minimal VII) are indicated for the prevention and control of hemorrhagic episodes in hemophilia B patients. Nevertheless, the use of 3F-PCC for correcting hemostasis following warfarin overdose has been discussed but the lack of factor VII in these 3F-PCC products has raised questions about effi cacy. Conclusion In conclusion, 4F-PCC treatment eff ectively decreased apixaban-induced hemorrhage at a clinically relevant dose range. References . Herzog et al. Anesthesiology. Forthcoming 2014. 1. Herzog et al. Anesthesiology. Forthcoming 2014. 1. Herzog et al. Anesthesiology. Forthcoming 2014. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. References 1. Varga C, et al. Transfusion. 2013;53:1451-8. 2. Junagade P, et al. Hematology. 2007;12:439-40. g g p Results Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration with maximum bleeding signals seen at 1,200 μg/kg. Treatment with 4F-PCC resulted in a statistically signifi cant reversal in apixaban-induced bleeding time (all doses) and volume (doses ≥12.5 IU/kg). Of the coagulation parameters measured, thrombin generation initiated using phospholipids only was the in vitro coagulation parameter most sensitive to 4F-PCC-mediated bleeding reversal, although statistically signifi cant 4F-PCC-mediated reductions in the prothrombin time and whole blood clotting time were also observed.f P347 Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation E Herzog, F Kaspereit, W Krege, P Niebl, G Dickneite CSL Behring GmbH, Marburg, Germany Critical Care 2015, 19(Suppl 1):P347 (doi: 10.1186/cc14427) p Reference 1. Dickneite. Thromb Res. 2007;119:643-51. f Results The INR was reduced to <2 in 100% of patients in the 4F-PCC group versus 84.6% of patients in the factor IX group (P <0.05). The INR was reduced to <1.6 in 90.8% of patients in the 4F-PCC group versus 50% in the factor IX group (P <0.05). Mean pre-reversal INRs were 3.5 and 4 and ranged from 1.1 to 10 and from 1.3 to 10 in the 4F-PCC and factor IX group respectively (P = 0.29). On average, a medication cost savings of US$802.63 dollars per patient was calculated from using a fi xed 1,500 unit dose over traditional dosing of 4F-PCC. There was a trend toward a shorter mean ICU LOS in the PCC group when compared with the factor IX group (5.8 vs. 2.8 days) and shorter mean hospital LOS (10.7 vs. 5.7 days), although neither outcome was statistically signifi cant. No diff erence in adverse event rates was observed.ii P348 Four-factor prothrombin complex concentrate (Beriplex® P/N) mediated reversal of apixaban-induced bleeding in a rabbit model E Herzog, F Kaspereit, W Krege, J Mueller-Cohrs, B Doerr, P Niebl, G Dickneite CSL Behring GmbH, Marburg, Germany Critical Care 2015, 19(Suppl 1):P348 (doi: 10.1186/cc14428) Introduction This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can eff ectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].i Introduction This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can eff ectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].i gif Conclusion A fi xed dose of 1,500 units of 4F-PCC was signifi cantly more eff ective at lowering the INR to a threshold of less than either 2 or 1.6 when compared with a combination of factor IX complex and vitamin K with or without FFP. Further research is needed to investigate clinical outcomes and a possible reduction in ICU and hospital LOS. References Methods For dose-fi nding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800 to 1,600 μg/kg). In a subsequent study phase, anesthetized rabbits were treated with apixaban (1,200 μg/kg) followed by 4F-PCC (6.25 to 100 IU/kg). Bleeding signals were quantifi ed following a standardized kidney incision by measurement of the volume of blood loss and time to hemostasis over an observation period of 30 minutes. Blood samples were collected for monitoring of coagulation parameters. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) In parallel, PT and aPTT were prolonged from 8.9 to 29.9 seconds and from 14.5 to 25.5 seconds, respectively. Treatment with 4F-PCC was able to fully and statistically signifi cantly reverse bleeding, achieving average bleeding times of 676 seconds. In parallel, the elevation in PT was reduced to 15.1 seconds. In contrast, the two 3F-PCC products were not or only partially able to reduce coumarin-induced bleeding with average bleeding times of 1,398 and 1,708 seconds post treatment, respectively. This also correlated with inferior reductions in PT which achieved minimum levels of 23.8 and 29.5 seconds, respectively. There was no reduction in aPTT seen for any treatment option.i Introduction FDA-approved dosing of four-factor prothrombin complex concentrate (4F-PCC) in the USA is based on an INR and weight; however, there are data suggesting that a fi xed dose of 4F-PCC may be suffi cient for INR reversal and hemostasis [1,2]. The objective of this study was to assess the effi cacy and safety of a fi xed dose of 1,500 units of 4F-PCC. Historically, warfarin reversal included a combination of factor IX complex, vitamin K, and fresh frozen plasma (FFP). Using a fi xed dose of 4F-PCC may also provide signifi cant cost savings when compared with traditional dosing. y p Conclusion In conclusion, this fi rst direct comparison of 4F-PCC and 3F-PCCs for the reversal of VKA anticoagulation in a rat model of acute bleeding suggests that replenishment of all four vitamin K-dependent coagulation factors including factor VII as achieved using a 4F-PCC may result in superior effi cacy compared with the use of 3F-PCCs. R f Methods This retrospective chart review compared 26 admitted adults who received a fi xed dose of 1,500 units 4F-PCC with 26 patients who received a combination of factor IX complex and vitamin K, with or without FFP, for warfarin reversal from 1 January 2012 to 1 November 2014. Primary outcomes included reversal to an INR of <2 and reversal to an INR of <1.6. Secondary outcomes included ICU and hospital length of stay (LOS), change in INR, INR nadir, potential cost savings from 4F-PCC versus traditional dosing, and major adverse eff ects. Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model M Honickel1, T Braunschweig1, J Van Ryn2, R Rossaint1, O Grottke1 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co KG, Biberach, Germany Critical Care 2015, 19(Suppl 1):P351 (doi: 10.1186/cc14431) Results In the ShBl injury strand there was a signifi cant reduction in ATC in Early compared with Late PRBC:FFP treatment (TEG R and K times) in both the prehospital (P = 0.004 and P = 0.003 respectively, ANOVA) and early in-hospital (P = 0.002 and P = 0.005) phases, although clotting was normalised in the Late group within 60 minutes of initiating PRBC:FFP. Prehospital base defi cit (BD) was signifi cantly attenuated in ShBl Early versus Late (9.0 ± 2.1 vs. 14.4 ± 2.2 mM). BD improved in both Early and Late treatment groups during the in-hospital phase but remained greater in the Late group throughout (P <0.001). In the Bl injury strand the trend in coagulation was similar to that seen in the ShBl injury strand (but the diff erences between Early and Late did not attain statistical signifi cance). By contrast, Early versus Late PRBC:FFP treatment did not result in a diff erence in BD in the Bl strand. Finally, there was no diff erence in the total amount of PRBC:FFP used between the two treatments in either injury strand, but in both injury strands the Early treatment groups required signifi cantly less saline (P <0.001). Conclusion Prehospital use of PRBC:FFP may attenuate ATC and improve physiological status. Furthermore the amount of crystalloid may be reduced with potential benefi t of reducing the third-space eff ect and later tissue oedema. M Honickel1, T Braunschweig1, J Van Ryn2, R Rossaint1, O Grottke1 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co KG, Biberach, Germany Critical Care 2015, 19(Suppl 1):P351 (doi: 10.1186/cc14431) Introduction The anticoagulant eff ect of dabigatran can be reversed with idarucizumab or PCCs in porcine blood in vitro [1]. However, the impact on clinical parameters such as blood loss is not known. Thus, this study assessed the effi cacy of idarucizumab in comparison with PCC and aPCC in dabigatran-anticoagulated swine following polytrauma on clinically relevant endpoints. Reference 1. Grottke O, et al. Crit Care. 2014;18:R27. p p Results According to LFTEG, polytrauma patients had statistically signifi cant abnormalities in platelet aggregation (intensity of contact coagulation (ICC)), in coagulation (intensity of coagulation drive (ICD), clot maximum density (MA)) and in fi brinolytic activity (index of retraction and clot lysis (IRCL)). ICC in patients with multiple injuries was decreased by 27.51%, ICD was decreased by 34.68%, MA was decreased by 75.16%, IRCL was 91.06% above the norm. Patients of group 1 according to LFTEG had signifi cant changes in all parts of coagulation 24 hours after intensive care. Indicators of platelet hemostasis characterized by persistence of hypoaggregation: ICC was decreased by 24.51%, compared with the norm; parameters of coagulation and fi brinolysis had a reliable trend toward normal and decreasing the activity, the fi brinolysis index reached normal reference values. P350 Results Dabigatran levels were comparable between groups (571 ± 174 ng/ml) and resulted in altered coagulation variables. Blood loss was comparable 12 minutes post trauma between groups (801 ± 49 ml) and increased to 3,816 ± 236 ml in anticoagulated control animals post injury. Idarucizumab treatment reduced total blood loss to 1,086 ± 55 ml (P <0.005 vs. all), aPCC to 1,639 ± 104 ml (P <0.05 vs. control) and PCC to 1,797 ± 80 ml (P <0.05 vs. control) after 5 hours. All animals in the intervention groups survived, whereas control animals died within the observation period (mean survival: 89 minutes, range: 62 to 145 minutes). In histopathology no signs of thromboembolic events were present. Altered coagulation variables returned to baseline levels after idarucizumab application and were also signifi cantly, although inconsistently and to a lesser extent, ameliorated following PCCs. Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model y p Methods After ethical approval, 28 male pigs were administered dabigatran etexilate (30 mg/kg twice daily p.o.) for 3 days. Dabigatran was administered intravenously in anaesthetised animals on day 4 to achieve consistent high concentrations. Animals were randomised to receive idarucizumab (60 mg/kg, n = 7), PCC (50 U/kg; n = 7), aPCC (50 U/kg; n = 7) or placebo (n = 7). Intervention started 12 minutes after bilateral femur fractures and a standardised blunt liver injury. The primary endpoint was blood loss (observation period 300 minutes). Further, histopathology, haemodynamics and several coagulation variables were also assessed. Data were analysed by repeated-measures ANOVA (mean ± SD). Acknowledgements © Crown copyright 2014. Published with the permission of the Dstl on behalf of the Controller of HMSO. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of prehospital versus immediate in-hospital packed red blood cells and fresh frozen plasma (PRBC:FFP) in two models of severe battlefi eld injury. of prehospital versus immediate in-hospital packed red blood cells and fresh frozen plasma (PRBC:FFP) in two models of severe battlefi eld injury. Patients in group 2 had hypoaggregation and hypocoagulation state with increased activity of fi brinolysis: ICC was reduced by 25.62%, ICD decreased by 19.76%, MA was decreased by 22.34%, IRCL was increased by 24.52%. Clinically, patients of group 1 had reduced indicators for infectious complications, reducing the term of mechanical ventilation and reducing the volume of blood transfusions. j y Methods This is a prospective randomised controlled trial using in vivo models of injury conducted in accordance with the Animals (Scientifi c Procedures) Act, 1986. Two injury strands were investigated in 43 terminally anaesthetised Large White pigs: whole body blast exposure (Bl) or no blast (ShBl) plus soft tissue injury and haemorrhage. Thirty minutes later animals were randomly allocated to a 60-minute simulated prehospital hypotensive resuscitation with either PRBC:FFP (1:1 ratio) or 0.9% saline (Early and Late groups respectively). This was followed by 150 minutes of simulated in-hospital resuscitation with a revised normotensive target whereby PRBC:FFP was initiated in the Late group and continued in the Early group.i g Conclusion Patients with multiple injuries have violation in all parts of blood coagulation. The use of prothrombin complex concentrate can reduce the severity of pathological changes in the hemostatic system in patients with polytrauma. Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) g p y O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) Introduction To compare the eff ectiveness of prothrombin complex concentrate and fresh frozen plasma (FFP) in patients with multiple injuries, complicated with coagulopathy bleeding. Introduction To compare the eff ectiveness of prothrombin complex concentrate and fresh frozen plasma (FFP) in patients with multiple injuries, complicated with coagulopathy bleeding. Methods The study involved 51 patients who entered Odessa Regional Hospital with traumatic injuries (concomitant skeletal trauma) complicated with hypocoagulation. Patients were divided into two groups: in the fi rst group (26 patients), as a treatment for coagulopathy, was administered PCC in a dose of 1 ml/kg (25 IU/kg); in the second group (25 patients) was administered FFP in a dose of 15 ml/kg. Evaluation of the functional state of the hemostasis system was carried out using low-frequency thromboelastography (LFTEG) on admission to hospital and 24 hours after the patient’s admission to the ICU. y g Conclusion All medical interventions were associated with reduced blood loss and increased survival. However, idarucizumab, a specifi c antidote to dabigatran, reduced total blood loss more prominently and normalised coagulation parameters to a greater degree as compared with either PCC or aPCC. Reference 1. Grottke O, et al. Crit Care. 2014;18:R27. Reference Benefi cial eff ects of prehospital versus immediate in-hospital blood products during resuscitation in two models of severe military injury S Watts, G Nordmann, C Wilson, A Carter, H Poon, E Kirkman Dstl, Salisbury, UK Critical Care 2015, 19(Suppl 1):P349 (doi: 10.1186/cc14429) Methods Rats received an oral dose of 2.5 mg/kg phenprocoumon. At 15.75 hours post dosing, animals were treated with a single intravenous dose of saline, 4F-PCC (Beriplex® P/N, Kcentra®; CSL Behring) or 3F-PCC (Bebulin® VH; Baxter and Profi lnine® SD; Grifols). Study endpoints included bleeding following tail clip, activated partial thromboplastin Introduction Acute trauma coagulopathy (ATC) is seen in 30 to 40% of severely injured trauma casualties. Early use of blood products is thought to attenuate ATC. This study determined the potential impact S123 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P352 Coagulation support algorithm with rapid TEG and functional fi brinogen TEG in critical bleeding: more results and less time E De Blasio, C Pellegrini, A Federico, V Rocco, M Fumi, Y Pancione, S Sale, D Liberti Hospital G. Rummo, Benevento, Italy Critical Care 2015, 19(Suppl 1):P352 (doi: 10.1186/cc14432) Hospital G. Rummo, Benevento, Italy Critical Care 2015, 19(Suppl 1):P352 (doi: 10.1186/cc14432) Introduction Early coagulation support is essential in massively bleeding patients. A Coagulation Support Algorithm (CSA), integrating rapid TEG (r-TEG) and functional fi brinogen TEG (ff -TEG) could shorten the time to a tailored treatment (Figure 1). Introduction Early coagulation support is essential in massively bleeding patients. A Coagulation Support Algorithm (CSA), integrating rapid TEG (r-TEG) and functional fi brinogen TEG (ff -TEG) could shorten the time to a tailored treatment (Figure 1). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 S124 , pp http://ccforum.com/supplements/19/S1 T d s e s e A e e l , g Table 1 (abstract P353). Results of the cost-eff ectiveness model Total medical Total costs/ Treatment QALYs costs ($) QALY ($) Antibiotics + albumin 2.45 7,628 3,111 Antibiotics 1.48 7,682 5,182 Results Total costs were decreased when using albumin, and the improved survival resulted in an additional QALY for patients on albumin, decreasing the cost per QALY. See Table 1 and Figure 2. Conclusion The use of albumin in the treatment of SPB is cost-eff ective. References 1. Poca M, et al. Clin Gastroenterol Hepatol. 2012;10:309-15. 2. Wells CD, et al. Dig Dis Sci. 2004;49:453-8. P354 Estimation of the latent therapeutic demand for albumin in the USA: a focus on three indications 1 l2 Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 1 (abstract P352). Coagulation Support Algorithm. Figure 1 (abstract P352). Coagulation Support Algorithm. Table 1 (abstract P352). Comparison of time to results Test k-TEG r-TEG r-TEG + ff -TEF SCT r (minutes) 13.8 ± 7.1 2.6 ± 2 – ACT (seconds) 265.7 ± 171.9 – 105.2 ± 46.3 TMA (minutes) 42.6 ± 12.4 25.4 ± 14.1 – CSAT (minutes) 21 ± 7.4 Data presented as mean ± SD. ACT, activated clotting time; CSAT, Coagulation Support Algorithm total time; SCT, standard coagulation tests; TMA, time to maximum amplitude. P352 r: r-TEG versus k-TEG, P = 0.0000003; r-TEG versus SCT, P = 0.000000001; k-TEG versus SCT, P = 0.000000009; TMA: r-TEG versus k-TEG, P = 0.0001; r-TEG versus SCT, P = 0.00000004; k-TEG versus SCT, P = 0.000002; ACT versus r of k-TEG (seconds), P = 0.000004; CSAT versus k-TMA, P = 0.00000005. Table 1 (abstract P352). Comparison of time to results Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Methods A retrospective comparison of the time to available TEG and Standard Coagulation Tests (SCT: INR, aPTTr, fi brinogen level) results in two groups of bleeding and coagulopathic patients using citrate kaolin-TEG (k-TEG) or the CSA protocol (r-TEG/ff -TEG). Statistical analysis was performed with Student’s t test for unpaired samples. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Results Twenty-three patients for each k-TEG and CSA group were compared. The time to available results was shorter using the CSA protocol in comparison with k-TEG (Table 1). The diff erences were both statistically (P <0.00001) and clinically (mean reduction time 21 minutes) signifi cant. SCT needed the longest time to obtain the fi nal results. Table 1 (abstract P353). Results of the cost-eff ectiveness model Total medical Total costs/ Treatment QALYs costs ($) QALY ($) Antibiotics + albumin 2.45 7,628 3,111 Antibiotics 1.48 7,682 5,182 Conclusion The implementation of a CSA, including r-TEG and ff -TEG, could shorten the time to a targeted treatment in critically bleeding patients. References Results Total costs were decreased when using albumin, and the improved survival resulted in an additional QALY for patients on albumin, decreasing the cost per QALY. See Table 1 and Figure 2. C l i Th f lb i i th t t t f SPB i t ff ti 1. Stensballe J, et al. Curr Opin Anesthesiol. 2014;27:212-18. 3. Kashuk JL, et al. Ann Surg. 2010;251:604-14. g g Conclusion The use of albumin in the treatment of SPB is cost-eff ective. f P353 Use of albumin in spontaneous bacterial peritonitis is cost-eff ective A Farrugia1, M Bansal2, P Caraceni3 P353 Use of albumin in spontaneous bacterial peritonitis is cost-eff ective A Farrugia1, M Bansal2, P Caraceni3 1University of Western Australia, Perth, Australia; 2Thought Semantics LLC, Sterling, VA, USA; 3University of Bologna, Italy Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) g 1University of Western Australia, Perth, Australia; 2Thought Semantics LLC, Sterling, VA, USA; 3University of Bologna, Italy Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) P353 1. Poca M, et al. Clin Gastroenterol Hepatol. 2012;10:309-15. 2. Wells CD, et al. Dig Dis Sci. 2004;49:453-8. Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) Introduction Adequate fl uid therapy is essential to the care of septic patients, aiming to optimize oxygen delivery without compromising microcirculation. In recent years, a few studies have suggested that albumin may be superior when compared with crystalloids in severe cases of septic shock. However, there are no data in the fi rst hours of resuscitation. The aim of this study is to evaluate whether albumin 4% solution compared with lactated Ringer decreases 30-day mortality in cancer patients with septic shock. p p Methods The Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study is a prospective, randomized, double-blind and controlled trial, with 360 patients. Until November 2014, at the Cancer Institute of University of São Paulo, we enrolled 110 patients with cancer and septic shock to receive as resuscitation fl uid in the fi rst 12 hours of ICU an admission bolus of albumin 4% solution or lactated Ringer. The primary outcome was 30-day mortality. Secondary outcomes include ICU mortality, ICU and hospital length of stay, 90-day mortality, daily SOFA score, rates and length of mechanical ventilation, renal replacement, needing of vasopressor drugs, status performance and fl uid balance. Results From 650 eligible patients, 110 patients were included in the study – 50 patients in the albumin group and 60 in the Ringer group. The mean age was 63 (57 to 70) years in the albumin group and 61 (51 to 71) in the Ringer group, P = 0.508. Most patients were male (58% in the albumin group vs. 56.1% in the Ringer group, P = 0.846). The ECOG was similar between the albumin and Ringer groups ((0) 26% vs. 8%, (1) 38% vs. 36.8%, (2) 20% vs. 38.6%, (3) 16% vs. 15.8%, P = 0.05). The SAPS 3 admission score was 51 ± 13 in the albumin group and 49 ± 10 in the Ringer group, P = 0.492. The total amount of administered fl uid in the fi rst 12 hours of resuscitation was 1,000 ml (1,000 to 1,500) in the albumin group and 1,000 ml (1,000 to 1,000) in the Ringer group, P = 0.59. The 12-hour fl uid balance was 1,053 ml (385 to 1,700) in the albumin group and 990 ml (200 to 1,525) in the Ringer group. The 30- day mortality was similar in both groups (60% in the albumin group and 50.9% in the Ringer group, P = 0.34). Reference Reference 1. Caironi P, et al. N Engl J Med. 2014;370:1412-21. Methods A decision analysis model was constructed using Excel. The model is based on the relationships of the epidemiological and clinical factors shown in the infl uence diagram (exemplifi ed in Figure 1 for sepsis). Data for the individual factors were obtained from the literature. One-way sensitivity analysis was used to generate Tornado diagrams (exemplifi ed in Figure 2 for albumin use in sepsis) to determine the relative contribution of diff erent factors to the LTD. Probabilistic sensitivity analysis was used to generate a probability distribution and calculate a mean level for the LTD of each indication. p g p y p E Scotti, M Ferrari, M Chiodi, F Zadek, I Belloni, L Zazzeron, T Langer, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436) E Scotti, M Ferrari, M Chiodi, F Zadek, I Belloni, L Zazzeron, T Langer, L Gattinoni, P Caironi One-way sensitivity analysis was used to generate Tornado diagrams (exemplifi ed in Figure 2 for albumin use in sepsis) to determine the relative contribution of diff erent factors to the LTD. Probabilistic sensitivity analysis was used to generate a probability distribution and calculate a mean level for the LTD of each indication. Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436) g , , y Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436 Results On average, albumin use was calculated as 104 g per 1,000 inhabitants in severe sepsis, 157 g per 1,000 inhabitants in liver diseases and 61 g per 1,000 inhabitants in CABG. This shows a total LTD of 322 g per 1,000 use of albumin in the US annually. Introduction The induction of ECMO may result in metabolic acidosis [1] due to circuit priming with chloride-rich fl uids, and the sudden decrease in plasma strong ion diff erence (SID). This eff ect can be attenuated using balanced solutions with a SID equal to the patient’s plasma bicarbonate concentration (HCO3 –) [2]. We aimed to compare the eff ects of a novel balanced solution (SID equal to patients’ HCO3 –) with those of commonly employed crystalloids for circuit priming in patients undergoing venovenous ECMO. y Conclusion Albumin consumption in the USA currently averages 479 g per 1,000 population [3]. Hence, the LTD of these three evidence-based indications represents 67% of current usage. Further work is needed to assess the LTD for albumin in other, less well-defi ned areas. R f Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) No signifi cant diff erences in the other secondary outcomes were observed between the two groups. Methods The Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study is a prospective, randomized, double-blind and controlled trial, with 360 patients. Until November 2014, at the Cancer Institute of University of São Paulo, we enrolled 110 patients with cancer and septic shock to receive as resuscitation fl uid in the fi rst 12 hours of ICU an admission bolus of albumin 4% solution or lactated Ringer. The primary outcome was 30-day mortality. Secondary outcomes include ICU mortality, ICU and hospital length of stay, 90-day mortality, daily SOFA score, rates and length of mechanical ventilation, renal replacement, needing of vasopressor drugs, status performance and fl uid balance. Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Figure 2 (abstract P354). Relative importance of inputs into the LTD model for sepsis. g Conclusion In cancer patients with septic shock, resuscitation with albumin 4% as compared with lactated Ringer did not improve the rate of survival at 30 days. allocation, but many of the clinical and epidemiologic variables are subject to uncertainty. Decision analysis [2] may assist in generating an assessment of the demand for albumin. 2. Goel V. CMAJ. 1992;147:413-7. Estimation of the latent therapeutic demand for albumin in the USA: a focus on three indications g y g y Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) Introduction Assessing the cost-eff ectiveness of therapeutic interven- tions is increasingly crucial for health decision-making. Spontaneous bacterial peritonitis (SBP) is one of the major complications of liver cirrhosis. The use of albumin in conjunction with antibiotics has been shown to be eff ective through clinical trials [1]. Introduction The use of albumin in therapeutics is controversial in several areas and requires assessment based on evidence for eff ective resource allocation. Supported indications include sepsis, areas of hepatic diseases and coronary artery bypass grafts (CABG). Latent therapeutic demand (LTD) [1] is the underlying evidence-based demand ensuring ample supplies of drugs are available and aff ordable. Estimating the LTD would assist decision-making and resource Methods A decision tree (TreeAge®) (Figure 1) was populated from published sources for clinical, cost and epidemiologic variables. The perspective taken was that of the US payer. The robustness of the model was checked using one-way and probabilistic sensitivity analyses. The clinical course was followed for 3 months or until death. Total medical costs and quality-adjusted life years (QALYs) [2] were calculated. S125 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury Conclusion During the resuscitation phase of the burn patients, the use of HES (130/0.4/6%) at low doses does not seem to cause more risk or injury according to RIFLE or AKIN criteria than those reported by studies in burn patients resuscitated without HES. However, the need for RRT is associated with a high mortality, although in many cases the display is terminal. Introduction The aim of our study is to investigate the eff ect of intraoperative use of gelatin in living donor liver transplantation on postoperative acute kidney injury (AKI). It has been demonstrated that ischemia and chloride-liberal fl uid management cause AKI in liver transplantation [1]. Gelatin has minimal side eff ects on renal functions [2]; however, it might be a reason for postoperative AKI. g Methods A total of 154 liver transplantation patients were retrospectively evaluated between September 2011 and September 2013, and among these, 128 patients were included in the study. The patients who were under 18 years old, transplanted from cadaveric donors and needed preoperative renal replacement therapy were excluded. The patients were divided into two groups as GI (without gelatin administration) and GII (with gelatin administration). The patient’s age, gender, actual body weight, diagnoses, MELD score, APACHE II score, duration of operation, total clamping time, noradrenalin infusion rate, amount of erythrocyte suspension, fresh frozen plasma (FFP) and thrombocyte suspension used, intraoperative fl uid balance, intraoperative and total clamping diuresis, serum creatinine levels on the postoperative 1st, 2nd, 4th and 7th days, duration of mechanical ventilation, length of ICU and hospital stay, hospital and 1-year mortality rate were recorded. The changes in creatinine levels on the 1st, 2nd, 4th and 7th days were evaluated according to the KDIGO guideline for AKI [3]. Infl uence of anaesthetic factors on skin graft viability in a burns ICU C Isitt, KA McCloskey, A Cabello, P Sharma, MP Vizcaychipi Chelsea and Westminster Hospital, London, UK Critical Care 2015, 19(Suppl 1):P359 (doi: 10.1186/cc14439) Introduction Graft failure is a major cause of morbidity in patients with burns, resulting in increased length of hospital stay and increased number of operations. At our regional burns unit we collated the data from anaesthetic charts of patients admitted to our burns ICU who required skin grafting. The aim was to analyse whether any anaesthetic variables contribute to graft failure. g Methods Thirty-fi ve patients were included in the analysis with a total of 191 operations. References Methods We randomly assigned patients with acute respiratory failure in need of ECMO to receive either NaCl 0.9% (NS, SID = 0), Ringer lactate (RL, SID = 28), or a novel balanced solution (Solution X, SID equal to the patient’s HCO3 –) for circuit priming solution. Arterial blood gases and laboratory parameters were collected at 0, 5, 30, 60, 90, and 1. Stonebraker J, et al. J Clin Immunol. 2014;34:233-44. 2. Goel V. CMAJ. 1992;147:413-7. 3. Market Research Bureau. The plasma proteins market in the USA (2013). http://marketingresearchbureau.com/list-of-reports/ the-plasma-proteins-market-in-the-united-states/. S126 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated. total clamping time was longer and amount of blood products used during surgery was more than the other group. Which of these factors is associated with AKI has to be revealed with further studies. 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated. total clamping time was longer and amount of blood products used during surgery was more than the other group. Which of these factors is associated with AKI has to be revealed with further studies. Results We enrolled 20 patients (23 priming procedures – RL, n = 8; NS, n = 8; Solution X, n = 7). ECMO was initiated for ARDS (45%), bridge to lung transplant (25%), acute graft failure after transplant (15%), and acute on chronic respiratory failure (15%). Average priming volume was 10 ± 5 ml/kg; patients’ baseline HCO3 – was 28 ± 6 mEq/l. During the fi rst 2 hours after ECMO initiation, arterial pH raised similarly in all groups (P = 0.39) due to CO2 removal. In contrast, BE decreased starting after 5 minutes in both the NS and RL groups (BE variation, –2.2 ± 1.7 and –1.9 ± 1.3 mEq/l, P <0.001 vs. baseline; P = 0.04 for interaction, two-way ANOVA, 2-hour period). No BE changes were observed in the Solution X group (0.3 ± 0.8 mEq/l). In the NS group, BE reduction was associated with a reduction in SID (from 39 ± 8 to 34 ± 6 mEq/l at 5 minutes, P = 0.008), entirely due to an increase in Cl (103 ± 7 vs. 108 ± 6 mEq/l, P = 0.001). References In the RL group, BE and SID reductions (40 ± 8 vs. 36 ± 8 mEq/l, P = 0.008) were associated with an increase in both Cl (105 ± 7 vs. 107 ± 7 mEq/l, P = 0.01) and lactate (1.4 ± 0.6 vs. 2.2 ± 1.0 mEq/l, P = 0.008). No changes were observed in other electrolyte concentrations. Dilution did not diff er between groups (P = 0.25 for Atot variation). The acidifying eff ect of NS and RL was amplifi ed in patients with higher baseline HCO3 –. References 1. Nadeem A, et al. Crit Care. 2014;18:625. 2. Eremenko AA, et al. Anesteziol Reanimatol. 2001;3:58-61. 3. KDIGO AKI Work Group. Kidney Int. 2012;2 Suppl:1–138. 1. Nadeem A, et al. Crit Care. 2014;18:625. 2. Eremenko AA, et al. Anesteziol Reanimatol. 2001;3:58-61. 3. KDIGO AKI Work Group. Kidney Int. 2012;2 Suppl:1–138. Incidence of acute kidney injury in critically burned patients resuscitated with crystalloid and colloid according to parameters of transpulmonary thermodilution, diuresis and lactic acid P Extremera Navas, M Sanchez Sanchez, I Pozuelo Echegaray, A Agrifoglio Rotaeche, A Robles Caballero, A García de Lorenzo Hospital Universitario la Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P358 (doi: 10.1186/cc14438) Introduction The purpose was to study the incidence of acute kidney injury (AKI) according to RIFLE and AKIN criteria in critically ill burn patients resuscitated with Ringer’s solution and supplements of lower molecular weight hydroxyethyl starch (HES)130/0.4/6%, and to determine the relationship between RRT indication and mortality. 3 Conclusion As compared with NS and RL, the use of a novel balanced solution with a SID equal to the patient HCO3 – level for ECMO priming uniquely avoids the addition of metabolic acidosis to patients with uncompensated hypercapnia. Methods We studied 165 consecutive patients admitted to the critical care burn unit. Resuscitation was performed using lactated Ringer’s solution and HES at a low dose to achieve urine output, lactate levels, and transpulmonary thermodilution parameters. The contributions of colloids and crystalloids were measured, and renal function was evaluated. Statistical analysis was performed using the Spearman test. Results The average total body surface area (TBSA) burned was 30 ± 15%, and the median of the total volume needed in the fi rst 24 hours was 4.01 ml/kg/% TBSA burned. According to the RIFLE criteria, 10 (6.1%) patients presented with risk, 11 (6.7%) presented with injury, and 11 (6.7%) presented with failure. References 1. Liskaser, et al. Anesthesiology. 2000;93:1170-3. 2. Langer, et al. Intensive Care Med. 2012;38:686-93. 1. Liskaser, et al. Anesthesiology. 2000;93:1170-3. 2. Langer, et al. Intensive Care Med. 2012;38:686-93. References According to the AKIN criteria: 9.7% presented stage I, 3% stage II and 10.3% stage III. Replacement therapy (RRT) was performed in 15 patients (9.1%). In six of these patients RRT was employed in the fi nal stages of multiorgan failure. In the remaining nine patients, for various reasons only one survived. P357 Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury HK Atalan1, B Gucyetmez2, S Aslan1, M Berktas3, KY Polat1 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P357 (doi: 10.1186/cc14437) Association of elevated levels of plasma chloride, in severity and mortality, in adult patients in the ICU M Aguilar Arzapalo Hospital O’Horan, Mérida, Mexico Critical Care 2015, 19(Suppl 1):P360 (doi: 10.1186/cc14440) Introduction For a long time, many investigators have tried to demon- strate increased mortality associated with acid–base distur bances. In this study, we sought to determine the association of hyperchloremia measured at ICU admission and whether this electrolyte disturbance is associated with an increase in morbidity and mortality. Methods A retrospective study was conducted. Patients with a diagnosis of AKI according to KDIGO creatinine criteria and available urinary chemistry at one point during their ICU stay were evaluated. Day 0 was defi ned as the day when SIDu was calculated from urinary spot analysis (SIDu = Na+U + K+U – Cl–U). Patients were followed and staged for AKI in the next 3 days. AKI reversibility was defi ned according to the lack of criteria for AKI. y y Methods Data were retrospectively collected for consecutive adult patients admitted to Agustin O’Horan Hospital ICU, between January 2011 and July 2014, who underwent inpatient medical treatment using electronic fi les. Results In total, 143 critically ill patients with a diagnosis of AKI were included. SIDu at day 0 did not diff er between diff erent AKI stages at day 0. SIDu at day 0 was statistically diff erent between diff erent AKI stages at days 1, 2, 3 (Table 1). SIDu at day 0 was statistically diff erent between reversible and not reversible AKI at days 1, 2, 3 (Table 2). A conventional receiver-operating curve was generated to assess the accuracy of SIDu to predict AKI reversibility at day 1. AUC for SIDu was 0.82 (P <0.0001; 95% CI: 0.75 to 0.88). i Results The dataset consisted of 936 medical fi les and serum chloride concentration values on admission, 853 being eligible. Hyperchloremia (serum chloride >110 mmol/l) is quite common, with an incidence of 47.71%. Patients were propensity matched based on their association with death and hyperchloremia. Of the 853 patients collected, patients with hyperchloremia after admission (n = 446, 52.3%), patients were matched to patients who had normal serum chloride levels after admission. These two groups were well balanced with respect to all variables collected. The hyperchloremic group was at increased risk of mortality at ICU discharge, relative risk ratio = 1.81; 95% confi dence interval, 1.41 to 2.51 risk increase of 25.31%. Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury These were a combination of debridement, split skin grafts (SSG) and change of dressings. All patients were admitted to our burns ICU between January 2009 and October 2013. Exclusion criteria were death prior to discharge and initial surgery at a diff erent hospital. Sixteen patients had good graft viability (Group A) and 19 patients had poor graft viability (Group B). Logistical regression was performed using SPSS (Version 22.0). Hosmer and Lemeshow testing was used to confi rm goodness of fi t. Independent variables were age, sex, preoperative haemoglobin, intraoperative fl uid resuscitation, blood products, inotropes, volatile agents and temperature. Poor graft viability was defi ned as requiring at least one additional skin graft. Analysis was performed on all operations and then by subtype of operation (that is, SSG and debridement, SSG only). Results In total, 128 patients were categorized as GI (58, 45%) or GII (70, 55%). Total clamping time, intraoperative diuresis, intraoperative crystalloid use, intraoperative fl uid balance, operation bleeding, erythrocyte suspension, FFP and thrombocyte suspension use and postoperative lactate levels of GII were statistically signifi cantly higher than GI (P <0.001 for each). According to the KDIGO guideline, AKI in GII on the 1st, 2nd, 4th and 7th days (11.4%; 20%; 24.3%; 17.1%) was statistically signifi cantly higher than GI (P <0.001 for each). Conclusion In patients who received gelatin, kidney dysfunction in the postoperative period was observed more frequently. Also in this group, S127 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results There was no signifi cant diff erence in age, %total burn surface area or Belgian Outcome Burns Injury score between the groups. For all operation data, use of colloids was found to signifi cantly contribute towards poor graft viability (P = 0.035, 95% CI). When analysis was performed on only SSG and debridement operations, colloids remained signifi cant (P = 0.034, 95% CI) and metarminol use was found to signifi cantly contribute (P = 0.028, 95% CI) to poor graft viability. Overall use of inotropes was not signifi cant between the two groups. Other variables including minimum and maximum temperature, preoperative haemoglobin and blood transfusion were not found to be signifi cant. References 1. Gauthier PM, Szerlip HM. Metabolic acidosis in the intensive care unit. Crit Care Clin. 2002;18:289-308. 1. Gauthier PM, Szerlip HM. Metabolic acidosis in the intensive care unit. Crit Care Clin. 2002;18:289-308. 2. Eisenhut M. Causes and eff ects of hyperchloremic acidosis. Crit Care. 2006;10:413. 2. Eisenhut M. Causes and eff ects of hyperchloremic acidosis. Crit Care. 2006;10:413. 3. Wooten EW. Science review: quantitative acid–base physiology using the Stewart model. Crit Care. 2004:8:448-52. 3. Wooten EW. Science review: quantitative acid–base physiology using the Stewart model. Crit Care. 2004:8:448-52. 36 Urinary strong ion diff erence and acute kidney injury: an early marker of renal dysfunction? i Conclusion Our results suggest that the use of colloids is a contributor to poor graft viability in burns. This was found to be independent of temperature and overall inotrope use; however, the use of metarminol may be a contributing factor. y P Balsorano1, A De Gaudio1, Stefano Romagnoli1, Ipsita Krishnan2 1AOUC Careggi, Florence, Italy; 2Rhode Island Hospital, Providence, RI, USA Critical Care 2015, 19(Suppl 1):P361 (doi: 10.1186/cc14441) Introduction Kidneys play a crucial role in the regulation of electrolytes and acid–base homeostasis. Impaired renal function is associated with greater urinary strong ion diff erence (SIDu) in patients with metabolic acidosis [1]. In critically ill patients, several factors, such as infused fl uids and acid endogenous production, would lead to changes in plasma SID and acid–base homeostasis without renal regulation of urinary electrolytes and SIDu [2]. Hence, AKI can be highlighted as an inability to address acid–base metabolic disturbances, which may be detected before major increases in creatinine or decreases in urine output. We evaluated the eff ects of renal function on urinary strong ion excretion using the Stewart approach to acid–base in critically ill patients with AKI. P360 Association of elevated levels of plasma chloride, in severity and mortality, in adult patients in the ICU Admission hyperchloremia was associated with increased morbidity, mortality and higher scores in severity scales; this association was statistically important. See Figure 1. Conclusion This retrospective cohort trial demonstrates an association between hyperchloremia and poor ICU admission outcome (death). Additional studies are required to demonstrate a causal relationship between these variables. Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission AKI stage 3 2 1 0 P value Day 1 48.1 (21) 46 (22) 37.9 (20) 17.3 (22) <0.001 Day 2 40.2 (23) 45.9 (20) 45 (23) 29 (22) 0.004 Day 3 40.3 (26) 47.2 (18) 53.2 (23) 31 (23) 0.006 Table 2 (abstract P361). SIDu (mEq/l) between reversible versus not reversible AKI at days 1, 2, 3 Reversible Not reversible P value Day1 16.8 (23) 43.9 (21) 0.0001 Day2 28.5 (24) 45.3 (22) 0.0001 Day3 30 (24) 47.3 (21) 0.0001 Conclusion SIDu identifi ed patients with reversible AKI with good accuracy. SIDu can be a promising, simple and cost-eff ective tool in AKI patient evaluation. Further research is needed to assess SIDu capability to early detect patients with renal dysfunction before increases in creatinine or decreases in urine output. References 1. Moviat M, et al. J Crit Care. 2012;27:255-60. 2. Masevicius FD, et al. Crit Care Resusc. 2010;12:248-54. Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission AKI stage 3 2 1 0 P value Day 1 48.1 (21) 46 (22) 37.9 (20) 17.3 (22) <0.001 Day 2 40.2 (23) 45.9 (20) 45 (23) 29 (22) 0.004 Day 3 40.3 (26) 47.2 (18) 53.2 (23) 31 (23) 0.006 Table 2 (abstract P361). SIDu (mEq/l) between reversible versus not reversible AKI at days 1, 2, 3 Figure 1 (abstract P360). Group mortality, high and low chlorine. Reference Reference 1. Ridley SA, et al. Anaesthesia. 2004;59:1193-200. 1. Ridley SA, et al. Anaesthesia. 2004;59:1193-200. P366 Incidence and predisposing factors for the development of disturbed glucose metabolism and diabetes mellitus after intensive care admission: the DIAFIC study S Van Ackerbroeck, K Janssens, P Jorens, T Schepens, W Verbrugghe, V Van Hoof, L Van Gaal, C De Block University Hospital Antwerp, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P366 (doi: 10.1186/cc14446) Low serum 25-hydroxyvitamin D at critical care initiation is associated with sepsis and morbidity in Dutch critically ill patients K De Haan Erasmus MC, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P365 (doi: 10.1186/cc14445) Low serum 25-hydroxyvitamin D at critical care initiation is associated with sepsis and morbidity in Dutch critically ill patients K De Haan Erasmus MC, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P365 (doi: 10.1186/cc14445) Introduction Vitamin D defi ciency may frequently occur in critically ill patients and may be associated with sepsis and increased mortality. We therefore evaluated the prevalence of 25-hydroxyvitamin D defi ciency in a Dutch ICU, and its relationship with sepsis, morbidity and mortality. Methods We conducted a prospective observational study in a 10-bed mixed ICU. A total of 1,372 patients were admitted between July 2011 and June 2013 including 198 readmissions, of which 940 patients were studied. 25-Hydroxyvitamin D levels were determined within 24 hours after admission. 25-Hydroxyvitamin D levels were judged as suffi ciency (>50 nmol/l), insuffi ciency (30 to 50 nmol/l) and defi ciency (<30 nmol/l). Results The prevalence of defi ciency and insuffi ciency was 36% and 38%, respectively. Only 26% of the patients had suffi cient vitamin D levels. Vitamin D defi ciency is associated with sepsis (P <0.001) at ICU admission. Patients with defi cient levels had higher mean APACHE IV scores, 64 versus 52 (P <0.001), and longer length of hospital stay, 12 versus 9 days (P <0.001), respectively, as compared with patients with suffi cient levels. Patients with defi cient vitamin D levels had an odds ratio for in-hospital mortality of 1.4 (95% confi dence interval of 0.84 to 2.29, P = 0.2) relative to patients with suffi cient vitamin D levels.i Conclusion Implementation of i.v. potassium replacement guidelines improved the use of i.v. potassium in the ICU by reducing the requirement for i.v. potassium supplementation and increasing the overall time patients spent without hypokalaemia. Whilst nursing staff found the guideline useful and felt it increased safe use of i.v. potassium, more work is needed to ensure nurse workload is not increased signifi cantly. fi Conclusion 25-Hydroxyvitamin D defi ciency frequently occurs in Dutch critically ill patients. Although relating to sepsis, disease severity and morbidity, vitamin D defi ciency is not an independent predictor of mortality in these patients, which was otherwise relatively low. References 1. Moviat M, et al. J Crit Care. 2012;27:255-60. Masevicius FD, et al. Crit Care Resusc. 2010;12:248-54. S128 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P362 Evaluation of the eff ect of guidelines to reduce intravenous potassium infusions in ICU patients MC Law Min1, RS Bourne1, S Burd2, M Stone2 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2De Montfort University, Leicester, UK Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) P362 P362 Evaluation of the eff ect of guidelines to reduce intravenous potassium infusions in ICU patients MC Law Min1, RS Bourne1, S Burd2, M Stone2 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2De Montfort University, Leicester, UK Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) almost every patient and at every studied time point. Moreover, these levels were signifi cantly higher than in controls or compared with referenced literature. The chronology of exposure was demonstrated: the preoperative urine and serum levels of the DEHP metabolites were often below the detection limit. Medical devices are the source of these chemicals: patients on hemofi ltration, extracorporeal membrane oxygenation or both showed serum levels 100-fold or 1,000-fold higher than the general population or workers in plastic industry. The serum and some of the urinary levels of the DEHP metabolites are the highest ever reported in humans; some at biologically highly relevant concentrations of even ≥10 to 50 μM. almost every patient and at every studied time point. Moreover, these levels were signifi cantly higher than in controls or compared with referenced literature. The chronology of exposure was demonstrated: the preoperative urine and serum levels of the DEHP metabolites were often below the detection limit. Medical devices are the source of these chemicals: patients on hemofi ltration, extracorporeal membrane oxygenation or both showed serum levels 100-fold or 1,000-fold higher than the general population or workers in plastic industry. The serum and some of the urinary levels of the DEHP metabolites are the highest ever reported in humans; some at biologically highly relevant concentrations of even ≥10 to 50 μM. Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) Introduction The aim was to evaluate whether guidelines for intra- venous (i.v.) potassium replacement improved plasma potassium homeostasis in ICU patients. Prompt and eff ective treatment of hypokalaemia is an important intervention in the ICU, but concentrated i.v. potassium solutions may cause serious harm if used inappropriately [1]. There were previously no formalised guidelines on i.v. potassium supplementation in the ICU at Sheffi eld Teaching Hospitals. Practice was reviewed and guidelines were introduced to improve patient safety, plasma potassium homeostasis and reduce i.v. potassium supplementation requirements. Conclusion Adult ICU patients are exposed to plastic softeners, in particular PMs. Despite the continuously tightening regulations, BPA and DEHP are still present in medical devices. Because patient safety is a concern in the ICU, further research into the (possibly toxic and clinical) eff ects of chemicals released from medical devices should be undertaken. pp q Methods A before and after evaluation of plasma potassium homeo- stasis in ICU patients requiring i.v. potassium supplementation was conducted over a period of 8 months (August 2013 to May 2014). Patient data on plasma potassium levels, i.v. and oral potassium supplements administered were obtained from the clinical information system. Clinical appropriateness of i.v. potassium acetate prescriptions, fl uid and chloride intake related to potassium infusions and cost linked to the guidelines were also compared pre/post implementation. Impact of the guidelines on nurses’ practice was assessed using questionnaires. Results Median i.v. potassium replacement dose per patient was signifi cantly reduced in the post-guidelines group from 215 (IQR: 94; 485) to 80 (IQR: 40; 160) mmol; P <0.001. Although the percentage time per group for patients who were hypokalaemic was less in the post group (18.2% vs. 14.8%), there was no diff erence in mean patient values (24.2 (20.3)% vs. 22.1 (17.5)%; P = 0.228). The duration of hyperkalaemia was increased. Prescribing of i.v. potassium acetate was not always appropriate. Median patient fl uid-related dose was increased (107.5 (IQR: 47.1; 242.4) vs. 250 (IQR: 100; 600) ml; P <0.001), whilst chloride doses were reduced (170.7 (IQR: 91.3; 438.3) vs. 110 (IQR: 55; 250) mmol; P <0.009). Nurses were satisfi ed with the new practice, reporting it was safe, eff ective and clinically useful. However, compared with baseline practice, they perceived the guidelines as less eff ective and felt the workload was higher. Associations between the degree of correction of hypoglycemia and ICU mortality g g Results The median glucose in the EndoTool group (141.5 mg/dl) was lower than in the Adult ICU group (159.9 mg/dl) (P <0.0001). The standard deviation of glucose in the EndoTool group (32.3 mg/ dl) was lower than the Adult ICU group (39.5 mg/dl) (P = 0.0001). The proportion of patients in each group with 10% or higher of measurements at a severe hyperglycemia level (≥200 mg/dl) in the EndoTool group (35.2%) was lower than the Adult ICU group (64.1%) (P <0.0001). The proportion of patients who had at least one moderate hypoglycemic measurement (<70 mg/dl) was not signifi cantly diff erent between the EndoTool group versus the Adult ICU group (11.73% vs. 9.3%, respectively; P = 0.34). However, there was a higher overall incidence of hypoglycemia in the EndoTool group (5.65 hypoglycemic measurements/100 person-protocol days) compared with the Adult ICU group (3.43/100 person-protocol days) (RR = 1.65, 95% CI = 1.09 to 2.45, P = 0.014). Severe hypoglycemia (<40 mg/dl) was rare, only occurring in 1/179 (0.56%) in the EndoTool group and 4/580 (0.69%) in the Adult ICU group. R Van Hooijdonk1, JM Binnekade1, A Abu-Hanna1, j , , , F Van Braam Houckgeest2, LS Hofstra3, J Horn1, MA Kuiper4, f F Van Braam Houckgeest2, LS Hofstra3, J Horn1, MA Kuiper4, f NP Juff ermans1, HL Van den Oever5, JP Van der Sluijs6, PE Spronk7, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Tergooi Hospitals, Hilversum, the Netherlands; 3Scheper Hospital, Emmen, the Netherlands; 4Medical Centre Leeuwarden, the Netherlands; 5Deventer Hospital, Deventer, the Netherlands; 6Medical Center Haaglanden, The Hague, the Netherlands; 7Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P367 (doi: 10.1186/cc14447) Introduction It is conjectured that transition of hypoglycemia to hyperglycemia may be more harmful than hypoglycemia itself. We investigated the association between the degree of correction of hypoglycemia and ICU mortality in patients under moderately strict to strict glycemic control. Methods This is a retrospective analysis from a pooled cohort from seven ICUs in the Netherlands over 6 years. ICU patients who developed hypoglycemia (<70 mg/dl) were included. We excluded patients who were readmitted, and patients with hypoglycemia in whom no follow- up blood glucose measurement was performed within 8 hours. Marked exposure to the endocrine-disrupting chemicals phthalates and bisphenol A in the ICU J Huygh, P Jorens Antwerp University Hospital, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P364 (doi: 10.1186/cc14444) J Huygh, P Jorens J Huygh, P Jorens Introduction Care for ICU patients has benefi ted from medical devices. Bisphenol A (BPA) and phthalates can leach from the plastic matrix. We hypothesized that ICU patients are exposed to BPA and phthalates through medical devices. Introduction Stress hyperglycaemia (SH) is commonly observed during hospitalisation in the ICU and adversely infl uences outcome [1]. When SH occurs in previously nondiabetic patients, this might refl ect a latent disturbance of glucose metabolism and predict future risk of diabetes. We wanted to assess the incidence of disturbed glucose metabolism (DGM) and identify predictors for future diabetes risk. This could support timely diagnosis, prevention, and early treatment of impending diabetes mellitus (DM). Methods Serum (n = 118) and urinary (n = 102) samples of adult (n = 35) ICU patients were analyzed for total BPA and di(2-ethylhexyl)phthalate (DEHP) and other phthalate metabolites (PMs). We also enrolled patients preoperatively before scheduled thoracic surgery and repeat samples were taken on days 1 to 4 during the ICU stay. Control data came from 44 healthy controls or from referenced literature. Methods In this prospective observational study, we enrolled 338 patients without known DM, who were admitted for at least 36 hours to the ICU of the Antwerp University Hospital between September 2011 and March 2013. A 75 g oral glucose tolerance test was performed Results Our results show that adult ICU patients are continuously exposed to phthalates (that is, DEHP) as well as to BPA, albeit to a lesser extent, resulting in detectable serum and urinary levels in S129 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Not the transition to hyperglycemia, but insuffi cient recovery from hypoglycemia is associated with an increased ICU mortality in patients under moderately strict or strict glucose control with insulin. 6 to 9 months post ICU admission to screen for disturbed glucose metabolism. Furthermore, we examined whether post-discharge glucose disturbances could be predicted by the FINDRISC questionnaire [2], patient demographics, comorbidities, HbA1c at ICU admission, and by parameters related to ICU stay (glucose parameters, insulin need, caloric intake, disease severity). y Results In total, 246 patients (73%) experienced SH during their ICU stay. Eight months post ICU admission, glucose metabolism was disturbed in 119 (35%) subjects. Computer versus paper insulin protocol for managing hyperglycemia in three ICUs A Peckham Oregon Health & Science University, Portland, OR, USA Critical Care 2015, 19(Suppl 1):P368 (doi: 10.1186/cc14448) Computer versus paper insulin protocol for managing hyperglycemia in three ICUs A Peckham Oregon Health & Science University, Portland, OR, USA Critical Care 2015, 19(Suppl 1):P368 (doi: 10.1186/cc14448) Introduction The purpose of this study was to compare a computer protocol against a paper protocol in managing three domains of glucose control. Hyperglycemia is common in critically ill patients, and their risk of death is associated with hyperglycemia, hypoglycemia, and glucose variability. A safe and eff ective insulin protocol must minimize hyperglycemia and glucose variability while also avoiding hypoglycemia. Computer-based insulin protocols promise better performance by adjusting to each individual’s sensitivity to insulin. Conclusion Stress hyperglycaemia is frequent in nondiabetic patients and has a tendency towards future disturbances in glucose metabolism and DM. Glucose metabolism was disturbed in 35% of subjects 8 months post ICU admission, of whom 7% was diagnosed with diabetes mellitus. Predictors of elevated risk included a high FINDRISC score, high SAPS 3 score, and a lower daily caloric intake during ICU stay. R f Methods This is a historical cohort study with 759 patients admitted to three ICUs (medical/cardiac, trauma, and neuroscience) at an academic tertiary care hospital. All adult patients from January 2012 to October 2013 on one of two continuous insulin protocols for at least 8 hours were included. At the start of the study period the paper protocol in use (Adult ICU) had a target glucose of 140 to 180 mg/dl and was used for any patient with a glucose higher than 180 mg/dl. In June 2013 this was replaced by a computer-based insulin protocol (EndoTool) that had the same criteria for initiation and had a target glucose of 150 mg/dl. The primary exposure was the insulin protocol, and the primary outcome was performance in maintaining glucose control. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Schwarz PE, et al. Horm Metab Res. 2009;41:86-97. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Schwarz PE, et al. Horm Metab Res. 2009;41:86-97. Associations between the degree of correction of hypoglycemia and ICU mortality We determined the association between three measures of correction of hypoglycemia within 8 hours after hypoglycemia and ICU mortality: predefi ned ranges of the ‘highest blood glucose level’ (<80 mg/dl; 80 to 110 mg/dl; 110 to 150 mg/dl (reference category); 150 to 180 mg/ dl; and >180 mg/dl); quartiles of the ‘delta glucose’, defi ned as the diff erence between minimum and maximum blood glucose level with the third quartile as reference category; and quartiles of the ‘standard deviation’ of the blood glucose level with the third quartile as reference category. Conclusion Patients on the computer protocol had a lower median glucose, less variability, and less hyperglycemia than patients on the paper protocol. There was a higher risk of moderate but not severe hypoglycemia in the computer group. Marked exposure to the endocrine-disrupting chemicals phthalates and bisphenol A in the ICU J Huygh, P Jorens Antwerp University Hospital, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P364 (doi: 10.1186/cc14444) Of these, 27 (8%) had impaired fasting glucose, 43 (13%) had impaired glucose tolerance, 25 (7%) had impaired fasting glucose and impaired glucose tolerance, and 24 (7%) were diagnosed with DM. A disturbed glucose metabolism tended to be more prevalent in subjects who experienced SH during ICU stay as compared with those without SH (38% vs. 28%, P = 0.065). HbA1c on admission correlated with the degree of SH (r = 0.308, P <0.001). The FINDRISC score (9.5 vs. 11, P = 0.001), SAPS 3 score (median of 42 in both groups, P = 0.003) and daily caloric intake during ICU stay (222 vs. 197, P = 0.011) were associated with a DGM. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf yp g y y Results Of the 1,059 patients admitted with sepsis, 526 (55.8%) had admission glucose levels within the normal range, 270 (25.5%) had mild hyperglycemia and 202 (19.1%) severe hyperglycemia. Patients with severe hyperglycemia were older, had higher APACHE IV scores and were more often diabetics compared with euglycemic patients. Shock on admission was more common in patients admitted with euglycemia. Crude mortality increased with increased admission glucose and a Cox regression analysis showed increased risk for 30- day (HR = 1.67, CI = 1.24 to 2.23), 60-day (HR = 1.42, CI = 1.08 to 1.87) and 90-day (1.31, CI = 1.02 to 1.70) mortality in patients admitted with severe hyperglycemia compared with euglycemia. The association between mortality and severe hyperglycemia on admission was only present in patients without known diabetes but not in patients with a history of diabetes (30-day mortality HR = 1.67, CI = 1.15 to 2.43 vs. 1.84, CI = 0.97 to 3.49). Severe hyperglycemia was associated with a blunted proinfl ammatory cytokine response (IL-6 and IL-8) on admission in patients without, but not in patients with diabetes. g y p R Van Hooijdonk, JH Leopold, T Winters, JM Binnekade, NP Juff ermans, J Horn, JC Fischer, EC Van Dongen-Lases, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P370 (doi: 10.1186/cc14450) Introduction There is a need for continuous glucose monitoring in critically ill patients. The objective of this trial was to determine the point accuracy and reliability of a device designed for continuous monitoring of interstitial glucose levels in ICU patients (Sentrino; Medtronic MiniMed, Northridge, CA, USA). Methods Critically ill patients with an anticipated life expectancy >96 hours were eligible for participation, if the platelet count was >30 × 1012 ml. Device readings were compared with glucose measurements in arterial blood using blood gas analyzers (RapidLab Siemens Healthcare Diagnostics, The Hague, the Netherlands). We used a linear mixed model to determine which factors aff ect point accuracy. In addition, we determined the reliability, including duration of device start-up and calibration, skips in data acquisition, and premature disconnections of sensors. Conclusion Severe hyperglycemia on admission is associated with increased 30-day, 60-day and 90-day mortality in sepsis patients without a history of diabetes mellitus. Results We included 50 patients, aged 65 (56 to 72) years with an APACHE II score of 23 (17 to 26). P370 Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patients R Van Hooijdonk, JH Leopold, T Winters, JM Binnekade, NP Juff ermans, J Horn, JC Fischer, EC Van Dongen-Lases, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P370 (doi: 10.1186/cc14450) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 The number of skips in data acquisition was low, resulting in availability of real-time data during 95 (89 to 98)% of the connection time per sensor. glucose data prove in such routine use?’ Using actual case data, we have shown how comparing the mean absolute relative diff erence (MARD) and integration of the area under the curve (AUC) from the continuous glucose monitoring and intermittent measurement can be used to measure patient risk. Conclusion The point accuracy of the device was relatively low in critically ill patients. The device reliability was relatively good, although sensors were removed prematurely for a variety of reasons. p Methods The analysis used aggregated case data generated from our recent clinical trials, where a GlySure sterile, single-use sensor and dedicated monitoring system was used to measure the blood glucose concentration in patients continuously and in real time. The measurement of risk was compared using the MARD, an accepted error calculation tool, and the AUC was calculated using an AUC analysis software program. g References 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442.i 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442.i 2. Gopal PB, Mulavisala KP. ESICM 2014. Abstract 0264. http://react-profi le.org/ Download/ESICM2014_Abstract_Book_fi nal_version.pdf. P370 Eff ect of admission hyperglycemia in sepsis patients with or without a history of diabetes g Results When MARD from the GlySure sensor and intermittent measurement using the hospital’s existing protocol was compared, the measure of risk to the patient (that is, the uncertainty regarding the patient’s absolute blood glucose status) for the GlySure sensor was 50.5% lower than the intermittent measurement. The results also showed that as the variability of the BG data increases, the benefi t of continuous monitoring increases by signifi cantly reducing patient risk. The continuous monitoring reduces the patient’s risk by 88%, 73%, and 69% respectively in high, medium and low variability situations. Introduction Hyperglycemia is common and often multifactorial in critically ill patients. The association of hyperglycemia with adverse outcome has repeatedly been established in a variety of settings. The objective of this study was to investigate whether hyperglycemia on admission to the ICU impacts presentation and outcome of sepsis patients and whether this eff ect is diff erent for patients with a history of diabetes mellitus. p y g y Conclusion It is more and more evident that continuous glucose technology will be instrumental in driving safe and eff ective glucose management protocols that will support more consistent glycemic management standards within ICUs and across institutions. Methods A two-center, prospective observational cohort study was conducted including all consecutive critically ill patients admitted to the ICU between January 2011 and July 2013. Sepsis patients were identifi ed using strict clinical and diagnostic criteria. The fi rst glucose measurement within a time window of 4 hours before up to 4 hours after ICU admission was categorized into euglycemia (71 to 140 mg/dl), mild hyperglycemia (141 to 200 mg/dl) or severe hyperglycemia (>200 mg/ dl), patients with hypoglycemia were excluded. A multivariable Cox proportional hazard model was used to determine the eff ect of admission hyperglycemia on mortality corrected for covariates. Continuous blood glucose monitoring reduces the risk to ICU patients KP Mulavisala1, J Norrie2, B Crane3, N Barwell3 1CARE Hospitals, Hyderabad, India; 2SumStats Ltd, Edinburgh, UK; 3GlySure Ltd, Abingdon, UK g , Critical Care 2015, 19(Suppl 1):P369 (doi: 10.1186/cc14449) Results In total, 4,516 ICU patients developed at least one episode of hypoglycemia. In three separate multivariate analyses for each of the three measures we adjusted for the respective confounders. The category 80 to 110 mg/dl of the ‘highest blood glucose level’ was associated with increased mortality compared with the reference category (odds ratio (OR) = 1.31, 95% confi dence interval (CI) = 1.06 to 1.61). The lowest quartile of the ‘delta glucose’ (OR = 1.32, 95% CI = 1.03 to 1.69) and the lowest quartile of the ‘standard deviation’ (OR = 1.55, 95% CI = 1.23 to 1.96) were associated with higher ICU mortality than their reference categories. Introduction GlySure Limited (Abingdon, UK) has developed a continuous intravascular glucose monitoring system (CIGMS) to simplify the application of hospital protocols for optimal glucose control at the point of care. We have previously reported on the early results achieved in cardiac patients [1] and MICU patients [2]. This initial success has been sustained and demonstrated in further patient groups. We have now reached the point where we can conjecture upon the regular application of the GlySure CIGMS in day-to-day ICU practice. This in turn prompts the question, ‘How eff ective will continuous blood S130 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P371f Eff ect of admission hyperglycemia in sepsis patients with or without a history of diabetes LA Van Vught1, MA Wiewel1, PM Klein Klouwenberg2, AJ Hoogendijk1, DS Ong2, OL Cremer2, MJ Bonten2, MJ Schultz1, T Van der Poll1 1Academic Medical Center, Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P371 (doi: 10.1186/cc14451) 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf Admission types were medical (62%), elective surgery (22%) and emergency surgery (16%), and 22% had diabetes. For the accuracy analyses we had 929 comparative samples from 100 sensors in 45 patients (11 (7 to 28) samples per patient) during 4,639 hours (46 (27 to 134) hours per patient and 46 (21 to 69) hours per sensor). The Bland–Altman plot showed a bias of –0.6 mg/dl with limit of agreement between –57.2 and 56 mg/dl. Glucose prediction error analysis showed 60% of the glucose values <75 mg/dl within ±15 mg/dl and 75.8% of the glucose values ≥75 mg/dl within 20% of the comparative RapidLab results. Clarke error grid analysis showed 75.3% in zones A and 23.5% of the paired measurements in zones B, 0.3% of the paired measurements in zones C and 0.9% of the paired measurements in zones D. Point accuracy did not meet the ISO14971 standard for dosing accuracy, but improved with increasing numbers of calibrations, and was better in patients who did not have diabetes mellitus. Sixty out of 105 sensors were removed prematurely for a variety of reasons. The device start-up time was 49 (43 to 58) minutes. Acknowledgement This research was performed within the framework the Center for Translational Molecular Medicine (http://www.ctmm.nl), project MARS (grant 04I-201). Point and trend accuracy of continuous glucose monitoring using intravenous microdialysis in critically ill patients JH Leopold, RT Van Hooijdonk, M Boshuizen, T Winters, LD Bos, A Abu-Hanna, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P372 (doi: 10.1186/cc14452) Introduction Insulin infusion in critically ill patients mandates frequent measurements of the blood glucose level [1]. Microdialysis is a well- established technology that off ers the opportunity to sample blood analytes with high accuracy, without the need for drawing blood samples [2,3]. We aimed to determine point and trend accuracy of S131 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 values and arterial glycemia correlated well with 98.6% of data falling in regions A and B of error grid analysis. microdialysis-based continuous glucose monitoring (CGM) (EIRUS®; Maquet Critical Care AB, Solna, Sweden). g g y Conclusion In our study, the use of RT-CGM neither improved glucose control and variability, nor did it reduce hypoglycemic events. y g References 2. Schierenbeck F, et al. Diabetes Technol Ther. 2015;15:26-31. y y p y Results Ninety-nine FP patients were admitted between April 2008 and January 2014. Median age was 73 (IQR 61 to 79), with a female preponderance (53.5%). The median ICU length of stay (LOS) was 5 days (IQR 2 to 16). On admission to critical care, clinical data included (all medians): temperature 36.6°C (IQR 36 to 37.2), systolic blood pressure (BP) 113 mmHg (IQR 104 to 136), diastolic BP 56 mmHg (IQR 49 to 67), lactate 2.3 mmol/l (IQR 1.5 to 3.7), bilirubin 12 μmol/l (IQR 9 to 20), haemoglobin 104 g/l (IQR 93 to 116), haematocrit 31 (IQR 28 to 36), creatinine 88 μmol/l (IQR 66 to 152), prothrombin time 13.1 seconds (IQR 11.9 to 14.4). In 86 patients the initial operation was an emergency laparotomy, with primary perforation in 53 cases. Subsequent anastomotic dehiscence and need for relaparotomy happened in 24 and 33 cases respectively. Forty per cent of patients underwent more than one surgical abdominal intervention. The most common antibiotic used was tazobactam and fl uconazole was the commonest antifungal. The percentages of patients receiving mechanical ventilation, renal replacement therapy and inotropic/vasopressor support during ICU stay were 72.7%, 25.3% and 84.8% respectively. The ICU and hospital mortality rates were 23.5% and 26.1%, respectively, increasing to 26.7% at 28 days, 28.4% at 90 days and 32.2% at 1 year. None of the surgical factors or diabetes infl uenced survival. The strongest independent risk factors associated for ICU mortality were systolic BP on ICU admission (OR = 1.05, 95% CI = 1.01 to 1.09, P = 0.015), acute kidney injury (AKI) within the fi rst 24 hours of ICU admission (OR = 0.15, 95% CI = 0.03 to 0.9, P = 0.026) and lactate on ICU admission (OR = 0.62, 95% CI = 0.39 to 1, P = 0.05). , ; 4. Krouwer JS, et al. J Diabetes Sci Technol. 2010;4:75-83. 5. Clarke WL, et al. Diabetes Care. 1987;10:622-8. 6. Kovatchev, et al. Diabetes Care. 2004;27:1922-8. P373 P373 Real-time continuous glucose monitoring in the ICU J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) P373 Real-time continuous glucose monitoring in the ICU J Gios1, B Manuel-y-Keenoy2, P Rogiers3 J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) J Gios , B Manuel y Keenoy , P Rogiers 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) Introduction Hyperglycemia occurs in 50 to 85% of patients admitted to a medical ICU (MICU) and has been associated with poor prognosis [1,2]. Whether applying intensive insulin therapy to achieve tight glycemic control in critically ill patients is benefi cial remains controversial [2]. Another important observation is a link between glycemic variability and mortality [3]. We performed a pilot study hypothesizing that when implementing intensive insulin therapy, real-time continuous glucose monitoring (RT-CGM) may help to safely achieve tight glucose control, while avoiding hypoglycemia and reducing glycemic variability in MICU patients. Methods This two-center randomized controlled pilot study was performed during a 3-year period. To be included, patients had to be severely ill (APACHE II score ≥20) and CGM monitoring had to be started within 48 hours after admission in the MICU. Thirty-fi ve patients (age 66 ± 10 years; nondiabetic/diabetic patients 27/8; APACHE II score 28 ± 6) were randomly assigned to RT-CGM (n = 16) or to blinded CGM. In both groups a microdialysis-based glucose sensor (GlucoDay®S) was used during a 96-hour period of glucose monitoring. Insulin infusion was performed using a modifi ed Yale protocol. Outcome measures were percentage of time in normoglycemia and in hypoglycemia, glycemic variability, and CGM accuracy. Conclusion In this cohort of critically ill FP patients the ICU and 12-month mortality rates were 23.5% and 32.2%, respectively. The most consistent predictors of mortality across all time points were AKI within 24 hours of ICU admission and admission lactate. fi 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. References 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Kavanagh BP, et al. N Engl J Med. 2010;363:2540-6. 3. Hermanides J, et al. Crit Care Med. 2010;38:838-42. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Kavanagh BP, et al. N Engl J Med. 2010;363:2540-6. 3. Hermanides J, et al. Crit Care Med. 2010;38:838-42. Results Three-hundred and fi fty-four paired samples were obtained from seven patients (66 (59 to 79) years old, APACHE II score 23 (20 to 28), 51 (19 to 77) samples per patient). Point accuracy: 91% of paired values were in zone A, with the remaining 9% of the values in zone B in the Clarke error grid. In the Bland–Altman, bias was 5.4 mg/dl with an upper limit of agreement of 32.5 mg/dl and a lower level of agreement of –21.8 mg/dl. Glucose prediction error analysis showed that 91% of the values ≥75 mg/dl within 20% of the values measured by the blood gas analyzer were within range. Trend accuracy: in the rate error grid of the continuous glucose error grid analysis, 96% of the paired values were in zone A, 3.7% were in zone B and 0.3% were in zone C. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf On the other hand we can state that our insulin infusion protocol already led to overall tight glucose control without a signifi cant hypoglycemia risk, leaving little space for improvement. R f Methods Patients with an expected stay in the ICU of >48 hours needing an arterial catheter and a central venous catheter (CVC) were eligible. For a maximum of 3 days, during 8 hours per day, 125 μl blood was drawn from the arterial line every 15 minutes. Point accuracy was expressed using Clarke error grids, Bland–Altman plots and glucose prediction error analysis [4,5]. Trend accuracy was expressed using continuous glucose error grid analysis [6].i Critically ill patients with faecal peritonitis: a 5-year review in a tertiary centre Critically ill patients with faecal peritonitis: a 5-year review in a tertiary centre V Paul1, A Tridente2, P Kaur1, M Mahmood1, R Mellors1, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454) V Paul1, A Tridente2, P Kaur1, M Mahmood1, R Mellors1, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454) y Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454 Introduction Faecal peritonitis (FP) is a common cause of sepsis and admission to the ICU [1]. We report a review of all patients admitted to our ICU over 5 years with FP. The aim was to defi ne the clinical characteristics, outcomes and risk factors for mortality in ICU patients with FP. Introduction Faecal peritonitis (FP) is a common cause of sepsis and admission to the ICU [1]. We report a review of all patients admitted to our ICU over 5 years with FP. The aim was to defi ne the clinical characteristics, outcomes and risk factors for mortality in ICU patients with FP. Conclusion Point and trend accuracy of the tested microdialysis-based CGM are good in critically ill patients. Acknowledgement Maquet Critical Care AB provided two CGM systems and disposables for the duration of the study, but had no infl uence on study design or study reporting. y Methods Data were extracted retrospectively from electronic case fi les. The primary outcome was ICU mortality. Secondary outcomes were hospital, 28-day, 90-day and 1-year mortality. Logistic regression analysis was used to identify independent risk factors for mortality. P377 Results We included 349 patients: age 67.5 ± 10.8 years, M/F sex ratio 252/97, preoperative left ventricle ejection fraction 58.8 ± 10.6%, bypass/valve ratio 234/154, number of grafts 2.7 ± 0.9, mammal arteries 1.8 ± 0.5. In univariate analyses, bypasses received more anaesthetic drugs (P <0.01), had shorter extracorporeal circulation duration, 67 ± 27 versus 75 ± 24 minutes (P <0.01), and received less blood products (P <0.0001). Bypasses had lower postoperative levels of troponin (3.9 ± 7.6 vs. 8.1 ± 21 pg/ml, P <0.01) and LDH (330 ± 162 vs. 420 ± 175 pg/ ml). In contrast, the intra-abdominal pressure (IAP) was higher and related to the number of grafts at day 0 (Figure 1) and day 1 (P = 0.01 and 0.02 respectively), and to the number of mammal grafts at day 0 and day 1 (P = 0.01 and 0.04 respectively). The TTFE was longer but did not reach signifi cance (P = 0.13) as well as the occurrence of abdominal ischaemia (P = 0.22). The occurrence of pneumonia was higher (P = 0.01). In multivariate analysis, the IAP at day 0 and day 1 was related to propofol quantities only. The predictors of pneumonia were: duration of mechanical ventilation, peak lactate in the postoperative 24 hours, and coronary bypass: OR = 163, 2.6, and 4.2 respectively. P377 Disseminated intravascular coagulation score predicts mortality in critically ill patients with liver cirrhosis A Drolz, T Horvatits, K Rutter, K Roedl, S Kluge, V Fuhrmann University Medical Center Hamburg-Eppendorf, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P377 (doi: 10.1186/cc14457) Introduction The disseminated intravascular coagulation (DIC) score is a predictor of outcome in critically ill patients [1,2]. Yet disturbances of coagulation and hemostasis, as refl ected by the DIC score, are a common fi nding in patients with liver cirrhosis. Thus, it is unclear whether the DIC score has prognostic value in critically ill patients with liver cirrhosis. The aim of this study was to assess the applicability and prognostic impact of the DIC score in critically ill patients with liver cirrhosis. Methods Patients with liver cirrhosis admitted to the medical ICU were analyzed for this study. Detailed laboratory analyses including platelet count, D-dimer, fi brinogen and prothrombin index were performed on admission and the DIC score was calculated. Survival was assessed on site or by contacting the patients or the attending physician. Bowel and related complications after cardiac surgery Results In the RT-CGM group the percentage of time at the target glycemia (80 to 110 mg/dl) was 37 ± 12% versus 34 ± 10% in the control group (NS) and glycemia averaged 119 ± 17 mg/dl versus 122 ± 11 mg/ dl respectively (NS). Time spent in hypoglycemia (<60 mg/dl) was not statistically diff erent between the group assigned to RT-CGM (0.6 ± 1.6% of the time) versus those with blinded CGM registration (2.4 ± 4.3% of the time). Parameters of glucose variability (standard deviation of mean glucose value, coeffi cient of variation, mean amplitude of glucose excursions) did not diff er between the groups. The GlucoDay®S Bowel and related complications after cardiac surgery CK Kerneis, AL Lafarge, LL Larnier, F Scalbert, AB Brusset, PE Estagnasie, PS Squara Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P375 (doi: 10.1186/cc14455) CK Kerneis, AL Lafarge, LL Larnier, F Scalbert, AB Brusset, PE Estagnasie, PS Squara q Clinique Ambroise Paré, Neuilly-sur-Seine, France q Clinique Ambroise Paré, Neuilly-sur-Seine, France Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P375 (doi: 10.1186/cc14455) Introduction Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus. Introduction Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S132 Figure 1 (abstract P375). Figure 1 (abstract P375). Results Eighty-seven patients (69.9%) were females. Mean age was 48.9 years. Primary cancer was colorectal in 42 patients (32.5%), ovarian in 39 (30%), appendiceal in 29 (22%), others in 15.5%. Average operative time was 11 ± 2.1 hours. Average intraoperative crystalloids given were 12,217 ± 4,359 ml, packed RBCs were 2 ± 2.3 units, colloids 1,083 ± 898 ml, average blood loss was 1,108 ± 785 ml. All patients were admitted to the ICU post procedure. The average fl uid balance during the OR was 9,481 ± 4,694 ml. Patients stayed in the ICU for an average of 6 ± 5.3 days. All patients survived the ICU stay. The duration of mechanical ventilation was 57 ± 83 hours, total fl uid balance while in the ICU was 1,467 ± 3,399 ml. Hypomagnesemia was the most frequent electrolyte abnormalities in 79 (61%). 1. Taylor FB, Toh CH, Hoots WK, Wada H, Levi M, Scientifi c Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Towards defi nition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost. 2001;86:1327-30. 2. Angstwurm MWA, Dempfl e C-E, Spannagl M. New disseminated intravascular coagulation score: a useful tool to predict mortality in comparison with Acute Physiology and Chronic Health Evaluation II and Logistic Organ Dysfunction scores. Crit Care Me. 2006;34:314-20; quiz 328. ICU outcome of patients undergoing cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy: a single-center study A Nadeem, A Al-Tarifi King Faisal Specialist Hospital, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P376 (doi: 10.1186/cc14456) g y A Nadeem, A Al-Tarifi King Faisal Specialist Hospital, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P376 (doi: 10.1186/cc14456) overt DIC was 70% compared with 40% in those with a DIC score <5. Conclusion Disturbances in coagulation and hemostasis are found in the majority of cirrhotic patients admitted to the ICU. The DIC score is a suitable predictor of 28-day mortality in critically ill patients with liver cirrhosis. Introduction Peritoneal carcinomatosis (PC) is associated with poor prognosis. The advent of complete cytoreductive surgery (CRRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has shown promise in improved survival for locally advanced intra- abdominal carcinomatosis. Such patients are routinely admitted to the ICU postoperatively. Little is known about the natural course of such patients while in the ICU. P377 Conclusion The number of coronary grafts and of mammal artery used in cardiac surgery is associated with higher IAP and higher risk of pneumonia. However, whether this is due to direct bowel ischaemia or longer anaesthesia remains to be studied in larger trials. Results In total, 150 admissions to the ICU with liver cirrhosis were analyzed. Thirty-nine percent were female. Median age was 56 (IQR 49 to 63) years. The median SOFA score on admission was 9 (6 to 13), median MELD score 26 (IQR 18 to 36). Twenty-eight-day mortality was 59%. Median DIC score on admission was 5 (IQR 4 to 6). Overt DIC (DIC score ≥5) was found in 65%. DIC score was signifi cantly higher in nonsurvivors compared with survivors (5 (IQR 4 to 7) vs. 4 (IQR 3 to 6); P <0.01). AUROC for the DIC score in prediction of 28-day mortality was 0.68 (95% CI = 0.59 to 0.77). Overt DIC on admission was signifi cantly associated with 28-day mortality (OR = 3.4 (95% CI = 1.69 to 6.84), P <0.01). The 28-day mortality rate in admissions with cirrhosis and overt DIC was 70% compared with 40% in those with a DIC score <5. P376 Bowel and related complications after cardiac surgery Pleural eff usions in 48 (37%), of which three patients only required drainage, Seven patients (5.6%) developed pneumonia, no patient required renal replacement therapy. Average hospital LOS was 33.7 ± 29 days. Only two patients died in the hospital. When the fi rst 65 patients were compared with the last 64 patients, the duration of MV, ICU LOS and hospital LOS were all signifi cantly shorter in the latter group (72 vs. 43 hours, 6.8 vs. 5.0 and 40 vs. 27 days respectively; P <0.01 for all). Methods In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P <0.1 for inputs P <0.05 for outputs. Methods In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P <0.1 for inputs P <0.05 for outputs. Conclusion With proper selection of patients, CRS with HIPEC can be done safely with no major complications. There is a signifi cant reduction in ICU utilization and shorter hospital LOS with more experience in such procedure, suggesting a learning curve as well as better utilization of resources by referring such patients to a high-volume center. P377 References 1. Taylor FB, Toh CH, Hoots WK, Wada H, Levi M, Scientifi c Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Towards defi nition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost. 2001;86:1327-30. Methods The procedure was introduced in our hospital in 2008 as the fi rst regional center performing such therapy. A retrospective chart review of 129 cases of CRS-HIPEC admitted to a 22-bed surgical ICU in a tertiary care academic center between November 2008 and March 2014. Primary outcomes were ICU length of stay (LOS) and duration of mechanical ventilation (MV). Secondary outcomes were hospital LOS and hospital mortality. 2. Angstwurm MWA, Dempfl e C-E, Spannagl M. New disseminated intravascular coagulation score: a useful tool to predict mortality in comparison with Acute Physiology and Chronic Health Evaluation II and Logistic Organ Dysfunction scores. Crit Care Me. 2006;34:314-20; quiz 328. S133 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P378 Conclusion In the present study, it was observed that the sero- prevalences of HBsAg, anti-HCV and anti-HIV were not higher than in our city population. However, taking the safety precautions of the healthcare workers during surgical or invasive procedures such as catheterization, intubation or tracheostomy without any information about the serological test results of the patients will reduce the contamination of these agents. P378 Warm ischemia time, postreperfusion syndrome and initial poor function after liver transplantation: are they connected? E Scarlatescu, G Manga, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P378 (doi: 10.1186/cc14458) Introduction Factors associated with initial poor graft function (IPGF) after liver transplantation are still under debate. Although the initial insult to the graft begins during the cold ischemia time (CIT), recent studies showed that most injuries occur during rewarming. Ischemic– reperfusion (I/R) injuries are present in all grafts and may be responsible for postoperative graft dysfunction. Along with other factors, I/R injury may also play a role in the development of postreperfusion syndrome (PRS) after revascularization of the liver graft. The aim of this study was to assess whether longer warm ischemia time (WIT) is associated with PRS or with IPGF after liver transplant. Outcomes of decompensated chronic liver disease in a UK district general hospital critical care setting E Ahmadnia1, F Manneh2, K Raveendran2 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) , , 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) , , 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) p p Methods This retrospective observational study included 60 liver transplant patients. We excluded from the study group patients with retransplant procedures, and the recipients of divided grafts and of grafts from extended criteria donors. We recorded: demographic data, intraoperative PRS, CIT, WIT, ALT, AST levels and standard coagulation tests on postoperative days (POD) 1 to 5. Statistical analysis was performed using SPSS Statistics v.19.1 with signifi cant P value under 0.05. Results We used the criteria of Nanashima and colleagues for the diagnosis of IPGF (ALT and/or AST level above 1,500 IU/l within 72 hours after OLT). The study group included 33 men (55%) and 27 women. Mean (±SD) age was 50.56 (±13.26) years. WIT longer than 60 minutes correlated signifi cantly with ALT and AST levels in POD 1 to 3 (P <0.0001 for ALT in POD 1 to 3, P = 0.001 for AST in POD 1, P = 0.007 and 0.013 for AST in POD 2 and 3) and with prothrombin time (P = 0.008 in POD 1, P = 0.03 in POD 2 and P = 0.015 in POD 3). We could not fi nd a correlation between PRS and WIT (P = 0.566), CIT (P = 0.439) or transaminase levels on POD 1 to 3. The correlation between WIT >60 minutes and IPGF was confi rmed using the Pearson chi-square test (P <0.0001). The same test was used to correlate IPGF with PRS with nonsignifi cant results (P = 0.876). Introduction Patients with decompensated cirrhosis admitted to the ICU have historically had a very high mortality rate [1]. It has been suggested that improving patient selection can improve ICU outcomes in patients with cirrhosis [2]. The aim of this study was to determine the mortality and evaluate the risk factors that may infl uence the outcome of this group of patients in a large UK district general hospital with a view to introducing selection criteria for future ICU admission of patients with decompensated liver disease. p p Methods A retrospective analysis was performed of all adult patients with decompensated chronic liver disease admitted to a general (nontransplant) critical care unit between January 2012 and December 2013. References The secondary aim was to investigate whether patients with intraoperative PRS have a higher risk for postoperative IPGF. P379 Seroprevalence of hepatitis B, hepatitis C and HIV in ICU patients H Bayir, I Yildiz, E Kocoglu, A Kurt, H Kocoglu Abant Izzet Baysal University, Medical School, Bolu, Turkey Critical Care 2015, 19(Suppl 1):P379 (doi: 10.1186/cc14459) Conclusion Those with decompensated chronic liver disease admitted to the ICU have a signifi cant ICU/hospital mortality, which is increased in alcoholic liver disease. Sepsis and AKI were the most common acute diagnoses in this cohort. Alcoholic liver disease patients requiring organ support have a very high mortality, and the outlook for multiorgan failure requiring RRT in this group is dismal. Introduction Healthcare workers are at risk for infections caused by hepatitis B (HBV), hepatitis C (HCV) and human immunodefi ciency (HIV) viruses that transmit via blood and body fl uids. In the present study, it was aimed to investigate the seroprevalences of HBsAg, anti- HCV and anti-HIV in patients admitted to the ICU. References References 1. Olson JC, et al. Hepatology. 2011;54:1864-72. 2. Sauneuf B, et al. Crit Care. 2013;17:R78. Methods HBsAg, anti-HCV and anti-HIV test results and demographical data of the patients admitted to the Reanimation ICU between January 2012 and December 2014 were evaluated retrospectively. HBsAg, anti-HCV and anti-HIV tests were assayed with a macro-ELISA method (Axsym-Abbott, Architect i2000; Abbott, USA). Statistical analysis was performed with the chi-square test. References 1. Erdem K, Tas T, Tekelioglu UY, Bugra O, Akkaya A, Demirhan A, et al. The hepatitis B, hepatitis C and human immunodefi ciency virus seroprevalence of cardiac surgery patients. SDÜ Tip Fak Derg. 2013;20:14-17 1. Erdem K, Tas T, Tekelioglu UY, Bugra O, Akkaya A, Demirhan A, et al. The hepatitis B, hepatitis C and human immunodefi ciency virus seroprevalence of cardiac surgery patients. SDÜ Tip Fak Derg. 2013;20:14-17 y 2. Tekin A, Deveci O. Seroprevalences of HBV, HCV and HIV among healthcare workers in a state hospital. J Clin Exp Invest. 2010;1:99-103. 2. Tekin A, Deveci O. Seroprevalences of HBV, HCV and HIV among healthcare workers in a state hospital. J Clin Exp Invest. 2010;1:99-103. Outcomes of decompensated chronic liver disease in a UK district general hospital critical care setting E Ahmadnia1, F Manneh2, K Raveendran2 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) Data were collected regarding demographics, ICU mortality, hospital mortality, aetiology of chronic liver disease, severity scores, acute diagnoses, and organ support requirements.i Results Thirty-seven patients were identifi ed, with a median age of 57 years, predominantly male (62%). Seventy-six per cent had alcohol-related cirrhosis. Overall ICU mortality was 29.7% and hospital mortality was 48.6% – these values were higher in the alcoholic group (39.3% and 57.1% respectively). All ICU deaths were in those with alcoholic liver disease. Median scores were: APACHE III 93, SOFA (day 1) 9, Child–Pugh 11, MELD 21. Seventy per cent were treated for sepsis, 22% had a GI bleed, 57% had encephalopathy, 24% had suspected/ confi rmed spontaneous bacterial peritonitis, and 70% had an acute kidney injury. Organ support requirements were: 35% respiratory (non-invasive or invasive ventilation), 38% vasoactive agent support, 24% renal replacement therapy (RRT). Alcoholic liver disease patients requiring respiratory or cardiovascular support had an ICU mortality of 64%, and those requiring RRT had a mortality of 75%. Alcoholic liver disease patients requiring combined respiratory, cardiovascular, and RRT support had 100% mortality. Conclusion Our study showed that PRS is not a risk factor for IPGF after liver transplantation. WIT over 60 minutes does not infl uence the development of PRS, but is associated with IPGF after liver transplantation. Close monitoring of liver tests in the early postoperative period is very important especially in recipients of grafts with WIT over 60 minutes. Further eff orts to decrease WIT may prove useful for the reduction of IPGF in liver transplant patients. First clinical experience with a new type of albumin dialysis: the HepaWash® system First clinical experience with a new type of albumin dialysis: the HepaWash® system First clinical experience with a new type of albumin dialysis the HepaWash® system B Henschel, R Schmid, W Huber TU-München Klinikum rechts der Isar, Munich, Germany Critical Care 2015, 19(Suppl 1):P383 (doi: 10.1186/cc14463) B Henschel, R Schmid, W Huber TU-München Klinikum rechts der Isar, Munich, Germany Critical Care 2015, 19(Suppl 1):P383 (doi: 10.1186/cc14463) Introduction Liver failure (LF) is associated with prolonged hospital stay, increased cost and substantial mortality. With regard to a limited number of donor organs, extracorporeal liver support is an appealing concept to bridge to transplant or to avoid transplant in case of recovery. A new type of albumin dialysis, the HepaWash® system, was recently introduced. The HepaWash® system provides rapid re- generation of toxin-binding albumin by secondary circuits altering binding capacities of albumin by biochemical (changing pH) and physical (changing temperature) modulation of the dialysate.i Results Eight children (fi ve male/three female), 8.6 ± 5.9 years old (range 2 to 15.6 years), BW 32 ± 21 kg, GFR 71 ± 20 ml/minute/1.73 m2, with an uncuff ed double lumen dialysis catheter (8 to 14 Fr Femoralis (n = 6) and 9 Fr Jugularis (n = 2)) were treated according to this protocol. In total, 19 sessions were executed using FX40 (n = 13), FX50 (n = 3), and FX60 (n = 3) dialysers during 6.5 ± 0.9 hours. Blood fl ow was 149 ± 45 ml/minute, albumin fl ow 226 ± 49 ml/minute, and ultrafi ltration fl ow 432 ± 517 ml. RRs were 70 ± 15% (urea), 34 ± 14% (Crea), 44 ± 16% (bili), and 36 ± 10% (NH3). Primary survival rate was 100%. Four patients were transplanted (bridge to transplant) of which, however, one died within 30 days after discharge from the ICU. The fi fth patient died due to primary disease 9 months after treatment, and the remaining three patients fully recovered (bridge to recovery). y y Methods We evaluated the fi rst 14 patients treated with the HepaWash® system with regard to safety and effi cacy. Seven patients were treated in the context of the run-in phase of the studies (HEPATICUS 1 and HEPATICUS 2) and seven patients were treated since the HepaWash® system received the CE certifi cate in July 2013. P381 Prometheus® liver therapy in children with acute liver failure J Vande Walle, S Claus, E Snauwaert, J De Rudder, A Raes, M Dick, A Prytula, W Van Biesen, S Eloot Ghent University Hospital, Gent, Belgium Critical Care 2015, 19(Suppl 1):P381 (doi: 10.1186/cc14461) Results The records of 462 patients admitted to our ICU were reviewed. The results of 36 patients could not be reached, so 426 patients were evaluated in the study. Among 426 patients, 169 (39.7%) were female and 257 (60.3%) were male. The mean age was 63.7 ± 18.7. HBsAg was positive in nine (2.1%) patients; all of these nine were male. Anti-HCV was positive in four (0.9%) patients; among these, three were male and one was female. Only one patient was positive for anti-HIV. Introduction The Fractionated Plasma Separation and Adsorption System Prometheus® (Fresenius Medical Care, Germany) aims at being S134 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Close observation and treatment of coagulopathy and electrolyte disturbances is essential when treating patients with MARS. MARS can reduce and stabilize ammonium and other biomedical markers in patients listed for urgent liver transplantation with high MELD score and liver encephalopathy. It seems that, in some cases and with our settings, the detoxifi cation properties of MARS may be insuffi cient. a supportive therapy as a bridge to transplantation or recovery in adults with liver failure. The system off ers specifi c challenges when applied in children due to the large extracorporeal volume (700 to 750 ml). We therefore developed an adapted protocol for the application in children. Methods Priming of the blood circuit is performed using 2 l isotonic saline, whereas the plasma circuit, containing both adsorption devices, is fi lled with 2 U fresh frozen plasma or 400 ml stabilized solution of human plasma proteins. Next, for children with body weight (BW) <25 kg, a solution of 60 to 65% packed cells (PC) is infused in the inlet blood line at 40 ml/minute. The volume of PC needed is calculated based on the circuit priming volume and the maximum allowed extracorporeal blood volume of the child (= 8 ml/kg × BW). After the priming phase, blood and plasma fl ow are increased to at least 100 ml/ minute and 200 ml/minute, respectively, and dialysate fl ow is set at 300 ml/minute. P381 Regional citrate anticoagulation is done with a calcium- free dialysate, while, eventually, heparin is added to the priming solution. Post treatment, the circuit volume is either not reinfused (BW <25 kg) or reinfused using isotonic saline (BW >25 kg), with a volume depending on the hydration status and the originally infused volume of PC. Reduction ratios (RRs, %) of urea, creatinine (Crea), bilirubin (bili), and ammonia (NH3) were calculated from pretreatment and posttreatment serum concentrations. Primary and secondary patient outcome was evaluated.i P383 Molecular adsorbent recirculating system treatment in 69 patients listed for liver transplantation P Oli H H Molecular adsorbent recirculating system treatment in 69 patients listed for liver transplantation P Olin, H Haugaa Oslo University Hospital, Oslo, Norway Critical Care 2015, 19(Suppl 1):P382 (doi: 10.1186/cc14462) Introduction The molecular adsorbent recirculating system (MARS) is used to remove circulating albumin-bound toxins in patients with liver failure. However, the application of MARS has not demonstrated improved survival in randomized clinical trials and the clinical utility has not been fi nally established. In our department, the use of MARS is now restricted to the most critically ill patients with acute or acute on chronic liver failure. We aimed to explore MARS effi cacy in removing toxicity parameters and the safety of the system. Conclusion So far the HepaWash® system has proven to be a safe and feasible procedure to eff ectively eliminate water and protein-bound toxins in humans with LF. First clinical experience with a new type of albumin dialysis: the HepaWash® system Patients treated suff ered under acute on chronic LF (n = 9) or secondary LF which resulted from nonhepatic diseases such as sepsis (n = 5). Primary endpoint: comparison of serum bilirubin, creatinine and serum BUN before and after the fi rst treatment with the HepaWash® system. Statistics: IBM SPSS Statistics version 22. The Wilcoxon test for paired samples was used to detect signifi cant treatment eff ects. gif Results A total of 254 treatments (1 to 101 per patient) were performed in 14 patients (six female, eight male). Mean age 54 ± 13. MELD score 33.7 ± 7.0, CLIF-SOFA 14.6 ± 2.7. Main underlying disease: nine acute- on-chronic LF; fi ve secondary LF. While bilirubin did not change signifi cantly on the day before HepaWash® treatment (26.2 ± 15.4 vs. 26.0 ± 15.4 mg/dl; P = 0.116), serum bilirubin levels were signifi cantly decreased by the HepaWash® procedure (26.0 ± 15.4 vs. 17.7 ± 10.5 mg/ dl; P = 0.001). Similarly, serum creatinine (2.2 ± 0.8 vs. 1.6 ± 0.7 mg/dl; P = 0.005) and serum BUN (49.4 ± 23.3 vs. 31.1 ± 19.7 mg/dl; P = 0.003) were signifi cantly lowered by the HepaWash® procedure. There were no serious adverse events observed in conjunction with the HepaWash® treatment. Conclusion This adapted Prometheus® protocol is promising for the treatment of children with liver failure. P382 Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) to c ty pa a ete s a d t e sa ety o t e syste . Methods Since 2005, we have treated 69 patients (30 males/39 females with median age of 39 years ranging from 1 month to 70 years) listed for liver transplantation with MARS. The median Model of End Stage Liver Disease (MELD) score in patients older than 12 years of age (n = 56) was 33 (interquartile range 26 to 39). The fl ow rate was 35 to 40 ml/ kg/hour and treatment kits were changed every 8 to 12 hours. The patients were treated for a median of 31 hours (range 1 to 240 hours). Results Fifty-fi ve patients (79%) were successfully bridged to transplantation. Nine died before they could be transplanted, and fi ve patients recovered without liver transplantation. Forty-four (81%) of the transplanted patients were alive 30 days after transplantation. Ammonium decreased modestly from a median of 148 to 124 μM (P = 0.03) during MARS treatment. We detected worsening of coagulopathy with signifi cant decreases in platelet count and fi brinogen concentrations, and increase in International Normalized Ratio. Phosphate and magnesium decreased signifi cantly during MARS treatment. J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow UK J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Introduction Hospital admission and mortality rates for patients with cirrhosis in the UK are rising [1]. Cirrhotic patients are physiologically challenged and at increased risk of sepsis and death [2]. Mortality rates for cirrhosis in nontransplant ICUs are up to 37% [3]. Increased availability of medical therapies and public expectation places pressure on limited intensive care resources. There is a lack of research into factors used to decide which patients to admit or refer to the ICU Introduction Hospital admission and mortality rates for patients with cirrhosis in the UK are rising [1]. Cirrhotic patients are physiologically challenged and at increased risk of sepsis and death [2]. Mortality rates for cirrhosis in nontransplant ICUs are up to 37% [3]. Increased availability of medical therapies and public expectation places pressure on limited intensive care resources. There is a lack of research into factors used to decide which patients to admit or refer to the ICU. Methods A prospective survey was sent to all consultant gastroenterologists and consultant intensivists in Scotland. Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? g A Vaz, M Eusebio, A Antunes, A Sousa, P Perez, R Ornelas, C Granja, H Guerreiro Centro Hospitalar do Algarve, Faro, Portugal Critical Care 2015, 19(Suppl 1):P385 (doi: 10.1186/cc14465) Methods A prospective study of 132 consecutive patients admitted to the Critical Burn Unit between October 2008 and October 2011. In all of them resuscitation was performed by objectives: blood pressure (>65 mmHg), hourly diuresis (0.5 to 1 cm3/kg), lactic acid clearance and thermodilution transpulmonary parameters (CI >2.5 l/minute/m2, ITBI: 600 ml/m2). We performed measurements of IAP with a bladder catheter every 8 hours in the fi rst 72 hours. Introduction Cirrhotic patients admitted to the ICU are usually regarded as having a particularly poor prognosis when compared with other groups of critically ill patients. The aim of our study was to evaluate the prevalence, case mix and outcomes of patients with cirrhosis admitted to the general ICU of a nontransplant center. yi Results Ninety-eight men and 34 women were studied. Mean age 48 ± 18 years and a TBSA of 35 ± 22%. The fl uid provided by %TBSA in the fi rst 8 hours was less than predicted by Parkland (4.05 ml/kg), although the total contribution in the fi rst 24 hours was similar. The evolution of the intra-abdominal pressure was: admission 9.7 mmHg, 8 hours 11, 16 hours 10.5, 24 hours 12.1, 32 hours 12.0, 40 hours 12.0, 48 hours 11.1, 56 hours 10.3, 64 hours 10.0 and 72 hours 10.0. A total of 44 patients (33.3%) had a determination higher than 12 mmHg, distributed: 15 patients between 12 and 15 mmHg (IAHT I grade), 14 between 16 and 20 mmHg (II), nine between 21 and 25 mmHg (III) and six >25 mmHg (IV). See Figures 1 and 2.l g p Methods Data were collected from a running ICU database. We studied cirrhotic patients admitted to the ICU between January 2013 and November 2014. Results A total of 30 patients with cirrhosis were admitted, accounting for 3% of total ICU admissions. Mean age was 54.5 years, with a male preponderance (76.7%). The main cause for cirrhosis was alcohol (53.3%), followed by alcohol plus chronic hepatitis C virus (HCV) infection (20%) and HCV virus infection alone (13.3%). The most common causes for admission were sepsis/septic shock (26.7%), surgical (23.4%), gastrointestinal bleeding and hepatic encephalopathy (16.7% each). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 decision. Recipients listed additional criteria used in their own practice and asked whether they would admit or refer individual grades of Child–Pugh cirrhosis with either a gastrointestinal bleed or sepsis.i decision. Recipients listed additional criteria used in their own practice and asked whether they would admit or refer individual grades of Child–Pugh cirrhosis with either a gastrointestinal bleed or sepsis.i C in the Child–Pugh score; mean Acute Physiology and Chronic Health Evaluation (APACHE II) 29.2 ± 8.7 and new Simplifi ed Acute Physiology Score (SAPS II) 62.7 ± 29. Regarding organ failure at admission, the mean Sequential Organ Failure Assessment score was 12.8 ± 4.5. The ICU mortality of these patients was 43.3% and hospital mortality was 53.3%. The variables at admission that related signifi cantly with ICU mortality were: all scores described except for Child–Pugh score, bilirubin, the International Normalized Ratio, creatinine, bicarbonate, lactate, pH and the use of renal replacement therapy during the ICU stay (P <0.05). The mortality rate of cirrhotic patients was superior to the general ICU mortality (43% vs. 26%). However, patients with cirrhosis presented signifi cantly higher severity scoring systems (APACHE II; SAPS II) at admission compared with noncirrhotics, with high prevalence of organ dysfunction as assessed by SOFA score. Results Thirty-fi ve consultant gastroenterologists and 65 intensive care consultants responded, representing a response rate of 34% and 45% respectively. The only criterion given an average rating of 5 by both gastroenterologists and intensivists was Child–Pugh score when stable. Presence on the transplant list, referral secondary to bleeding varices, recent discharge from the ICU, abstinence from alcohol, nutritional status, age under 30 and more than one additional organ failure all scored 4 or 5 from both groups. Sex, employment, smoking or drug use, deprivation and positive virology status did not infl uence the decision to refer or admit patients. Clinicians reported compliance with medication and outpatient appointments plus an obvious precipitant factor as important features in their decision. The majority of respondents would refer or admit all grades of Child–Pugh cirrhosis with gastrointestinal bleeding. Most would refer or admit Child–Pugh A or B with sepsis. A total 76.5% of gastroenterologists would refer Child–Pugh C cirrhosis with sepsis but only 33.3% of intensivists would accept. P385 Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? A Vaz, M Eusebio, A Antunes, A Sousa, P Perez, R Ornelas, C Granja, H Guerreiro Centro Hospitalar do Algarve, Faro, Portugal Critical Care 2015, 19(Suppl 1):P385 (doi: 10.1186/cc14465) Introduction The aim was to study the evolution and incidence of intraabdominal hypertension in critical burn patients using a slightly restrictive fl uid therapy protocol based on monitoring transpulmonary thermodilution and lactic acid. Intraabdominal pressure in critical burn patients PM Millan 2. Foreman MG, et al. Chest. 2003;124:1016-20. , ; 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. Hospital Universitario La Paz, Madrid, Spain Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P386 (doi: 10.1186/cc14466) p , , p Critical Care 2015, 19(Suppl 1):P386 (doi: 10.1186/cc14466) Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) Each recipient rated the signifi cance of 18 physiological and social criteria on their decision to refer or admit a patient to intensive care from 1 to 5, with 1 being no infl uence and 5 denoting signifi cant impact on the Results Fifty-fi ve patients (79%) were successfully bridged to transplantation. Nine died before they could be transplanted, and fi ve patients recovered without liver transplantation. Forty-four (81%) of the transplanted patients were alive 30 days after transplantation. Ammonium decreased modestly from a median of 148 to 124 μM (P = 0.03) during MARS treatment. We detected worsening of coagulopathy with signifi cant decreases in platelet count and fi brinogen concentrations, and increase in International Normalized Ratio. Phosphate and magnesium decreased signifi cantly during MARS treatment factors used to decide which patients to admit or refer to the ICU. Methods A prospective survey was sent to all consultant gastroenterologists and consultant intensivists in Scotland. Each recipient rated the signifi cance of 18 physiological and social criteria on their decision to refer or admit a patient to intensive care from 1 to 5, with 1 being no infl uence and 5 denoting signifi cant impact on the S135 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The high severity of disease in conjunction with the high mortality rate observed in this group of patients should make us consider the possible benefi ts of earlier referring/admission to the ICU, ideally before multiorgan failure arises. On the other hand, in nontransplant centers where cirrhotic patients constitute a small percentage of total ICU admissions, the complexity and peculiarities of the management of these patients should prompt their early transfer to a specialized center. Conclusion Referral and admission decisions for patients with cirrhosis are multifactorial. Child–Pugh status when stable appears to be of greatest signifi cance. The diff erence in opinion of admission of patients with Child–Pugh C with sepsis requires further evaluation. References 1. Thomson SJ, et al. Alcohol Alcohol. 2008;43:416-22. 2. Foreman MG, et al. Chest. 2003;124:1016-20. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. 1. Thomson SJ, et al. Alcohol Alcohol. 2008;43:416-22. P387 Intraabdominal hypertension in burn patients Results are depicted as mean values ± SD in Tables 1 and 2. y y Results Twenty patients were enrolled in the study. The mean age was 36 ± 13 years. There were 14 males and six females. The average TBSA was 44 ± 17%. Screening and monitoring of IAP were applied by: oliguria (42%), abdominal distension (31.5%) and gastrointestinal trouble (21%). IAH occurred between day 2 and day 3 after early burn resuscitation, respectively in 52% and 63%. IAH was observed in 69% of cases in patients admitted to the ICU with a delay of 1.6 days post burn injury. IAH was noted in 13 patients; of these, fi ve patients developed an abdominal compartment syndrome. The mean IAP was 16 ± 7 mmHg. Patients were assigned into two groups: G1 (IAH+; n = Conclusion Both sepsis and IAH have negative eff ects on respiratory mechanics. However, their combination has even more detrimental eff ects, which do not ameliorate after deinsuffl ation. Table 1 (abstract P388). P387 Intraabdominal hypertension in burn patients Methods Sixteen pigs were divided into two groups of eight (G-A/ G-B). All animals received general anesthesia and were mechanically ventilated. Parameters recorded included respiratory system, chest wall and lung compliance (CRS, CCW, CL) and respiratory system and chest wall inspiratory and expiratory resistances (RRSisp, RRSexp, RCWisp, RCWexp). After baseline measurements (0 minutes), intra- abdominal pressure IAP was raised by helium insuffl ation to 25 mmHg in both groups and remained at that level for the whole study. In G-B, sepsis was induced 60 minutes after IAP increase, by i.v. administration of Escherichia coli endotoxin. Parameters were recorded every 20 minutes. The last measurement was made at 180 minutes, right after deinsuffl ation, and IAP return to baseline levels.i Intraabdominal hypertension in burn patients A Mokline, I Rahmani, L Gharsallah, A Hachani, S Tlaili, R Hammouda, B Gasri, A Ksontini, AA Mesadi Trauma and Burn Centre of Tunis, Tunisia Critical Care 2015, 19(Suppl 1):P387 (doi: 10.1186/cc14467) Introduction Intra-abdominal hypertension (IAH) is frequent in the ICU and has been associated with adverse outcomes and worse prognosis. The purpose of our study was to assess risk factors for IAH and prognosis of major injured patients during burn resuscitation. Introduction Intra-abdominal hypertension (IAH) is frequent in the ICU and has been associated with adverse outcomes and worse prognosis. The purpose of our study was to assess risk factors for IAH and prognosis of major injured patients during burn resuscitation. j j p g Methods Adult burned patients with a burn injury exceeding 20% of total body surface area, from 1 April to 30 November 2013, were included. IAP was measured when IAH was suspected, according to the Kron method via the Foley catheter. Monitoring of IAP was performed every 6 hours during 5 days until normalization. fl Results CRS decreased statistically signifi cantly in both groups after IAP increase and increased after deinsuffl ation only in G-A. Similarly, CCW decreased in both groups but returned to baseline values in both groups after deinsuffl ation. CL decreased more signifi cantly in G-B and returned to baseline values only in G-A. RRSisp increased only in G-B and did not decrease after deinsuffl ation, whereas RRSexp increased in both groups, in a more signifi cant manner in G-B, and decreased only in G-A after deinsuffl ation. RCWisp and RCSesp did not show any alterations during the study period. P388 Eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension Figure 2 (abstract P386). IAH classifi cation. Introduction The aim of our study was to investigate the eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension (IAH). aSepsis induction. Comparison with baseline: P <0.05, ** P<0.01. Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? At admission, these patients presented an average Model for End- Stage Liver Disease score of 23.5 ± 10.4 with 70% classifi ed as grade Conclusion IAH incidence when a slightly restrictive fl uid protocol used is less than expected. Figure 1 (abstract P386). Intraabdominal pressure. S136 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 2 (abstract P386). IAH classifi cation. Figure 2 (abstract P386). IAH classifi cation. 13) and G2 (IAH–; n = 7). Comparative study of the two groups shows that HIA increases signifi cantly body weight gain within the fi rst 5 days after injury: 8 kg for G1 versus 2 kg for G2 (P = 0.04), occurrence of ARDS (70% for G1 vs. 16.7% for G2, P = 0.02), respiratory failure (77% for G1 vs. 28.5% for G2, P = 0.06), shock (70% for G1 vs. 16.7% for G2, P 0.02) and mortality (61.5% vs. 50%). 13) and G2 (IAH–; n = 7). Comparative study of the two groups shows that HIA increases signifi cantly body weight gain within the fi rst 5 days after injury: 8 kg for G1 versus 2 kg for G2 (P = 0.04), occurrence of ARDS (70% for G1 vs. 16.7% for G2, P = 0.02), respiratory failure (77% for G1 vs. 28.5% for G2, P = 0.06), shock (70% for G1 vs. 16.7% for G2, P 0.02) and mortality (61.5% vs. 50%). Conclusion IAH was frequent in early burn resuscitation of major injured patients. It seems to be associated with fl uid overload in burns and contributes to organ damage. P388 P388 Eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension B Fyntanidou, K Kotzampasi, M Kyparissa, G Stavrou, E Oloktsidou, X Mpesi, K Papapostolou, V Grosomanidis Aristotle Medical School, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P388 (doi: 10.1186/cc14468) aSepsis induction. Comparison with baseline: P <0.05, * P<0.01. P387 Intraabdominal hypertension in burn patients Compliance alterations during the study CRS (ml/cmH2O) CCW (ml/cmH2O) CL (ml/cmH2O) Minutes A B P value A B P value A B P value 0 35 ± 8 31 ± 4 NS 58 ± 20 46.1 ± 11 NS 99 ± 22 98 ± 14 NS 20 14 ± 4 12 ± 3 NS 20 ± 6 17.7 ± 3 NS 52 ± 21 35 ± 11 NS 40 13 ± 4 12 ± 1 NS 19 ± 6 16.8 ± 3 NS 47 ± 23 36 ± 13 NS 60a 12 ± 3 11 ± 2 NS 20 ± 5 18.4 ± 4 NS 41 ± 20 28 ± 7 NS 80 13 ± 3 10 ± 1 NS 20 ± 6 18.2 ± 44 NS 41 ± 21 24 ± 6 <0.05 100 12 ± 3 10 ± 2 NS 21 ± 8 19.9 ± 5 NS 37 ± 20 19 ± 4 <0.05 120 12 ± 3 10 ± 1 NS 20 ± 6 21.9 ± 7 NS 37 ± 20 17 ± 3 <0.05 140 12 ± 3 9 ± 1 NS 21 ± 7 21.8 ± 6 NS 37 ± 20 17 ± 5 <0.05 160 12 ± 3 9 ± 1 NS 21 ± 7 22.3 ± 7 NS 37 ± 19 16 ± 4 <0.05 180 33 ± 7 16 ± 5 <0.001 50 ± 18 77.0 ± 18 <0.05 81 ± 20* 21 ± 7** <0.001 aSepsis induction. Comparison with baseline: P <0.05, * P<0.01. Table 1 (abstract P388). Compliance alterations during the study S137 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 2 (abstract P388). P389 P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G Tsaousi1, EJ Giamarellos-Bourboulis2 1Aristotle University of Thessaloniki, Greece; 2 University of Athens Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P390 (doi: 10.1186/cc14470) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G Tsaousi1, EJ Giamarellos-Bourboulis2 1Aristotle University of Thessaloniki, Greece; 2 University of Athens Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P390 (doi: 10.1186/cc14470) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery Introduction CD73/ecto-5’-nucleotidase is an enzyme that generates adenosine, which dampens infl ammation and improves vascular barrier function in several disease models. CD73 also circulates in a soluble form in the blood [1]. We studied whether levels of soluble form of CD73 and cytokines/chemokines predict the development of organ failure in acute pancreatitis [2,3]. Introduction Heterogeneous published results led us to conduct a clinical trial to assess the effi cacy of a new formulation of four probiotics (P) as prophylaxis for complications after colorectal surgery. p Methods Altogether, 161 patients with acute pancreatitis (107 were subclassifi ed according to the revised Atlanta criteria into mild, 29 into moderately severe and 25 into severe forms) were studied. Serum and blood cell samples were collected at admission. Protein levels of soluble form of CD73 in serum were determined using a novel enzyme- linked immunosorbent assay, activity of soluble form of CD73 using radioactive enzyme assays, and CD73 messenger RNA levels from leukocytes using quantitative PCR. Serum levels of 48 cytokines and growth factors were determined using Bio-Plex Pro Human Cytokine Assay 21-plex and 27-plex magnetic bead suspension panels. y g y Methods A double-blind, placebo-controlled randomized study was conducted enrolling patients undergoing colorectal cancer surgery. Placebo or a formulation of L. acidophilus, L. plantarum, B. lactis and S. boulardii was administered starting 1 day before operation and continuing for 15 days post operation. Patients were followed-up for 30 days with the development of postoperative complications as the primary outcome. PAXGene tubes and serum were collected on postoperative day 4 for measurement of gene expression and serum cytokines (ClinicalTrials.gov NCT02313519). 1. Salmi M, Jalkanen S. Semin Immunopathol. 2014;36:163-76. 2. Maksimow M, et al. Crit Care Med. 2014; 42:2556 3. Nieminen A, et al. Crit Care. 2014 [Epub ahead of print]. P387 Intraabdominal hypertension in burn patients Respiratory system resistance alterations during the study RRSisp (cmH2O/l/minute) RRSexp (cmH2O/l/minute) Minutes A B P value A B P value 0 8.1 ± 0.8 8.3 ± 0.7 NS 13.6 ± 4.1 15.5 ± 3.7 NS 20 7.8 ± 0.6 8.1 ± 0.7 NS 17.1 ± 5.2 19.3 ± 2.2 NS 40 7.8 ± 0.8 7.6 ± 0.9 NS 18.1 ± 5.4 20.6 ± 3.1 NS 60a 7.6 ± 0.9 7.5 ± 1.1 NS 18.9 ± 4.4 19.9 ± 4.7 NS 80 7.8 ± 1.2 8.2 ± 1.2 NS 18.6 ± 4.2 21.1 ± 4.12 NS 100 7.8 ± 0.9 7.9 ± 0.9 NS 18.2 ± 4.5 22.7 ± 4.5 NS 120 8 ± 0.6 9.1 ± 1.1 <0.05 18.6 ± 4.3 20.9 ± 2.2 NS 140 7.7 ± 0.6 9.5 ± 1.2 <0.01 18.8 ± 3.5 22.9 ± 3.2 <0.05 160 7.3 ± 0.7 9.6 ± 1.5* <0.01 17.7 ± 3.5 22.2 ± 2.3 <0.01 180 8.1 ± 0.8 9.7 ± 1.2* <0.01 14.9 ± 3.3 18.9 ± 2.3* <0.05 aSepsis induction. Comparison with baseline: P <0.05, P <0.01. Table 2 (abstract P388). Respiratory system resistance alterations during the study P389 Severity markers in acute pancreatitis S Jalkanen University of Turku, Finland Critical Care 2015, 19(Suppl 1):P389 (doi: 10.1186/cc14469) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G T i1 EJ Gi ll B b li 2 P389 y g Results Administration of P signifi cantly decreased the rate of all postoperative major complications (28.6% vs. 48.8% of placebo, P = 0.010, odds ratio: 0.42). Major benefi t was found in the reduction of the postoperative pneumonia rate (2.4% vs. 11.3%, P = 0.029), of wound infections (7.1% vs. 20.0%, P = 0.020), of anastomotic leakage (1.2% vs. 8.8%, P = 0.031) and of the need for mechanical ventilation (20.2% vs. 35.0%, P = 0.037). The time until hospital discharge was shortened as Results Activity and protein concentration of soluble form of CD73 and messenger RNA level of CD73 all decreased along with the disease severity (P ≤0.01 for all). The activity of soluble form of CD73 at admission predicted the development of severe pancreatitis in diff erent groups of the patients. Especially, activity of soluble form of CD73 was better than C-reactive protein or creatinine in predicting the severity of pancreatitis in the group of patients without any signs of organ failure at admission. In subgroup analyses of patients with severe pancreatitis and without organ dysfunction upon admission, IL- 8, hepatocyte growth factor and granulocyte colony-stimulating factor (G-CSF) levels predicted the development of severe pancreatitis, with G-CSF being the most accurate cytokine. Figure 1 (abstract P390). Figure 1 (abstract P390). y Conclusion Activity of soluble form of CD73 and levels of certain cytokines at admission to the hospital have prognostic value in predicting the development of the severe form of acute pancreatitis. The possibility that combining them with other prognostic markers might improve prognostic accuracy requires further studies. References 3. Nieminen A, et al. Crit Care. 2014 [Epub ahead of print]. Figure 1 (abstract P390). S138 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 well. Gene expression of SOCS3 was positively related with circulating IL-6 in the P group but not in the placebo group (Figure 1). Conclusion The studied P formulation signifi cantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism. thiamine 200 mg or placebo i.v. twice/day for 7 days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Role of ultrasonography in detection of the localization of the nasoenteric tube R Dagli1, H Bayir2, Y Dadali1, T Tokmak1, Z Erbesler1 1Ahi Evran University Education and Research Hospital, Kirsehir, Turkey; 2Abant Izzet Baysal University, Medical School, Bolu, Turkey Critical Care 2015, 19(Suppl 1):P391 (doi: 10.1186/cc14471) Introduction In this study, we aimed to determine the success rate of nasoenteric tube (NET) insertion into the postpyloric area by ultrasonography (USG) and compare it with the commonly used method, direct abdominal radiography. g p y Methods Patients admitted to an adult ICU between April and July 2014 with an indication for NET insertion for enteral feeding were included in the study after informed consent was given from patients’ relatives. Nasoenteric feeding tubes were placed using the blind bedside method by a single anesthesiologist. Any motility stimulant agent was not used. The outside of the polyurethrane 8 F with unweighted NET (Bexen, Spain) and its guiding wire were lubricated with gel. The NET was inserted into the nostril after determination of the mouth– posterior ear–xiphoid distance and pushed on at least such a distance. Followed by auscultation of the gastric area and air infusion of 30 to 50 ml into the tube, the patient was positioned on their right side and the tube was advanced 20 to 30 cm more. Then the guiding wire inside the NET was removed. The patient was then brought to the supine position and NET was visualized by two radiologists simultaneously by M5 portable USG (Mindray, PRC), with a 3.5 MHz convex probe whether it passes through the postpyloric area or not. Localization of the tube was confi rmed with abdominal radiography in all patients. During the fi rst insertion of the NET, the ratios for inaccurate localization and correct placements through the postpyloric area were recorded and results were compared with abdominal radiography. Conclusion Thiamine defi ciency is prevalent in septic shock. Thiamine did not decrease overall median lactate levels at 24 hours. In the patients with thiamine defi ciency, there were statistically signifi cant lower lactate levels at 24 hours in the thiamine group and a large, although nonsignifi cant, diff erence in mortality. P392 Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial M Donnino1, LW Andersen1, M Chase1, KM Berg1, TA Giberson1, H Smithline2, M Tidswell2, R Wolfe1, M Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Critical Care 2015, 19(Suppl 1):P392 (doi: 10.1186/cc14472) Unraveling the link between malnutrition and adverse clinical outcomes: association of acute and chronic malnutrition measures with blood biomarkers from diff erent pathophysiological systems S Felder, N Braun, A Kutz, M Batschwaroff , P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P393 (doi: 10.1186/cc14473) Introduction Malnutrition is common in hospitalized medical patients and is associated with poor clinical outcomes. Whether malnutrition has a direct link to adverse outcomes or is rather a mirror of the severe patient condition remains debated. Our aim was to study the association of acute and chronic malnutrition status with blood biomarkers from diff erent pathophysiological concepts to better understand the underlying mechanisms of malnutrition. Results In this study, the bedside blind method was used for NET insertion into 34 patients. Eleven of the tubes were detected passing through the postpyloric area by USG. In one case the NET could not be seen in the postpyloric area by USG, but it was detected in the postpyloric area by control abdominal radiography. In 22 patients, NETs were detected in the stomach with control abdominal radiography. Success for NET placement with the bedside blind method and USG imaging was 35% versus 91.6%, respectively. Methods We prospectively followed consecutive adult medical inpatients hospitalized between February 2013 and October 2013 in a tertiary care Swiss hospital. Nutritional risk was assessed using the Nutritional Risk Screening (NRS 2002) score, which incorporates acute and chronic measures of malnutrition. Multiadjusted regression models were used to investigate the associations between acute and chronic nutritional risk and biomarkers mirroring infl ammation (CRP, PCT, proADM, leucocytes), stress (copeptin), renal dysfunction (creatinine, urea), nutritional status (vitamin D25, albumin, calcium, glucose), and hematological function (platelets, INR, Hb, RDW). Biomarker levels were transformed into deciles due to skewed distributions. Conclusion The success rate of the bedside blind method in the NET placement was low. It is clear that if any other placement techniques with high success rate will be applied, USG will be useful in a higher number of patients reducing the need for abdominal radiography. P389 Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher’s exact test. Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefi ned plan. We performed a preplanned analysis in those with baseline thiamine defi ciency (≤7 nmol/l). well. Gene expression of SOCS3 was positively related with circulating IL-6 in the P group but not in the placebo group (Figure 1).i thiamine 200 mg or placebo i.v. twice/day for 7 days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher’s exact test. Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefi ned plan. We performed a preplanned analysis in those with baseline thiamine defi ciency (≤7 nmol/l). Conclusion The studied P formulation signifi cantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism. yi y ( ) Results We enrolled 88 patients; 43 received thiamine and 45 placebo. Baseline characteristics were similar between groups. We found no overall statistical signifi cant diff erence in 24-hour lactate levels between thiamine and placebo groups (2.5 (IQR: 1.5 to 3.4) vs. 2.6 (IQR: 1.6 to 5.1), P = 0.40). Fewer patients in the thiamine group had lactate levels >4 mmol/l at 24 hours (21% vs. 38%, P = 0.10) and this was statistically signifi cant if only evaluating survivors at 24 hours (7% vs. 33%, P = 0.03), although our preplanned analysis was to impute data. We found no diff erence in 24-hour SOFA score or mortality. A total of 28 (35%) patients were thiamine defi cient. Of the defi cient patients, those receiving thiamine had statistically signifi cant lower lactate levels at 24 hours (2.1 (IQR: 1.4 to 2.5) vs. 3.1 (IQR: 1.9 to 8.3), P = 0.03) and more patients in the placebo group had a lactate >4 mmol/l (38% vs. 7%, P = 0.07). Mortality in the thiamine and placebo groups was 13% and 46%, respectively (P = 0.10).i Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial We considered target calories to be 25 kcal/kg/day and target proteins to be 1.5 g/kg/day. Introduction The provision of nutrition in the critical care unit (CCU) has shifted from nutrition support to nutrition therapy, and the potential benefi ts derived from this in the recovery of the critically ill is being explored [1]. We audited the management of nutrition in the CCU in a South African Hospital against the American Society of Parenteral and Enteral Nutrition Guidelines. Furthermore, we reviewed the knowledge and confi dence of healthcare providers in the management of nutrition in the CCU. g y g p g g y Results Patients received more calories (78.3% vs. 59.1%) and more proteins (70.2% vs. 54.6%) post implementation. The mean percentage of patients in the post group who achieved >70% of required calories was 80.1% versus 30.9% in the pre group. The mean percentage of patients who achieved >70% of required proteins was 58.3% versus 32.1% in the pre group. p g p Conclusion The multipronged approach of the quality improvement methodology helped to increase the provision of calories and proteins in our population of critically ill surgical patients. However, there is still room for improvement in terms of achieving optimal enteral nutrition targets early in our population. There is also a need to look into sustaining such results. Methods Retrospective data collection of patients admitted to a four- bed CCU over a 4-month period in 2013. A survey was distributed to diff erent disciplines involved in patient nutrition in the CCU. Results Seventy-two patients were admitted to the CCU during this time period, and notes were able for 44. Three paediatric patients were excluded. Twenty-nine patients stayed for 2 or more days (the audit population). The median age of the audit population was 38, 19 were female. Sixteen were postoperative admissions. The median APACHE II score of the patients with suffi cient available data (n = 16) was 14 (range 6 to 34). The audit found that 21 of the patients had nutrition started in the CCU, with 15 having nutrition started within 48 hours. Only eight patients had a nutritional assessment done. A total of 45 responded to the survey: eight anaesthetists, 25 from surgical disciplines, seven CCU nurses, and fi ve dieticians. All agreed that nutrition should be started in the fi rst 48 hours, except from the surgeons only 14 (56%) agreed. Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial The average self-rating of knowledge of nutrition management in the CCU (1 = lowest, 5 = highest) was 2.1 with the dieticians and CCU nurses showing the highest confi dence with 3.4 and 2.6, respectively. The anaesthetists rated their knowledge at 1.9 and the surgeons rated themselves at 1.8. P397 Introduction Optimizing enteral nutrition early has been shown to be benefi cial in critically ill patients. However, underfeeding is still a common problem. The critically ill surgical patient often presents with additional challenges to optimal enteral feeding. The objective of this study was to improve enteral feeding practices in a surgical ICU. P395 P395 Quality improvement project to optimize enteral nutrition in a tertiary hospital’s surgical ICU J Li, LY Koh, JH Yang, C Khoo, T Ter, BH Tan National University Health System, Singapore Critical Care 2015, 19(Suppl 1):P395 (doi: 10.1186/cc14475) g p g Conclusion Perioperative optimal nutrition support for at least 7 days could modulate the infl ammatory status and clinical outcome of severe malnourished surgical neoplasic patients. Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial Results A total of 529 patients (mean age 72 years, 57.1% male) were included. Overall, there was a signifi cant association of NRS and most biomarkers of infl ammation, stress, renal function, nutrition and the hematological system (coeffi cient and 95% CI): CRP 0.021, P = 0.0021, PCT 0.28, P = 0.003, proADM 0.4, P <0.001, copeptin 0.44, P <0.001, urea 0.28, P = 0.002, vitamin D25 –0.23, P = 0.012, albumin –0.6, P <0.001, hemoglobin –0.5, P <0.001, RDW 0.46, P <0.001. These associations remained robust after adjustment for sociodemographics (model 1), comorbidities (model 2) and main medical diagnosis (model 3). Subgroup analysis suggested that mainly the acute part of malnutrition and not chronic malnutrition was associated with an increase in biomarker levels. H Smithline2, M Tidswell2, R Wolfe1, M Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Introduction The objective was to determine whether the administration of thiamine mitigates elevated lactate levels in patients with septic shock. Thiamine is essential for aerobic metabolism and we have found that thiamine levels are low and inversely correlated with lactate levels in patients with sepsis. Conclusion Acute malnutrition was associated with a pronounced infl ammatory response and an increase in biomarkers from diff erent pathophysiological systems which may partly explain the link between malnutrition and adverse medical outcomes. However, interventional trials are needed to prove causal relationships. Methods We performed a randomized, double-blind, placebo- controlled, two-center trial from January 2010 to October 2014. We enrolled patients with septic shock, elevated lactate (≥3 mmol/l) and no obvious competing cause of lactate elevation. Patients received S139 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P394 Evaluation of the provision of nutrition in a South African provincial hospital S Kudsk-Iversen, R Matos-Puig, R Naidoo, S Moorad Stanger Provincial Hospital, Stanger, South Africa Critical Care 2015, 19(Suppl 1):P394 (doi: 10.1186/cc14474) interventions were conducted with lectures to physicians and nurses. Visual aids in the form of screensavers at each bedside computer served as reminders to the team to optimize feeding. A subsequent audit was then conducted to determine the improvement in achieving the desired outcomes, namely the amount of calories and proteins received as well as the proportion of patients who achieved >70% of their target calories and proteins. Could preoperative and postoperative optimal nutrition support modulate the infl ammatory response and clinical outcome of severe malnourished surgical patients with gastrointestinal neoplasia? p L Mirea, D Pavelescu, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P396 (doi: 10.1186/cc14476) p L Mirea, D Pavelescu, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P396 (doi: 10.1186/cc14476) p L Mirea, D Pavelescu, I Grintescu Introduction Our aim was to assess whether perioperative and postoperative optimal 7-day nutrition support could modulate the infl ammatory status and clinical outcome of severe malnourished patients with surgery for gastrointestinal neoplasia. Methods A prospective randomized study of 64 patients with gastrointestinal neoplasia, severe malnourished BMI <18.5, albumin level <3 g/dl, BW loss >10%, NRS >3, scheduled for surgery, allocated into two groups. Group A: 32 patients, minimal enteral nutrition in the postoperative period according to tolerance, medium 500 kcal/day. Group B: 32 patients received optimal parenteral nutrition support (25 kcal/kg/day) 3 days before surgery and continued for at least 4 days postoperatively. We measured CRP, fi brinogen, IL-6, TNF, albumin level preoperative and at 96 hours, the incidence of complications, and the length of ICU stay.i Conclusion We found that there is poor management of nutrition in the CCU. This is paired with limited knowledge and low confi dence in management amongst the attending staff . Evidence would suggest that the development and dissemination of clear hospital guidelines could improve rates of correct management [2]. However, the lack of uniform guidance based on strong evidence from the leading global authorities on nutrition suggests that, in order to improve implementation of adequate nutrition, more research is urgently required. References 1. McClave S, et al. JPEN J Parenter Enteral Nutr. 2009;33:277-316. 2. Martin CM, et al. CMAJ. 2004;170:197-204. Results There was a signifi cant decrease in the values of CRP, IL-6, TNF, albumin at 96 hours in group B. No diff erence in fi brinogen. A signifi cantly lower rate of complications and a shorter time of ICU stay were observed in group B. See Figures 1 and 2 (overleaf). 1. McClave S, et al. JPEN J Parenter Enteral Nutr. 2009;33:277-316. 2. Martin CM, et al. CMAJ. 2004;170:197-204. Does discontinuation of the use of hydroxyethyl starches in the critically ill cardiac surgery patient have an impact on caloric intake? E De Waele, K De Bondt, S Mattens, J Czapla, J Nijs, M La Meir, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Brussels, Belgium Critical Care 2015, 19(Suppl 1):P397 (doi: 10.1186/cc14477) Methods The Clinical Practice Improvement Programme is a local quality improvement initiative involving a multidisciplinary team aimed at identifying and improving defi ciencies in the process of delivery of care. A team led by an intensivist, consisting of doctors, surgeons, nurses and a pharmacist, was formed to improve enteral feeding practices in a surgical ICU. The quality improvement methodology was employed. An audit was carried out to determine the problem of underfeeding in the unit. Root cause analyses were conducted and team members identifi ed key barriers to optimal feeding and areas for improvement. Protocols were developed to standardize and encourage early enteral feeding as well as to reduce the time feeds are interrupted for patients who were going for surgeries or for various other reasons. Educational Introduction After research revealed unwanted eff ects of the use of starches in critically ill patients, its use in the immediate postoperative period of cardiac surgery patients came to an abrupt ending. However, they constitute an important source of non-intended calories, providing 4 calories per gram. We investigated whether this phenomenon (involuntary) attributed to an increase in caloric debt for this critically ill patient population. Methods We retrospectively searched a database of 417 elective cardiac surgery patients, representing 5,004 observation-days. Caloric S140 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 Figure 1 (abstract P396). Results 1. Figure 2 (abstract P396). Results 2. Figure 1 (abstract P396). Results 1. Figure 1 (abstract P396) Results 1 Figure 1 (abstract P396). Results 1. Figure 2 (abstract P396). Results 2. Figure 2 (abstract P396). Results 2. by aggressive nutrition therapy, in the general population of an ICU. Cancer patients are more prone to be at nutritional risk due to the disease and treatment complications. Our aim was to characterize NUTRIC score behavior in the population of patients admitted to an oncologic ICU. intake was evaluated in the group of patients before and after the cessation of starch use. Results Patient characteristics and caloric needs were comparable: 2,054 ± 395 kcal/day and 2,056 ± 347 kcal/day. Does discontinuation of the use of hydroxyethyl starches in the critically ill cardiac surgery patient have an impact on caloric intake? The 140 patients who in the immediate postoperative period had volume resuscitation without the use of starches had a mean non-intended fl uid caloric intake of 69 (± 36.3) kcal/day. The group of 277 patients who received starches in the postoperative period had a mean non-intended fl uid caloric intake of 105 (± 100.2) kcal/day. g Methods Between January and June 2014 we applied the NUTRIC score to all patients, age >18 years, without cerebral death criteria and with a length of stay (LOS) >72 hours. Data were collected and analyzed using SPSS v20.0. To evaluate the impact on mortality we used logistic regression. y Conclusion Withdrawal of the use of starches resulted in a 34% decrease of non-intended caloric intake by fl uids, contributing to caloric debt. Whether outcome is infl uenced and/or whether these fi ndings are clinically relevant needs further research. g Results Sixty-nine patients were included, 23 women (33.3%) and 46 men (66.7%). Most patients were aged between 50 and 75 years (72.5%) and had normal range weight 58% (n = 40). The mean LOS was 11.56 (minimum: 3 to maximum: 69). The most common motive for admission was sepsis (7.7%, n = 26). APACHE II score was above 15 in 77% of the patients (n = 53) and SOFA score was superior to 6 in 56.5% (n = 30). The NUTRIC score was low risk in 42% (n = 29) of the patients and high in 58% (n = 40). Twenty-eight-day mortality was 26.1% (n = 18). A high NUTRIC score corresponded to a 22-fold increased odds of dying in the fi rst 28 days (P <0.001). Both APACHE II and SOFA were mortality predictors alone, with an increase of 1 point in APACHE score corresponding to an increase of 14% (P = 0.002) and an increase of Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) Conclusion The NUTRIC score is a good tool in cancer patients to predict 28-day mortality. Nevertheless, the only compounds of the NUTRIC score that correlated independently with mortality were APACHE II and SOFA scores. Further investigation towards the inclusion of other categories such as tumor staging and the type of tumor could be useful to develop a specifi c prognostic tool for this population. Introduction Critically ill patients experience hypermetabolism in- creas ing substrate utilization, especially glucose oxidation. Glycogen is the main source of glucose in the body, being 85% and 15% stored in skeletal muscle and liver respectively. Since glycogen stores are limited we evaluated the hypothesis that critical illness could be associated with glycogen depletion leading to skeletal muscle catabolism for gluconeogenesis and eventually resulting in cachexia, an important determinant of future ICU survival and ICU-acquired weakness. Methods Nine critically ill patients (58.75 ± 25 to 75 years old) with an ICU stay from 1 day to 5 weeks were evaluated for skeletal muscle glycogen content using a rapid, non-invasive high-frequency ultrasound methodology (MuscleSound®, Denver, CO, USA). Scans were obtained from the rectus femoris and vastus lateralis muscles. Glycogen content was measured with a score from 0 to 100 according to the MuscleSound® scale. Patients had a variety of primary diagnoses including septic shock (n = 3), hemorrhagic shock/abdominal hypertension (n = 1), hypovolemic shock/post major oncologic surgery (n = 1), trauma (n = 3), and burn injury (n = 1). Early calorie-dense immune nutrition in haemodynamically compromised cardiac patients S Efremov, V Lomivorotov Research Institute of Circulation Pathology, Novosibirsk, Russia Critical Care 2015, 19(Suppl 1):P399 (doi: 10.1186/cc14479) Introduction The aims of present study were to test the hypothesis that early enteral nutrition (EN) with calorie-dense food supplemented with glutamine improves recovery of nutritional status in critically ill cardiac patients and to evaluate their resting energy expenditure (REE). Results Six out of nine patients had no glycogen present in the muscle (score = 0). The other three patients had glycogen scores between 5 and 15 which are well below scores of healthy individuals (reference 50 to 70). Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) As a comparison we collected post-competition levels in competitive athletes, which decrease their glycogen stores (score 15 to 25) but are well above those of most critically ill patients we have studied. p g gy p Methods A prospective randomised study of 40 adult cardiac patients undergoing elective cardiopulmonary bypass surgery no more than 24 hours before eligibility assessment, complicated with acute heart failure syndrome. Patients were randomised to receive either standard isocaloric isonitrogenic early EN (standard group, n = 20) or immunomodulating early EN (immune group, n = 20). The daily energy target was set using REE measured by indirect calorimetry (CCM Express; Medgraphics, St. Paul, MN, USA). Serum prealbumin, transferrin, C-reactive protein, blood lactate and clinical characteristics were analysed. Conclusion This is the fi rst time that muscle glycogen stores have been evaluated in critical illness. Our data show severe glycogen depletion in ICU patients which probably leads to muscle catabolism necessary for gluconeogenesis, eventually resulting in cachexia. This fi nding poses severe metabolic challenges for ICU patients in which interfering with recovery can contribute to poor survival. In light of our fi ndings, re-evaluation of nutritional protocols and potential anabolic/anti- catabolic therapy to decrease muscle catabolism may improve survival. Diff erent therapeutics that may prevent hypermetabolism (such as beta-blockers) should be re-evaluated along with anabolic agents (that is, oxandrolone) which could counteract the severe catabolic response in critical illness. Monitoring of muscle glycogen repletion could signal the transition from the catabolic to anabolic phase. y Results The actual REE was an average of 6.8 and 7.5 kcal/kg/day higher than the REE calculated using the Harris–Benedict equation and empiric approach (25 kcal/kg/day), respectively (Figure 1). Early EN with immune formula was associated with higher levels of prealbumin concentration on the 14th day (0.13 ± 0.01 g/l and 0.21 ± 0.1 g/l; P = 0.04) and transferrin on the 3rd, 5th, 7th, and 14th day (P <0.05) after surgery. Conclusion Haemodynamically compromised cardiac patients have increased REE, which in the absence of indirect calorimetry should be set at 30 kcal/kg/day. Early EN using a calorie-dense immune formula leads to better recovery of nutritional status as assessed by serum protein levels. Plasma glutamine after acute or elective admission on the ICU H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) NUTRIC score in oncologic patients A Patrão, L Bei, F Coelho A Patrão, L Bei, F Coelho Instituto Português de Oncologia – Porto, Portugal g g , g Critical Care 2015, 19(Suppl 1):P398 (doi: 10.1186/cc14478) Introduction The NUTRIC score is a tool designed to quantify the risk of critically ill patients developing adverse events that may be modifi ed S141 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1 point in SOFA corresponding to an increase in the odds of being dead at 28 days (P = 0.002). Body mass index, age, number of comorbidities, and days in the ICU did not correlate with mortality. Conclusion The NUTRIC score is a good tool in cancer patients to predict 28-day mortality. Nevertheless, the only compounds of the NUTRIC score that correlated independently with mortality were APACHE II and SOFA scores. Further investigation towards the inclusion of other categories such as tumor staging and the type of tumor could be useful to develop a specifi c prognostic tool for this population. 1 point in SOFA corresponding to an increase in the odds of being dead at 28 days (P = 0.002). Body mass index, age, number of comorbidities, and days in the ICU did not correlate with mortality. Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis y p Methods Sepsis was induced by E. coli LPS. GLN was infused: i.v. through the femoral vein (0.5 g/kg); enterally (E) through jejunostomy (0.5 g/kg); and i.v. + E. Blood was drawn continuously from the femoral artery and the portal vein for GLN plasma levels in systemic-S and portal-P circulation. P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) p Results In healthy swine, GLN levels remained stable, both in S and P; i.v. infusion, and even more i.v. + E signifi cantly increased GLN in the S circulation (P <0.001), whereas E infusion failed to do so (P = 0.4). On the contrary, GLN P levels were signifi cantly increased after i.v. + E infusion, as well as after E infusion (P <0.001) and to a lesser extent, after i.v. (P <0.001). In sepsis, both S and P GLN levels decreased signifi cantly. Introduction Intravenous fi sh oil (FO) lipid emulsions (LEs) are rich in ω-3 polyunsaturated fatty acids, which exhibit anti-infl ammatory and immunomodulatory eff ects. We previously demonstrated that FO- containing emulsions may be able to decrease mortality and ventilation days in the critically ill. Over the last year, several additional randomized controlled trials (RCTs) of FO-based LEs have been published. Therefore, the purpose of this meta-analysis was to update our systematic review aimed to elucidate the effi cacy of FO-based LEs on clinical outcomes in the critically ill. Figure 1 (abstract P404). Figure 1 (abstract P404). y Methods We searched computerized databases from 1980 to 2014. Overall mortality was the primary outcome and secondary outcomes were infections, ICU and hospital length of stay (LOS), and mechanical ventilation (MV) days. We included RCTs conducted in critically ill adult patients that evaluated FO-based LEs in parenteral nutrition (PN) or enterally fed patients. We analyzed data using RevMan 5.1 (Cochrane IMS, Oxford, UK) with a random eff ects model. f Results A total of 10 RCTs (n = 733), including four trials published over the last year, met inclusion criteria. Plasma glutamine after acute or elective admission on the IC H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) Figure 1 (abstract P399). Indirect calorimetry measured signifi cantly higher resting energy expenditures. Introduction Low plasma glutamine concentrations are associated with unfavourable outcome at acute ICU admission. We questioned whether there is a diff erence in plasma glutamine level after acute or elective ICU admission. Methods We performed a single-centre prospective observational study in a 22-bed mixed ICU. Exclusion criteria were age <18 years and total parental nutrition at admission. Patients were divided into two groups: elective surgery and acute admissions. Blood samples were taken at ICU admission and daily at 6.00 a.m. Glutamine levels were measured using the Bioprofi le 100 plus analyser (Nova Biomedical UK, Cheshire, UK). A Mann–Whitney U test was used to detect diff erences between groups and a Bonferroni method to correct for multiple comparisons. Results We included 88 patients after elective surgery (76 cardiac and 12 general surgery) and 90 patients after acute admission (27 sepsis, 17 acute surgery, two trauma and 44 medical). Baseline characteristics are presented in Table 1. Plasma glutamine levels at admission were signifi cantly lower in acute patients compared with elective surgery, 0.25 mmol/l (IQR 0.09 to 0.37) versus 0.43 mol/l (IQR 0.33 to 0.55) Figure 1 (abstract P399). Indirect calorimetry measured signifi cantly higher resting energy expenditures. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S142 Table 1 (abstract P402) Elective Acute Age (years) 68 ± 10 59 ± 17 APACHE IV 46 ± 14 67 ± 29* LOS ICU 2 (2 to 2) 4 (2 to 7)* Survival (n) 88 85* Mean ± SD or median (IQR). P <0.01. 0.17, heterogeneity I2 = 0%), and hospital LOS (WMD –4.06; 95% CI, –10.14 to 2.03; P = 0.19, I2 = 89%, P <0.00001), without eff ect on ICU LOS. See Figure 1. Conclusion FO-based LEs may be associated with a reduction in infections, as well as clinically important reductions in duration of ventilation, and hospital LOS. Nevertheless, according to current literature there is inadequate evidence to give a fi nal recommendation on the routine use of FO-containing emulsions in PN and/or as a pharmaconutrient strategy in enterally fed critically ill patients. Further large-scale RCTs which should aim to consolidate potential positive treatment eff ects are warranted. (P <0.001). There appeared to be a signifi cant correlation between the APACHE IV score and glutamine levels (R = 0.52, P <0.001). P403 P403 Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) P404 Glutamine administration in sepsis: enteral, parenteral or both? Experimental study in swine G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) Plasma glutamine after acute or elective admission on the IC H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) Moreover, in a backward linear regression analysis this correlation was independently associated with APACHE IV scores and the presence of infection, but not with the type of admission.i Glutamine administration in sepsis: enteral, parenteral or both? Experimental study in swine G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) yp Conclusion Plasma glutamine levels were signifi cantly lower after acute admission compared with elective surgery. In both groups a considerable amount of patients had decreased glutamine levels, but this was not independently associated with the type of admission. In contrast to previous studies we found that glutamine levels were determined by severity of illness and the presence of an infection. p Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) Introduction Glutamine (GLN) is recommended in the critically ill for i.v. administration but enteral use is not quite clarifi ed. We decided to measure GLN plasma levels in healthy and septic swine after GLN given by i.v., enteral or both routes, over a 3-hour period. P405 Methods The SALOMON algorithm is based on 53 common presentation fl owcharts using specifi c discriminators to triage calls into four categories according to the level of care required: emergency medical services, nonemergent visit to local emergency department, PCP home visit or PCP delayed consultation. Using an appropriate statistical test, we assessed the accuracy of presentation fl owchart and triage category selections attributed to 130 clinical scenarios, by 10 diff erent nurses, in comparison with references established by a local team of experts, at two diff erent time periods: immediately after training (T0) and 3 to 6 months after algorithm practice (T1).l Prehospital factors associated with an ICU admission from the emergency department P406 P406 Reliability of a new French-language triage algorithm for out-of- hours primary care calls: the SALOMON rule E Brasseur1, A Ghuysen1, AF Donneau2, V D’Orio1 1C.H.U. of Liège, Belgium; 2University of Liège, Belgium Critical Care 2015, 19(Suppl 1):P406 (doi: 10.1186/cc14486) Figure 2 (abstract P404). Introduction Because of increased workloads associated with primary care physician (PCP) shortage, many western countries have been facing the diffi cult challenge of optimizing their out-of-hours primary care services. PCPs initially gathered in small rotation groups, and then further collaborations led to larger-scale cooperatives. In such models, implementation of patient call triage is mandatory to increase the effi ciency, quality and safety of care [1]. Organization models diff er, from the PCP performing all patient calls to nurses and nurse assistants answering calls and performing triage, but no validated triage algorithm has been reported to date. We developed a specifi c French- language triage algorithm called SALOMON (Système Algorithmique Liégeois d’Orientation pour la Médecine Omnipraticienne Nocturne) in order to guide nurse triage PCP out-of-hours calls. The present study tested this algorithm reliability. As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P = 0.08). See Figures 1 and 2. As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P = 0.08). See Figures 1 and 2. Conclusion In our experimental early sepsis model, a combination of E and i.v. GLN seems to be the most appropriate; this results in high GLN levels for the functional needs, including those of the gut mucosa. Reference 1. Crit Care. 2014;18:R76. Reference Reference 1. Huibers L, et al. Scand J Prim Health Care. 2011;29:198-209. 1. Huibers L, et al. Scand J Prim Health Care. 2011;29:198-209. Prehospital factors associated with an ICU admission from the emergency department TA Williams1, J Finn1, D Fatovich2, D Brink3, KM Ho2, H Tohira4 1Curtin University, Bentley, Australia; 2Royal Perth Hospital, Perth, Australia; 3St John Ambulance – WA, Belmont, Australia; 4 Curtin University Health Sciences, Bentley, Australia Critical Care 2015, 19(Suppl 1):P405 (doi: 10.1186/cc14485) Introduction This study aimed to describe the patient characteristics and prehospital factors associated with an ICU admission from the ED. There is a paucity of information about the early recognition of critical illness by paramedics; especially in the Australian prehospital setting. Methods A retrospective cohort study, July 2012 to June 2014, conducted in the Perth metropolitan area, which is served by a single ambulance service. Adult patients were included if transported to a public hospital ED that used the ED information system (EDIS) (seven of eight EDs) and were admitted to the ICU from the ED (ED-ICU group). Patients aged <16 years, those from rural areas or transfers were excluded. We used existing ambulance clinical data linked to EDIS data. Prehospital cohort characteristics are described using univariate statistical techniques. Logistic regression was conducted with admission to the ICU from the ED (critical illness surrogate) as the outcome variable. Variables included in regression models were age, sex, paramedic-identifi ed urgency, that is the time patients should be seen by a doctor based on the Australasian Triage Scale, paramedic- identifi ed patient problem and the time taken from the ambulance service receiving the call to hospital arrival. Physiological variables: systolic blood pressure (SBP), heart rate (HR), respiratory rate (RR), temperature, oxygen saturation, and GCS were included in the logistic models. Results Overall selection of fl owcharts was accurate for 94.1% of scenarios at T0 and 98.7% at T1. Triage category selection was correct for 93.3% of scenarios at T0 and 98.4% at T1. Both fl owchart selection and triage category were correct in of 89.5% case in T0 and at 97.5% T1. When an incorrect fl owchart was used, triage category remained accurate in 64.9% and 70.5% respectively. Both fl owchart and triage selection accuracy improved signifi cantly from T0 to T1 (P <0.0001). y gi y Conclusion The results of the present study revealed that using the SALOMON algorithm is reliable for out-of-hours PCP call triage by nurses. Validity of this rule may be further evaluated through its actual implementation in real-life conditions. Reference Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis There was considerable heterogeneity in interventions tested in these trials. No eff ect on overall mortality was found. When the results of fi ve RCTs that reported infections were aggregated, FO-containing emulsions signifi cantly reduced infections (RR 0.64; 95% CI, 0.44 to 0.92; P = 0.02, heterogeneity I2 = 0%). Furthermore, FO-based LEs were associated with a trend toward a reduction in MV days (WMD, –1.41; 95% CI, –3.43 to 0.61; P = Figure 1 (abstract P404). Figure 1 (abstract P403). Eff ects of parenteral fi sh oil lipid emulsions on infections. S143 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P = 0.08). See Figures 1 and 2. Conclusion In our experimental early sepsis model a combination of Figure 2 (abstract P404). (all P <0.001) except the time taken from receiving the call to hospital arrival (P = 0.48). Figure 2 (abstract P404). Conclusion Three-quarters of the ED-ICU patients were transported to the ED with high urgency. Currently no prehospital severity of illness or early warning system (EWS) is used in our ambulance service. Given the small proportion of ED-ICU patients who presented with abnormal observations, it is unlikely that introducing an EWS would alter practice or patient outcome. P407 Adequacy of trained assistance, emergency equipment and drugs at emergency calls in the ICU and in remote areas of the hospital I Kolic1, S Unell2, A Watts2, A Barry2, J Short2 1South London Healthcare NHS Trust, London, UK; 2Lewisham and Greenwich NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P407 (doi: 10.1186/cc14487) Results Of the 142,448 eligible patients transported by ambulance, 1,076 (0.75%) were admitted to the ICU from the ED: the ED-ICU group was younger (mean 53 vs. 61 years, P <0.001). Seventeen percent of ICU patients were transported as Urgency 1 (resuscitation/immediate) and 58% as Urgency 2 (within 10 minutes) while 70% of non-ICU patients were transported as Urgency 3 to 5 (P <0.001). Thirteen percent of ICU patients had a SBP <90 mmHg, 15% had a HR ≥130 and 19% had a RR >30. Drug overdose (21%) and respiratory conditions (18%) were the most common ICU conditions identifi ed by paramedics for the ED-ICU group. All variables entered into the logistic models were signifi cant Introduction We aimed to identify the adequacy of assistance provided and to assess correct anaesthetic equipment and drug availability at emergency calls made in the ICU and in remote areas of the hospital. Emergency calls often involve managing critically ill patients with the highest mortality results. The importance of a clinical team with the necessary competencies and the right level of resources are paramount. Methods We undertook a prospective survey of all adult patients with emergency calls put out to the anaesthetic team in a London district S144 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 general hospital over a 6-week period. We performed a snapshot audit of equipment in resuscitation trolleys across each ward and in the radiology department. We compared the data collected on available equipment with the standard set by the Resuscitation Council (UK) Recommended Minimum Equipment Checklist [1]. The survey addressed the availability, clinical competency and appropriate duration of stay of the anaesthetic assistant at the emergency calls. Further qualitative data were collected on the availability of required emergency drugs. general hospital over a 6-week period. We performed a snapshot audit of equipment in resuscitation trolleys across each ward and in the radiology department. Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P408 (doi: 10.1186/cc14488) g g p J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P408 (doi: 10.1186/cc14488) J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK f Results The intervention signifi cantly increased satisfaction amongst staff regarding: identifi cation of the team leader and other key staff members at the response; and time out eff ectiveness in reducing repetition and improving staff understanding of the patient’s status and medical issues. We found no signifi cant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response. Introduction There is widespread concern regarding excess mortality for patients admitted to hospital out of hours. We introduced an electronic track and trigger system (Patientrack) with automated alerts in a large university teaching hospital between 2010 and 2012. The system computes the patient’s early warning score and alerts medical staff via a pager. It is operational 24 hours a day, 7 days a week and could be an eff ective tool to reduce variations in mortality throughout the working week. Conclusion Utilising a low-cost intervention in a regional setting, we were successful in improving staff perceptions of role allocation and communication within our MET call responses. The intervention also led to signifi cantly increased overall satisfaction with the MET call system. Through our surveys we have identifi ed other facets of the MET call response that also require attention. Given our encouraging results we are designing a follow-up intervention incorporating structured multidisciplinary training in MET call scenarios. g Methods We extracted hospital outcome data for all admissions during the fi nancial years between 2007 and 2014. We identifi ed variables that predicted mortality and incorporated them into a multivariate logistic regression model to assess risk of death for admissions in hours (9:00 am to 5:00 pm, Monday to Friday) versus out of hours (all other times). P409 Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) P409 Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) Introduction Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation. Introduction Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation. Methods We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identifi cation of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient’s current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success. Results The intervention signifi cantly increased satisfaction amongst staff regarding: identifi cation of the team leader and other key staff members at the response; and time out eff ectiveness in reducing repetition and improving staff understanding of the patient’s status and medical issues. We found no signifi cant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response. Conclusion Emergency calls require standards to be met involving the competency of responding team members and adequate resources. This leads us to question whether guidelines should exist regarding the clinical competency and timeliness of the assistant available to the physician at emergency calls. Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia C l C (S l ) P (d / ) 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Methods We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identifi cation of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient’s current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success.i Reference 1. Resuscitation Council (UK). Recommended minimum equipment for in hospital adult resuscitation (October 2004). https://www.resus.org.uk/pages/ eqipIHAR.html. P407 We compared the data collected on available equipment with the standard set by the Resuscitation Council (UK) Recommended Minimum Equipment Checklist [1]. The survey addressed the availability, clinical competency and appropriate duration of stay of the anaesthetic assistant at the emergency calls. Further qualitative data were collected on the availability of required emergency drugs. sex, unplanned admission and admission from supportive care. Risk of death for out-of-hours admissions was not signifi cantly diff erent to in- hours for any year (1.01 (0.92 to 1.11), P = 0.784). There was a signifi cant fall in risk of death over the 7-year period compared with baseline values in 2007/08 (Table 1). Conclusion In our cohort there was no evidence of increased mortality for patients admitted out of hours compared with in hours. This remained true after adjustment for age, sex, emergency admissions and admission source. Our data demonstrated an overall fall in risk of death over the study period. The introduction of Patientrack could have contributed to this reduction in mortality. Results During the study period 44 emergency calls were attended. Twenty-three (52%) of these calls were in the accident and emergency department, and four (9%) in the ICU. Survey results demonstrated two cases where no anaesthetic assistant arrived at the emergency call put out to them. In cases where timely assistance was available, the assistant did not have the adequate clinical and anaesthetic skills required by the attending physician. In 6% of cases where skilled assistance was required (n = 2), it was felt that the assistant did not stay for the clinically required length of time. Emergency drugs required were found to not be available in 11% of cases (n = 5) and in 17% of cases (n = 6) the necessary emergency equipment was not available. Data were collected on equipment from 17 resuscitation trolleys. The inadequacies identifi ed were the oxygen cylinders were fi lled less than 75% full in 41% cases (n = 7) and end-tidal capnography was identifi ed to be absent. P409 Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital P410 S Results Data were available for 1,180,268 hospital admissions, of which 7,264 (0.6%) died. Predictors for hospital mortality included: age, male P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) Among true cardiac arrest events, 92.6% was due to pulseless electrical activity/asystole and 7.7% was due to ventricular fi brillation (VF)/pulseless ventricular tachycardia (VT); both of these did not have any diff erence on the initial outcome. But having an initial rhythm of VF/pulseless VT had 90% more chance for discharge from the hospital, with P = 0.04. Although arrival time of the CBT did not have any infl uence on the fi nal outcome, duration of resuscitation ≤20 minutes had an odds ratio of 10.6 with P <0.001 favoring return of spontaneous circulation over death after controlling for age. Of the 203 patients who had true cardiac arrest events, 43 (21.2%) were discharged from the hospital. Good neurological outcome at discharge was seen among 22 (10.8%) of the patients based on Cerebral Performance Category Score. Conclusion Our experience shows that out of every 1,000 patients admitted to our hospital, about fi ve sustained cardiac arrest, of whom only 11.3% survived to hospital discharge with good neurological recovery. Variation in the eff ectiveness of the cardiopulmonary resuscitation quality in comparison with world data could be due to the inherent diff erence in the severity of the primary illness in the patients and diversity in the reported data. P411 Evaluation of emergency call Code Blue over a 5-year period N Bakan, G Karaoren, S Tomrk, S Keskin Istanbul Umraniye Training and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P411 (doi: 10.1186/cc14491) Introduction Code systems are the emergency call and management systems for rapid response in healthcare institutions. The main aim of these systems is to provide common institutional understanding of what is necessary to be done immediately at the time of an event. Code Blue (CB), which is used throughout the world and was described in the 2008 service quality standards of Turkey, defi nes the necessary emergency intervention in cases of respiratory or cardiac arrest. This study aimed to evaluate the clinical and application data of patients for whom a CB call was made between 2009 and 2013. Methods After approval of local ethics committee, retrospective examination was made of CB forms. P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. Methods All Code Blue activations from 1 January 2012 to 31 December 2012 were analyzed retrospectively. Data were gathered from the Code Blue form and fi ner details of individual patients were linked through their medical records. Code Blue was activated only for events that happened outside the medical and surgical ICUs. Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. f Methods All Code Blue activations from 1 January 2012 to 31 December 2012 were analyzed retrospectively. Data were gathered from the Code Blue form and fi ner details of individual patients were linked through their medical records. Code Blue was activated only for events that happened outside the medical and surgical ICUs. Conclusion The MET has been successfully implemented, with demand for its services having increased by 32.7% in 1 year. The unadjusted immediate mortality rate of patients for whom a MET/cardiac arrest call is activated is 3.64%. Response time had no infl uence on mortality, most probably due to the rapid response time. Immediate mortality was low, probably as a result of early adequate intervention. Further evaluation of overall hospital mortality is warranted for future studies. pp g Results A total of 260 Code Blue activations were made, out of which there were 203 true cardiac arrest events among 40,168 in-patients; the cumulative incidence of the same was 0.51%. Mean (SD) duration of arrival of the Code Blue Team (CBT) to the scene was 64.5 (27.7) seconds. Cardiovascular illness was the predominant baseline morbidity but none of the baseline illness showed increased risk of mortality in this group. P410 Successful implementation of a medical emergency team: 2-year experience in a teaching hospital A Tridente, J Elmore, R Varia, T Mahambrey St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P410 (doi: 10.1186/cc14490) Table 1 (abstract P408). Overall risk of death ratios compared to 2007/08 Year ROD 95% CI 2008/09 1.01 0.73 to 1.5 2009/10 1.03 0.74 to 1.5 2010/11 0.46 0.33 to 0.7 2011/12 0.31* 0.22 to 0.4 2012/13 0.27* 0.2 to 0.39 2013/14 0.26* 0.2 to 0.37 P <0.001. Table 1 (abstract P408). Overall risk of death ratios compared to 2007/08 Introduction A medical emergency team (MET) was introduced in our institution in January 2012 to provide timely response to the needs of acutely ill inpatients and cardiac arrest calls. The MET assesses the patient and prescribes a management plan for the responsible team to follow; promptly stabilising and transferring patients to a place of safety where required. We aimed at evaluating the eff ects of introducing the MET on clinically relevant processes and outcomes. Methods Prospective data were analysed using STATA 10.1. The primary outcome measure was immediate mortality (defi ned as mortality at S145 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The time taken to reach patients conformed with the global standard mean 2 to 3 minutes [1]. The rates of erroneous CB and time to reach patients reduced each year due to more staff experienced and knowledgeable in CB and the structuring of the emergency clinic. Conclusion The time taken to reach patients conformed with the global standard mean 2 to 3 minutes [1]. The rates of erroneous CB and time to reach patients reduced each year due to more staff experienced and knowledgeable in CB and the structuring of the emergency clinic. conclusion of the MET intervention); the secondary outcome measures were admission to critical care after a MET call and cardiac arrest. conclusion of the MET intervention); the secondary outcome measures were admission to critical care after a MET call and cardiac arrest. Results A total of 5,763 MET calls were made between 9 January 2012 and 4 March 2014, of which 5,310 (92.1%) were MET calls, 349 (6.1%) cardiac arrest calls, 36 (0.6%) false alarms and 68 (1.2%) unclassifi ed. The number of calls increased by 32.7% from 2,255 in 2012 to 2,993 in 2013, with all month-specifi c comparisons showing signifi cant increases in MET activity (ranging from 0.5% to 103.6% increases). P410 Successful implementation of a medical emergency team: 2-year experience in a teaching hospital A Tridente, J Elmore, R Varia, T Mahambrey St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P410 (doi: 10.1186/cc14490) MET activity displayed cyclical yearly changes, with the winter months and the month of August (junior doctors’ changeover period) being particularly busy. Median response time (interquartile range) was 1 (1 to 2) minutes, with 99.1% calls attended to within 3 minutes. There were 210 (3.64%) immediate deaths (with no signifi cant diff erences between years), 112 (1.9%) patient transfers to critical care, 233 (4%) patients were transferred to other locations (other than critical care) while 4,697 (81.5%) patients remained on the ward of origin. In 408 cases (7.1%) a do-not-resuscitate order was instituted. On multiple logistic regression analyses, when the type of call was taken into consideration, the response time had no infl uence on primary (mortality OR = 0.83, 95% CI = 0.63 to 1.09, P = 0.18) and secondary outcomes (admission to critical care OR = 0.85, 95% CI = 0.62 to 1.17, P = 0.33; subsequent cardiac arrest OR = 0.57, 95% CI = 0.27 to 1.2, P = 0.14). References 1. Abella BS, et al. JAMA. 2005;293:305-10. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. 1. Abella BS, et al. JAMA. 2005;293:305-10. 1. Abella BS, et al. JAMA. 2005;293:305 10. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. Results From CB calls for a total of 1,195 patients over the 5-year period, 1,035 (86.6%) were evaluated. The rate of erroneous CB was 36.9%. Patients comprised 413 (39.9%) females and 622 (60.1%) males with a mean age of 59.73 ± 23.13 years (range, 0.1 to 102 years). The distribution of total cases over the 5 years (2009 to 2013) was 15.5%, 25.2%, 26.5%, 19% and 13.8% respectively. Distribution according to clinic was emergency internal (37.5%), internal (16.5%) and emergency surgical (9.5%). Clinical diagnosis was cardiac 28.8%, neurological 15.6% and end-stage cancer 13.5%. A total 19.9% of the patients were those discharged from intensive care. The total survival rate was 59.6%. The duration of CPR in survivors was statistically longer than in nonsurvivors (P <0.01). There was no statistically signifi cant relationship between the duration of CPR and age (P >0.05). The overall mean time taken to reach the patient was 102.71 ± 22.47 seconds, which reduced to 93.64 ± 19.91 seconds in 2013. The APACHE II–PRISM scores and mortality rates were low in the cases of erroneous CB (P <0.05). P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) The age and gender of the patient, diagnosis, department to which admitted, time of CB call, reason for CB, whether or not CB was appropriate, whether or not CPR was applied, duration of CPR if applied, APACHE II and PRISM scores and predicted mortality were recorded from the hospital automated record system and the CB form. Patients who refused treatment or who could not reach the necessary parameters for the calculation of APACHE II and PRISM scores were excluded. References Reference 1. Nolan JP, et al. European Resuscitation Council guidelines for resuscitation 2010 section 1. Executive summary. Resuscitation. 2010;81:1219-76. P412 P413 Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools J Salciccioli, D Marshall, M Sykes, A Wood, S Joppa, M Sinha, PB Lim Imperial College London, UK Critical Care 2015, 19(Suppl 1):P413 (doi: 10.1186/cc14493) TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest Methods We conducted a registered audit of CPR and AED training in London schools. Secondary schools were identifi ed via web links for each of the London Borough Councils. Telephone interviews with school staff familiar with CPR and AED training practices were conducted prospectively using a standardized web-based survey. All survey response data were captured electronically. We defi ned universal training as any programme which delivers CPR and AED training to all students in the school. We used simple descriptive statistics to summarise the results. J Callerova1, R Skulec2, J Knor3, V Cerny3 1Emergency Medical Service of Central Bohemian Region, Beroun, Czech Republic; 2Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015, 19(Suppl 1):P416 (doi: 10.1186/cc14496) J Callerova1, R Skulec2, J Knor3, V Cerny3 1Emergency Medical Service of Central Bohemian Region, Beroun, Czech Republic; 2Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015 19(Suppl 1):P416 (doi: 10 1186/cc14496) Introduction Implementation of point-of-care ultrasound examination into cardiopulmonary resuscitation (CPR) may increase diagnostic accuracy for determining the cause of cardiac arrest. However, current protocols either do not refl ect all causes detectable by ultrasound or are too complicated for prehospital use. Thus, we decided to construct and validate a new protocol TRACE (ThoRacic and Abdominal sonography in Cardiac arrEst) for ultrasound examination during out-of-hospital cardiac arrest (OHCA). Results A total of 51 schools completed the survey covering an estimated student population of 54,037. There were four (8%) schools that provide universal training programmes and an additional 23 (45%) off er optional training programmes for students. There were 16 (31%) schools which have an AED available on the school premises. The most common reasons for not having a universal CPR training programme is the requirement for additional class time (15/51; 29%) and that funding is unavailable for such a programme (12/51; 24%). There were three students who died from sudden cardiac arrest over the period of the past 10 years. Methods We designed a new protocol for ultrasound examination during OHCA to increase the success rate for the establishment of OHCA cause. Body position during transport in a refractory cardiac arrest porcine model p Methods Data were prospectively collected from January 2011 to January 2012 from 336 female medical and pharmacy students undergoing CPR training at the Lithuanian University of Health Sciences. During the training process, the instructors performed a simple 5-second intervention (Andrew’s manoeuvre) with all of the rescuers in the study group. The instructor pushed 10 times on the shoulders of each trainee while she performed CCs to achieve the maximal required compression depth. Immediately after training, the participants were asked to perform a 6-minute BLS test on a manikin that was connected to a PC with SkillReporter™ System software (Laerdal, Norway); the quality of the participants’ CPR skills was then evaluated. Body position during transport in a refractory cardiac arrest porcine model J Belohlavek1, M Mlcek2, M Huptych3, T Boucek1, T Belza2, P Krupickova2, O Kittnar2 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) Introduction Cardiac arrest patients are not transported only supine. The eff ect of body position on resuscitability and cerebral perfusion in a 30° and 60° incline is not known.i Results The CC depth in the study group increased by 6.4 mm (P <0.001) compared with the control group (52.9 vs. 46.6 mm). A regression analysis showed that Andrew’s manoeuvre increased the depth of the CCs among women by 14.87 × (1 – 0.01 × weight) mm.i Methods Twenty-fi ve female pigs were subjected to a simulated cardiac arrest (3 minutes no fl ow, 5 minutes mechanical CPR). Next, animals were randomly assigned to one of the three groups: GROUP 60 (n = 8), 60° incline for 3 minutes to simulate transport in space restricted elevator; GROUP 30 (n = 8), 30° incline for 8 minutes to simulate staircase transport; and GROUP 0, with no incline. During subsequent standard CPR including rescue ECMO, resuscitability and cerebral perfusion were assessed. Conclusion Andrew’s manoeuvre during CPR training signifi cantly improved the performance of the female rescuers and helped them achieve the CC depth required by 2010 resuscitation guidelines. It is most eff ective among the women with the lowest body weight. Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools Introduction Mortality from cardiac arrest remains high [1]. Bystander cardiopulmonary resuscitation (CPR) and the use of automated external defi brillators (AED) are two of the most important factors favouring survival [2]. CPR/AED training in schools is a recommended intervention for signifi cantly improving training rates across a large population [3]. The current practice for CPR/AED training in London schools is unknown. The primary aim of this study was to assess current Introduction Mortality from cardiac arrest remains high [1]. Bystander cardiopulmonary resuscitation (CPR) and the use of automated external defi brillators (AED) are two of the most important factors favouring survival [2]. CPR/AED training in schools is a recommended intervention for signifi cantly improving training rates across a large population [3]. The current practice for CPR/AED training in London schools is unknown. The primary aim of this study was to assess current S146 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 practices relating to CPR and AED training in London secondary schools. 1. Krikscionaitiene A, et al. Magical manoeuvre: a 5-s instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth. Eur J Emerg Med. 2014;21:424-8. Magical manoeuvre: a 5-second instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth Magical manoeuvre: a 5-second instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth A Krikscionaitiene, A Pranskunas, M Dambrauskiene, N Jasinskas, Z Dambrauskas, E Vaitkaitiene, J Vencloviene, D Vaitkaitis Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P415 (doi: 10.1186/cc14495) A Krikscionaitiene, A Pranskunas, M Dambrauskiene, N Jasinskas, Z Dambrauskas, E Vaitkaitiene, J Vencloviene, D Vaitkaitis Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P415 (doi: 10.1186/cc14495) Conclusion Implementation of the TRACE protocol to the CPR process was feasible, required minimal interruption of cardiac compressions and resulted in a high recognition rate for the cause of OHCA. Introduction Adequate chest compression (CC) depth is crucial for resuscitation outcomes. Lightweight rescuers, particularly women, are often unable to achieve the required 5 to 6 cm CC depth. This nonrandomised cohort study investigated new strategies to improve CC performance. TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest The subcostal view was performed during planned rhythm check to assess the presence of cardiac tamponade and size of the right and left ventricle and inferior caval vein. Thereafter, during ongoing cardiac compressions, Morrison’s pouch and right pleural space were investigated to exclude intraperitoneal and inrapleural free fl uid. The same procedure was applied on the left side of the body. Finally, the anterior thoracic view was done to exclude pneumothorax. Working diagnosis was compared with the fi nal in-hospital diagnosis or autopsy. Conclusion CPR and AED training rates in London secondary schools are low. The majority of schools do not have an AED available on premises. The most common reason for not providing CPR training is the requirement for additional class time. These data highlight an opportunity to vastly improve CPR training rates in a large population. Future studies should assess programmes which are cost-eff ective and which do not require signifi cant amounts of additional class time. References 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87. 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87. Results We examined 40 consecutive OHCA patients. Correct cause of OHCA during CPR was recognised in 38 patients (95%). Leading causes were acute coronary syndrome (55.0%), pulmonary embolism (15.0%) and complication of chronic heart failure (10.0%). Incorrect recognition was performed in one patient with respiratory cause, originally considered as pulmonary embolism, and in another with pulmonary embolism, considered as respiratory cause. One rhythm check was suffi cient to perform TRACE in 31 patients, in the other two interruptions of cardiac compressions were required. Return of spontaneous circulation was achieved in 15 (37.5%) patients, favourable neurological outcome at hospital discharge in eight (20%) patients. Specifi c therapy to aff ect the cause of OHCA was applied during OHCA in 12 (30%) patients. 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. P417 Body position during transport in a refractory cardiac arrest porcine model J Belohlavek1, M Mlcek2, M Huptych3, T Boucek1, T Belza2, P Krupickova2, O Kittnar2 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Ch 1 ZM Li 1 JH T 1 S S h 1 M H i Bi I k d 1 B L 2 Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Chua1, ZM Lin1, JH Tan1, S Surentheran1, M Haris Bin Iskander1, B Leong2 1National University of Singapore, Singapore; 2National University Hospital, Singapore Critical Care 2015 19(Suppl 1):P418 (doi: 10 1186/cc14498) Introduction Despite several large studies concerning out-of-hospital cardiac arrests in recent years, it is not clear whether their in-hospital counterparts have benefi ted from advances in resuscitation as well as post-resuscitation care. Introduction Despite several large studies concerning out-of-hospital cardiac arrests in recent years, it is not clear whether their in-hospital counterparts have benefi ted from advances in resuscitation as well as post-resuscitation care. p Methods We identifi ed all cases of in-hospital cardiac arrest (IHCA) occurring in the National University Hospital in Singapore from 1 June 2008 to 31 May 2009. Patients for which IHCA occurred but where no resuscitation was attempted were excluded. Key outcomes were classifi ed as primary (survival to discharge) and secondary (return of spontaneous circulation). Additionally, various arrest characteristics were analysed to identify predictive factors for survival to discharge with level of signifi cant set at P <0.05. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. Figure 2 (abstract P419). Comparison complications of CPR between the OHCA group and the IHCA group. gi Results Among 353 unique cases of IHCA analysed, 63 patients (17.8%) had a shockable rhythm (ventricular fi brillation and pulseless ventricular tachycardia) of which 17 (27.0%) survived to discharge. While 290 (82.2%) patients presented with nonshockable rhythm (asystole or pulseless electrical activity), only 32 patients (11%) survived to discharge. For patients who survived to discharge, univariate analysis showed that event location (P = 0.016), nationality (P = 0.035), paying class (P = 0.038), use of ECG monitoring (P = 0.048), initial cardiac rhythm (P = 0.000) and presence of a house offi cer (P = 0.005) were statistically signifi cant. Multivariate analysis showed that patients with shockable rhythms were 2.52 times more likely to survive but other factors were not signifi cant. For patients who attained ROSC, univariate analysis showed that time of day (P = 0.006), event location (P = 0.000), and number of adrenaline doses administered (P = 0.000) were statistically signifi cant. 9 Comparison of complications secondary to cardiopulmonary resuscitation between out-of-hospital cardiac arrest and in-hospital cardiac arrest M Seung Y Park M Seung, Y Park Yonsei University Severance Hospital/Yonsei University College of Medicine, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P419 (doi: 10.1186/cc14499) M Seung, Y Park Yonsei University Severance Hospital/Yonsei University College of Medicine, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P419 (doi: 10.1186/cc14499) Introduction Chest compression during cardiopulmonary resuscitation (CPR) could bring out unintended complications which are mainly composed of chest injuries. The aim of this study was to assess whether there was a signifi cant diff erence in the complications of CPR between out-of-hospital cardiac arrest (OHCA) and in-hospital cardiac arrest (IHCA) survivors using multidetector computed tomography (MDCT). g p g p y Methods We performed a retrospective cohort study in the emergency departments of two academic tertiary care centres from January 2009 to May 2014. We enrolled both OHCA and IHCA patients who underwent successful CPR. The enrolled patients had undergone a chest CT within 48 hours after ROSC. We evaluated the MDCT fi ndings of the CPR-related chest injuries and compared complications between OHCA and IHCA patients. Figure 1 (abstract P417). Figure 1 (abstract P417). (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1. (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1. p Results A total of 148 patients were fi nally enrolled in this study, OHCA were 89 (60.1%) and IHCA were 59 (39.8%). The mean CPR time, both in-hospital and total, was longer in OHCA survivors. Rib fractures were Conclusion Positional changes during simulated refractory cardiac arrest in this experimental model signifi cantly aff ect resuscitability and brain perfusion. Animals subjected to shorter time in a more inclined (GROUP 60) position were more easily resuscitated; however, cerebral blood fl ow was better preserved in GROUP 30. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. P418 Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Chua1, ZM Lin1, JH Tan1, S Surentheran1, M Haris Bin Iskander1, B Leong2 1National University of Singapore, Singapore; 2National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P418 (doi: 10.1186/cc14498) Body position during transport in a refractory cardiac arrest porcine model Reference Results Attainment of ROSC (3, 5, 5 in respective groups, P = 0.021), time to ROSC (15:24 (13:26; 16:02) vs. 19:19 (18:28; 19:37) vs. 9:10 minutes (8:28; 9:41), respectively, P = 0.005) signifi cantly diff ered. Changes in carotid blood fl ow according to the respective periods of the protocol 1. Krikscionaitiene A, et al. Magical manoeuvre: a 5-s instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth. Eur J Emerg Med. 2014;21:424-8. S147 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1 Figure 1 (abstract P417). Figure 1 (abstract P417). Impact of intra-arrest fl uid loading with diff erent doses of crystalloid infusion on hemodynamics in experimental cardiac arrest R Skulec1, A Truhlar1, R Parizkova1, Z Turek1, D Astapenko1, J Dudakova2, V Cerny3 R Skulec1, A Truhlar1, R Parizkova1, Z Turek1, D Astapenko1, J Dudakova2, V Cerny3 y 1Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 2Emergency Medical Service of Central Bohemian Region, Kladno, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic l ( l ) (d ) 1Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 2Emergency Medical Service of Central Bohemian Region, Kladno, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015, 19(Suppl 1):P420 (doi: 10.1186/cc14500) Introduction Fluid loading during cardiopulmonary resuscitation for nonhypovolemic cardiac arrest remains controversial. Thus, we conducted an experimental study comparing the impact of two diff erent doses of balanced crystalloid infusion on hemodynamics in a porcine model of ventricular fi brillation.i Conclusion Although our study has a number of methodological limitations, these results about incidence in cardiac arrest survivors corroborate previous retrospective reports. It is possible that every cardiac arrest survivor has had to live a NDE, regardless of brain mechanisms associated with experience, but only some patients remember it. If some chronic medications, such as benzodiazepine, may decrease memorization, the role of the elements of the clinical context about NDE during resuscitation is speculative References i Methods Ventricular fi brillation was induced for 15 minutes in 19 anesthetized domestic pigs. Before induction, the animals were randomized to receive either 1,000 ml (34 ± 3 ml/kg, group A, n = 7) or 500 ml (16 ± 2 ml/kg, group B, n = 7) of balanced crystalloid solution or to undergo no fl uid loading during CPR (group C, n = 5). After spontaneous circulation (ROSC) was restored, the animals were observed for 90 minutes. 1. Van Lommel P, van Wees R, Meyers V, Elff erich I. Near-death experience in survivors of cardiac arrest: a prospective study in the Netherlands. Lancet. 2001;358:2039-45. Results In all groups, signifi cant increase of intracranial pressure followed by its decrease after ROSC was observed. While in groups B (from 12 ± 2 to 18 ± 2 mmHg, P <0.05) and C (from 13 ± 1 to 18 ± 3 mmHg, P <0.05) it was comparable (P >0.05), the rise of intracranial pressure in group A was signifi cantly higher (from 12 ± 3 to 23 ± 3 mmHg, P <0.05). Impact of intra-arrest fl uid loading with diff erent doses of crystalloid infusion on hemodynamics in experimental cardiac arrest Whereas coronary perfusion pressure was lower in group A than in control group C during volume loading, fl uid infusion induced its mild increase in group B (group A: 12.1 ± 2.4, group B: 16.0 ± 2.6, group C: 13.6 ± 2.8 mmHg, P = 0.043). Decrease of cerebral perfusion pressure was equal in all groups. Cardiac index 10 minutes after ROSC signifi cantly diff ered among all groups (group A: 8.9 ± 2.2, group B: 7.1 ± 1.3, group C: 4.9 ± 1.9 l/minute/m2, P = 0.007) and the dose of crystalloid infusion during cardiac arrest positively correlated with cardiac index increase (r = 0.815, P <0.001).i 2. Parnia S, Spearpoint K, de Vos G, Fenwick P, Goldberg D, Yang J, et al. AWARE – AWAreness during REsuscitation – a prospective study. Resuscitation. 2014;85:1799-805. 3. Greyson B. The near-death experience scale. Construction, reliability, and validity. J Nerv Ment Dis. 1983;171:369-75. 3. Greyson B. The near-death experience scale. Construction, reliability, and validity. J Nerv Ment Dis. 1983;171:369-75. f References 1. Kim EY, Yang HJ, Sung YM, et al. Multidetector CT fi ndings of skeletal chest injuries secondary to cardiopulmonary resuscitation. Resuscitation. 2011;82:1285-8. Kim EY, Yang HJ, Sung YM, et al. Multidetector CT fi ndings of skeleta Methods In a retrospective study from 2005 to 2012, 295 consecutive cardiac patients who were successfully resuscitated after cardiac arrest were enrolled. In total, 204 (69%) were alive during the research period (mean delay: 55 months). A total of 118 (40%), over 18 years, able to answer a short standardized interview were included in the study when they accepted to participate. Demographic, medical, pharmacological and psychological data were recorded and we used the Greyson NDEs scale to identify and characterize NDEs. Descriptive and unifactorial analysis was performed using the Jacknife method and Wald test according to low event frequency. 2. Kim MJ, Park YS, Kim SW, et al. Chest injury following cardiopulmonary resuscitation: a prospective computed tomography evaluation. Resuscitation. 2013;84:361-4. g y Results From our 118 reports, 20 described a core experience and 18 (15.3%) met the criteria for NDEs (Greyson NDEs total score >6/32 (7 to 19)). Only one patient recounted a negative experience. Regarding the risk factors for NDEs, using univariate analysis, we found for demographic data: woman (CI: 1.11 (1.10 to 1.12), P <0.0001), age under 60 (CI: 1.23 (1.21 to 1.24), P <0.0001), prior knowledge of NDEs (CI: 1.97 (1.95 to 1.99)) and previous NDE (CI: 5.82 (4.19 to 8.08)). According to the history of previous disease, we found an increased risk for pulmonary disease (CI: 1.75 (1.73 to 1.77)), rheumatic disease (CI: 3.79 (3.75 to 3.84)), endocrine disease (CI: 1.45 (1.43 to 1.46)), and a decrease for cardiac disease (CI: 0.65 (0.64 to 0.66)), psychiatric disease (CI: 0.71 (0.69 to 0.72)) and digestive tract disease (CI: 0.71 (0.69 to 0.72)). For previous pharmacological treatment we found a decrease of risk for all classes and particularly when two drugs were simultaneously given (CI: 0.37 (0.36 to 0.38)). Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Centre Hospitalier Universitaire, Reims Cedex, France Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Centre Hospitalier Universitaire, Reims Cedex, France Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) p Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) Introduction Near-death experiences (NDEs) are increasingly being reported as a clear reality of clinical signifi cance. Previous studies, essentially, have been trying to estimate their incidence in various populations, notably after cardiac arrest resuscitation, and to understand the implication of resuscitation characteristics [1,2]. Using the Greyson NDE scale [3], the present retrospective study aimed at exploring cardiac arrest survivors and the correlations between NDE and physiological, medical, psychological and pharmacological context. i Conclusion Frequency of rib fractures and multiple rib fractures were higher in OHCA survivors. Further investigation is needed into the relation between the location of CPR and the CPR-related injuries, eff orts to reduce the complications after CPR. Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Ch 1 ZM Li 1 JH T 1 S S h 1 M H i Bi I k d 1 B L 2 Multivariate analysis showed that an arrest occurring in the ICU setting was 2.9 times more likely to attain ROSC (95% CI: 1.02 to 5.59, P = 0.044). Conclusion The results of this study have described some key predictive factors regarding positive outcomes in IHCA in Singapore. These are vital in understanding important features regarding IHCAs and will aid in developing policies to help improve care and survival in this group of patients. S148 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P421 detected more in OHCA survivors. Frequency of multiple rib fractures was higher in OHCA survivors. Frequency of sternum fractures was higher in OHCA survivors, showing no signifi cant diff erence. In lung injuries, lung contusion and pneumothorax account for the large part, and OHCA survivors had higher incidence in both complications but statistically insignifi cant. Major complications occurred in eight cases in OHCA survivors and three cases in IHCA survivors during the study period. After adjusting for the time factor in multiple logistic regression analysis, rib fractures and multiple rib fractures became statistically signifi cant in OHCA survivors. See Figures 1 and 2. Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest Eighty-three percent (n = 45) of survivors admitted to the ICU went to the cardiac cathlab before ICU admission compared with 53% (n = 39) of nonsurvivors. Forty-three percent of survivors had PPCI compared with 26% of nonsurvivors. Eighty- one percent (n = 44) of survivors received therapeutic hypothermia compared with 62% (n = 48) of nonsurvivors. Introduction Mild hypothermia and fever control have been shown to improve neurological outcomes post cardiac arrest. Common methods to induce hypothermia include body surface cooling and intravascular cooling; however, a new approach using a catheter placed into the esophagus has recently become available. p g y Methods We report the fi rst three cases of temperature control using an esophageal cooling device (ECD). The ECD was placed orally in a similar fashion to orogastric tubes. Temperature reduction was achieved by connecting the ECD to a commercially available heat exchange unit (Blanketrol II or III). Conclusion Over 3 consecutive years our annual case mix, ICU and hospital mortalities for OOHCA patients admitted to the ICU have remained stable, while our annual pre-ICU cathlab and PPCI utilisation have risen consistently in both survivors and nonsurvivors. ICU survivors were more likely to have had a shockable rhythm, been to the cathlab, and received PPCI and TH, but all may simply refl ect selection bias. Any benefi t these conferred to cardiac patients may have been off set by our liberal ICU admission policy to OOHCAs with nonshockable rhythms. Access to 24–7 PPCI in this group may determine survival and we suggest that OOHCA patients should be taken directly to regional heart attack centres. Results The fi rst patient, a 59-year-old male (73 kg), was admitted after successful resuscitation from a protracted out-of hospital cardiac arrest. His initial temperature was 35°C, which is within our current institutional protocol of 34 to 36°C. Several hours after arrival, his temperature slowly increased to 35.8°C despite application of a cooling blanket and ice packs to the groin and axilla. The ECD was inserted and a reduction of temperature to 34.8°C was achieved within 3 hours. The patient expired on day 3. The second patient, a 54-year-old female (95 kg), was admitted after resuscitation from an out-of-hospital PEA arrest. Despite initiating our cooling protocol with surface-cooling blankets and cold intravenous saline, she mounted a fever peaking at 38.3°C shortly after admission. Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, V Achan, M Davies, M Peck Frimley Park Hospital, Frimley, UK y p y Critical Care 2015, 19(Suppl 1):P422 (doi: 10.1186/cc14502) y p y Critical Care 2015, 19(Suppl 1):P422 (doi: 10.1186/cc14502) Conclusion Fluid loading during CPR had signifi cant impact on hemodynamics in our experimental model. While a high dose led to unintentional increase of intracranial pressure and decrease of coronary perfusion pressure, a low dose did not aff ect intracranial pressure and was associated with mild increase of coronary perfusion pressure during cardiac arrest. Introduction Our 700-bed hospital has a 24–7 cathlab service that routinely investigates patients with indications prior to ICU admission following out-of-hospital cardiac arrest (OOHCA). Our aim was to compare survivors and nonsurvivors and evaluate utilisation and S149 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 nonshockable rhythms. However, access to 24–7 PPCI may determine survival and we suggest that all OHCA patients should be taken directly to regional heart attack centres. nonshockable rhythms. However, access to 24–7 PPCI may determine survival and we suggest that all OHCA patients should be taken directly to regional heart attack centres. prognostic impact of angiography and primary percutaneous coronary intervention (PPCI) in this patient group. Methods A retrospective analysis using Trust electronic databases (Symphony, WardWatcher, PICIS, PRISM) of all OOHCA patients admitted to our ICU over 3 consecutive years between 1 November 2011 and 31 October 2014. P424 Targeted temperature management after cardiac arrest and fever control with an esophageal cooling device A Hegazy, D Lapierre, E Althenayan University of Western Ontario, London, ON, Canada Critical Care 2015, 19(Suppl 1):P424 (doi: 10.1186/cc14504) Results A total of 351 patients presented to our emergency department (ED) following OOHCA in this period, and of these 50% died in the ED, 37% were admitted to the ICU and 13% were admitted elsewhere. Of the 129 patients admitted to the ICU, median age was 66 (range 18 to 93), 71% were male, 68% had a shockable presenting rhythm, median ICU LOS was 3.75 (range 1 to 34 days) and ICU and hospital mortalities were 50% and 60% respectively. Eighty-nine percent (n = 48) of OOHCA survivors admitted to the ICU had a shockable rhythm compared with 55% (n = 41) of nonsurvivors. P425 P425 Is selective nasopharyngeal brain cooling detrimental to neuroprotection? M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P425 (doi: 10.1186/cc14505) P423 Utilisation and prognostic impact of angiography and primary percutaneous coronary intervention prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, M Davies, V Achan, M Peck Frimley Park Hospital, Frimley, UK Critical Care 2015, 19(Suppl 1):P423 (doi: 10.1186/cc14503) P423 Utilisation and prognostic impact of angiography and primary percutaneous coronary intervention prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, M Davies, V Achan, M Peck Frimley Park Hospital, Frimley, UK Critical Care 2015, 19(Suppl 1):P423 (doi: 10.1186/cc14503) Introduction Our 700-bed hospital has a 24–7 cathlab service that routinely investigates patients with indications prior to ICU admission following out-of-hospital cardiac arrest (OHCA). Our aim was to compare ICU survivors and nonsurvivors and evaluate the utilisation and prognostic impact of angiography and primary percutaneous coronary intervention (PPCI) in this patient group. f Conclusion The ECD is a novel technology that can be used for temperature management post cardiac arrest and for fever control in critically ill patients. Despite patients mounting a febrile response, temperature control was achieved and maintained successfully. The device was reported as being easy to use, by both physicians and nurses. y p g p Methods A retrospective analysis using Trust electronic databases (Symphony, WardWatcher, PICIS, PRISM) of all OHCA patients admitted to our ICU over 3 consecutive years between 1 November 2011 and 31 October 2014. Results A total of 351 patients presented to our hospital following OHCA in this period, and of these 50% died in the ED, 37% were admitted to the ICU and 13% elsewhere. Of the 129 patients admitted to the ICU, median age was 66 (range 18 to 93), 71% were male, 68% had a shockable presenting rhythm, median ICNARC score was 31 (range 10 to 66), median ICU LOS was 3.75 (range 1 to 34 days) and ICU and hospital mortality were 50% and 60% respectively. ICU survivors were more likely to have had a shockable rhythm (89 vs. 55%), been to the cathlab (83 vs. 53%), received PPCI (43 vs. 26%) and TH (82 vs. 62%) and had lower median ICNARC scores (26 vs. 35) than nonsurvivors. Over the 3 consecutive years, pre-ICU cathlab and PPCI utilisation increased annually in ICU survivors (73 vs. 86 vs. 89% and 45 vs. 54 vs. 59% respectively) and nonsurvivors (40 vs. 38 vs. 50% and 20 vs. 27 vs. 31% respectively). However, our annual ICU and hospital mortality remained unchanged (46 vs. 51 vs. 51% and 60 vs. 57 vs. 62% respectively). Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest After ECD insertion and confi rming the external heat exchanger connection, her temperature gradually dropped to 35.7°C over a period of 4 hours. She subsequently made a favorable neurological recovery and was discharged home at day 23. The third patient, a 47-year-old male patient (86 kg) presented with an ongoing fever secondary to necrotizing pneumonia in the postoperative period after coronary artery bypass grafting. His fever was unresponsive to empiric antibiotic therapy, antipyretics, cooling blankets, and ice packs. ECD insertion resulted in a decrease in temperature from 39.5°C to 36.5°C in less than 5 hours. The patient eventually made a full recovery and was discharged home after 59 days. In all three patients, placement of the device occurred in less than 3 minutes and ease of use was reported as excellent by nursing staff and physicians. P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) Introduction Uncontrolled shivering may have negative consequences by increasing metabolic demand and subsequently neutralize the benefi ts of therapeutic normothermia [1]. Previous anti-shivering protocols that utilize the least sedation have been described in therapeutic temperature modulation (TTM) [2]. Our aim is to describe and evaluate an anti-shivering protocol that emphasizes the least sedating regimen with the least number of pharmacologic agents for patients undergoing therapeutic normothermia. brain temperature, intracranial pressure (ICP), core temperatures and vital signs were continuously recorded. Cooling was terminated once the brain reached 32°C and the animals were allowed to passively rewarm. Results In the SNBC group, the brain target temperature was reached in 54 ± 11 minutes. The mean ICP dropped precipitously to a nadir of –15 mmHg. TCD showed signifi cant vasospasm in the MCA, compared with the internal carotid artery (ICA), during the entire cooling phase (Table 1). Upon termination of cooling, the brain temperature spontaneously rewarmed to core temperature in 13 ± 4 minutes. Rewarming was associated with hyperemia and elevation of ICP. In group 2, there was no cerebral vasospasm or hyperemia during cooling and rewarming respectively.i Methods This retrospective chart review includes patients with neurologic injury who underwent TTM from March 2013 to November 2014 and were evaluated for the following outcomes: percentage of total patient hours in each score of the Bedside Shivering Assessment Scale (BSAS) at 72 hours, 168 hours, and total duration of TTM; percentage of total patient days in each tier of the anti-shivering protocol at 72 hours, 168 hours, and total duration of TTM; de- escalation of anti-shivering agents with or without the necessary need for re-escalation; ICU and hospital length of stay (LOS); rescue agents utilized; and hospital mortality. Conclusion SNBC is associated with signifi cant vasospasm of the MCA. In addition, spontaneous and rapid rewarming of the brain, vasodilation, rapid reperfusion, and rebound elevation of ICP, all occurring minutes after termination of SNBC, are likely to be detrimental to an already ischemic brain. y Results Evaluation of 47 patients who underwent TTM resulted in a total of 505 patient-days of TTM with 6,967 BSAS hours. Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) 2 yg Methods A prospective, observational study during therapeutic hypothermia (24 hours –33°C) in 82 post-CA patients monitored with near-infrared spectroscopy. Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) y Results Forty-three patients (52%) survived in CPC 1 to 2 until 180 days post CA. The mean MAP range associated with maximal survival was 76 to 86 mmHg (OR = 2.63, 95% CI (1.01; 6.88), P = 0.04). The mean SVO2 range associated with maximal survival was 67 to 72% (OR = 8.23, 95% CI (2.07; 32.68), P = 0.001). In two separate multivariate models, a mean MAP (OR = 3.88, 95% CI (1.22; 12.33), P = 0.02) and a mean SVO2 (OR = 8.79, 95% CI (1.69; 18.36), P = 0.01) in the optimal range persisted as independently associated with increased survival after correction for presence of early bystander CPR and presenting shockable rhythm. Based on more than 1,625,000 data points, we found a strong linear relation between SVO2 (range 40 to 90%) and average cerebral saturation (R2 = 0.86) and between MAP and average cerebral saturation for MAPs between 40 and 87 mmHg (R2 = 0.70). Based on our hemodynamic model, the optimal MAP and SVO2 were determined to be 87 mmHg and 72%. Introduction Therapeutic hypothermia (TH) is the international standard of care for all comatose patients after cardiac arrest, but criticism focuses on poor outcomes. We sought to develop criteria to identify American urban patients more likely to benefi t from TH. Introduction Therapeutic hypothermia (TH) is the international standard of care for all comatose patients after cardiac arrest, but criticism focuses on poor outcomes. We sought to develop criteria to identify American urban patients more likely to benefi t from TH. Methods A retrospective chart review of 107 consecutive adults under- going TH in downtown New Orleans from 2010 to 2014 yielded records for 99 patients with all 44 survivors or families contacted up to 4 years. Results Sixty-nine males and 38 females with a mean age of 60.2 years showed 63 dead (58%) and 44 survivors (42%). Presenting cardiac rhythm was divided into shockable (pulseless ventricular tachycardia, ventricular fi brillation) and nonshockable (pulseless electrical activity, asystole). Presenting in shockable rhythms with ROSC <20 minutes were 21 patients with 15 (71%) survivors (P = 0.001). P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) Overall, patients spent 85.5% of total hours at BSAS 0, 11.4% of total hours at BSAS 1, 2.5% of total hours at BSAS 2, and 0.6% of total hours at BSAS 3. Patients were in tier 0 of the anti-shivering protocol 33.1% of the time, in tier 1 of the anti-shivering protocol 20.6% of the time, in tier 2 of the anti-shivering protocol 43.6% of the time, and in tier 3 of the anti- shivering protocol 2.8% of the total duration of TTM. There were 487 rescue doses of fentanyl and 243 rescue doses of meperidine that were required for shivering. Patients had a mean ICU LOS of 19 days, mean hospital LOS of 21 days, and a mortality rate of 23.4%.fi P426 Conclusion This study demonstrates a high level of effi cacy of our protocol and the feasibility of de-escalation to limit the number of pharmacologic interventions. With our patient population spending a large percentage of time without shivering, it would suggest that this protocol could be revised further by utilizing rescue agents more frequently in order to prevent escalation of therapy to the next tier. References Hemodynamic targets during therapeutic hypothermia after cardiac arrest: a prospective observational study K Ameloot, I Meex, C Genbrugge, W Boer, F Jans, B Ferdinande, W Mullens, M Dupont, C Dedeyne, J Dens ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P426 (doi: 10.1186/cc14506) 1. Badjiatia N, et al. Crit Care Med. 2009;37:S250-7. 2. Choi HA, et al. Neurocrit Care. 2011;14:389-94. 1. Badjiatia N, et al. Crit Care Med. 2009;37:S250-7. 2. Choi HA, et al. Neurocrit Care. 2011;14:389-94. Introduction In analogy with sepsis, current postcardiac arrest (post- CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. Introduction In analogy with sepsis, current postcardiac arrest (post- CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. Methods A prospective, observational study during therapeutic hypothermia (24 hours –33°C) in 82 post-CA patients monitored with near-infrared spectroscopy. P428 Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) Is selective nasopharyngeal brain cooling detrimental to neuroprotection? M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P425 (doi: 10.1186/cc14505) M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Introduction Clinical outcomes vary depending on the method used to induce therapeutic hypothermia following stroke or cardiac arrest. In swine, we tested the hypothesis that selective nasopharyngeal brain cooling (SNBC), in contrast to systemic hypothermia, adversely impacts cerebral perfusion. Methods In two groups of animals (34 to 35 kg), blood fl ow in the right middle cerebral artery (MCA) was measured using transcranial Doppler (TCD). In group 1, SNBC was initiated using perfl uorohexane aerosol (1 ml/kg/minute) and oxygen (1 l/kg/minute) through a nasopharyngeal catheter atomizer. In group 2, the animals were body surface cooled using water-circulating blankets (4°C). In both groups, Conclusion ICU survivors were more likely to have had a shockable rhythm, been to the cathlab, received PPCI and TH and been less sick than nonsurvivors, but these may simply refl ect selection and other biases. Any benefi t these factors did confer to cardiac patients may have been off set by our liberal ICU admission policy to OHCAs with S150 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 brain temperature, intracranial pressure (ICP), core temperatures and vital signs were continuously recorded. Cooling was terminated once the brain reached 32°C and the animals were allowed to passively rewarm. Results In the SNBC group, the brain target temperature was reached in 54 ± 11 minutes. The mean ICP dropped precipitously to a nadir of –15 mmHg. TCD showed signifi cant vasospasm in the MCA, compared with the internal carotid artery (ICA), during the entire cooling phase (Table 1). Upon termination of cooling, the brain temperature spontaneously rewarmed to core temperature in 13 ± 4 minutes. Rewarming was associated with hyperemia and elevation of ICP. In group 2, there was no cerebral vasospasm or hyperemia during cooling and rewarming respectively. Conclusion SNBC is associated with signifi cant vasospasm of the MCA. In addition, spontaneous and rapid rewarming of the brain, vasodilation, rapid reperfusion, and rebound elevation of ICP, all occurring minutes after termination of SNBC, are likely to be detrimental to an already ischemic brain. Is selective nasopharyngeal brain cooling detrimental to neuroprotection? P426 Hemodynamic targets during therapeutic hypothermia after cardiac arrest: a prospective observational study K Ameloot, I Meex, C Genbrugge, W Boer, F Jans, B Ferdinande, W Mullens, M Dupont, C Dedeyne, J Dens ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P426 (doi: 10.1186/cc14506) Introduction In analogy with sepsis, current postcardiac arrest (post- Table 1 (abstract P425). Cerebral vasospasm during SNBC MCA fl ow ICA fl ow velocity velocity Lindegaard Pulsatility ICP Intervention (cm/second) (cm/second) ratio ratio (mmHg) Baseline 62 41 1.51 0.77 13 SNBC cooling 128 52 2.46 0.48 –15 SNBC rewarming 96 44 1.74 1.12 21 Table 1 (abstract P425). Cerebral vasospasm during SNBC Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest p g C Endisch, C Storm, CJ Ploner, C Leithner C Endisch, C Storm, CJ Ploner, C Leithner Methods With IRB approval, we prospectively measured rSO2 during ALS in consecutive OHCA patients. One sensor of the Equanox™ 7600 (NONIN) was applied on the patient’s forehead’s right side when the medical emergency team arrived in an OHCA. ROSC was defi ned as ROSC >20 minutes. Charité Universitätsmedizin Berlin, Germany Charité Universitätsmedizin Berlin, Germany Critical Care 2015, 19(Suppl 1):P431 (doi: 10.1186/cc14511) y Critical Care 2015, 19(Suppl 1):P431 (doi: 10.1186/cc14511) Introduction Electrical median nerve stimulation elicits a burst of high-frequency oscillations (HFOs) superimposing onto the cortical short-latency potential. Digital fi ltering of somatosensory evoked potentials (SSEPs) enables non-invasive analysis of these HFOs. The late HFO component following the cortical N2O peak is ascribed to spiking activity of cortical neurons. Results We included 88 prehospital cardiac arrest patients between December 2011 and October 2014 with eight (9%) patients with CPC 1 or 2. Twenty-seven patients of the nonsurvivors had ROSC >20 minutes and one patient had CPC 3 at hospital discharge. We observed no signifi cant diff erence between both groups in age (P = 0.161), time between emergency call and start of ALS (P = 0.788) and duration of basic life support performed by bystanders, general practitioners or paramedics (P = 0.649). The initial rhythm was asystole in one (12.5%) survivor and in 50 (62.5%) nonsurvivors (P = 0.009), ventricular fi brillation in six (75%) survivors and 13 (16%) nonsurvivors (P = 0.001), and pulseless electrical activity in one (12.5%) survivor and 17 (21%) nonsurvivors (P = 1.00). The cardiac arrest was witnessed in all survivors (100%) and in 49 (61%) nonsurvivors (P = 0.046). First measured rSO2 was 38% (27 to 67) in the survivor group compared with 22% (8 to 32) in the nonsurvivor group (P = 0.004). Also a signifi cant diff erence was found in mean rSO2 until 1 minute before ROSC between survivors and nonsurvivors, respectively 46% (40 to 74) and 34% (22 to 42). We observed no signifi cant diff erence in increase of rSO2 until 1 minute before ROSC between survivors 12.5% (5 to 21) and nonsurvivors 11% (5 to 18) (P = 0.719). Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) Time >20 minutes until ROSC in shockable rhythms had fi ve patients with three survivors (78%, P = 0.001). Presenting in nonshockable rhythms with ROSC <20 minutes were 54 patients with 18 survivors (33%, P = 0.001). ROSC >20 minutes in nonshockable rhythms had 19 patients with two survivors (8%, P = 0.001). Survivors of shockable rhythms showed 19 (100%) living post TH. Fifteen survivors (79%, n = 19, P = 0.001) had CPC g Conclusion The optimal SVO2 (72%) and MAP (87 mmHg) derived from our hemodynamic model matched with the observed SVO2 (67 to 72%) and MAP (76 to 86 mmHg) associated with maximal survival. Prospective interventional studies to reach or maintain these targets are needed to confi rm these fi ndings. S151 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of this study were to investigate the association between hemoglobin, cerebral oxygenation (SctO2) and outcome in post-CA patients. score 1 or 2 with four survivors (21%, n = 19) having a CPC score of 3. A total of 25 survived nonshockable rhythm. Acute survival of patients with nonshockable rhythm showed 18 expired <72 hours (72%, n = 25) with long-term survival of four patients (5%, n = 74) and CPC scores of 1 or 2 (P = 0.001). Interestingly, patients with time to ROSC <20 minutes exhibiting more than one loss of sustained ROSC showed 100% mortality (P = 0.001). Patients presenting with shockable >20 minutes ROSC had overall survival of 70% (P = 0.001), but those undergoing >3 cardiac rhythm changes had 100% mortality (P = 0.001). yg 2 Methods A prospective observational study in 82 post-CA patients during hypothermia in the fi rst 24 hours of ICU stay. Hemoglobin was determined hourly together with a corresponding SctO2 by NIRS and SVO2 in patients with a pulmonary artery catheter (n = 62). 2 Results Based on 2,099 paired data, we found a strong linear relationship between hemoglobin and average SctO2 (SctO2 = 0.70 × hemoglobin + 56 (R2 = 20.84, P = 10–6)). Given the previously suggested SctO2 target between 66 and 68%, hemoglobin levels below 10 g/dl generally resulted in suboptimal brain oxygenation. Forty-three patients (52%) had a good neurological outcome (CPC 1 to 3) at 180 days post CA. P432 P432 One-size-fi ts-all or patient-tailored hemodynamic targets in post-cardiac arrest patients: an observational near-infrared spectroscopy study on cerebral autoregulation C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) P432 One-size-fi ts-all or patient-tailored hemodynamic targets in post-cardiac arrest patients: an observational near-infrared spectroscopy study on cerebral autoregulation C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest y Methods We retrospectively studied late HFO components of median nerve SSEPs obtained 24 hours to 4 days after cardiac arrest in patients treated with mild hypothermia (33°C for 24 hours). Cortical average recordings were digitally fi ltered at 450 to 750 Hz and noise- corrected maximum peak-to-peak amplitudes of the cortical late HFO bursts determined. Outcome upon ICU discharge was dichotomized according to the Cerebral Performance Category (CPC) scale. CPC 1 to 3 was classifi ed as good outcome, CPC 4 to 5 (unresponsive wakefulness syndrome and death) as poor outcome. Results Of 307 consecutive patients, 153 (50%) achieved good outcome (CPC 1 to 3) and 154 (50%) had poor outcome. Late HFO bursts were present in 102 (33%) recordings. Among patients with late HFO amplitudes above 0.1 μV, 26 had CPC 1 to 3, none had CPC 4 and eight died. Case review indicated causes of death other than hypoxic encephalopathy in all patients who died despite HFO amplitudes above 0.1 μV. Conclusion We found cortical late HFO bursts, obtained by digital fi ltering of standard SSEP recordings, in a signifi cant proportion of patients after cardiac arrest treated with mild hypothermia. Our data indicate that the presence of late HFO bursts with amplitudes above 0.1 μV may confi rm absence of severe hypoxic encephalopathy early after cardiac arrest with high specifi city. Conclusion We observed a signifi cant diff erence in fi rst measured rSO2 and mean rSO2 until 1 minute before ROSC between patients with good neurological outcome (CPC 1 or 2) at hospital discharge and patients with worse neurological outcome or nonsurvivors (CPC 3 or 4 or 5). However, no signifi cant diff erence was observed in the increase between both groups. Larger studies are necessary to confi rm these results. Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) There was a signifi cant association between average hemoglobin above 12.3 g/dl and good neurological outcome (OR = 2.88, 95% CI = 1.02; 8.16, P = 0.04). In a multivariate model, this association persisted after correction for comorbidities and age by the modifi ed Charlson score (OR = 2.99, 95% CI = 1.05; 8.53, P = 0.03). This association was entirely driven by results obtained in patients with an average SVO2 below 70% (OR = 17.55, 95% CI = 1.67; 184.41, P = 0.01). Conclusion Patients presenting with shockable rhythms undergoing TH had overall acute survival of 70% followed by long-term survival of 100% after 4 years. In contrast, patients presenting with nonshockable rhythm had long-term survival of 5%. TH is not recommended. Diff erence in cerebral saturation during cardiopulmonary resuscitation between survivors with favorable neurological outcome and compromised neurological outcome at hospital discharge C Genbrugge1, W Boer1, I Meex2, F Jans1, C Deyne1, J Dens1 1ZOL, Genk, Belgium; 2Hasselt University, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P429 (doi: 10.1186/cc14509) C Genbrugge , W Boer , I Meex , F Jans , C Deyne , J Dens 1ZOL, Genk, Belgium; 2Hasselt University, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P429 (doi: 10.1186/cc14509) Conclusion There is a steep linear relationship between hemoglobin and SctO2 in post-CA patients with hemoglobin levels below 10 g/dl generally resulting in cerebral desaturation. Average hemoglobin below 12.3 g/ dl was independently associated with worse neurological outcome 180 days post CA. An interventional trial is necessary to investigate whether maintaining higher hemoglobin would improve outcome. Introduction During out-of-hospital cardiac arrest (OHCA) monitoring possibilities are limited. Recently, the role of cerebral oximetry, using near-infrared spectroscopy, during ALS was investigated. In this study we determined whether the magnitude of increase in cerebral saturation (rSO2) or mean rSO2 during prehospital ALS was associated with good neurological outcome at hospital discharge (Cerebral Performance Category (CPC) 1 or 2). P431 Response of regional oxygen saturation technologies during hypoxia UB Borg, AM Neitembach Covidien, Boulder, CO, USA Critical Care 2015, 19(Suppl 1):P433 (doi: 10.1186/cc14513) Results Thirty-four simplifi ed EEG samples were analysed. According to standard EEG, 11 patients showed a DS pattern, three had CI, six showed BS, four showed PEDs and 10 had an SE. Neurophysiologists interpreted all samples with a high accuracy. Mean sensitivity was 82.12% and mean specifi city was 91.88%. Only one SE was missed by one neurophysiologist. Unfortunately, only one PED was confi rmed by both neurophysiologists. Interobserver reliability was high (κ = 0.843). High correlations were found for the comparison of full and simplifi ed EEG for both neurophysiologists (r = 0.809). Further, the two inexperienced physicians identifi ed SE with a sensitivity of 85% and specifi city of 98%. Introduction The purpose of this study was to determine the rate and magnitude of response to hypoxia for three diff erent regional oxygen saturation (rSO2) devices. rSO2 technologies are focused on absolute accuracy without consideration of response characteristics. Current rSO2 technologies assume that the oxygen saturation is a fi xed ratio of arterial and venous blood. Cerebral arteries have an oxygenation- related vasoactivity that may change the arterial/venous ratio during hypoxia. Thus, absolute rSO2 accuracy may be less important compared with sensitivity to changes in cerebral rSO2. pi y Conclusion Simplifi ed EEG monitoring, using BIS, resulted in high accuracy of a simple classifi cation system in post-CA patients. Not only neurophysiologists, but also treating physicians were capable to identify SE, which may play an important role in the early detection of SE. We suggest using BIS as a screening tool in post-CA patients to save valuable time in the detection of SE, without replacing the need for full EEG monitoring for confi rmation. 2 Methods Ten subjects completed the study and are included in the analysis. One INVOS sensor (SAFB-SM) was placed on the left side and one Equanox (8000CA) or Foresight (1 July 2007 or 1 July 2005) sensor (alternated between subjects) was placed on the right side of the forehead for bilateral monitoring. Desaturation was induced by adjusting the inspiratory gas mixture of O2/N2. Desaturation was titrated from room air to achieve a plateau of 70% arterial oxygen saturation (SpO2). Resaturation was induced by rapid change in FiO2 to 1.0. Diff erences in cerebral saturation measured during prehospital advanced life support, between patients with presumed cardiac origin and noncardiac origin of cardiac arrest C Genbrugge1, W Boer1, K Anseeuw2, I Meex3, F Jans1, J Dens1, C De Deyne1 2 g p Results The rate of rSO2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was signifi cantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P <0.05). There was signifi cant diff erence in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P <0.005) and between INVOS compared with Foresight (20 ± 5%) (P <0.05). Conclusion All rSO2 devices followed the SpO2 slope during desaturation as expected. The diff erences between the devices in terms of total rSO2 change reached statistical signifi cance. There were also signifi cant diff erences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change 2 Results The rate of rSO2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was signifi cantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P <0.05). There was signifi cant diff erence in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P <0.005) and between INVOS compared with Foresight (20 ± 5%) (P <0.05). Introduction During out-of hospital cardiac arrest (OHCA) cerebral saturation may provide relevant information on cerebral oxygenation. In this study we examined the time course in cerebral saturation (rSO2) during prehospital ALS comparing patients with a presumed cardiac origin (survivor = Sc, nonsurvivor = NSc) of arrest and noncardiac origin (survivor = Snc, nonsurvivor = NSnc) of arrest. Conclusion All rSO2 devices followed the SpO2 slope during desaturation as expected. The diff erences between the devices in terms of total rSO2 change reached statistical signifi cance. There were also signifi cant diff erences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change Methods With IRB approval, we prospectively measured rSO2 from the start of ALS in consecutive OHCA patients. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients (4) The time under the individual optimal MAP was negatively associated with survival (OR = 0.97, 95% CI (0.96; 0.99), P = 0.02). The time under previously proposed fi xed targets (65, 70, 75, 80 mmHg) was not associated with a diff erential survival rate. Introduction Assessment of prognosis in postcardiac arrest (post- CA) patients became very challenging since the introduction of therapeutic hypothermia (TH). Continuous EEG monitoring has been proposed to improve prognostication; however, its use is limited due to diffi culties in ready interpretation. Thus emerges the need for a simple EEG montage. The bispectral index (BIS) monitor is a simplifi ed EEG system, mainly calculating an index ranging from 0 (isoelectric EEG) to 100 (full consciousness) to provide information on hypnotic depth of anaesthesia. The aim of the study was to validate the accuracy of simplifi ed EEG monitoring in a CA setting. pi g g Methods BIS monitoring (BIS VISTATM) was applied to collect frontotemporal data in TH-treated CA patients. A standard 19-channel EEG was performed after return to normothermia. Afterwards, small EEG frames coincident with the time of full EEG registration were extracted from the BIS monitor. We asked two neurophysiologists to indicate the presence of status epilepticus (SE), cerebral inactivity (CI), burst suppression (BS), periodic epileptiformic discharges (PEDs) or a diff use slowing pattern (DS). In addition, these samples were analysed by two inexperienced physicians, who were asked to indicate the presence of SE. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients g , yg neurologic outcome in postcardiac arrest patients I Meex, K Ameloot, C Genbrugge, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P430 (doi: 10.1186/cc14510) I Meex, K Ameloot, C Genbrugge, M Dupont, B Ferdinande, J Dens C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P430 (doi: 10.1186/cc14510) p py y g C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) Introduction The safety of a restrictive transfusion threshold of 7 g/dl applied in all critically ill patients can be questioned in postcardiac arrest (post-CA) patients since these are phenotypically clearly distinct. The aims Introduction A subgroup of post-CA patients with disturbed cerebral autoregulation might benefi t from higher mean arterial pressures S152 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 in seconds and the magnitude of absolute change may result in better resolution to detect physiological changes. Clinical studies are required to elucidate the clinical relevance of the diff erences. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. Methods A prospective observational study in 51 post-CA patients monitored with near-infrared spectroscopy. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. p Methods A prospective observational study in 51 post-CA patients monitored with near-infrared spectroscopy. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients P434 P434 Use of bispectral index EEG monitoring for a fast and reliable detection of epileptic activity in postcardiac arrest patients J Haesen1, L Desteghe1, I Meex2, C Genbrugge2, J Demeestere2, J Dens2, L Ernon2, C De Deyne2 1Universiteit Hasselt, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium Critical Care 2015, 19(Suppl 1):P434 (doi: 10.1186/cc14514) p py Results (1) In multivariate analysis, patients with preserved auto- regulation (33.65%) had a signifi cant higher 180-day survival rate (OR = 4.62, 95% CI (1.06; 20.06), P = 0.04). (2) Phenotypically, a higher proportion of patients with disturbed autoregulation had pre- CA hypertension (31 ± 47 vs. 65 ± 49%, P = 0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood fl ow to higher blood pressures. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3) An individual optimal MAP could be determined in 33/51 patients. (4) The time under the individual optimal MAP was negatively associated with survival (OR = 0.97, 95% CI (0.96; 0.99), P = 0.02). The time under previously proposed fi xed targets (65, 70, 75, 80 mmHg) was not associated with a diff erential survival rate. Conclusion Cerebral autoregulation was shown to be disturbed in 35% of post-CA patients of which a majority had pre-CA hypertension. Disturbed cerebral autoregulation within the fi rst 24 hours after CA is associated with a worse outcome. In contrast to uniform MAP goals, the time spent under a patient-tailored optimal MAP, based on an index of autoregulation, was negatively associated with survival. Results (1) In multivariate analysis, patients with preserved auto- regulation (33.65%) had a signifi cant higher 180-day survival rate (OR = 4.62, 95% CI (1.06; 20.06), P = 0.04). (2) Phenotypically, a higher proportion of patients with disturbed autoregulation had pre- CA hypertension (31 ± 47 vs. 65 ± 49%, P = 0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood fl ow to higher blood pressures. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3) An individual optimal MAP could be determined in 33/51 patients. Response of regional oxygen saturation technologies during hypoxia After 5 minutes of SpO2 100%, the process was repeated by desaturation to SpO2 70% and rapid return to SpO2 100%. Cerebral and pulse oximetry data were recorded during the study and the time of each FiO2 change and plateau was recorded. rSO2 levels at 10, 20, 40, 60 and 80% of the total SpO2 response were calculated for each device to assess the rate of rSO2 change. The rate of rSO2 change in seconds and total rSO2 change were compared. P436 Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) C Storm, CJ Ploner, C Leithner Introduction Bilaterally absent cortical somatosensory evoked potentials (SSEPs) predict poor outcome after cardiac arrest. A threshold for the amplitude of early cortical SSEPs above which patients may survive with good outcome has not been determined. Thus, tolerable noise levels for the interpretation of cortical SSEPs are poorly defi ned. Furthermore, it has not been systematically investigated whether high amplitudes of cortical SSEPs may exclude severe hypoxic encephalopathy incompatible with re-awakening. Conclusion In patients with cardiac arrest and targeted temperature management at 33°C, an NSE serum concentration of >90 μg/l strongly indicates poor outcome. NSE producing tumors, acute brain diseases, severe hematologic diseases, use of an intra-aortic balloon pump and blood transfusions need to be considered as potential confounders. An NSE serum concentration of <17 μg/l largely excludes hypoxic encephalopathy incompatible with re-awakening. p p y p g Methods We prospectively studied SSEPs after median nerve stimulation obtained 24 hours to 4 days after cardiac arrest in patients treated with targeted temperature management at 33°C for 24 hours. Amplitudes of cortical SSEPs were determined, if at least two peripheral, spinal and cortical recordings per side were available, spinal potentials were bilaterally reproducible and cortical noise level was below 0.25 μV. Cortical SSEP amplitude was defi ned as largest amplitude of a reproducible cortical SSEP of four cortical recordings (two per side) within 50 milliseconds after stimulation. Outcome was assessed upon ICU discharge using the Cerebral Performance Category (CPC) scale. CPC 1 to 3 was defi ned as good outcome, CPC 4 to 5 as poor outcome. Results Of 318 consecutive patients examined, 293 had complete SSEP recordings with reproducible spinal potentials and cortical noise levels below 0.25 μV. Of those, 137 (47%) had good outcome and 156 (53%) had poor outcome. The lowest amplitude of the early cortical SSEPs in a survivor with good outcome was 0.62 μV. All 79 patients with amplitudes below this threshold had poor outcome. None of 27 patients who survived with CPC 4 (unresponsive wakefulness syndrome) had cortical SSEP amplitudes above 2.5 μV. Twenty-four patients with amplitudes above this limit died. Detailed case review indicated a cause of death other than hypoxic encephalopathy in these patients. P436 Results Of 601 included patients, 309 (51%) had good outcome. An NSE serum concentration threshold of 90 μg/l predicted poor outcome with a positive predictive value of 0.98 and a sensitivity of 0.51. Three patients survived with good outcome despite an NSE serum concentration >90 μg/l. In two of these patients NSE elevations had been documented prior to cardiac arrest. One patient had a neuroendocrine tumor of the pancreas, the other patient suff ered from encephalitis of unknown etiology and an osteomyelofi brosis. Potential confounders in the third patient were an ovarian carcinoma, the use of an intra-aortic balloon pump and blood transfusions shortly after cardiac arrest. Only 16 of 205 patients with NSE <17 μg/l had poor outcome, the majority of these patients died from causes other than hypoxic encephalopathy. Diff erences in cerebral saturation measured during prehospital advanced life support, between patients with presumed cardiac origin and noncardiac origin of cardiac arrest One sensor (Equanox™ 7600; Nonin) was applied on the patient’s forehead’s right side when S153 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 the medical emergency team arrived at the OHCA setting. ROSC was defi ned as ROSC >20 minutes. Retrospectively, included patients were divided into two groups with respect to their presumed origin of arrest. Results Between December 2011 and October 2014, 113 OHCA patients were included. We observed a signifi cant diff erence in asystole and VF as initial rhythm between NSc and NSnc, respectively (P = 0.035 and P = 0.001). In both groups of NS, duration of ALS was signifi cant longer compared with the two S groups (P = 0.001 in both comparisons). We observed no signifi cant diff erence in fi rst measured rSO2, mean rSO2 until 1 minute before ROSC and increase in rSO2 until 1 minute before ROSC (respectively P = 0.123, P = 0.501, P = 0.265) between Sc and Snc. However, when we compare the nonsurvivors of cardiac with noncardiac origin, we observed a signifi cant diff erence in mean rSO2 until 1 minute before ROSC, 35% (27 to 44) in the NSc group and 27 (21 to 34) in the NSnc group (P = 0.026). First measured rSO2 was 24.5% (13 to 34) in the NSc group and 14 (4 to 28) in the NSnc group (P = 0.069) trending to be signifi cantly diff erent. No signifi cant diff erence was observed in increase until 1 minute before ROSC between both groups of NS (P = 0.920). Signifi cant diff erences was observed in mean rSO2 until 1 minute before ROSC and increase in rSO2 between Snc and NSnc (P = 0.033; P <0.001) and between Sc and NSc (P = 0.001; P <0.001).i Prognostic value of neuron-specifi c enolase after cardiac arrest and targeted temperature management Prognostic value of neuron-specifi c enolase after cardiac arrest and targeted temperature management g p g K Streitberger, C Leithner, CJ Ploner, C Storm Charité Universitätsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P437 (doi: 10.1186/cc14517) K Streitberger, C Leithner, CJ Ploner, C Storm Charité Universitätsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P437 (doi: 10.1186/cc14517) Introduction The serum concentration of neuron-specifi c enolase (NSE) has been established as a highly specifi c predictor of poor outcome after cardiac arrest. Recent studies have indicated that patients treated with targeted temperature management at 33°C for 24 hours may have good outcome despite NSE serum concentrations considerably higher than the cutoff established for normothermic patients. The threshold above which survival with good outcome becomes very unlikely, its positive predictive value and sensitivity for prediction of poor outcome have not been established in this patient group. Furthermore, a threshold below which hypoxic encephalopathy may be largely excluded has not been determined. Methods From 2006 through 2014 we prospectively included in- hospital and out-of-hospital cardiac arrest patients treated with targeted temperature management at 33°C for 24 hours. The NSE serum concentration was determined 3 days after cardiac arrest and the outcome was assessed according to the Cerebral Performance Category (CPC) upon ICU discharge. CPC 1 to 3 was defi ned as good outcome and CPC 4 to 5 as poor outcome. Individual case review was performed in patients with good outcome despite very high NSE serum concentration and in patients with poor outcome despite very low NSE serum concentration. Conclusion We can conclude that NSc have a signifi cant higher mean rSO2 and trend to have a signifi cant diff erence in fi rst measured rSO2 compared with NSnc. However, no signifi cant diff erence was observed between Sc and Snc. P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522 Results One hundred and ninety-three patients (110 males, 83 females, mean age 48.2 ± 18.3 years) were studied. The mean time interval between collapse and onset of resuscitation was 2.3 ± 2.1 minutes. A total of 65.3% arrests were witnessed. Sustained ROSC occurred in 36.8% patients and the SOHD was 24.9%. The initial rhythm recorded during CPR was asystole in 133 patients, pulseless electrical activity in 21 patients and ventricular fi brillation/tachycardia (VF/VT) in 39 patients. SOHD for these rhythms was 8.3%, 33.3% and 76.9%, respectively. On univariate analysis, type of rhythm, witnessed arrests and time to resuscitation were associated with sustained ROSC and SOHD. On multivariate analysis, only type of rhythm, VF/VT (P = 0.000) and PEA (P = 0.017), were signifi cantly associated with SOHD, while witnessed arrest and time to resuscitation were not. Introduction The association between blood alcohol level (BAL) on mortality and neurologic outcome in patients with polytrauma and head injury is not clear and the data in the literature are sometimes confl icting. Some studies suggest a possible neuroprotective eff ect of alcohol and increased survival while others show the opposite. The rationale for this study was to investigate whether BAL has any impact on presentation, neurologic outcome and survival in patients with combination of polytrauma and head injury. y j y Methods This is a retrospective study of 43 polytraumatized patients with head injury who were intubated and treated by the prehospital unit and transported to the trauma center. Patients were grouped according to their BAL into BAL+ (>0.5 mg/l) and BAL– (≤0.5 mg/l). Inclusion criteria were age ≥18, Injury Severity Score ≥16 and head Abbreviated Injury Scale (AIS) ≥3. Physiological parameters and outcome with respect to survival to hospital discharge (STHD) and functional outcomes were analyzed. Severity of injuries was measured using the Trauma–Injury Severity Score (TRISS) and head injury using AIS. Functional outcome was measured using the Glasgow Outcome Scale (GOS), Cerebral Performance Category (CPC) and Glasgow Coma Conclusion Sustained ROSC occurred in 36.8% patients and the SOHD was 24.9%. P440 Outcome after CPR: when we cannot save lives, we can save organs C Caestecker, J Froyman, P Lormans, W Stockman AZ Delta, Roeselare, Belgium Critical Care 2015, 19(Suppl 1):P440 (doi: 10.1186/cc14520) Introduction Patients resuscitated after cardiac arrest (CA) who suff er bad neurologic outcome or become brain dead might become organ donors (OD) when well managed and identifi ed. We report on the cohort of patients resuscitated after in-hospital or out-of-hospital CA becoming OD in a tertiary community hospital with an intensive donor identifi cation program. i g Methods Data from our 28-bed mixed medical/surgical adult ICU in a 900-bed tertiary hospital were analyzed from 2010 to 2014. y y Results Our ICU admitted 2,320 patients/year. Overall ICU mortality in this period was 7.4%. A summary of the results is presented in Table 1. Of the 219 patients admitted after CA, 21 (10%) became OD. This resulted in 55 successfully transplanted organs (28 kidneys, 17 livers, seven lung pairs, three hearts). Of note, good outcome (CPC 1 and 2) was achieved in 55 patients (25%). frequently suff ered cardiogenic shock, had more organ dysfunctions and died more frequently, respectively, with hospital mortality of 79.5% versus 29.1%, P <0.001; see also Figure 1. Table 1 (abstract P440) Organ Donors ICU admission Year donors after CA DCD/DBD after CA 2010 11 3 0/3 40 2011 9 5 3/2 41 2012 18 4 3/1 47 2013 17 5 4/1 53 2014 13 4 1/3 38 Total 68 21 (31%) 11/10 219 Conclusion Ten percent of patients resuscitated after CA and admitted to the ICU become OD, consisting of up to 31% of the total number of OD in our center. Patients resuscitated after CA who suff er severe irreversible brain damage or are brain dead can thus substantially expand the donor pool. This justifi es extensive resuscitation eff orts, if not to save lives, then to save organs. This might be reassuring for families, staff and the community. Table 1 (abstract P440) Organ Donors ICU admission Year donors after CA DCD/DBD after CA 2010 11 3 0/3 40 2011 9 5 3/2 41 2012 18 4 3/1 47 2013 17 5 4/1 53 2014 13 4 1/3 38 Total 68 21 (31%) 11/10 219 Conclusion In patients hospitalized for acute heart failure, both prehospital and postadmission resuscitated cardiac arrest is a severe complication associated with signifi cantly morbidity and mortality. Outcome of cardiopulmonary resuscitation in cancer patients in an Indian tertiary cancer center Conclusion Ten percent of patients resuscitated after CA and admitted to the ICU become OD, consisting of up to 31% of the total number of OD in our center. Patients resuscitated after CA who suff er severe irreversible brain damage or are brain dead can thus substantially expand the donor pool. This justifi es extensive resuscitation eff orts, if not to save lives, then to save organs. This might be reassuring for families, staff and the community. Introduction Cardiopulmonary resuscitation (CPR) after cardiac arrest in cancer patients is often discouraged as it is associated with poor outcome. In our 700-bed tertiary cancer hospital in Mumbai, India, the ICU runs an in-hospital cardiac arrest team (CAT). We reviewed our data to determine outcome from CPR, identify factors associated with improved outcomes and justify the presence of a CAT in our cancer hospital. p Methods All in-hospital patients from November 2012 to November 2014 (2-year period) with unanticipated cardiorespiratory arrests were included. Data were recorded prospectively using the Utstein template. Only patients with cardiac arrest rhythms were included. Patients with anticipated progression towards arrest, those with seizures, hypotension without dysarrythmias or other medical emergencies were excluded. The outcomes studied were return of spontaneous circulation (ROSC) and survival on hospital discharge (SOHD). Binary logistic regression analysis was performed to determine factors associated with ROSC and SOHD. P436 Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) P438 38 Resuscitated cardiac arrest in patients admitted with acute heart failure: analysis of a large prospective AHEAD network registry J Parenica1, J Belohlavek2, J Spinar1, G Dostalova2, S Havranek2, A Linhart2 R Miklik1, L Dusek3, J Jarkovsky3 1University Hospital Brno, Czech Republic; 2General University Hospital, Prague, Czech Republic; 3Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P438 (doi: 10.1186/cc14518) Introduction Heart failure is a frequent cause of cardiac arrest and vice versa, cardiac arrest frequently complicates acute heart failure episodes. We aimed to characterize the infl uence of cardiac arrest on outcome of acute heart failure patients admitted to hospital. Methods The AHEAD registry includes patients hospitalized for acute heart failure from 10 PCI and fi ve non-PCI centers in the Czech Republic. The data were collected from September 2006 to October 2012. Conclusion Our data indicate that the prognostic value of SSEP after cardiac arrest extends beyond a mere absent–present dichotomy. Bilaterally absent as well as very low amplitude SSEPs predict poor outcome with high positive predictive value. SSEPs should not be used for prognostication, if noise in cortical recordings could mask critically low amplitudes. High amplitudes of early cortical SSEPs strongly argue against severe hypoxic encephalopathy incompatible with re-awakening. Results In the respective period, a total of 6,242 patients were enrolled into the registry. Resuscitated cardiac arrest occurred in 313 patients prehospitally and in 484 after admission; the remaining 5,445 patients were not resuscitated during their index hospitalization. Patients resuscitated after admission in comparison with prehospitally resuscitated were older, had lower left ventricle ejection fraction, more S154 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P438). Overall mortality (including hospitalization). Total n = 6,242. Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Endothelial dysfunction in acute brain injury and the development of cerebral ischemia S Van Ierssel1, VM Conraads1, EM Van Craenenbroeck1, Y Liu2, AI Maas1, PM Parizel1, VY Hoymans1, CJ Vrints1, PG Jorens1 1University Hospital Antwerp, Edegem, Belgium; 2The Third Xiangya Hospital, Changsha, China Critical Care 2015, 19(Suppl 1):P443 (doi: 10.1186/cc14523) Introduction Since most patients with acute brain injury are treated head-up at 30 to 45°, there can be a height diff erence of up to 15 cm between the heart and the ear canal. This causes a diff erence between mathematical CPP and true CPP of up to 11 mmHg depending on the zero reference level used and the body length of the patient (Figure 1). We conducted a survey to analyze the current practice on CPP targets and zero reference levels in diff erent ICUs. Introduction Cerebral ischemia (CeI) is a major complicating event after acute brain injury (ABI) in which endothelial dysfunction is a key player. p y Methods We studied cellular markers of endothelial dysfunction and peripheral reactive hyperemia index (RHI) in 26 patients with ABI at admission and after 6 and 12 days, and compared these with healthy volunteers (n = 15). CeI was determined clinically or using computer tomography.i f Methods Neuro-ICU physicians were invited to participate in a survey on ICP and CPP targets and the level of measurement. Results The results of 30 centers are summarized in Table 1. Most centers (83.3%) use head-up elevation of 30 to 45° and consider an ICP of 20 mmHg as the threshold to start treating ICP (80%). More variation is noted in minimal and maximal CPP threshold. All centers measure ICP at the ear canal. Twenty-seven centers (90%) measure MAP at the heart, three centers measure MAP at the ear canal. These three centers use >50, 60 and 65 mmHg as minimal CPP and raise the bed to 30 to 45°. The two centers using minimal CPP >60 mmHg do not apply an upper limit. g p y Results In patients with ABI, RHI at admission was signifi cantly reduced compared with healthy subjects (P = 0.003), coinciding with a decrease in circulating endothelial progenitor cells (EPC) (P = 0.002) (Table 1). The RHI recovered in eight patients without development of CeI (Figure 1, black), but failed to fully recover by day 12 in three out of four patients that developed CeI (Figure 1, red). P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) A reduced response time, witnessed arrest and type of rhythm are associated with ROSC and improved SOHD. The type of rhythm was independently associated with SOHD, with VF/VT and PEA having improved survival while asystolic patients had a poor outcome. These considerations justify the presence of a CAT in our cancer hospital. S155 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P443). Evolution in time of markers of endothelial dysfunction after acute brain injury Scale (GCS) at discharge. Diff erences among groups were analyzed using Student’s t test, the Mann–Whitney U test and the chi-square test. Results BAL did not have a signifi cant eff ect on presenting physiological parameters. There was a signifi cant diff erence in the age of the groups, showing that patients in the BAL+ group were younger (32.6 vs. 56.8 years; 95% confi dence level; P = 0.000). BAL had a lasting eff ect on functional measures of neurological outcome which showed better results in the BAL+ group; it had signifi cantly better GOS (4.7 vs. 3.9; 95% confi dence level; P = 0.027), and GCS at discharge was on the border of statistical signifi cance (15 vs. 14; 95% confi dence level; P = 0.05). Other variables (TRISS, AIS, STHD, CPC) did not show statistically signifi cant diff erences among groups. gif g g p Conclusion Presence of alcohol in the blood had a positive eff ect on neurological outcome but there was no signifi cant diff erence in survival. However, the positive results in neurologic outcome in the BAL+ group may also be due to the fact that this group was younger. The small number of patients in the study is another limiting factor of the interpretation. Therefore, the neuroprotective role of alcohol needs further clarifi cation. Conclusion Patients with ABI exhibit impaired microvascular endo thelial function measured as RHI and a decreased circulating level of EPC. P444 P444 P444 Survey on ICP, target CPP and MAP measurement level in patients with severe acute brain injury in diff erent ICUs M Van Laer1, K Deschilder2, W Stockman2 1UZ Brussel, Brussels, Belgium; 2AZ Delta, Roeselare, Belgium Critical Care 2015, 19(Suppl 1):P444 (doi: 10.1186/cc14524) Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Despite recovery Figure 1 (abstract P443). y Conclusion Considering the infl uence of position on CPP, the variations among centers and the narrow range of CPP thresholds, future studies and guidelines should describe where MAP is measured. Alternatively, we propose a new formula for CPP: true CPP = MAP(heart) – ICP(ear) – height diff erence (heart to ear canal (cm)) × 0.73. Conclusion Considering the infl uence of position on CPP, the variations among centers and the narrow range of CPP thresholds, future studies and guidelines should describe where MAP is measured. Alternatively, we propose a new formula for CPP: true CPP = MAP(heart) – ICP(ear) – height diff erence (heart to ear canal (cm)) × 0.73. Figure 1 (abstract P443). Figure 1 (abstract P444). Table 1 (abstract P444) ICP threshold (mmHg); n (%) >15; 2 (6.7%) >18; 1 (3.3%) >20; 24 (80%) >25; 3 (10%) Minimal CPP (mmHg); n (%) >50; 6 (20%) >55; 4 (13.3%) >60; 16 (53.3%) >65 to 70; 4 (13.3%) Maximal CPP (mmHg); n (%) <70 to 75; 8 (26.7%) <80; 8 (26.7%) <85; 2 (6.7%) No limit; 12 (40%) Figure 1 (abstract P444). Figure 1 (abstract P444). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S156 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P445 with traumatic brain injuries defi ned as a Glasgow Coma Score <8 upon admission. Patients included were 18 to 59 years of age and were mechanically ventilated with intracranial pressure monitoring for greater than 72 hours. The primary outcome evaluated was the number of patients treated for confi rmed infections. Secondary outcomes included the antibiotic length of therapy (LOT), antibiotic days of therapy (DOT), number of cultures, and ICU and hospital length of stay (LOS). DOT was defi ned as the sum of days on which each antibacterial drug was administered. P445 Comparison of 15oxygen positron emission tomography and near-infrared spectroscopy for measurement of cerebral physiology J Simpson1, N Sudhan1, H Hare2, J Donnelly1, X Liu1, F Aigbirhio1, T Fryer1, G Stocks-Gee1, P Smielewski1, D Bulte2, J Coles1 1University of Cambridge, UK; 2University of Oxford, UK Critical Care 2015, 19(Suppl 1):P445 (doi: 10.1186/cc14525) Introduction The gold standard technique for imaging cerebral blood fl ow (CBF) and metabolism is 15oxygen positron emission tomography (15O PET). g References 1. Zweifel C, et al. Stroke. 2010;41:1963-8. 1. Zweifel C, et al. Stroke. 2010;41:1963-8. 2. Coles JP, et al. JCBFM. 2004;24:202-11. 3. Culver JP, et al. JCBFM. 2003;23:911-24. f Results Sixty-four treatments (32 HTS, 32 Man) were studied among 26 patients (19 TBI, seven SAH; age 42 ± 17 years, time from injury to treatment 2.6 ± 1.9 days). Man and HTS eff ectively decreased ICP (coeffi cient = –2.5 mmHg, 95% CI = –3.2 to –1.8 mmHg and –2.9 (–3.8 to –2.0) respectively; both P < 0.0001). No signifi cant diff erence was found in CMD lactate, pyruvate, glucose and PbtO2 after HTS or Man treatment. CMD glutamate decreased signifi cantly after Man (–0.73 ( –1.41 to –0.052); P <0.05), but not after HTS. Eff ect of osmotherapy with mannitol or hypertonic saline on cerebral oxygenation and metabolism in patients with intracranial hypertension after severe brain injury y j T Suys, H Quintard, C Patet, M Oddo Introduction Osmotherapy with mannitol (Man) or hypertonic saline (HTS) is currently used to treat elevated intracranial pressure after severe acute brain injury (sABI); however, their eff ect on cerebral oxygenation and metabolism has not been extensively evaluated. Methods A retrospective analysis of a cohort of patients with sABI after traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) monitored with cerebral microdialysis (CMD), brain oxygen (PbtO2) and ICP, who were treated with Man (20%, 0.5 g/kg) or HTS (7.5%, 100 ml) for ICP >25 mmHg. Osmotherapy was administered over 20 minutes and each patient’s individual response to intervention was analyzed up to 120 minutes following infusion. Only episodes where no other hypertonic solute was administered within 2 hours before or after treatment were selected. Variables analyzed included CMD lactate, pyruvate, glucose, glutamate, lactate/pyruvate ratio, and main brain physiologic variables ICP, PbtO2, CPP. Analysis was conducted using mixed-eff ects multilevel regression. Introduction Osmotherapy with mannitol (Man) or hypertonic saline (HTS) is currently used to treat elevated intracranial pressure after severe acute brain injury (sABI); however, their eff ect on cerebral oxygenation and metabolism has not been extensively evaluated. Conclusion We found no relationship between NIRS and baseline physiology as determined by 15O PET. Further studies should assess the dynamic response of NIRS to a measured change in physiology in patients. Further confi nes of NIRS include its limited and focal brain coverage. Conclusion We found no relationship between NIRS and baseline physiology as determined by 15O PET. Further studies should assess the dynamic response of NIRS to a measured change in physiology in patients. Further confi nes of NIRS include its limited and focal brain coverage. Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Continuous near-infrared spectroscopy (NIRS) has been used to assess adequacy of cerebral oxygenation following stroke, traumatic brain injury and subarachnoid haemorrhage [1], and measurements have been compared with jugular oxygen saturation. In this study we compared NIRS with 15O PET within healthy volunteers. g Results A total of 23 patients treated with normothermia and 119 patients in the control group were evaluated between January 2009 and September 2014. The number of patients treated for confi rmed infections was similar between groups (normothermia: 73.9%, control: 80%, P = 0.803). Empiric antibiotic therapy was more commonly utilized in the normothermia group at 34% versus 20.5% (P = 0.173). Antibiotic LOT and DOT were 13.8 versus 10.8 days (P = 0.157) and 18.3 versus 16.2 days (P = 0.575) in the normothermia versus control groups, respectively. Total culture rate was lower in the normothermia group with 13.2 versus 8.78 (P = 0.0002) cultures per patient. No signifi cant diff erence in hospital LOS (normothermia: 23 days, control: 18 days, P = 0.158) or ICU LOS (normothermia: 17 days, control: 15 days, P = 0.185) was demonstrated.i y Methods Fifteen healthy subjects were recruited (12 male, average age 38 years); PET precluded females of reproductive age. Steady-state 15O PET with arterial sampling was performed to measure CBF, cerebral metabolic rate of oxygen (CMRO2), oxygen extraction ratio (OEF) and cerebral blood volume (CBV) [2]. Simultaneously, NIRS data were collected using a Hamamatsu NIRO 200 with sensors on either side of the forehead. NIRS OEF was calculated from tissue oxygen saturation, SaO2 and an assumed arterial/venous blood volume ratio of 30/70 [3]. Results The frontal region 15O PET CBF, CMRO2, OEF and CBV were mean (SD) 44.9 (10) ml/100 ml/minute, 158.7 (24.7) μmol/100 ml/minute, 45.8 (7.3)%, and 2.8 (0.8) ml respectively, and there was no relationship between NIRS and 15O PET (Figure 1). Conclusion Rates of confi rmed infections and number of antibiotic days were similar between the normothermia and control groups, suggesting that normothermia does not increase infection risk. However, the number of cultures obtained in the control group was signifi cantly greater than the normothermia group with a trend toward increased empiric antibiotic use. Figure 1 (abstract P445). Linear correlation between NIRS and PET OEF. Reference Figure 1 (abstract P445). Linear correlation between NIRS and PET OEF. Reference 1. Greer DM, et al. Stroke. 2008;39:3029-35. 1. Greer DM, et al. Stroke. 2008;39:3029-35. Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate C Patet1, H Quintard1, T Suys1, L Pellerin2, P Magistretti3, M Oddo1 1CHUV – Lausanne University Hospital, Lausanne, Switzerland; 2University of Lausanne, Switzerland; 3Brain Mind Institute, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P448 (doi: 10.1186/cc14528) Introduction In patients with acute brain injury (ABI), increased cerebral energy demand is frequent, potentially leading to cerebral glucose depletion (GD) and poor outcome. In this setting, lactate may act as supplemental fuel. We examined dynamics of cerebral lactate supply during prolonged GD in ABI. Results ICP registrations were made parallel with all TCD measurements in 51 patients. Intraparenchymal ICP monitoring was inserted within the fi rst 3 hours after trauma and there was no complication (infections, intracranial hemorrhage, or technical failure) related to invasive ICP monitoring. Increased ICP was noted upon transducer insertion in 38 children with male prevalence (10 girls, 28 boys). Median GCS was 6, indicating the magnitude of injury in this group of patients. The overall results of the 38 patients showed a strong correlation between the ICP and PI and during outbursts of ICP with a correlation coeffi cient of r = 0.89 (ICP >20 mmHg) and r = 0.90 (ICP <20 mmHg). The relation between ICP and PI was estimated by the linear regression equation: ICP = 22,299 × PI – 10,705 (ICP >20 mmHg) and ICP = 38,592 × PI – 16,972 (ICP <20 mmHg). The CPP and PI were correlated signifi cantly during the changes in intracranial pressure. However, a better correlation was found when ICP >20 mmHg and PPC <50 mmHg (PI was 2.4 ± 0.89 when CPP was 35.96 ± 4.48 with a correlation coeffi cient of Pearson r = 0.80) than when ICP <20 mmHg and CPP >50 mmHg (PI was 0.78 ± 0.14 when CPP was 57.11 ± 9.62 with a correlation coeffi cient of Pearson r =0.76). y Methods We retrospectively analyzed severe ABI (18 TBI, eight SAH) monitored with brain oxygen and cerebral microdialysis (CMD) to measure hourly levels of cerebral extracellular glucose, lactate, pyruvate and lactate/pyruvate ratio (LPR). Variations of CMD variables were analyzed as a function of GD (defi ned as spontaneous decreases of CMD glucose from normal to low (<1.0 mM), at least 2 hours) and increased cerebral energy demand (LPR >25). Results During GD (60 episodes; 26 patients), we found an increase of CMD lactate (4 ± 2.3 vs. 1. Paolin A, Manuali A, Di Paola F, Boccaletto F, Caputo P, Zanata R, et al. Reliability in diagnosis of brain death. Intensive Care Med. 1995;21:657-62. 2. Gan TJ, Glass PS, Windsor A, Payne F, Rosow C, Sebel P, et al. Bispectral index monitoring allows faster emergence and improved recovery from propofol, alfentanil, and nitrous oxide anesthesia. Anesthesiology. 1997;87:808-15. Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury g Conclusion The BIS is a noninvasive, simple, and easy to interpret method, showing a perfect correlation with the other diagnostic methods. BIS can be used in severely comatose patients as an assessment of brain death. j y H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate 5.4 ± 2.9 mM) and LPR (27 ± 6 vs. 35 ± 9; all P <0.005) while brain oxygen and blood lactate remained normal. Dynamics of lactate and glucose supply were studied by analyzing the relationship between blood and CMD samples. No correlation between blood and brain lactate was found when brain glucose and LPR were normal (r = –0.12, P = 0.48; Figure 1), while this correlation became linear during GD, progressively rising to r = 0.53 (P <0.0001) when energy demand increased, suggesting increased cerebral lactate availability. The correlation between blood and brain glucose changed in the opposite direction, decreasing from r = 0.78 to 0.37 (P <0.0001) during GD and at LPR >25. Conclusion The absolute value of the PI is a reliable noninvasive indicator of ICP in pediatric severe TBI. A strong correlation between PI and ICP was demonstrated. Therefore, PI may be of guiding value in the invasive ICP placement decision in the neurointensive care patient when ICP monitoring is not systematically performed. In particular, ICP monitoring remains as grade C in the latest guidelines of management of STBI in children published in 2012. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. P449 Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Figure 1 (abstract P448). Correlations between blood and brain lactate. Figure 1 (abstract P448). Correlations between blood and brain lactate. P450 P449 P449 Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Evaluation of infection risk and antibiotic exposure in traumatic brain injury patients treated with therapeutic normothermia h k , , Hennepin County Medical Center, Minneapolis, MN, USA Hennepin County Medical Center, Minneapolis, MN, USA p y p Critical Care 2015, 19(Suppl 1):P446 (doi: 10.1186/cc14526) Introduction The purpose of this study is to assess the rate of confi rmed infections, antibiotic exposure, and monitoring practices with normothermia protocol utilization for traumatic brain injury patients. Treatment and prevention of fever is a focus of therapy for patients with severe neurological injury as fever has been identifi ed as an independent risk factor for morbidity and mortality [1]. Conclusion Osmotherapy with Man and HTS treatment had no eff ect on cerebral oxygenation and metabolism. Man, but not HTS, favorably reduced brain glutamate. These fi ndings support further investigation to test the value of alternative osmotic agents (such as hypertonic lactate) that may reduce ICP and at the same time improve cerebral metabolism after sABI. Methods The retrospective chart review analyzed outcomes of maintaining normothermia at 36.5°C versus a similar population without temperature control as a standard of care in patients admitted Acknowledgements Supported by grants from the Swiss National Science Foundation and the Novartis Foundation for Biomedical Research. S157 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P448 Methods A prospective and descriptive study conducted in our PICU from June 2011 to December 2013. We investigated 51 children with severe trauma brain injury (TBI). The TCD measurements were routinely performed bilaterally on the middle cerebral artery parallel to the ICP registration. The ICP and CPP data were correlated to PI and the correlation coeffi cient calculated. To control the linear correlation, the residuals were tested for normal distribution around the regression line. P448 Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate C Patet1, H Quintard1, T Suys1, L Pellerin2, P Magistretti3, M Oddo1 1CHUV – Lausanne University Hospital, Lausanne, Switzerland; 2University of Lausanne, Switzerland; 3Brain Mind Institute, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P448 (doi: 10.1186/cc14528) Bispectral index as a predictor of unsalvageable traumatic brain injury Introduction The aim was to evaluate the accuracy of bispectral index (BIS) monitoring for the diagnosis of brain death in severely comatose patients. We aimed to determine the utility of the BIS as a tool for clinical evaluation of the moment of brain death. Methods A prospective observational study with waiver of consent was conducted in the trauma ICU for 2 years from October 2012 to September 2014. Monitoring of BIS occurred during patient stay in the ICU. Population: 62 severely comatose patients (Glasgow Coma Score less than 6) admitted to the ICU mainly because of intracerebral hemorrhage, head injury, or postanoxic coma. BIS was recorded continuously during the hospitalization in the ICU. Where necessary, clinical brain death was confi rmed by EEG or brain stem test. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Results Twenty-nine patients were already clinically brain dead at the time of admission, and their individual BIS values were 0. Twenty-four patients were not clinically brain dead at the time of admission, and their individual BIS values were between 20 and 30. These patients became brain dead, and their individual BIS values dropped to 0 in a few hours to a few days. Seventeen patients who did not become brain dead during their hospitalization in the ICU had persistent electrocerebral activity on EEG, and their average BIS values remained above 31. References Introduction This study was designed to see whether there is a correlation between the transcranial Doppler (TCD) parameters and the CCP and intracranial pressure (ICP) monitoring during the cerebral hemodynamic changes and to evaluate ICP indirectly by TCD. S158 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P451 P451 Neuromonitoring of patients with severe traumatic brain injury at the bedside M Aries1, JG Regtien1, M Czosnyka2, J Donnelly2, P Smielewski2 1UMCG, Groningen, the Netherlands; 2Addenbrookes Hospital, University of Cambridge, UK Critical Care 2015, 19(Suppl 1):P451 (doi: 10.1186/cc14531) PbtO2 decreased and the microdialysis showed continuous signs of ischemia and cellular degradation. Conclusion This experimental study documents that during protracted pronounced hemorrhagic shock, cerebral energy metabolism was severely compromised and exhibited a biochemical pattern typical of ischemia and cellular degradation. After retransfusion this pattern continued. From intravenous microdialysis in the sagittal sinus, it is possible to achieve semiquantitative information of the intracerebral redox state. Accordingly, it might be possible to monitor the global cerebral energy state continuously with a strictly extracerebral technique. This technique might be valuable in various severe conditions during critical care when cerebral energy metabolism may be compromised; for example, resuscitation after cardiac standstill, open heart surgery, multitrauma and so forth. Interestingly the study also showed that after reinfusion of blood other parts of the body recovered, evaluated by microdialysis, but the brain showed signs of damage, making the brain the limiting organ in hemorrhagic shock. Introduction Measurement of intracranial pressure (ICP) and arterial blood pressure is used to derive cerebral perfusion pressure (CPP) and to guide targeted therapy of severe traumatic brain injury (TBI) necessitating ICU admission. In this review we discuss the evidence for ICP monitoring, CPP calculation, and ICP/CPP-guided therapy after severe TBI. Despite its widespread use, there is currently no class I evidence that ICP/CPP-guided therapy improves outcomes. Similarly, no class I evidence can currently advise the ideal CPP. y Methods A review of current literature with special focus on autoregulation (PRx)-guided CPP treatment in TBI patients.i Results Optimal CPP is probably patient, time, and pathology specifi c and related to cerebral autoregulation status. P453 P453 Amantadine sulfate treatment in cases with brain injury in the ICU: a retrospective clinical trial S Goksu Tomruk, N Bakan, G Karaören, A Salvarcý Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P453 (doi: 10.1186/cc14533) P452 y Results The patients’ diagnoses included two post-CPR, fi ve intracranial and one subdural hematoma, one CVA, one postoperative aneurysm, one subarachnoid hemorrhage and one brain contusion. Table 1 (overleaf) presents the fi ndings. The AS therapy was initiated between days 3 and 33 of admission in all patients other than Patients 2 and 8. A dramatic improvement was observed in a patient with both GCS and JFK- CRS score of 5 when AS therapy was initiated in month 5 and the patient was discharged for home care. In Patient 9, AS therapy was withdrawn on day 5 due to persistent thrombocytopenia (TP) despite exclusion of other reasons; subsequently, improvement was observed in TP. The complications were relatively less severe with average acceptability. Amantadine sulfate treatment in cases with brain injury in the ICU: a retrospective clinical trial S Goksu Tomruk, N Bakan, G Karaören, A Salvarcý Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P453 (doi: 10.1186/cc14533) Introduction Improvement of recovery is a challenging process in cases with varying degrees of severe brain injury (BI) requiring intensive care. Amantadine sulfate (AS) is recommended for use in cases with brain injury. The Coma Remission Scale (JFK-CRS) consists of auditory–visual– motor–mouth–tongue functions, communication and awareness scales; provides a score between 0 and 23; and shows numeric recovery from coma. The aim of this study was to evaluate outcomes and eff ects of AS used for neurorecovery on the Glasgow Coma Scale (GCS) and JFK-CRS in our ICUs during the last 5 months. Conclusion The management of TBI is likely to become increasingly based on a more comprehensive monitoring and management approach rather than relying on absolute numbers of ICP and CPP in isolation. This will allow individual optimization of perfusion and therefore of oxygen and energy substrate delivery. We await further robust, high-quality evidence to support the benefi ts of using more sophisticated monitoring tools like the autoregulation- guided CPP concept during the ICU management of TBI. For the near future, more important is a greater understanding of the underlying pathophysiology. Methods After approval of the Ethics Committee, we recruited 12 patients with brain injury resulted from trauma or hemorrhage who had initial GCS of ≤9 and received AS (500 mg, twice daily over 10 days) during the recovery period. In all subjects, age, gender, diagnosis, initial APACHE II score, time of initiation of AS therapy, JFK-CRS and GCS scores, aspartate aminotransferase, alanine aminotransferase, BUN, creatinine, platelet count, electrocardiography fi ndings, electrolyte values and arterial blood gas values on days 1, 6, 10 and 14 were recorded.i p p y References References 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2. Jaeger M, et al. Crit Care Med. 2010;38:1343-7. 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2. Jaeger M, et al. Crit Care Med. 2010;38:1343-7. 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2 Jaeger M et al Crit Care Med 2010;38:1343-7 Technique for continuous bedside monitoring of the global cerebral energy state gy R Jakobsen1, A Granfeldt2, TH Nielsen1, P Toft1, CH Nordström1 1Odense University Hospital, Odense, Denmark; 2Regional Hospital of Randers, Denmark Critical Care 2015, 19(Suppl 1):P452 (doi: 10.1186/cc14532) Introduction In the present experimental study we explore whether cerebral venous lactate/pyruvate ratio (LP ratio) measured by intra- vascular microdialysis during induced hemorrhagic shock may be used as a surrogate marker for compromised cerebral oxidative metabolism. Methods Six female pigs were anesthetized and vital parameters was recorded. Microdialysis catheters were placed in cerebral hemisphere parenchyma, the superior sagittal sinus and femoral artery. Brain tissue oxygenation (PbtO2) and intracranial pressure (ICP) was recorded. Hemorrhagic shock was achieved by bleeding the animals to a mean arterial pressure (MAP) of approximately 35 mmHg. Animals were kept at a MAP of about 30 to 40 mmHg for 90 minutes. The animals were resuscitated with reinfusion of shed blood followed by 3 hours of observation. Conclusion We suggest that an AS dose of 1,000 mg/day (over 10 days) seems to improve neurorecovery in BI patients with good tolerability. Prospective controlled studies with large, homogeneous BI populations will better defi ne the role of AS for recovery and complications. P454 References The fact that optimal CPP and autoregulation status varies between individual patients and over time makes it an attractive bedside tool to serve as a (simplifi ed) model to investigate the use of autoregulation (PRx) status to fi ne tune or feedback clinical treatments in individual sedated TBI patients (optimal CPP concept) [1]. Evidence is emerging for the role of other monitors (representing (local) metabolism, oxygen supply/use, perfusion, neuronal functioning) that enable the intensivist to employ an individualized multimodality monitoring approach in TBI [2]. P453 Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) P454 Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Results In the cerebral hemisphere, hemorrhagic shock caused a marked increase in the LP ratio, while a signifi cant but minor increase was observed in the sagittal sinus. The LP ratio increased and continued doing so to a very high level. In the femoral artery, the shock period was associated with a slight increase of the LP ratio. The increase in the LP ratio in the sagittal sinus was markedly and signifi cantly higher than in the arterial blood. Further, the dynamic changes in the LP ratio in the sagittal sinus followed that of the parenchyma, not the arterial blood. After infusion of blood ICP increased, cerebral perfusion pressure and M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Introduction Traumatic brain injury (TBI) prognostication is a developing fi eld striving to identify indicators, notably neurochemical S159 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P453). Findings of 12 patients receiving amantadine sulfate Table 1 (abstract P453). P454 The main purpose of this study was to determine the impact of CB2R inhibition on leukocyte activation within the microcirculation following endotoxin challenge in an experimental stroke model. Methods This was a prospective, randomized animal study. Five experimental groups (male C57BL/6 mice, age: 6 to 8 weeks) were subjected to the following treatments: control; endotoxemia (LPS 5 mg/kg, i.v.); transient cerebral hypoxia–ischemia (HI) + endotoxemia; HI + endotoxemia + CB2R antagonist (AM630 2.5 mg/kg, i.v.). HI was induced by unilateral carotid artery occlusion, followed by 50-minute exposure to a low oxygen atmosphere (8% O2). The CB2R antagonist was given 15 minutes prior to LPS administration. Intravital microscopy was carried out 2 hours after LPS administration. Brains were then extracted and stained with tetrazolium chloride to calculate the infarct volume. The primary outcome measurement in this study regarding the immune response after stroke was the quantifi cation of leukocyte adhesion following endotoxin challenge in submucosal venules of the gut – an important organ in the development of multiorgan failure in endotoxemia and sepsis. Methods The MEDLINE, Embase, The Cochrane Library and BIOSIS electronic databases, conference abstracts and existing narrative reviews were searched from their inception to July 2013. Cohort studies including patients with moderate or severe TBI having evaluated the prognostic value of UCHL-1 according to mortality or the Glasgow Outcome Scale (GOS) were considered. Data concerning patients, outcomes, study methods, and laboratory methods were abstracted. Pooled results were planned to be presented using mean diff erences and analyzed using random eff ect models, as well as sensitivity analyses to explain potential heterogeneity.i Results Our search strategy yielded 2,257 articles, of which fi ve studies corresponded to our inclusion criteria (n = 730). All studies were performed by the same group of researchers. Five studies reported mortality (n = 515), two studies reported GOS (n = 58). Results from all included studies observed that UCHL-1 was a signifi cant predictor of mortality. However, only two studies represented a unique study population, thus precluding a meta-analysis. Results Compared with endotoxemic animals without CNS injury, mice subjected to HI displayed reduced leukocyte activation in intestinal submucosal venules indicative of CIDS. Administration of the CB2R antagonist in animals with CIDS challenged with endotoxin restored peripheral leukocyte recruitment without a detrimental impact on infarct size. P455 Cannabinoid 2 receptor antagonism reverses central nervous system injury-induced immune defi ciency syndrome IB Burkovskiy, J Zhou, G Robertson, C Lehmann Dalhousie University, Halifax, NS, Canada Critical Care 2015, 19(Suppl 1):P455 (doi: 10.1186/cc14535) P454 Findings of 12 patients receiving amantadine sulfate Initiation of Patient number Sex/age (years) APACHE II GCS A/B/C AS therapy (day) CRS score D/E/F/G Complications Outcome 1 M/14 24 3/5/7 6 0/4/5/6 H, J Still in ICU 2 F/78 33 8/8/10 84 0/0/0/0 K Ex 3 F/75 35 5/6/4 4 1/1/0/Ex K, L Ex 4 M/24 29 3/3/3 6 0/0/Ex K, L Ex 5 M/27 27 4/4/15 6 1/13/18/22 L, M, J Discharged 6 F/70 45 3/3/3 20 0/0/0/0 – Ex 7 F/72 22 5/5/12 3 4/7/11/17 K, L, I Discharged 8 F/53 27 3/5/10 150 5/7/10/14 K Discharged 9 F/38 34 4/4/4 33 0/0/1/1 H, J Still in ICU 10 M/50 26 4/6/13 8 0/7/18/19 L, J Discharged 11 M/63 24 8/10/12 14 6/8/8/12 – Discharged 12 M/50 31 4/6/11 6 2/5/7/9 L, J Still in ICU F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, convulsion. F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, l i F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, convulsion. biomarkers, of long-term outcomes. Among these, ubiquitin carboxy- terminal hydrolase L1 (UCH-L1) is currently being investigated to defi ne its potential prognostic value. The objective of this systematic review is to determine the ability of UCH-L1 to predict prognosis following a moderate or severe TBI. CIDS. P454 Conclusion In this systematic review, we observed that all published studies on UCHL-1 were conducted by the same group of investigators and presented results from an intersecting cohort of patients. Due to the paucity of data, we could not perform a pooled analysis and conclude on the association of this biomarker with long-term prognosis. Assays using UCHL-1 were only recently developed and further studies done by diff erent research teams will be needed to determine the reproducibility and validity of UCH-L1 as a potential prognostic tool. Conclusion CB2R-related modulation of leukocyte activation is involved in the impaired immune response following CNS injury. Future studies will explore the CB2R pathway in order to develop novel therapies to improve the immune response in CIDS. P456 Implementation process of a large multicenter study in trauma AF Turgeon, TBI-Prognosis Team in collaboration with the CCCTG CHU de Québec, QC, Canada Critical Care 2015, 19(Suppl 1):P456 (doi: 10.1186/cc14536) Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Results Overall, 33.4 weeks (95% CI = 24.8 to 42.1) were required on average from the start-up package mailing to centers and the start of the screening process. Data sharing and fi nancial agreement were mainly responsible for this delay with an average of 28.6 weeks (95% CI = 20.4 to 36.7) needed to complete the agreement with the coordinating center. REB approval was obtained on average 17.5 weeks (95% CI = 13.8 to 21.2) from the shipping of the study package to the participating centers. Eighty percent of the REBs had members with prior experience in multicenter clinical research, and more than half with specifi c clinical expertise in critical care medicine or neurology/ neurosurgery. A standardized electronic REB submission process was used in most REBs (60%). All centers had experience in implementing multicenter clinical studies. PIs had experience conducting from zero to 40 prior clinical studies and from 3 to 24 years of research experience with protected research time ranging from 5 to 75%. RCs had managed from zero to more than 50 clinical studies. Most centers (87%) organized specifi c presentation of the study to the critical care medical staff (93%), while some (60%) presented the study to other medical teams. Agreements from other departments such as radiology (87% of centers), electrophysiology (80%) and clinical imaging (73%) were requested in most centers. p L Tsentsiper, E Kondratyeva, S Kondratyev, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P458 (doi: 10.1186/cc14538) p L Tsentsiper, E Kondratyeva, S Kondratyev, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P458 (doi: 10.1186/cc14538) Introduction Currently, the most common way to predict the outcome of acute brain damage is to study the level of protein S-100 in the serum. This method lacks specifi city as the concentration of protein S-100 signifi cantly increases with age, more for men than women, and there are no data on prognostically signifi cant changes in the level of S-100 after removal of the tumor and cerebral hemorrhages. Endothelins, vasopressin, some cytokines, excess sodium or calcium in serum, activation of the sympathoadrenal system, and tachycardia are the stimulants of brain natriuretic peptide production. Levels of N-terminal pro-brain natriuretic peptide in brain injury patients The rise of the natriuretic peptide level in cases of acute brain damage has a functionally adaptive nature, based on vasodilation, diuretic action peptide and ability to reduce sympathoadrenal system activity. Thus, we can suppose that the more severe the damage, the higher the stimulation of natriuretic peptide. In this study we investigate the level of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with severe brain damage and fi nd correlation between the level of peptide and outcome. Endothelins, vasopressin, some cytokines, excess sodium or calcium in serum, activation of the sympathoadrenal system, and tachycardia are the stimulants of brain natriuretic peptide production. The rise of the natriuretic peptide level in cases of acute brain damage has a functionally adaptive nature, based on vasodilation, diuretic action peptide and ability to reduce sympathoadrenal system activity. Thus, we can suppose that the more severe the damage, the higher the stimulation of natriuretic peptide. In this study we investigate the level of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with severe brain damage and fi nd correlation between the level of peptide and outcome. Conclusion The implementation of a large Canadian multicenter and multifaceted clinical study in the trauma population involved a signifi cant amount of time and energy from both the coordinating and the participating sites. The variable experience of participating sites and teams, as well as the involvement of diff erent medical disciplines, may have had an impact on study implementation time. Delays for REB approval but also for data sharing and fi nancial agreement must be taken into consideration in the timeline for implementing large multicenter clinical studies in trauma. Methods We studied 110 patients having brain injuries of various origins. All patients were divided into four groups. All patients were 20 to 72 years old, 58 men and 52 women. Group 1 (n = 17) – acute TBI, group 2 (n = 29) – patients operated on for the brain tumor, group 3 (n = 36) – hemorrhagic stroke, group 4 (n = 28) – vegetative state. We measured the level of brain natriuretic peptide on days 1 to 3, and then every 7 days for 21 days. Results All patients with severe acute brain damage (groups 1, 2, 3) had a level of NT-proBNP higher than normal (normal 0 to 125 pg/ml). Signifi cant diff erence in values between the groups was not observed. P458 the ongoing Pan-Canadian TBI-Prognosis Study. Descriptive statistics were used. the ongoing Pan-Canadian TBI-Prognosis Study. Descriptive statistics were used. P457 Optic nerve sheath diameter by bedside ultrasound is a reliable screening test for cerebral edema in the comatose ICU patient A Mohamed, N Alharbi, N Salahuddin, I Hussain, O Solaiman King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P457 (doi: 10.1186/cc14537) Conclusion In cases of acute severe brain damage the level of NT- proBNP signifi cantly increased. Correlation between the level of NT-proBNP and etiology of acute brain damage was not observed. If the level of NT-proBNP is above 700 pg/ml and/or in the absence of its reduction to normal, then poor outcome of the disease – severe disability or death – can be predicted. Level of NT-proBNP cannot be used as an indicator for the severity of the status for patients in a vegetative state in contrast to patients with acute brain damage. Introduction ICU patients may remain comatose after resolution of critical illness. Frequently this is due to delayed sedative clearance but may also result from increases in intracranial pressure (ICP) and cerebral edema. We proposed that measurement of the optic nerve sheath diameter (ONSD) is a rapid, bedside screening test that can be used to quickly identify patients with cerebral edema and increased ICP. y y Methods This was a prospective, observational study carried on consecutive patients admitted to a multidisciplinary medical and surgical ICU. Stable patients with unexplained coma and scheduled for brain imaging were included. Patients with obvious ocular trauma or on sedative, narcotic infusions were excluded. ONSD was measured using a 7.5 to 10 MHz linear array ultrasound transducer probe placed on the closed eye in the transverse axis. The ONSD was measured at a predefi ned point 3 mm posterior to the globe. Both eyes were measured and the mean value used. The study protocol was approved by the Hospital Research Ethics Committee (RAC Prop No. 2141 103). Statistical analysis was carried out using SPSS version 20.0. Cannabinoid 2 receptor antagonism reverses central nervous system injury-induced immune defi ciency syndrome IB Burkovskiy, J Zhou, G Robertson, C Lehmann Dalhousie University, Halifax, NS, Canada Critical Care 2015, 19(Suppl 1):P455 (doi: 10.1186/cc14535) Introduction Our purpose was to evaluate the time from shipping of the study start-up package to study screening, as well as conditions that may impact this process, in the context of a large-scale multifaceted and multicenter clinical study in trauma. Introduction Our purpose was to evaluate the time from shipping of the study start-up package to study screening, as well as conditions that may impact this process, in the context of a large-scale multifaceted and multicenter clinical study in trauma. Introduction Central nervous system (CNS) injury, such as stroke, is known to increase susceptibility to infections that adversely aff ect clinical outcome. This impaired immune response to infection is termed CNS injury-induced immune defi ciency syndrome (CIDS). Activation of the cannabinoid 2 receptor (CB2R) suppresses immune function suggesting that antagonism of this receptor may overcome Methods We designed a survey questionnaire based on four domains: REB characteristics and process, centers’ characteristics, experience of the study and clinical teams, and center-specifi c implementation approaches. The questionnaire was self-administered to all lead research coordinators of the 17 level 1 participating trauma centers in S160 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Level of NT-proBNP above 700 pg/ml and/or the absence of its reduction to normal dynamic indicators was marked by an unfavorable outcome of the disease – severe disability (n = 25) or death (n = 18). For patients from group 4 regardless of their age, sex, severity of condition and treatment results in a level of NT-proBNP below 250 pg/ml. P459 C b Cerebral oximetry monitoring in pediatric seizure patients in the emergency department T Abramo1, B Schnieder1, E Storm1, N Hobart-Porter1, Z Hu1, N Todd1, L Crawley1, M Meredith2, S Godbold1 1University of Arkansas School of Medicine, Little Rock, AR, USA; 2University of Tennessee School of Medicine, Memphis, TN, USA Critical Care 2015, 19(Suppl 1):P459 (doi: 10.1186/cc14539) Introduction During ictal/post-ictal events, altered cerebral physiology occurs: increased neuronal activity causes signifi cant increase in cerebral metabolism with changes in ipsilateral cerebral blood fl ow. Standard PED seizure monitoring is by O2SAT and ETCO2 which yield no direct data about regional cerebral oxygenation/physiology (rSO2). Signifi cant abnormal hemispheric cerebral physiology resulting in neurological injury can occur without knowing because the current monitoring system could not detect the abnormal hemispheric abnormality. Cerebral oximetry can provide a rapid, non-invasive detection of each hemisphere’s cerebral physiologic changes during ictal/post-ictal phases. The aim was to study left and right rSO2 values in patients in the pre and post seizure periods and in nonseizing controls. Methods An observational study of seizing and nonseizing patient’s left and right rSO2 readings compared with nonseizure patients. Results ONSD was measured in 43 patients; mean age was 62 ± 19.2 years, 48% (n = 20) were female. Admitting diagnosis was sepsis in 24% (n = 10), intracranial vascular event in 21% (n = 9), cardiac arrest in 12% (n = 5), hepatic encephalopathy in 7% (n = 3), malignancy with metastases in 7% (n = 3) and other causes in 28% (n = 12). The ONSD measured correlated highly between eyes, Spearman’s ρ = 0.799, P ≤0.001. The area under the ROC curve for detecting cerebral edema was 0.812 (95% CI = 0.667 to 0.957). Using a 0.58 cm cutoff ONSD diameter, the sensitivity was 86%, specifi city 74%, negative predictive value 96% and the positive likelihood ratio = 3.3. Conclusion This study suggests that bedside measurement of ONSD by ultrasound performs well as an initial screening test for cerebral edema. The identifi ed cutoff value of 0.58 cm can be used to detect cerebral edema with reasonable accuracy. S161 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P459). Results No diff erence for ictal left and right rSO2 readings across ages. See Figure 1. of our institute. In post-anoxic comatose patients we recorded EEG during hypothermia to assure burst suppression. P460 Neurophysiological tests in the neuro ICU Neurophysiological tests in the neuro ICU A Marudi1, Y Valzani2, F Valzania1, M Carpentiero1, C Parenti1, S Scacchetti1, S Baroni1, E Bertellini1 1Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy; 2University of Modena and Reggio Emilia, Modena, Italy Critical Care 2015, 19(Suppl 1):P460 (doi: 10.1186/cc14540) P459 C b In post-traumatic brain-injured patients with a persistent comatose state we use EEG to detect nonconvulsive states which potentially can increase secondary brain injury if untreated. In malignant epilepticus status we use EEG to monitor the eff ect of therapy and to modify it. In patients who present profound weakness of legs and hands we performed EMG to distinguish primary peripheral myopolyneuropathy (Guillian Barrè, miastenia gravis) from secondary illness acquired in the ICU (critical polyneuropathy, critical myopathy) [3]. Conclusion We have demonstrated abnormal hemispheric cerebral physiology during focal or generalized ictal activity. In patients with generalized seizures, the left and right rSO2 values were signifi cantly decreased. In patients with focal seizures, the ipsilateral rSO2 values were signifi cantly diff erent from the contralateral rSO2 readings and correlated to the hemisphere experiencing the focal seizure. In certain patients, during the ictal phase their rSO2 readings rose and stayed or rose then dropped. Overall, cerebral oximetry has shown great monitoring potential for actively seizing patients in the emergency department. Conclusion NPT can improve management of patients admitted to the neuro ICU. The data provided can modify therapeutic strategies and improve outcome in these settings of patients. R f References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery Patients were randomized into two groups: MAP group, in which MAP S162 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 was maintained above 70 mmHg or within 20% from baseline (n = 78); or CPP group, in which CPP was maintained above 60 mmHg or within 20% from baseline (n = 54). ICP, MAP and CPP were assessed every hour of anesthesia. Time of recovery of consciousness, incidence of POD and length of stay in the ICU and in the hospital were also evaluated. ischemia-operated animals with normothermia (normothermia + ischemia group); sham-operated animals with hyperthermia (hyperthermia + sham group); and ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia (39.5 ± 0.2°C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30 minutes before and during ischemia–reperfusion.i y Results Initial ICP was 14 ± 3 mmHg in the MAP group and 15 ± 2 mmHg in the CPP group. During the anesthesia it was stable without any signifi cant change. Decreasing of MAP after induction of anesthesia was similar in two groups and it was stable during the anesthesia. The frequency of use of vasopressors and infusion rate was higher in the CPP group. Time of recovery of consciousness in the MAP group was higher (28 ± 7 minutes vs. 18 ± 5 minutes (P <0.05)). The incidence of postoperative delirium was higher in the MAP group (18% vs. 11% in the CPP group (P <0.05)). There were no signifi cant diff erences between two groups in other complications. Total length of stay in the ICU and in the hospital was higher in the MAP group (6 ± 2 days vs. 4 ± 2 (P <0.05) and 15 ± 3 days vs. 12 ± 2 in the N group (P <0.05)). Results In the normothermia + ischemia group, a signifi cant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5 days after ischemia–reperfusion. In the hyperthermia + ischemia group, neuronal death in the SP of the CA1 occurred at 1 day post ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2 days post ischemia. In addition, we examined activation of astrocytes and microglia using immunohistochemistry for anti-glial fi brillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). Hydroquinone shows neuroprotective potential in an experimental ischemic stroke model via attenuation of blood–brain barrier disruption CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P464 (doi: 10.1186/cc14544) JH Cho, CW Park, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P462 (doi: 10.1186/cc14542) Introduction Therapeutic exercise is an integral component of rehabilitation for patients with stroke. Despite the high prevalence of cerebral ischemia in the older population, the mechanisms linking restorative exercise to memory recovery from ischemic stroke have not been completely understood in aged animals. In this study, we investigated eff ects of long-term exercise on neuronal death and memory recovery in the aged gerbil hippocampus after transient cerebral ischemia. We also investigated changes in gliosis, ischemia- induced myelin repair, microvessels, neurogenesis, and growth factor immunoreactivity in the hippocampus to study possible mechanisms of restorative exercise in memory recovery. Introduction Hydroquinone (HQ), a major benzene metabolite, occurs naturally in various plants and food, and is also manufactured for commercial use. Although many studies have demonstrated the various biological eff ects of HQ, the neuroprotective eff ects of HQ following ischemic stroke have not been investigated. Therefore, in this study, we fi rst examined the neuroprotective eff ects of HQ against ischemic damage in a focal cerebral ischemia rat model. Methods It was proven that pre and post treatment with 100 mg/ kg HQ protects from ischemia-induced cerebral damage, which was confi rmed by evaluation of neurological defi cit, positron-emission tomography and 2,3,5-triphenyltetrazoliumchloride staining. Methods The gerbils were divided into four groups (n = 12 in each group): the sham-operated group (Sham), 4-week sedentary group following ischemia (SD4), 1-week treadmill group following ischemia (TR1) and 4-week treadmill group following ischemia (TR4). Treadmill exercise was stared at 5 days after ischemia/reperfusion (I/R) and lasted for 1 or 4 weeks, and the animals were sacrifi ced 31 days after ischemia. Results In this study, 4 weeks of treadmill exercise facilitated memory recovery despite neuronal damage in the CA1 region after I/R. On the other hand, the long-term treadmill exercise alleviated the increased gliosis in the CA1 region, and increased the myelin repairing and microvessels in the CA1 region and DG, and enhanced the ischemia- induced cell proliferation, neuroblast diff erentiation, neuronal maturation of the newly generated cells, and BDNF expression in the ischemic DG of the aged gerbil. Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery Introduction Neurophysiological tests (NPTs) are important prog- nostic and diagnostic tools for patients admitted to the modern neuro ICU (NICU) [1]. Electroencephalography (EEG), somasensorial evoked potentials (SSEP), auditory brainstem response (ABR) and electromyography (EMG) complete clinical examination and radio- logical fi ndings in patients suff ering from post-traumatic brain injury, post-anoxic brain injury, refractory male epiletticus status, and neuromuscular illness. We evaluate the spread of NPTs in our NICU. Methods We collected data from patients admitted to our NICU from January 2014 to November 2014. We recorded the admission diagnosis and the NPT applied. y j I Zabolotskikh, N Trembach I Zabolotskikh, N Trembach Kuban State Medical University, Krasnodar, Russia Kuban State Medical University, Krasnodar, Russia Critical Care 2015, 19(Suppl 1):P461 (doi: 10.1186/cc14541) y Critical Care 2015, 19(Suppl 1):P461 (doi: 10.1186/cc14541) Introduction Increased intracranial pressure (ICP) adversely aff ects anesthesia due to a disturbed cerebral blood fl ow. In older patients this disturbance may increase the incidence of postoperative delirium (POD) and may lead to a poor outcome [1]. The standard hemodynamic protocol involves maintaining the mean arterial blood pressure (MAP), but in patients with intracranial hypertension it may not be enough to maintain adequate cerebral perfusion. The purpose of this study was to evaluate the protocol of maintaining cerebral perfusion pressure (CPP) in the prevention of postoperative delirium in older patients in abdominal surgery. Methods We collected data from patients admitted to our NICU from January 2014 to November 2014. We recorded the admission diagnosis and the NPT applied. Results From January 2014 to November 2014 we performed 521 EEG, 45 SSEP/ABR and 10 EMG. In post-anoxic and post-traumatic brain- injured comatose patients we performed EEG, SSEP and ABR 24 to 48 hours after the admission to predict later prognosis and expected neurological defi cit [2]. In the presence of a benign pattern no further evaluation was performed; in the presence of a malignant pattern the NPTs were repeated every 48 to 72 hours according to the protocol Methods A total of 132 ASA 3 patients, undergoing major abdominal surgery (duration 5.2 (4.3 to 6.5) hours) with ICP >12 mmHg evaluated by a venous ophthalmodynamometry [2], were included in our research. Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia + ischemia group than those in the normothermia + ischemia group.i y g p Conclusion A goal-directed hemodynamic protocol of maintaining CPP can decrease the incidence of POD in older patients with intracranial hypertension after major abdominal surgery compared with a protocol of maintaining MAP. Conclusion Based on our fi ndings, we suggest that experimentally hyperthermic precondition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus. Hydroquinone shows neuroprotective potential in an experimental ischemic stroke model via attenuation of blood–brain barrier disruption y y Results In addition, pre and post treatment with 100 mg/kg HQ signifi cantly attenuated ischemia-induced Evans blue dye extra- vasation, and signifi cantly increased the immunoreactivities and protein levels of SMI-71 and glucose transporter-1 (GLUT-1), which were well known as useful makers of endothelial cells, in ischemic cortex compared with a vehicle-treated group.l Conclusion Briefl y, these results indicate that pre and post treatment with HQ can protect from ischemic damage induced by transient focal cerebral ischemia, and the neuroprotective eff ects of HQ may be closely associated with the prevention of BBB disruption via increase of SMI-71 and GLUT-1 expressions. P462 P464 Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P464 (doi: 10.1186/cc14544) References 1. Zabolotskikh I, et al. Eur J Anesth. 2013;30:114-5. 1. Zabolotskikh I, et al. Eur J Anesth. 2013;30:114-5. 2. Firsching R, et al. J Neurosurg. 2011;115:371-4. 2. Firsching R, et al. J Neurosurg. 2011;115:371-4. P463 Impact of hyperthermia before and during ischemia reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P463 (doi: 10.1186/cc14543) Conclusion These results suggest that, in the aged gerbil, long-term treadmill exercise after ischemic stroke could restore the impaired short-term memory function through the cumulative eff ects of multiple neurorestorative processes. Prognostic value of blood lactate and glucose levels after aneurysmal subarachnoid hemorrhage y g S Dijkland1, K Van Donkelaar2, W Van den Bergh2, J Bakker1, D Dippel1, M Nijsten2, M Van der Jagt1 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) j g 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) Results A total of 307 patients were included in the validation cohort. The observed 60-day case fatality rate was 30.6%. Discrimination was good, and was considerably better for the model with WFNS grade at treatment decision (AUC = 0.89) compared with the model with WFNS grade on admission (AUC = 0.82). Calibration was poor, with mean predicted probabilities of 17.0% for the model with WFNS grade on admission and 17.7% for the model with WFNS grade at the time of treatment decision. Introduction In critically ill patients, blood lactate on admission is associated with outcome, but in patients with aneurysmal sub- arachnoid hemorrhage (SAH) this has not been investigated. We studied the association of early circulating lactate and glucose with unfavorable disease course. The prognostic role of both lactate and glucose was studied, hypothesizing that both may be increased due to sympathetic activation after SAH [1]. y p Methods In this retrospective cohort study we included consecutive patients with aneurysmal SAH admitted to the ICUs of two university hospitals in the Netherlands between November 2006 and December 2011. Exclusion criteria were: nonaneurysmal SAH, ICU admission >24 hours after ictus, death ≤48 hours after admission and no lactate measurement <24 hours after admission. Maximum blood lactate and glucose levels within the fi rst 24 hours after SAH were used for analyses. The outcomes were DCI, defi ned as a new hypodensity on brain CT due to DCI, and poor outcome, defi ned as a modifi ed Rankin Scale of 4, 5 or death 3 to 6 months after the ictus. We performed proportional hazard analyses to assess the associations of lactate and glucose with DCI, and logistic regression was used to assess the associations with poor outcome. Association between high arterial oxygen survival after intracerebral hemorrhage Introduction Hyperthermia can exacerbate the brain damage pro- duced by ischemia. In the present study, we investigated eff ects of hyperthermia before and during ischemia–reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofl uorescence staining. Methods The animals were randomly assigned to four groups: sham- operated animals with normothermia (normothermia + sham group); M Fallenius1, R Raj1, M Reinikainen2, S Bendel3, MB Skrifvars1 1Helsinki University Hospital, Helsinki, Finland; 2North Karelia Central Hospital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2015, 19(Suppl 1):P465 (doi: 10.1186/cc14545) M Fallenius1, R Raj1, M Reinikainen2, S Bendel3, MB Skrifvars1 1Helsinki University Hospital, Helsinki, Finland; 2North Karelia Central Hospital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2015, 19(Suppl 1):P465 (doi: 10.1186/cc14545) Introduction Liberal use of oxygen after brain insults remains controversial [1,2]. We studied whether high arterial oxygen tension S163 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Maximum lactate in the acute phase after aneurysmal SAH is associated with both DCI-related infarction and poor outcome. Once glucose was considered, early lactate remained independently associated with poor outcome, while glucose, instead of lactate, was associated with DCI. These routinely available laboratory measurements may improve identifi cation of patients at risk for complications or poor outcome after SAH. Confi rmation of the pathophysiological signifi cance of lactate and glucose in prospective research is warranted. Reference (PaO2) is associated with decreased long-term survival in patients with spontaneous intracerebral hemorrhage (ICH) treated in the ICU. (PaO2) is associated with decreased long-term survival in patients with spontaneous intracerebral hemorrhage (ICH) treated in the ICU. Methods Data on primary admissions for adult patients (>18 years) treated for ICH in Finnish ICUs between 2003 and 2012 were collected from a nationwide ICU database. Patients were divided into three groups according to the PaO2 value associated with the lowest measured PaO2/ FIO2 ratio during the fi rst 24 hours after ICU admission. High arterial oxygen tension was defi ned as PaO2 >19.9 kPa; intermediate as PaO2 13 to 19.9 kPa; and low as PaO2 <13 kPa. The primary outcome was 6-month mortality. 1. Kaukonen K, et al. Crit Care Med. 2014;42:1379-85. Association between high arterial oxygen survival after intracerebral hemorrhage Results Of the 3,033 patients, 63% (n = 1,923) had low PaO2, 29% (n = 892) intermediate PaO2, and 7% (n = 218) high PaO2. Forty-nine percent of the patients died during the 6-month follow-up. Of these, 75% died before discharge from hospital. Univariate analysis showed that 6-month mortality was higher in the high PaO2 group (61%) compared with the intermediate and low PaO2 groups (52% and 46% respectively, P <0.001). Multivariate analysis, however, showed no statistically signifi cant correlation between high PaO2 and mortality (with the low PaO2 group as the reference category, odds ratio for death (OR) for high PaO2 = 1.10, 95% confi dence interval (CI) = 0.76 to 1.58 and for intermediate PaO2 = 0.96, 95% CI = 0.78 to 1.17). P467 Prediction of 60-day case fatality after aneurysmal subarachnoid hemorrhage: external validation of a prediction model S Dijkland, B Roozenbeek, P Brouwer, H Lingsma, D Dippel, L Vergouw, M Vergouwen, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P467 (doi: 10.1186/cc14547) Introduction Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease with substantial morbidity and mortality. Prognostic modeling is an important instrument to identify high- risk patients in both clinical practice and research settings. Recently, a prognostic model to predict 60-day case fatality after aneurysmal SAH was developed with data from the International Subarachnoid Aneurysm Trial (ISAT) [1]. Our aim was to externally validate this model in a retrospective cohort of consecutive SAH patients. 2 Conclusion High PaO2 is not predictive of 6-month mortality in patients treated for spontaneous ICH in the ICU. Therefore, targeting higher PaO2 values appears to be a safe approach in order to avoid hypoxemia. References 1. Rincon F, Kang J, Maltenfort M, et al. Association between hyperoxia and mortality after stroke. Crit Care Med. 2014;42:387-96. 1. Rincon F, Kang J, Maltenfort M, et al. Association between hyperoxia and mortality after stroke. Crit Care Med. 2014;42:387-96. p p Methods We included consecutive aneurysmal SAH patients admitted to one university hospital between October 2007 and October 2011. Exclusion criteria were: age <18 years, hospital admission >28 days after SAH, nonaneurysmal SAH, explicit objection by the patient to view the medical data and missing data on 60-day case fatality. The model’s predictors were age, maximum lumen size of the aneurysm, Fisher grade and World Federation of Neurological Surgeons (WFNS) grade. Two versions of the model were validated: one with WFNS grade scored on admission and the other with WFNS grade assessed at the time of treatment decision, as a proxy to WFNS grade at randomization used in the ISAT. The outcome was 60-day case fatality. Model performance was assessed by studying discrimination, expressed by the area under the receiver operating characteristic curve (AUC), and calibration. 2. de Jonge E, Peelen L, Keijzers PJ, et al. Association between administered oxygen, arterial partial oxygen pressure and mortality in mechanically ventilated intensive care unit patients. Crit Care. 2008;12:R156. P467 P467 Prediction of 60-day case fatality after aneurysmal subarachnoid hemorrhage: external validation of a prediction model S Dijkland, B Roozenbeek, P Brouwer, H Lingsma, D Dippel, L Vergouw, M Vergouwen, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P467 (doi: 10.1186/cc14547) Reference 1. Risselada R, et al. Eur J Epidemiol. 2010;25:261-6. P468 Prognostic value of blood lactate and glucose levels after aneurysmal subarachnoid hemorrhage Multivariable analyses were adjusted for established predictors for DCI and poor outcome.i Conclusion Our results indicate that the ISAT prediction model is generalizable, since the model showed adequate performance in an independent, unselected cohort of aneurysmal SAH patients. The model discriminated well between patients who died and those who survived the fi rst 60 days after SAH. Additionally, use of WFNS grade at the time of treatment decision of the ruptured aneurysm improved model performance. However, since predicted probabilities were lower than observed probabilities, the ISAT prediction model needs to be adapted before use in clinical practice. Cortical spreading depolarizations in patients with intracerebral hemorrhage: preliminary datal Results Two hundred and eighty-fi ve patients were included in the analyses. DCI occurred in 84 patients (29%) and 106 patients (39%) had poor outcome. Lactate was independently associated with DCI (adjusted HR = 1.16, 95% CI = 1.04 to 1.30) and poor outcome (adjusted OR = 1.53, 95% CI = 1.25 to 1.94). Maximum lactate and glucose were strongly related (Spearman’s ρ = 0.55, P <0.001). In multivariable analyses including both lactate and glucose as independent variables, only lactate was independently related to poor outcome (OR = 1.42, 95% CI = 1.11 to 1.81), and only glucose was independently associated with DCI (HR = 1.10, 95% CI = 1.02 to 1.19). AJ Schiefecker1, R Beer1, M Kofl er1, B Pfausler1, I Unterberger1, P Lackner1, G Broessner1, P Rhomberg1, F Sohm1, M Mulino1, C Thome1, M Fabricius2, E Schmutzhard1, R Helbok1 1Medical University of Innsbruck, Austria; 2Rigshospitalet, Copenhagen, Denmark Critical Care 2015, 19(Suppl 1):P468 (doi: 10.1186/cc14548) Introduction Perihematomal edema (PHE) expansion contributes to increased morbidity and mortality after spontaneous intracerebral S164 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 positivity on admission is an independent prognostic predictor in acute ischemic stroke. Reference Figure 1 (abstract P469). hemorrhage (ICH). Pathophysiology of PHE is incompletely understood. Recently, the role of cortical spreading depolarizations (CSDs) in secondary brain injury was established in subarachnoid and traumatic brain injury patients. However, the value of CSDs after ICH is not known. Methods Patients with ICH fulfi lling the inclusion criteria were prospectively enrolled in the observational COSBID study (Co-operative Study on Brain Injury Depolarisations). g.BSamp (g.tec, Austria) connected to PowerLab and LabChart software (Adinstruments) was used for electrocorticography (EcoG). Electrocardiogram patches at the patient’s shoulder and bed served as groundings, and a surface reference electrode was glued on the mastoid. The duration of EcoG depressions was defi ned as the time between depression onset and start of EcoG recovery in the integral of power calculations (0.5 to 45 Hz; 60 seconds time constant decay). Brain tissue oxygen tension (PbtO2), cerebral blood fl ow (CBF), cerebral metabolism and intracranial pressure were monitored in the PHE region. Data are presented as median and interquartile range. q g Results Eighteen patients with ICH (ICH volume: 54 (33 to 69) ml) were analyzed. Hematoma evacuation was performed in 17 patients, one subject underwent craniectomy only. Troponin level as a predictor of prognosis in patients with acute ischemic stroke H Bayir1, R Dagli2, H Kaymaz2, I Yildiz1, H Kocoglu1 1Abant Izzet Baysal University, Medical School, Bolu, Turkey; 2Ahi Evran University Education and Research Hospital, Kirsehir, Turkey Critical Care 2015, 19(Suppl 1):P469 (doi: 10.1186/cc14549) H Bayir1, R Dagli2, H Kaymaz2, I Yildiz1, H Kocoglu1 1Abant Izzet Baysal University, Medical School, Bolu, Turkey; 2Ahi Evran University Education and Research Hospital, Kirsehir, Turkey Critical Care 2015, 19(Suppl 1):P469 (doi: 10.1186/cc14549) Methods The data from 1 million National Health Insurance bene fi ciaries were utilized. All adult benefi ciaries were followed from 1 January 2005 until 31 December 2012 to identify those who developed ischemic stroke. Meanwhile, each identifi ed patient with burn injury was matched with 100 unexposed patients based on the high-dimensional propensity score. Cox regression models were applied to compare the hazards of ischemic stroke in the matched cohorts. Introduction The aim of this study was to identify the association between troponin level and the outcome in patients with acute ischemic stroke. Introduction The aim of this study was to identify the association between troponin level and the outcome in patients with acute ischemic stroke. Methods We retrospectively investigated 152 patients admitted to our reanimation ICU for cerebrovascular accident between 1 January 2013 and 31 December 2013. Inclusion criteria were as follows: patients with acute ischemic stroke, measurement of serum troponin level and electrocardiography performed within 24 hours of admission. Not included were patients with intracerebral hemorrhage, no brain imaging or electrocardiography, previous myocardial infarction, stable or unstable angina pectoris before admission, previous coronary angioplasty or coronary bypass surgery. Figure 1 (abstract P470). Patient fl owchart of enrollment. g p y y yp g y Results Of 152 patients, 51 patients were excluded from the study because of the exclusion criteria. The serum troponin level was elevated in 81 patients. The patients were divided into two groups; patients in group 1 (n = 81) with serum troponin level >0.01, and those in group 2 (n = 20) with serum troponin level ≤0.01. For 1-month follow-up results of patients, death had occurred in 50.6% (n = 41) of patients in group 1 and in 25% (n = 5) of patients in group 2. There was a signifi cant positive correlation between the increase in troponin level and death within 1 month (r = 0.205; P = 0.040). P469 Introduction The results of studies attempting to assess the risks of ischemic stroke in patients with burn injury have been confl icting. We investigated the risks of ischemic stroke in hospitalized burn injury patients in Taiwan to evaluate whether the risk is higher compared with the general population.i Cortical spreading depolarizations in patients with intracerebral hemorrhage: preliminary datal Patients were 60 (55 to 67) years old and 38% female. Monitoring time per patient was 10 (6 to 14) days. A total of 129 CSDs with 16 (10 to 29) minutes of EcoG depression were observed. Eighty-four percent (n = 15) of patients showed expansion of PHE by 25 (10 to 50) ml within 3 to 6 days after bleeding. Neuromonitoring probes were 35 (23 to 58) mm distant from the EcoG strip. CSDs occurred in 73% (n = 11) of patients with PHE expansion. The interval between CSDs was 98 minutes (25 to 308). CSDs were associated with a signifi cant decrease of PbtO2 (–4 mmHg (–3; –7); duration 10 (5 to 23) minutes) in 68% (52/77), CBF changes in 95% (19/20) and metabolic derangement in 80% (80/100) of CSDs. PHE expansion was observed in all patients with spreading convulsions (n = 2) and patients with repetitive CSDs occurring as clusters (n = 3). positivity on admission is an independent prognostic predictor in acute ischemic stroke. positivity on admission is an independent prognostic predictor in acute ischemic stroke. Reference 1. Whiteley W, Chong WL, Sengupta A, Sandercock P. Blood markers for the prognosis of ischemic stroke: a systematic review. Stroke. 2009;40:e380-9. P470 Increased risk of ischemic stroke in patients with burn injury: a nationwide cohort study in Taiwan TY Hung1 YC Su2 Conclusion CSDs are common in ICH patients and associated with perihematomal PbtO2 decreases and metabolic derangement. Especially, clusters of CSDs might be associated with detrimental metabolic changes of the perihematomal brain tissue. TY Hung , YC Su 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) TY Hung , YC Su 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patientsf R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil fi y Results Ninety-nine of 204 included patients developed ICU-AW. Patients with ICU-AW had higher APACHE IV and SOFA scores, a longer duration of mechanical ventilation, longer ICU stay, and died more often on the ICU compared with ICU patients without ICU-AW. Maximal levels of IL-1β, IL-6, IL-8, IL-10, TNFα, IFNγ, fractalkine and sICAM-1 were higher in patients who developed ICU-AW compared with patients who did not develop ICU-AW (univariable analysis). PC 1 to 4 derived from maximal levels explained >69% of the total variance in the data. Multivariable logistic regression models showed that PC 1 (mainly loaded by IL-6, IL-8 and IL-10) and PC 4 (mainly loaded by sP-selectin) were signifi cantly higher in patients with ICU-AW compared with patients without ICU-AW (OR of 1.27 (95% CI = 1.02 to 1.60) and 1.55 (1.06 to 2.27) respectively). Introduction Coronary artery bypass grafting (CABG) surgery usually improves myocardial contractility, reducing cardiovascular events. However, it is a high-risk procedure associated with signifi cant neurological complications, including stroke, delirium and cognitive impairment. The pathophysiology of these complications is not very well known, and may include low fl ow state after surgery, low cardiac output, embolism and reperfusion lesion. The aim of this study is to prospectively evaluate the cerebral hemodynamics through transcranial color and spectral Doppler sonography in high-risk patients undergoing cardiac surgery with a pump. y Methods This was a prospective, single-center study, performed at the Heart Institute from University of São Paulo. From May to November 2014 we included 35 patients in the study, aged older than 18 years old, submitted to CABG with a pump, with EuroSCORE higher than 6 or left ventricular ejection fraction lower than 40%. Transcranial color and spectral Doppler sonography was performed 48 hours before surgery (T0), 7 days (T1) and 6 months after surgery (T2). We used a probe of 2.5 to 2 MHz (Doppler Box DWL/Compumedics, Singen, Germany). All recordings were taken with the patient in a supine position. P471f Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patients R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P471 (doi: 10.1186/cc14551) P472 ncreased early systemic infl ammation in patients with ICU-acquired weakness k h d ll h k E Witteveen, L Wieske, C Verhamme, T Van der Poll, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P472 (doi: 10.1186/cc14552) Introduction Infl ammation may be important in the pathogenesis of ICU-acquired weakness (ICU-AW) since SIRS, sepsis and multiple organ failure are the main risk factors. Local infl ammation has been found in muscle and nerve tissue of patients with ICU-AW, but little is known about the association with systemic infl ammation. We hypothesized that systemic infl ammation is increased in patients who develop ICU- AW compared with patients who do not develop ICU-AW. Figure 2 (abstract P470). Burn injury patients have higher adjusted hazard ratio of ischemic stroke. Results A total of 743,237 patients were enrolled. After matching, 1,763 burn injury patients and 176,300 unexposed patients were selected. The adjusted hazard ratio of ischemic stroke was signifi cantly increased in burn injury patients (1.84; 95% CI, 1.43 to 2.36). Such phenomenon remained signifi cantly after 12 months (1.54; 95% CI, 1.11 to 2.13). See Figures 1 and 2. p p p Methods Newly admitted ICU patients ≥48 hours on mechanical ventilation were included. Daily plasma samples were collected from leftover plasma. Muscle strength was evaluated as soon as patients were awake and attentive. ICU-AW was defi ned by a mean Medical Research Council score <4. IL-1β, IL-6, IL-8, IL-10, IL-13, TNFα, IFNγ, fractalkine, GM-CSF, sICAM-1, sE-selectin and sP-selectin were measured in plasma samples of days 0, 2 and 4 using cytometric bead arrays and FACS. Diff erences of maximum levels between patients with and without ICU- AW were calculated using Mann–Whitney U tests. Principal component (PC) analysis was used to avoid multicollinearity and to reduce the set of mediators into a smaller set of PCs. To investigate whether diff erent infl ammatory profi les are associated with development of ICU-AW, we used multivariable logistic regression models of selected PCs, corrected for a priori selected variables, being age, gender, BMI, sepsis, SOFA score, APACHE IV score, immune insuffi ciency and corticosteroids. Conclusion The risk of ischemic stroke is higher in patients hospitalized with burn injury than in the general population, and the eff ects may be extended longer than expected previously. f 1. Cho SJ, Minn YK, Kwon KH. Stroke after burn. Cerebrovasc Dis. 2007;24:261-3. Troponin level as a predictor of prognosis in patients with acute ischemic stroke The best cutoff point revealed by the ROC curve of troponin was 0.291 mg/l; at which the sensitivity was 73% and the specifi city was 79% when used for prediction of death within 1 month (area = 0.319, CI = 0.214 to 0.423, P = 0.021; Figure 1). Conclusion These results suggest that increased serum troponin g Conclusion These results suggest that increased serum troponin level at admission is associated with higher mortality rate. Troponin Figure 1 (abstract P470). Patient fl owchart of enrollment. S165 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 2 (abstract P470). Burn injury patients have higher adjusted hazard ratio of ischemic stroke. at T0, 0.85 ± 0.24 at T1 and 0.91 ± 0.25 at T2, P = 0.146). There was a signifi cant diff erence in the levels of hemoglobin (13.19 ± g/dl 1.97 at T0 and 9.64 ± 1.48 g/dl at T1, P = 0.002). However, this diff erence was not maintained at T2 (12.7 ± 2.02 g/dl at T2, P = 0.252). There were no diff erences regarding PETCO2 at the time points. f g g 2 p Conclusion After cardiac surgery with a pump in high-risk patients, improvement of cerebral hemodynamic occurs, perhaps due to the optimization of cardiovascular function. These fi ndings must be better investigated. Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patientsf We measured the middle cerebral artery mean fl ow velocity and pulsatility index. The end-expiratory pressure of CO2 (PETCO2) was measured with infrared capnography attached to a face mask. Blood pressure, hematocrit and axillary temperature was also recorded. Conclusion Development of ICU-AW is associated with increased systemic infl ammation in the fi rst days after ICU admission. li Acknowledgement This research was supported by the Center for Translational Molecular Medicine (MARS). P473f P472 P472 Increased early systemic infl ammation in patients with ICU-acquired weakness E Witteveen, L Wieske, C Verhamme, T Van der Poll, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P472 (doi: 10.1186/cc14552) Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture P Turton1, R Hay1, JK Taylor2, J Little2, J McPhee3, I Welters4 1Royal Liverpool and Broadgreen University Hospitals, Liverpool, UK; 2Warrington and Halton Hospitals NHS Trust, Warrington, UK 3Manchester Metropolitan University, Manchester, UK; 4University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P473 (doi: 10.1186/cc14553) P Turton1, R Hay1, JK Taylor2, J Little2, J McPhee3, I Welters4 1Royal Liverpool and Broadgreen University Hospitals, Liverpool, UK; 2Warrington and Halton Hospitals NHS Trust, Warrington, UK 3Manchester Metropolitan University, Manchester, UK; 4University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P473 (doi: 10.1186/cc14553) Results The mean age of patients was 64 years; most patients were male (74%). Middle cerebral artery mean fl ow velocity increased signifi cantly after cardiac surgery. It was 53.89 ± 17.23 m/second at T0, 61.48 ± 15.18 m/second at T1 and 59.27 ± 16.12 m/second at T2 (P = 0.029). The pulsatility index was similar at all time points (0.88 ± 0.25 Introduction Muscle wasting is a common consequence of long- term stay in the critical care environment, which may slow down the rehabilitation of survivors. Previous ultrasound studies have S166 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (35.6% vs. 15.2%, P <0.001). This association remained signifi cant after correction for weakness and other risk factors (OR: 2.463 (95% CI: 1.113 to 5.452), P = 0.026). Also among conscious/cooperative patients without weakness, reduced CMAP was independently associated with a higher likelihood of death within 1 year (HR: 2.818 (95% CI: 1.074 to 7.391), P = 0.035). (35.6% vs. 15.2%, P <0.001). This association remained signifi cant after correction for weakness and other risk factors (OR: 2.463 (95% CI: 1.113 to 5.452), P = 0.026). Also among conscious/cooperative patients without weakness, reduced CMAP was independently associated with a higher likelihood of death within 1 year (HR: 2.818 (95% CI: 1.074 to 7.391), P = 0.035). demonstrated a loss of cross-sectional area of lower limb muscles during a 10-day intensive care stay. In this study, we have looked at how markers of muscle architecture (muscle thickness, pennation angle and fascicle length) change in the lower limb, as well as looking at changes in muscle thickness in the upper limb. demonstrated a loss of cross-sectional area of lower limb muscles during a 10-day intensive care stay. Predictive value for weakness and 1-year mortality of screening electrophysiology tests in the ICU g Results A total of 115 ICU patients were included. The average age was 61 years and 67% of the patients were male. ICU admission diagnoses were 53% acute surgery, 14% elective surgery and 33% were admitted for medical nonsurgical reasons. Inter-rater reliability of the DEMMI was high: intraclass correlation coeffi cient (ICC) ranging from >0.91 (range 0.85 to 0.94) at ICU admission, >0.98 (range 0.96 to 0.99) at the MICU and >0.97 (range 0.96 to 0.98) at the general ward. Internal consistency reliabilities (Cronbach α) of the DEMMI were 0.84, 0.87 and 0.98 at the ICU, MICU and hospital ward respectively. Validity coeffi cients (Spearman’s rank correlations) with BI, Katz-ADL and MMT were 0.63, –0.45 and 0.62. Introduction Muscle weakness in long-stay ICU patients contributes to 1-year mortality [1]. Whether electrophysiological screening is an alternative diagnostic tool also in unconscious/uncooperative patients remains unknown. The aims of this study were to determine the diagnostic properties of abnormal compound muscle action potential (CMAP), sensory nerve action potential (SNAP) and spontaneous electrical activity (SEA) for Medical Research Council (MRC)-sum score defi ned weakness and their predictive value for 1-year mortality. i p y y Methods Data were prospectively collected during the EPaNIC trial (ClinicalTrials.gov NCT00512122) [2] from October 2008 to November 2010. From day 8 onwards, nerve conduction studies and electromyography were performed weekly in 642 long-stay and 88 randomly selected short-stay patients and muscle strength was assessed in cooperative patients using the MRC-sum score. The electrophysiologist was blinded for the clinical assessments of the physiotherapists and vice versa. The two primary outcomes were: sensitivity, specifi city, positive and negative predictive values of abnormal CMAP, SNAP and SEA for weakness (MRC-sum score <48); and the predictive value for 1-year mortality of abnormal fi ndings on fi rst electrophysiological screening. This association was assessed by univariate and multivariate analyses correcting for weakness and other risk factors, including baseline risk factors, comorbidities, illness severity and ICU exposures. Conclusion The DEMMI is a reliable, responsive and feasible measure ment instrument for the assessment of mobility in critically ill ICU patients. R f 1. Stevens RD. Intensive Care Med. 2007;33:1876-91. 2. de Morton NA, et al. Health Qual Life Outcomes. 2008;6:63. 2. de Morton NA, et al. Health Qual Life Outcomes. 2008;6:63. Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture In this study, we have looked at how markers of muscle architecture (muscle thickness, pennation angle and fascicle length) change in the lower limb, as well as looking at changes in muscle thickness in the upper limb. Methods Following ethical approval, patients who were intubated and ventilated in one of two critical care departments were assented to take part in the study by their next of kin. B-mode ultrasound scans of the right biceps, vastus lateralis and the medial head of gastrocnemius were performed on days 1, 5 and 10. Scans were not performed in patients once they were free of sedation. Muscle thickness (MT) was measured in all three muscles, with pennation angle (PA) being measured in the lower limb muscles. Fascicle length (FL) was derived from PA and MT. Conclusion The diagnostic properties of electrophysiological screening vary over time. Abnormal CMAP documented early during critical illness carries information about longer-term outcome, which should be further investigated mechanistically. Acknowledgement HVM and GH contributed equally. Acknowledgement HVM and GH contributed equally. References 1. Hermans G, et al. Am J Respir Crit Care Med. 2014;190:410-20. 2. Casaer M, et al. N Eng J Med. 2011;365:506-17. 1. Hermans G, et al. Am J Respir Crit Care Med. 2014;190:410-20. 2. Casaer M, et al. N Eng J Med. 2011;365:506-17. g Results Twenty patients were recruited, of which 15 were scanned on day 5, and eight were scanned on day 10. In the biceps, there were no alterations in MT over 5 or 10 days. MT of the vastus lateralis signifi cantly decreased on day 5 (1.77 ± 0.06 mm muscle loss, P = 0.03) and day 10 (5.58 ± 0.09 mm muscle loss, P = 0.01). There was also a signifi cant loss in PA over 5 days (1.48 ± 0.63°, P = 0.01) and 10 days (2.96 ± 0.72°, P = 0.01). However, FL was unchanged over time. There was a signifi cant relationship between size of PA and percentage loss of PA and FL in over 5 days. Loss of MT and PA (MT: 3.21 ± 2.08 mm lost, PA: 2.19 ± 1.64°) was observed in the medial gastrocnemius over 10 days, but did not approach signifi cance. Large fascicles on day 1 were associated with greater percentage loss of FL on day 5 (P = 0.012). P475 P474 y Methods The inter-rater reliability and validity were determined in a prospective observational study. Patients were included and assessed by two independent raters until hospital discharge. Reliability was expressed using the intraclass correlations (ICC). To evaluate the validity, the DEMMI scores were compared with the Barthel Index (BI), Katz-ADL and manual muscle testing (MMT). Predictive value for weakness and 1-year mortality of screening electrophysiology tests in the ICU H Van Mechelen1, G Hermans1, F Bruyninckx2, T Vanhullebusch1, B Clerckx1, P Meersseman2, Y Debaveye1, MP Casaer1, A Wilmer2, PJ Wouters1, I Vanhorebeek1, R Gosselink1, G Van den Berghe1 1KU Leuven, Belgium; 2UZ Leuven, Belgium Critical Care 2015, 19(Suppl 1):P474 (doi: 10.1186/cc14554) Psychometric properties of the de Morton Mobility Index in ICU patients J Sommers1, T Vredeveld2, J Horn1, RH Engelbert2, R Lindeboom1, M Vd Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2Amsterdam School of Health Professions (ASHP), University of Applied Sciences, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P475 (doi: 10.1186/cc14555) Conclusion In the lower limb, we have shown that MT and PA alterations occur in the fi rst 10 days. Patients with larger PA and FL appear to lose a greater percentage of angle and fascicle length in the fi rst 5 days. In contrast, we have demonstrated a sparing eff ect on the muscles of the upper limb compared with the lower limb. These fi ndings may have implications for rehabilitation and interventions to preserve muscle mass. Introduction Many ICU patients develop ICU-acquired muscle weak- ness (ICU-AW) due to inactivity and critical illness. ICU-AW is associated with short-term and long-term physical impairments and impaired functional status [1]. The de Morton Mobility Index (DEMMI) was developed to measure changes in mobility across clinical settings and proved to be reliable, feasible and sensitive to small but clinically relevant changes in functioning [2]. Our aim was to evaluate the psychometric properties of the DEMMI in ICU patients. Prevalence of psychiatric disorders in trauma patients: results from a major trauma unit M Adlam, A Feehan, V Metaxa Methods This was a prospective observational study of trauma patients from two critical care trauma centers. We excluded patients who had ICU stay <48 hours and those with severe traumatic brain injury (TBI) (GCS ≤8). Patients were followed until ICU discharge, resolution of delirium, death or ICU length of stay >28 days. Delirium was assessed daily using the Confusion Assessment Method for the ICU until the end of the follow-up period. Demographic and admission data, daily consumption of medications, and environmental factors (that is, presence of clock, TV/radio, and so forth) were collected daily. Univariate analysis was performed using Cox regression analysis to identify risk factors for delirium. The independent eff ect of modifi able risk factors was assessed using multivariate Cox regression analysis adjusting for severity of illness and nonmodifi able risk factors. King’s College Hospital, London, UK g g p Critical Care 2015, 19(Suppl 1):P477 (doi: 10.1186/cc14557) Introduction Mental illness has been recognised as a potential risk factor both for intentional and unintentional injury. About 50% of patients presenting with self-infl icting injuries in emergency departments had previous known psychiatric disorder (PD) [1,2], whereas individuals with mental illness were admitted for unintentional injury twice as often as those without [3]. We aimed to assess the prevalence of PD in trauma patients being admitted to a major trauma ICU and compare it with the nontrauma population. Methods We retrospectively reviewed all admissions from January 2010 to December 2013 in a tertiary, mixed ICU that serves a London major trauma centre (MTC) hospital. Data obtained were age, APACHE II score, reason for admission, length of stay (LOS), mortality and a diagnosis of depression, bipolar, self-harm, psychosis, schizophrenia and suicide attempt. j g yi Results We enrolled 150 trauma patients resulting mostly from falls (40%) and motor vehicle accidents (28.7%) over 14 months. Patients with TBI accounted for 56.7% while polytrauma patients without TBI accounted for 43.3%. Mean ICU length of stay was 8.1 ± 7.1 days, 69.3% required mechanical ventilation, 14.7% required a tracheostomy. Delirium developed in 58 patients (38.7%) (mean age 62.9 ± 15.7, mean APACHE score 15.4 ± 6.1, mean ISS score 23.4 ± 9.1). Univariate analysis revealed that delirium was signifi cantly associated with the following nonmodifi able risk factors: age (per 10-year range), APACHE II score (per 10-point increase), need of mechanical ventilation, presence of TBI and pre-existing diabetes. j References 1. Schecter WP, et al. Arch Surg. 2005;140:90. 1. Schecter WP, et al. Arch Surg. 2005;140:90. 2. Colman I, et al. Acad Emerg Med. 2004;11:136. 3 Wan JJ et al J Tra ma 2011 61 1299 2. Colman I, et al. Acad Emerg Med. 2004;11:136. 3. Wan JJ, et al. J Trauma. 2011;61:1299. B-type natriuretic peptide and estimated glomerular fi ltration rate at ICU admission as a predictor of delirium Introduction Delirium in the ICU is a predictor of mortality and cognitive impairment at hospital discharge. Although several pathways for delirium have been described, it is very diffi cult to predict the occurrence of delirium. In this study, we examined plasma biomarkers in delirious and nondelirious patients at admission and whether the biomarkers can predict onset of delirium. y Results A total of 730 patients were electrophysiologically screened, of which 432 were tested for weakness. On day 8, only normal CMAP excluded weakness with a high negative predictive value (80.5%). By day 15, abnormal SNAP and the presence of SEA revealed a high positive predictive value (91.7% and 80.0%, respectively). On day 8, only a reduced CMAP was associated with higher 1-year mortality S167 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We targeted 103 ICU patients in Okayama University Hospital between April 2013 and February 2014. Delirium was diagnosed using the Confusion Assessment Method – ICU. On admission, blood was obtained for biomarker analysis. Patients with severe head injury and under 16 years old were excluded. P <0.05 was considered statistically signifi cant. Conclusion Trauma patients with PD have increased mortality and LOS. MTCs provide a unique opportunity to identify mental illness during hospitalisation through screening and intervention programmes. Integration of mental health services into ICU care should be examined, as it might provide an effi cient and cost-eff ective way of decreasing the risk of reinjury. gi Results Thirty-seven delirious and 66 nondelirious patients were included. We found that delirious patients tented to have higher B-type natriuretic peptide (BNP) levels and to have lower estimated glomerular fi ltration rate (eGFR) (BNP: delirious patients 188.6 pg/ml, nondelirious patients 78.2 pg/ml (P = 0.001); eGFR: delirious patients 58.6 ml/ minute/1.73 m2, nondelirious patients 81.3 ml/minute/1.73 m2 (P = 0.020)). Procalcitonin (PCT) and D-dimer were almost the same between delirious and nondelirious patients (PCT: delirious patients 0.202 ng/ml, nondelirious patients 0.150 ng/ml (P = 0.613); D-dimer: delirious patients 5.25 ng/ml, nondelirious patients 5.35 ng/ml (P = 0.714)). Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study Conclusion BNP and eGFR in ICU admission was associated with delirium. PCT and D-dimer in ICU admission was not associated with delirium. BNP and eGFR might evaluate a predictor of delirium in ICU. References 1. Ely EW, et al. JAMA. 2004;291:1753-62. 1. Ely EW, et al. JAMA. 2004;291:1753-62. Introduction Delirium is associated with signifi cant morbidity and mortality in critically ill medical and surgical patients. However, patients suff ering from trauma are generally excluded from these studies. Our objectives were to assess the incidence of delirium and identify modifi able risk factors associated with delirium among critically ill trauma patients. y 2. van den Boogaard M, et al. Crit Care. 2011;15:R297. P478i Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study MA Duceppe1, A Elliott1, M Para1, MC Poirier1, M Delisle1, AJ Frenette2, D Deckelbaum1, T Razek1, M Desjardins2, JC Bertrand2, F Bernard2, P Rico2, L Burry3, D Williamson2, MM Perreault1 1The Montreal General Hospital, Montreal, QC, Canada; 2Hôpital du Sacré- Coeur de Montreal, QC, Canada; 3Mount Sinai Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P478 (doi: 10.1186/cc14558) Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study MA Duceppe1, A Elliott1, M Para1, MC Poirier1, M Delisle1, AJ Frenette2, D Deckelbaum1, T Razek1, M Desjardins2, JC Bertrand2, F Bernard2, P Rico2, L Burry3, D Williamson2, MM Perreault1 1The Montreal General Hospital, Montreal, QC, Canada; 2Hôpital du Sacré- Coeur de Montreal, QC, Canada; 3Mount Sinai Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P478 (doi: 10.1186/cc14558) Prevalence of psychiatric disorders in trauma patients: results from a major trauma unit In a multivariate analysis when adjusting for the nonmodifi able risk factors, opioids (adjusted HR = 0.37, 95% CI (0.14 to 1.0)), presence of a TV/radio in the room (adjusted HR = 0.28, 95% CI (0.12 to 0.67)), and number of hours mobilized (adjusted HR = 0.77, 95% CI (0.68 to 0.88)) had a protective eff ect on delirium; whereas use of physical restraints (adjusted HR = 2.20, 95% CI (1.11 to 4.35)) and active infection (adjusted HR = 2.08, 95% CI (1.16 to 3.71)) remained strongly associated with delirium. Results Of 978 trauma patients admitted to the ICU, 68 (7%) had a known PD. Their diagnoses are shown in Figure 1. Median APACHE II score and unadjusted mortality were 13 and 18% respectively in the PD group (15 and 12% in the entire cohort, P >0.05). Patients suff ering from more than one diagnosis or self-harm alone had increased median LOS (6 vs. 4 days in the entire cohort, P >0.05). Figure 1 (abstract P477). Conclusion Considering the long-term consequences of delirium, steps should be implemented to prevent its development in trauma and include optimizing opioids and mobilizing patients while limiting use of physical restraints. References References 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. References 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. P479 Evaluation of the PRE-DELIRIC delirium prediction tool on a general ICU J Hanison, S Umar, K Acharya, D Conway Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P479 (doi: 10.1186/cc14559) Introduction Delirium is a frequently occurring complication of critical care, occurring in approximately 45% of unplanned UK ICU admissions Introduction Delirium is a frequently occurring complication of critical care, occurring in approximately 45% of unplanned UK ICU admissions Figure 1 (abstract P477). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S168 Figure 1 (abstract P479). Range of PRE-DELIRIC scores. Methods A literature study via MEDLINE and Embase search with keywords ‘PRIS’, ‘lactic acid’, ‘propofol’ and ‘sedation’. All cases in English, French and Spanish were indentifi ed. Exclusion criteria were onset >48 hours, unclear description of time pattern and dose. p p Results Twenty-two cases of early PRIS were found. These concerned 10 pediatric versus 12 adult patients. Eleven were identifi ed in the ICU versus 11 in the operating room. The survival rate of early PRIS was 95.5%, and morbidity was restricted to four patients. In the adult subgroup, the mean propofol dose was 4.9 mg/kg/hour. Triggering factors such as use of catecholamines and corticosteroids were found in 36.4% and 45% of patients. In total, 3/22 cases matched Bray’s defi nition of PRIS. In 14/22 cases, lactic acidosis was interpreted as onset of PRIS. Conclusion Compared with a review by Fudickar [1], we found signifi cant diff erences in critical dose, risk factors, symptomatology and morbidity/mortality between PRIS and early PRIS cases. As criticisms are off ered, a question is whether these cases really are the onset of the fatal syndrome PRIS. Therefore, we completed diff erential diagnosis of lactic acidosis and found that not all possible causes (for example, hyperglycemia, ketonemia, pharmacologic confounders as biguanides, epinephrine) were ruled out in most cases. This is important since PRIS is an exclusion diagnosis. The existence of early PRIS should indeed be questioned and investigated by large, multicenter observational trials. Reference [1]. The presence of delirium in critical care is an independent risk factor for mortality; for every day of delirium, there is an additional 10% relative risk of death at 1 year [2]. A delirium prediction tool PRE- DELIRIC has been recently developed and calibrated in a multinational project [3]. This study aimed to determine the utility of PRE-DELIRIC on our ICU. Propofol sedation reduces contraction and motion of diaphragm in humans: preliminary results p y G Ranieri, M Luigi, F Belsito, M Rocco, RA De Blasi Sant’Andrea, Rome, Italy Results The PRE-DELIRIC scores predicted a mean rate of delirium of 39%. PRE-DELIRIC risk scores ranged from 4 to 93% (Figure 1). Six (15%) patients developed delirium in the fi rst 24 hours following extubation. Fifteen (37%) of patients were predicted 20% or less probability of delirium. Twelve (29%) patients developed delirium at any point during their ICU stay. This resulted in 36 total delirium bed-days. Introduction Among drugs used for sedation, propofol has a primary role [1]. Despite propofol being described to exert a relaxant eff ect on skeletal muscle, no data showing its action on diaphragm are reported. The aim of this observational study on humans is to apply ultrasound to assess propofol’s eff ect on diaphragmatic contraction and motion during endoscopic procedures. y y Conclusion Our observation that <30% of patients experienced delirium is less than the reported prevalence in similar settings and our own audits. This study demonstrates that there is some agreement between recorded rates of delirium and predicted rates using PRE- DELIRIC. We suggest that PRE-DELIRIC can be used in quality/audit work on UK ICUs in order to assess attempts to improve the management of delirium. Further work is required to assess the utility of PRE-DELIRIC as a risk assessment tool in individual patients. Methods We investigated seven consecutive patients undergoing gastroscopy or colonoscopy in the endoscopy unit of our hospital. Patients received propofol at a dose able to induce and maintain sedation to level 6 of the Ramsay Sedation Scale during the procedure. Measurements were obtained on right side of the thorax in millimeters; diaphragmatic motion (DM) and diaphragmatic motion at maximal inspiration (DM forced) were measured in M-Mode with a 3.5 MHz array convex probe placed on the midclavicular line using the liver acoustic window. Thickness at end inspiration (TEI) and thickness at end expiration (TEE) were measured in M-Mode with a 10 MHz vascular probe. The thickening fraction (TF) was calculated: (TEI – TEE) / TEE [2]. Time points of measurements were taken when the patient arrived in the surgery room (Baseline), 1 minute after level 6 of the Ramsey Sedation Scale was obtained (Sedation) and 5 minutes after the patient had a recovery to level 1 on the Ramsey Sedation Scale (Awakening). q Reference 1. Fudickar A. Propofol infusion syndrome in anaesthesia and intensive care medicine. Curr Opin Anaesthesiol. 2006;19:404-10. Methods This study prospectively investigated 41 patients. Medical and surgical general ICU patients were included after 24 hours of sedation and mechanical ventilation. The researchers calculated PRE-DELIRIC scores for each patient. PRE-DELIRIC involves recording 10 variables, submitted into an online algorithm that estimates the percentage risk of delirium. We diagnosed delirium with the CAM-ICU which was performed 12 hourly [4]. P484 Prolonged dexmedetomidine infusion and drug withdrawal in critically ill children A Haenecour, A Goodwin, W Seto, C Urbain, P Laussen, C Balit The Hospital for Sick Children, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P484 (doi: 10.1186/cc14564) Prolonged dexmedetomidine infusion and drug withdrawal in critically ill children A Haenecour, A Goodwin, W Seto, C Urbain, P Laussen, C Balit The Hospital for Sick Children, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P484 (doi: 10.1186/cc14564) Results One hundred and twenty-six of 174 respondents indicated that they practice in the ICU setting. Sixty-six per cent were specialists and mainly anaesthesiologists (42%), whilst 32% were critical care subspecialists. The respondents indicated that on average 30 ± 20% of their patients experience delirium. Eighty per cent of the respondents indicated that delirium impacts signifi cantly negatively on patient outcomes whilst 1% indicated that there was no such association. Delirium screening is achieved mainly by clinical assessment (77%). Twenty-four per cent utilise an objective tool to screen for delirium and amongst them the CAM-ICU is utilised by 80%. Amongst delirious patients the sedative of choice is dexmedetomidine in the majority. However, 20% prescribe midazolam as a fi rst choice in this setting. Conclusion The fi ndings are comparable with reports of similar surveys conducted in other regions. The delirium screening method is inadequate as the vast majority do not utilise an objective method. Introduction We investigated the incidence, symptoms and risk factors for withdrawal associated with prolonged dexmedetomidine use. Dexmedetomidine is an α2-adrenergic receptor agonist, with anxiolytic, analgesic and sedative properties. Intended for short-term use, there is increasing literature describing prolonged use for sedation. However, this raises the potential of withdrawal syndrome and there is no recommendation for the discontinuation of dexmedetomidine. Other goals included determining the hemodynamic eff ects of discontinuation of dexmedetomidine and role of clonidine in patients with prolonged dexmedetomidine use. g Methods A retrospective review of patients admitted to the critical care unit who had exposure to dexmedetomidine for longer than 48 hours, between 1 January 2014 and 15 July 2014. Data included patient demographics, dexmedetomidine exposure (bolus dose, total cumulative dose, duration), other sedative exposure, withdrawal symptoms measured by WAT-1 score, nursing subjective assessment and treatment given for withdrawal. Each potential withdrawal episode was reviewed by two reviewers. Hemodynamic parameters were analyzed to assess hemodynamic changes associated with discontinuation of dexmedetomidine. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. i Results During propofol administration TEI reduced 19% whereas after awakening it increased 14.5% but did not reach baseline. Conversely TEE did not change during the study. During propofol sedation, TF decreased 34% and returned to baseline after recovery. DM showed 29% reduction during propofol administration whereas the forced diaphragmatic motion tested when patients were conscious (forced DM) did not evidence any change. y g Conclusion In this observational study, ultrasound assessed that propofol causes a reduction of diaphragmatic contraction and motion during endoscopic procedures. P482 Delirium knowledge and assessment by ICU practitioners in South Africa: results of a national survey S Chetty, F Paruk University of the Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P482 (doi: 10.1186/cc14562) Delirium knowledge and assessment by ICU practitioners in South Africa: results of a national survey S Chetty, F Paruk University of the Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P482 (doi: 10.1186/cc14562) p g p Conclusion These results are consistent with the hypothesis of 2 days reduction of the median weaning time in the loxapine group, but the diff erence was not statistically signifi cant. Loxapine reduces the need for resuming sedation during weaning from MV. Given the quality of the data and methodology, these results may be useful in future meta-analyses. Introduction Delirium recognition in critically ill patients is considered to be important taking into account the poor outcomes associated with its occurrence. The purpose of this study was to evaluate knowledge pertaining to delirium as well as the implementation of screening practices. This study constituted a component of a survey that explored current sedation-related practices in South African ICUs. Methods Following approval from the University Human Research ethics committee, a validated questionnaire was distributed electronically to physician members of various medical databases in South Africa as South Africa does not have a formal registry of critical care practitioners. P484 Descriptive statistics were used with t test and chi-square test. Median and interquartile range (IQR) are reported. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. Results The trial was discontinued after 101 patients had been randomized because of insuffi cient enrollment. Fifteen patients withdrew consent, leaving 86 patients for analysis. Forty-seven patients were assigned to the loxapine group and 39 to the placebo group. Median time to successful extubation was 3.2 days in the loxapine group and 5 days in the placebo group (RR = 1.2, 95% CI = 0.75 to 1.88; P = 0.45). During the fi rst 24 hours, sedation was more frequently resumed in the placebo group (44% vs. 17%, P = 0.01). One patient had a transient seizure in the loxapine group. Data analyzed are reported in Table 1 and expressed as mean (SD). ANOVA was used to compare data for repeated measurements. Post hoc statistical comparison with Bonferroni’s test was used to identify signifi cant variations. than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. g References p g p yp Results The trial was discontinued after 101 patients had been randomized because of insuffi cient enrollment. Fifteen patients withdrew consent, leaving 86 patients for analysis. Forty-seven patients were assigned to the loxapine group and 39 to the placebo group. Median time to successful extubation was 3.2 days in the loxapine group and 5 days in the placebo group (RR = 1.2, 95% CI = 0.75 to 1.88; P = 0.45). During the fi rst 24 hours, sedation was more frequently resumed in the placebo group (44% vs. 17%, P = 0.01). One patient had a transient seizure in the loxapine group. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande University Hospitals Leuven, Belgium Critical Care 2015, 19(Suppl 1):P480 (doi: 10.1186/cc14560) University Hospitals Leuven, Belgium University Hospitals Leuven, Belgium y g Critical Care 2015, 19(Suppl 1):P480 (doi: 10.1186/cc14560) Table 1 (abstract P481) Variable Baseline Sedation Awakening P value Thickness end inspiration (mm) 3.25 (0.21) a2.66 (0.20) b,c3.22 (0.30) <0.001* Thickness end expiration (mm) 2.11 (0.20) d2.00 (0.15) 2.07 (0.15) 0.112* Thickening fraction 0.54 (0.07) e0.36 (0.10) f,g0.49 (0.11) <0.001* Diaphragmatic motion (mm) 18.66 (2.23) h13.27 (4.93) 15.31 (0.40) 0.055 Diaphragmatic motion forced 54.61 (19.34) – 56.34 (13.75) 0.298 (mm) aP <0.001 versus baseline. bP = 0.043 versus baseline. cP <0.001 versus sedation. dP = 0.030 versus baseline. eP =0.012 versus baseline. fP =0.353 versus baseline. gP = 0.041 versus sedation. hP = 0.022 versus baseline. Introduction Propofol infusion syndrome (PRIS) is a rare propofol complication, leading to cardiac failure. It was fi rst described in critically ill children and in adults with traumatic brain injury. Pathophysiology is unknown although common factors are the prolonged (>48 hours) use of high-dose (>5 mg/kg/hour) propofol combined with elevated levels of catecholamines and corticosteroids. Recently, case reports of early- onset PRIS during anesthesia and in the early postoperative setting were published. In many of these, lactic acidosis is interpreted as onset of PRIS. Criticism off ers that it might concern a poor diff erential diagnostic approach or an observational bias. Also, lactic acidosis is not an obligate PRIS symptom and incidence of lactic acidosis during propofol sedation is unknown. To gain insight into the incidence and characteristics of early PRIS, we performed a systematic review on early PRIS cases. S169 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. P487 Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients Introduction Recently, several animal studies observed that dexmedetomidine (DEX), a new sedative and α2-adrenoceptor agonist, inhibited the infl ammatory responses [1-3]. Moreover, DEX was reported to have anti-infl ammatory eff ects in patients [4,5]. However, these studies were the small-sized studies and there are few studies about the eff ects of long-term administration of DEX in severe septic patients. The present study evaluated the eff ects of long-term administration of DEX on infl ammatory responses and severity in severe septic patients. We hypothesize that the administration of DEX has benefi cial eff ects for severe septic patients. y y g y Methods We investigate the role of DEX as a sedative agent used during recovery from deep sedation and weaning from extracorporeal support in patients on vv-ECMO. From May 2014 to October 2014 we prospectively enrolled seven patients aff ected by ARDS of diff erent etiologies treated with vv-ECMO. The mean age was 53.7 ± 7.9 years and the mean ICU stay was 21.4 ± 11.5 days. Initially, all patients were sedated with association of opioids and GABA receptor agonists, following the internal protocol. At the time of weaning from ECMO, ruled out cardiovascular instability, we started the administration of DEX (0.7 μg/kg/hour, without initial bolus) with progressive decrease of the dose of other sedative drugs. if Methods In 66 patients (M/F 44/22, mean age 66 years) with severe sepsis, who were administered propofol (0.5 to 4.0 mg/kg/hour) only for sedation, 42 patients (M/F 28/14, mean age 67 years) were administered DEX (0.2 to 0.7 μg/kg/hour) for more than 24 hours in addition to propofol (DEX group). Twenty-four patients were not administered DEX (Control group). Primary outcome were changes in infl ammatory responses at 48 hours after the administration of DEX or none, and secondary outcomes were changes in APACHE II and SOFA scores at 48 hours after the administration of DEX or none. Results The mean duration of DEX infusion was 6.1 ± 4.8 days. Loxapine to control agitation during weaning from mechanical ventilation: a randomized controlled trial S Gaudry1, B Sztrymf2, R Sonneville3, B Megarbanne4, C Clec’h5, J Ricard1, D Hajage3, D Dreyfuss1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Béclère, Clamart, France; 3Hôpital Bichat, Paris, France; 4Hôpital Lariboisière, Paris, France; 5Hôpital Avicennes, Bobigny, France Critical Care 2015, 19(Suppl 1):P483 (doi: 10.1186/cc14563) Results A total of 53 patients accounted for 69 unique dexmedetomidine treatment courses. Median age at the time of dexmedetomidine infusion was 5 months (range 1 day to 3 years). Dexmedetomidine dose ranged from 0.1 to 2 μg/kg/hour with a median cumulative dose of 87 μg/kg (IQR 53, 156). Median duration of exposure to dexmedetomidine was 124 hours (IQR 76, 178) with a maximum duration of 466 hours. We identifi ed 24 separate episodes of withdrawal (incidence 35%). Most common symptoms were agitation (100%), fever (67%), vomiting/retching (46%), loose stools (29%) and decreased sleep (20%). Statistical analysis showed that factors signifi cantly associated Introduction Weaning from prolonged mechanical ventilation (MV) in the ICU may be impeded by the occurrence of agitation. Loxapine had the ability to control agitation without aff ecting the effi cacy of spontaneous ventilation in an observational study, justifying the implementation of a randomized controlled trial. Methods We conducted a multicenter, placebo-controlled, parallel group, randomized trial at fi ve French ICUs between November 2011 and November 2013. Patients (aged >18 years) under MV for more S170 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 to help tolerate intubation and reduce pain and anxiety. Propofol (Pr) is a widely used option, but other viable alternatives for short-term sedation (STS; that is, <24 hours) include benzodiazepines (BDZ) and dexmedetomidine (Dx). We aimed at pooling all available evidence on the comparative eff ects of Pr in terms of awakening and recovery times after STS in mechanically ventilated ICU patients. with withdrawal were cumulative dose (P = 0.01) and duration of use of dexmedetomidine (P = 0.02). Duration of opioids exposure prior to dexmedetomidine wean was also a risk factor for withdrawal (P = 0.01). Use of clonidine as a transition from dexmedetomidine did not protect against withdrawal (P = 0.59). g Conclusion This study showed that withdrawal syndrome is associated with prolonged infusion of dexmedetomidine. Patients with higher cumulative doses and longer duration of exposure were more at risk. P486 P486 Short-term sedation of mechanically ventilated ICU patients with propofol, benzodiazepines, or dexmedetomidine: systematic review and meta-analysis on awakening and recovery times A Feuersenger1, L Pradelli2, A Aliano2, JF Baron3, M Westphal1 1Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany; 2AdRes, Torino, Italy; 3Fresenius Kabi ELAMA, Paris, France Critical Care 2015, 19(Suppl 1):P486 (doi: 10.1186/cc14566) Conclusion In the present study, the long-term administration of DEX has benefi cial eff ects of infl ammatory responses and severity for severe septic patients. P485 Weaning from extracorporeal membrane oxygenation: experience with dexmedetomidine in seven adult ARDS patients M Cozzolino, A Franci, A Peris, L Tadini Buoninsegni, B Loriga A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P485 (doi: 10.1186/cc14565) Introduction Sedation in the ICU is a basic therapeutic procedure to increase tolerance of invasive treatments and reduce discomfort. Extracorporeal membrane oxygenation (ECMO) is a highly invasive treatment and prolonged sedation may be required. Patients undergoing ECMO represent a challenge with respect to sedation. Initially, deep sedation may be required to optimize ventilation and circuit–patient fl ows and to minimize oxygen consumption. The other critical phase is represented by weaning from ECMO support. Optimal sedation is not clearly defi ned, moreover there are no data on sedation practices with dexmedetomidine (DEX) in adult patients undergoing ECMO. In contrast to other sedatives, DEX has analgosedative eff ects without respiratory depression, and could be useful to facilitate spontaneous respiratory activity during recovery from sedation. i Conclusion In conclusion, Pr is associated with shorter awakening and recovery times after STS than BDZ, while no diff erence could be shown when Pr was compared with Dx. P487f P487 Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Except for one patient, who received DEX as a single drug after suspension of other sedatives, a low-dose infusion of another sedative (<50% compared with initial dose) was maintained. Three patients presented adverse events: two bradycardia and one hypotension. In four patients DEX was discontinued after recovery of respiratory function; in two patients deeper sedation for ventilatory dyssynchrony was needed so other sedative drugs were started. Only in one patient was the drug suspended for extreme bradycardia, resolved after suspension. Results The administration of DEX occurred for a mean 130 hours (24 to 433 hours) in the DEX group. White blood cell counts, C-reactive protein (CRP) and procalcitonin (PCT) in both groups signifi cantly decreased after the administration of DEX or none. However, CRP and PCT in the DEX group were signifi cantly lower than those in the control group: CRP 7.7 (5.0) versus 13.6 (7.9) mg/dl; P <0.05, PCT 7.6 (11.7) versus 18.6 (11.6) ng/ml; P <0.05, mean (SD). APACHE II and SOFA scores in both groups decreased after the administration of DEX or none, but APACHE II and SOFA scores in the DEX group were lower than those in the control group: APACHE II 10.8 (4.8) versus 15.2 (5.1); P <0.05, SOFA 3.6 (2.0) versus 5.8 (2.9); P <0.05, mean (SD). p y p Conclusion In our study, DEX allowed the reduction of doses of other sedative drugs during weaning from vv-ECMO; this may lead to a cooperative sedation, promoting spontaneous breathing. Side eff ects described and the cost–benefi t ratio must still be verifi ed extensively in patients during weaning from ECMO. Loxapine to control agitation during weaning from mechanical ventilation: a randomized controlled trial Our results suggested that clonidine use is not protective for withdrawal from dexmedetomidine. y Methods We planned a systematic literature review searching Medline and Scopus and performed a meta-analysis on direct comparisons reporting on weaning time (Tw), duration of mechanical ventilation (Tmv), time to extubation (Tex) and length of stay in the ICU (Ticu). The primary analysis considered only data from RCTs, while in a secondary analysis observational studies were also included.i y Results The literature search identifi ed 15 relevant RCTs, of which 11 versus BDZ, and a further fi ve observational studies, of which one versus BDZ. When compared with BDZ, Pr associated with signifi cantly reduced Tw (–1.6 hours, 95% CI: –2.5 to –0.8), Tmv (–2.0 hours, 95% CI: –3.7 to –0.2), and Ticu (–5.0 hours, 95% CI: –8.5 to –1.4); no statistically signifi cant diff erence resulted when comparing Pr and Dx. When nonrandomized evidence was included, results did not change signifi cantly. P490 Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) Results A total of 77 children with a median age of 15 (4 to 84) months, weight of 10 (5.7 to 20) kg and length of ICU stay of 8 (5 to 14) days received DEX, with a mortality rate of 9%. Indications were: weaning from mechanical ventilation (32.5%), neurosurgical postoperative (NCI) and upper airway surgery (VAS) (24.7%), non-invasive ventilation (13%), refractory tachycardia (6.5%) and other indications (23.3%). There was no diff erence between the initial and maximum doses and infusion length. There was a signifi cant decrease in MAP and HR after 6 hours infusion of DEX in the total group; however, no signifi cant diff erence occurred between groups when analyzing MAP and HR 24 hours after the start of infusion (P = 0.798 and 0.379, one-way ANOVA, respectively). In six patients (8%) DEX was suspended for possible side eff ects. Introduction To determine which of three methods of pain manage- ment provided the best pain control in severe ASA III older patients in the surgical ICU (SICU). As technology improves, more older patients benefi t from surgery and need SICU care. Older surgery patients frequently present two medical problems. First is unspecifi c symptoms and decreased pain sensation resulting in delayed diagnosis. Second, they usually are not given enough perioperative pain relief. Optimal pain management results in perioperative stable hemodynamic status and decreased morbidity, mortality, length of stay and medical costs. Methods A retrospective cohort study chart review of 1,872 all-cause patients in a 16-bed SICU during April 2011 to September 2012. Un- consciousness, uncooperative, ASA <III and <65-year-old patients were excluded. The primary point was to compare eff ectiveness of three diff erent methods of pain management: P.R.N. i.v. Demerol/NSAID (D/N), i.v. patient-controlled analgesia (PCA) and patient-controlled epidural analgesia (PCEA), in three diff erent conditions: rest, movement and coughing, with visual analogue scales (VAS 0 to 100). Secondary point was patient satisfaction. Introduction To determine which of three methods of pain manage- ment provided the best pain control in severe ASA III older patients in the surgical ICU (SICU). As technology improves, more older patients benefi t from surgery and need SICU care. Older surgery patients frequently present two medical problems. P490 Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) First is unspecifi c symptoms and decreased pain sensation resulting in delayed diagnosis. Second, they usually are not given enough perioperative pain relief. Optimal pain management results in perioperative stable hemodynamic status and decreased morbidity, mortality, length of stay and medical costs. Methods A retrospective cohort study chart review of 1,872 all-cause patients in a 16-bed SICU during April 2011 to September 2012. Un- consciousness, uncooperative, ASA <III and <65-year-old patients were excluded. The primary point was to compare eff ectiveness of three diff erent methods of pain management: P.R.N. i.v. Demerol/NSAID (D/N), i.v. patient-controlled analgesia (PCA) and patient-controlled epidural analgesia (PCEA), in three diff erent conditions: rest, movement and coughing, with visual analogue scales (VAS 0 to 100). Secondary point was patient satisfaction. f Conclusion Increased DEX indications have been observed in the pediatric population. In this study DEX was demonstrated to be a safe and tolerable drug with few side eff ects, especially related to the cardiovascular system. References 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 2. Afonso J RF. Dexmedetomidina: Papel Atual em Anestesia e Cuidados Intensivos. Rev Bras Anestesiol. 2012;62:118-33. 3. Panzer O, Moitra V, et al. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin. 2009;25:451-69. 4. Hsu CD CJ. Pharmacologic agents for pediatric procedural sedation outside of the operating room. UpToDate2014. http://www.uptodate.com/contents/ pharmacologic-agents-for-pediatric-procedural-sedation. 5. Tobias JD. Dexmedetomidine: applications in pediatric critical care and pediatric anesthesiology. Pediatr Crit Care Med. 2007;8:115-31. p p Results A total of 1,292 patients were excluded. VAS results are presented in Table 1 as mean ± SD. At rest, the PCEA group is signifi cantly better than the other two groups. When at movement, there is no diff erence between the PCEA group and the D/N group but both are better than the PCA group. While coughing, the PCEA group is worse than the D/N group, although there is no diff erence between the PCEA group and the PCA group. The PCEA group gets the best grades in patient satisfaction. 2. Afonso J RF. Dexmedetomidina: Papel Atual em Anestesia e Cuidados Intensivos. Rev Bras Anestesiol. 2012;62:118-33. 3. Panzer O, Moitra V, et al. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin. 2009;25:451-69. 4. Hsu CD CJ. Pharmacologic agents for pediatric procedural sedation outside of the operating room. UpToDate2014. http://www.uptodate.com/contents/ pharmacologic-agents-for-pediatric-procedural-sedation. 5. Tobias JD. Dexmedetomidine: applications in pediatric critical care and pediatric anesthesiology. Pediatr Crit Care Med. 2007;8:115-31. Psychometric comparison of three behavioral scales for the assessment of pain in critically ill patients unable to self-report p y p G Papakitsos1, A Kapsali1, T Papakitsou2 p , p , p 1GHA, Arta, Greece; 2General Hospital Messologi, Greece p g Critical Care 2015, 19(Suppl 1):P489 (doi: 10.1186/cc14569) Introduction Pain assessment is associated with important out omes in ICU patients but remains challenging, particularly in non communicative patients. Use of a reliable tool is paramount to allow any implementation of sedation/analgesia protocols in a multidisciplinary team. This study compared psychometric properties (inter-rater agreement primarily; validity, responsiveness and feasibility secondarily) of three pain scales: Behavioural Pain Scale (BPS/BPS-NI, that is BPS for non-intubated patients), Critical Care Pain Observation Tool (CPOT) and Non-Verbal Pain Scale (NVPS), the pain tool routinely used in this 16-bed medical ICU. Conclusion PCEA provided better pain control at rest than the other two methods, whereas P.R.N. Demerol/NSAID and PCEA were somewhat better than PCA when patients were moving. While coughing, P.R.N. Demerol/NSAID provided the best pain control. However, patient satisfaction was signifi cantly better with PCEA. p p References References 1. Taniguchi T, et al. Crit Care Med. 2004;32:1322-6. 2. Qiano H, et al. Crit Care. 2009;13:R136. 3. Wu Y, et al. Mediators Infl amm. 2013;2013:562154. 4. Pandharipande PP, et al. Crit Care. 2010;14:R38. 5. Ji F, et al. Circulation. 2013;127:1576-84. 1. Taniguchi T, et al. Crit Care Med. 2004;32:1322-6. 2. Qiano H, et al. Crit Care. 2009;13:R136.l Introduction Sedation in the ICU is crucial in reduction of patients’ discomfort, in particular in patients undergoing mechanical ventilation Introduction Sedation in the ICU is crucial in reduction of patients’ discomfort, in particular in patients undergoing mechanical ventilation S171 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P488 P488 Characteristics of the use of dexmedetomidine in critically ill children: a Brazilian study PL Lago, C Andreolio, J Piva, E Baldasso Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2015, 19(Suppl 1):P488 (doi: 10.1186/cc14568 FRAP values. We found that FRAP values were inversely correlated with APACHE II score (r = –0.266, P <0.01) suggesting that, in trauma patients, increased antioxidant response, as measured by the FRAP assay, could be a pathophysiological response to stress. Albumin and uric acid concentrations reproduced the FRAP trend with severity. Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, P <0.002) and head trauma (β = –0.219, P <0.045). These results accen- tuate the infl uence of trauma location and severity in TAC changes. The TAC response in ED patients reinforces the need for adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. Table 1 (abstract P490) Demerol/NSAID PCA PCEA (n = 449) (n = 62) (n = 69) P value VAS (rest) 21 ± 14 * 19 ± 9* 12 ± 10 <0.001 VAS (movement) 35 ± 20 41 ± 12* 33 ± 11 0.02 VAS (coughing) 40 ± 24* 58 ± 14 51 ± 14 <0.001 *Signifi cant diff erence. P488 Characteristics of the use of dexmedetomidine in critically ill children: a Brazilian study Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, P <0.002) and head trauma (β = –0.219, P <0.045). These results accen- tuate the infl uence of trauma location and severity in TAC changes. The TAC response in ED patients reinforces the need for adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. Introduction To describe the main indications, doses, infusion length and side eff ects of dexmedetomidine (DEX) administered to children and adolescents admitted to the pediatric ICU (PICU). p Methods A retrospective observational study including children (<18 years) admitted to a Brazilian PICU who received DEX between November 2011 and June 2014. Demographic data, indications, initial dose, maximum dose and time of infusion of DEX, side eff ects and impact on heart rate (HR) and mean arterial pressure (MAP) 6 and 24 hours after the start of infusion. P491 P491 Subanesthetic xenon increases erythropoietin levels in humans and remains traceable in the fi rst 24 hours after exposure: a randomized controlled trial J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) Methods In a prospective observational study of ED polytraumatized patients (n = 23, mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score of 11 ± 6) we measured (in the fi rst 24 hours) plasma TAC by the ferric reducing activity/antioxidant power (FRAP). For control subjects, we used age-matched and gender-matched volunteers (n = 32). We also evaluated the contribution of antioxidant molecules (uric acid, bilirubin, and albumin) to these values.f J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) Introduction The noble gas xenon was recently amended to the list of prohibited substances by the World Anti-Doping Agency as Introduction The noble gas xenon was recently amended to the list of prohibited substances by the World Anti-Doping Agency as Results Polytraumatized patients show diff erences in TAC with reference to control subjects. ED polytraumatized patients show high S172 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. system (Pall Medical), a new device for sedation in ICU patients [2]. The system delivers volatile anesthetics in mechanically ventilated patients. An open reservoir scavenger and a dedicated gas fi lter avoid residual volatile anesthetic halogenate escaping into the room air. g p g Methods Ten mechanically ventilated patients electively admitted for ICU postoperative monitoring were sedated with sevofl urane delivered with the MIRUS™ system. Two patients were excluded from the analysis because inclusion criteria had been lost during the study period. Analgesia was obtained with morphine sulfate: bolus 0.1 mg/ kg i.v. at the end of surgery and 0.2 to 0.4 mg/kg/24 hours. The primary endpoint was to achieve predefi nite levels of sedation (Riker scale 4). Secondary endpoints were the assessment of hemodynamic stability (MAP and HR), blood lactates, any type of side eff ects, and sevofl urane consumption. Data were collected at the following times: admission to the ICU (T1), 1 hour after initiation of sedation (T2), and 1 hour after sedation withdrawal (T3). i References Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. it is supposed to trigger the production of HIF-1α and subsequently erythropoietin. Subsequently, researchers and clinicians started a scientifi c discussion about the potential clinical benefi t in support of humans exposed to high demand, such as critically ill patients. The objective of this study was therefore to evaluate the eff ect of xenon on serum levels of erythropoietin in healthy volunteers. P491 Results were expressed as median (IQR) or mean (SD), where appropriate. Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. pp p Results The local ethical board approved the protocol. Median duration of sedation was 4 (5.5 to 2) hours. Predefi nite levels of sedation were achieved in all patients with a median MAC of sevofl urane of 0.5 (0.5 to 0.3)% and with a median gas consumption of 9.9 (14.3 to 5.3) ml/hour. MAP and HR values at T1 were 86.5 (97 to 80.8) mmHg and 81.5 (103.8 to 65) bpm, respectively; at T2, 74.5 (89 to 69.5) mmHg and 74 (82 to 58.3) bpm, respectively; and at T3 92.5 (101 to 76.8) mmHg and 74 (88.5 to 66.3) bpm, respectively. Lactates were always normal. Mechanical ventilation was interrupted 5.4 (3.1) minutes after withdrawal of sevofl urane and respiratory parameters always were within normal values. Finally, no side eff ects were registered at any phase of the study. Conclusion This pilot study shows that MIRUS™ is eff ective and safe in delivering sevofl urane for sedation at a predefi nite target level in postsurgical patients, without side eff ects. Further data with a larger number of patients and for a longer duration of sedation are required to confi rm these positive, preliminary observations. References Bispectral index-guided anesthesia on time to tracheal extubation after onpump cardiac surgery Bispectral index-guided anesthesia on time to tracheal extubation after onpump cardiac surgery E Kaval, P Zeyneloglu, A Camkiran, A Sezgin, A Pirat, G Arslan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P493 (doi: 10.1186/cc14573) y y Methods This is a monocenter, randomized, blinded, crossover trial, which was registered at ClinicalTrials.gov (NCT01285271). Healthy study test persons were spontaneously breathing randomly 1 hour of xenon 30% (Xe/O2 30%/65%) or control gas (N2/O2 30%/65%). The primary outcome parameter was the erythropoietin level 24 hours after exposure. Secondary outcome parameters are xenon’s elimination kinetics measured in blood and exhalation samples. Introduction Electroencephalographic-based cerebral monitors such as the bispectral index (BIS) have been used for titration of both inhalational and intravenous anesthetics during general anesthesia. Titration of anesthetics using these monitors may facilitate an earlier recovery from general anesthesia and less consumption of anesthetics. The primary aim of this study was to investigate whether BIS-guided anesthesia would reduce time to tracheal extubation when compared with minimum alveolar concentration (MAC)-guided anesthesia in patients undergoing onpump cardiac surgery. Results The application of xenon increases erythropoietin levels with a maximum 24 hours after exposure (1.32 (0.99 to 1.66) P = 0.033) compared with the baseline values and compared with control values (0.87 (0.68 to 1.05) P = 0.012, Figure 1). Xenon was gas chromatographically traceable in blood and exhalation probes up to 24 hours after exposure. Methods Fifty patients undergoing elective coronary artery bypass grafting surgery from a single tertiary referral university hospital were randomized to BIS-guided anesthesia (Group BIS, n = 25) and MAC-guided anesthesia (Group MAC, n = 25). The inspired desfl urane concentration was titrated to maintain a BIS value of 40 to 60 in Group BIS and an age adjusted minimum alveolar concentration of 0.7 to 1 was used in Group MAC. Time to tracheal extubation across the two groups was the primary outcome measure. Secondary outcomes included intraoperative desfl urane consumption, postoperative complications, and lengths of stay in the ICU and hospital. p Conclusion One hour of a subanesthetic level of xenon increases erythropoietin levels in healthy study test persons and remains gas chromatographically traceable in blood and exhalation probes 24 hours after exposure. These fi ndings may stimulate larger studies to confi rm these results and to open new avenues for the therapeutic use of xenon in critically ill patients. P492 MIRUS™, a new system for sedation with halogenates in the ICU: a preliminary study of feasibility in postsurgical patients P Mancinelli, S Romagnoli, C Chelazzi, G Zagli, E Bonicolini, A Belardinelli, AR De Gaudio Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P492 (doi: 10.1186/cc14572) Results Demographic features, logistic EuroSCOREs, duration of cardiopulmonary bypass and surgery were similar in both groups. Mean desfl urane consumption was signifi cantly lower in Group BIS (11.9 ± 1.7 ml/hour) compared with Group MAC (13.4 ± 3.0 ml/hour) (P = 0.031). Time to tracheal extubation was not signifi cantly diff erent between the groups (13.3 ± 9.6 hours vs. 17.0 ± 22.4 hours) (P = 0.68). Incidences of postoperative complications were similar and lengths of stay in the ICU and hospital were 2.4 ± 0.7 days versus 3.2 ± 2.7 days Introduction Sedation is standard practice in the ICU [1]. The aim of this study was to investigate the effi ciency and safety of the MIRUS™ Introduction Sedation is standard practice in the ICU [1]. The aim of this study was to investigate the effi ciency and safety of the MIRUS™ S173 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The study was approved by the appropriate institutional review board. After written informed consent, 63 ASA I to III patients undergoing elective hip or knee replacement surgery under general anaesthesia were included. Patients were randomly organised into three groups (20 to 22 each). Anaesthesia was induced with intravenous anaesthetics. After tracheal intubation MIRUS™ automatically adjusted the end-tidal VA concentration to 1.0 MAC. Patients were ventilated with the Puritan Bennett 840 ICU ventilator. After 1 hour of anaesthesia with 1.0 MAC the ventilator mode was switched from SIMV VC+ (totally controlled ventilation, passive patient, with a tidal volume of 8 ml/IBW) to proportional assist ventilation with 50% support (active patient). At the end of surgery the MIRUS™ system was stopped (MAC set to 0.0) and recovery times were measured. and 5.3 ± 1.2 days versus 6.5 ± 3.1 days in Group BIS and Group MAC respectively (P >0.05 for all). and 5.3 ± 1.2 days versus 6.5 ± 3.1 days in Group BIS and Group MAC respectively (P >0.05 for all). Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) y Results Patients were comparable in age, height, weight and operation time. In 60/63 patients a MAC of 1.0 was reached by MIRUS™. Therefore, ISO 11.2 ± 3.3 ml/hour, SEVO 24.3 ± 4.8 ml/hour or DES 41.7 ± 7.9 ml/ hour (mean ± SD; t test: P <0.001) were used during passive ventilation. During patients’ active ventilation, mean VA consumptions of ISO 9.6 ± 5.1 ml/hour, SEVO 19.4 ± 9.6 ml/hour or DES 35.5 ± 23.0 ml/hour were detected (NS between passive and active patients). ISO was the cheapest VA (€2.70 ± 3.10/hour passive patient, €1.90 ± 2.30 active patient), followed by SEVO (€8.40 ± 3.70 passive patient and €6.8 ± 3.8 active patient) and DES (€9.6 ± 4.1 passive patient and €8.6 ± 6.5 active patient). Recovery times were signifi cantly shorter after SEVO and DES compared with ISO (minutes:seconds; ISO 9:31 ± 6:04, SEVO 6:19 ± 2:56, DES 5:27 ± 1:59). Introduction The aim of this study is to present our experience with sevofl urane in the ICU, outline which patients were sedated with sevofl urane and present the safety profi le. Sevofl urane has some potential advantages over intravenous sedation: rapid elimination and few interactions. The optimal role of sevofl urane in ICU is not known. Methods We performed a retrospective study on adult patients who were sedated with sevofl urane in the medical ICU. The decision to use sevofl urane was left to the attending physician. Institutional ethics committee approval was obtained. The target mean alveolar concentration in all patients was 0.5 to 1%. The AnaConDa® device (Sedana Medical, Uppsala, Sweden) was used along with the Anastasia® (Sedana Medical) gas monitor. Data were obtained from patients’ medical records. P495 Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia V Vinnikov, D Drees, J Herzog-Niescery, P Gude, H Vogelsang, B Cevik, T Weber, M Bellgardt St. Josef-Hospital, Ruhr-Universität, Bochum, Germany Critical Care 2015, 19(Suppl 1):P495 (doi: 10.1186/cc14575) P492 Conclusion Intraoperative use of BIS monitoring in patients undergoing onpump cardiac surgery reduced desfl urane requirement but BIS- guided anesthesia did not facilitate time to extubation and lengths of stay in the ICU and hospital. P494 Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery JL Iribarren, JJ Jimenez, N Perez, M Brouard, R Perez, O Gonzalez, A Arbesu, R Martinez, ML Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P496 (doi: 10.1186/cc14576) Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery JL Iribarren, JJ Jimenez, N Perez, M Brouard, R Perez, O Gonzalez, A Arbesu, R Martinez, ML Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P496 (doi: 10.1186/cc14576) Introduction The use of etomidate is a risk factor for relative adrenal insuffi ciency in patients undergoing cardiopulmonary bypass (CPB) [1]. The objective was to determine the possible interaction between Conclusion We identifi ed sevofl urane as an appropriate sedation agent in a diverse group of patients. Sevofl urane advantages over intravenous sedation could be more pronounced in some patient groups (for example, successful resuscitation after cardiac arrest). The safety profi le of sevofl urane sedation was comparable with intravenous sedation [1]. Reference Figure 1 (abstract P496). 1. Mesnil et al. Intensive Care Med. 2011;37:933-41. 1. Mesnil et al. Intensive Care Med. 2011;37:933-41. Reference 1. Bomberg H, et al. Anaesthesia. 2014;69:1241-50. 1. Bomberg H, et al. Anaesthesia. 2014;69:1241-50. P496 Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Introduction The aim of this study is to present our experience with sevofl urane in the ICU, outline which patients were sedated with sevofl urane and present the safety profi le. Sevofl urane has some potential advantages over intravenous sedation: rapid elimination and few interactions. The optimal role of sevofl urane in ICU is not known. l Methods We performed a retrospective study on adult patients who were sedated with sevofl urane in the medical ICU. The decision to use sevofl urane was left to the attending physician. Institutional ethics committee approval was obtained. The target mean alveolar concentration in all patients was 0.5 to 1%. The AnaConDa® device (Sedana Medical, Uppsala, Sweden) was used along with the Anastasia® (Sedana Medical) gas monitor. Data were obtained from patients’ medical records. Conclusion This study showed that MIRUS™ could automatically control end-tidal VA concentrations in ventilated and spontaneously breathing patients. Using ISO reduces costs. Further studies must be taken to analyse feasibility, costs and recovery times of ISO, SEVO and DES used for sedation in an ICU setting. f Results We included 61 adult patients who were admitted from April 2012 to November 2014. Mean age was 62.6 ± 14.9 years, 39 (63.9%) were male. ICU mortality was 41%, hospital mortality was 43%. Mean duration of sevofl urane use was 3.56 ± 2.31 days. Admission diagnoses were: successful resuscitation after cardiac arrest (44.2%), sepsis (37.7%), cardiogenic shock (4.9%), pancreatitis (3.3%) and liver failure, acute exacerbation of COPD, asthma, tetanus and intracerebral hemorrhage (1.6% each). During treatment with sevofl urane, no patients developed malignant hyperthermia, new hyperkalemia or QT prolongation. In three (4.9%) patients, worsening liver function tests prompted sevofl urane discontinuation. Ischemic hepatitis was considered an alternative in all three patients. Seven (11.4%) patients developed renal failure while receiving sevofl urane. Sevofl urane was continued in all patients and renal failure was attributed to alternative diagnoses. No self-extubations were recorded. In seven (11.4%) patients, sevofl urane was discontinued because of worsening ventilation. In six (9.8%) patients, unexpected awakening occurred. Eight patients (13.1%) had symptoms of delirium after sevofl urane inhalations were concluded.il Reference Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia l V Vinnikov, D Drees, J Herzog-Niescery, P Gude, H Vogelsang, B Cevik, T Weber, M Bellgardt St. Josef-Hospital, Ruhr-Universität, Bochum, Germany Critical Care 2015, 19(Suppl 1):P495 (doi: 10.1186/cc14575) Introduction The new MIRUS™ system as well as the established AnaConDa® system uses a refl ector to conserve volatile anaesthetics (VA) [1]. Both systems act with commercially available ICU ventilators. In contrast to AnaConDa®, MIRUS™ includes an automated control of end-tidal VA concentrations. In this study we compared feasibility, costs and recovery times after anaesthesia with isofl urane (ISO), sevofl urane (SEVO) or desfl urane (DES) in ventilated and spontaneously breathing patients. S174 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of more signifi cant impact than medication errors. We assessed the incidence and characteristics of operation-related MEs to improve patient safety in such patients. etomidate and beta tumoral necrosis factor (TNFβ) polymorphism on hemodynamics after CPB. Methods A prospective cohort study on CPB patients who received etomidate or not during anesthetic induction during 2008 to 2011. Demographic and postoperative variables were collected. We tested the Hardy–Weinberg equilibrium in order to avoid selection bias. V18 SPSS was used. Methods The Japan Adverse Event (JET) study was a prospective cohort study which had evaluated AEs and MEs at two tertiary care hospitals. We included all adult patients aged ≥15 years old who had operations over a 2-month period. The primary outcome of this study was the operation-related MEs, defi ned as any deviation from appropriate process of an operation or perioperative care. Trained nurses placed at each participating hospital reviewed all charts daily on weekdays, along with laboratories, incident reports, and prescription queries to collect any potential event. They also collected the characteristics of the patients in the cohort. Some operation-related MEs are associated with operation-related AEs, which are operation-related preventable AEs. After those suspected events were collected, physician reviewers independently evaluated them and classifi ed them as operation- related MEs, AEs, or rule violations. Physician reviewers assessed and rated operation-related AEs according to the symptom and the severity of injury. Results We studied 433 patients undergoing CPB, 285 (65.8%) men and 148 (34.2%) women, 66 ± 6 years, EuroSCORE I 5.3 ± 4%. TNFβ was in Hardy–Weinberg equilibrium (χ2: 0.6; P = 0.42). Visualising patients’ dynamics in the ICU and predicting mortality in real-time using big data MK Komorowski, AF Faisal MK Komorowski, AF Faisal Imperial College London, UK Critical Care 2015, 19(Suppl 1):P499 (doi: 10.1186/cc14579) , Imperial College London, UK Critical Care 2015, 19(Suppl 1):P499 (doi: 10.1186/cc14579) Introduction As informatisation of hospitals continues to spread, increasing amounts of healthcare related data are being collected, and the ICU is no exception. Large datasets are being made available to the scientifi c community, and off er the potential to answer clinical questions and to develop the next generation of clinical tools. A demonstration of such a tool is presented here, built using data from the Multiparameter Intelligent Monitoring in Intensive Care II (MIMIC-II) open database. Methods All of the adult patients who died during their stay in the ICU were included, as well as a matched cohort of patients who survived for more than 28 days after discharge. Data regarding their vital signs, laboratory tests and demographics were collected. Using Matlab, a graphical method involving principal component analysis was developed. The expected mortality was computed using the k-nearest neighbours’ method and compared with several classifi cation algorithms (logistic regression, random forest, support vector machine, Gaussian mixture models). g Conclusion The fi ndings are comparable with reports of sedation surveys conducted in other countries. There is an evidence–practice gap that needs to be addressed. Sedation practices in South African ICUs: results of a national survey F Paruk, S Chetty University of Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P497 (doi: 10.1186/cc14577) Introduction There has been a paradigm shift in the approach to sedation of critically ill patients. The purpose of this study was to evaluate current sedation-related practices in South African ICUs. Conclusion Ninety-three percent of operation-related MEs resulted in operation-related AEs and 21% of them resulted in life-threatening events. Prevention of operation-related MEs should improve the mortality of surgical patients. p Methods A validated questionnaire was distributed electronically to physician members of various medical databases in South Africa as South Africa does not have a formal registry of critical care practitioners. Results One hundred and twenty-six of 174 respondents indicated that they practice in the ICU setting. Sixty-six per cent were specialists and mainly anaesthesiologists (42%), whilst 32% were critical care subspecialists. The public and private-sector representation was 64% and 46% respectively. A written sedation guideline is implemented by 42%. Forty-three per cent utilise a sedation scale, with the Ramsey Sedation Scale being the commonest in use. However, 38% of sedation scale users do so infrequently. Daily interruption of sedation is practiced by 75%. Light sedation is targeted by 42% and 14% do not follow any sedation targets. Upon admission and on subsequent days, sedation targets are achieved most of the time by 48% and 69% of the respondents respectively. Whilst a wide variety of sedatives are prescribed, midazolam constitutes the most commonly prescribed agent. Dexmedetomidine is the agent of choice for postcardiac surgery patients with cardiovascular comorbidities, delirious patients, during weaning and for non-invasive ventilation. Propofol is the agent of choice amongst neurological patients. The respondents indicated that there is a need for local sedation guidelines.i Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia A total of 254 (58.7%) patients received etomidate, 152 out of them required vasoactive drugs. Homozygous G was defi ned as unfavorable TNFβ versus the A allele [2]. Using the general linear model after adjusting for sex and amines dose at 4 hours, an independent association was observed between the systemic vascular resistance index (SVRI) at 4 hours and the use of etomidate (F: 18; P <0.001): 1,849 (95% CI: 1,673 to 2,024) versus 2,493 (95% CI: 2,258 to 2,729) dinas.seg/cm5.m2, the presence of homozygous G (F: 6.5; P = 0.01), and also showed a signifi cant etomidate–homozygous G interaction (F: 22.8: P <0.001): 1,687 (95% CI: 1,350 to 2,023) versus 3,041 (95% CI: 2,589 to 3,492) dinas seg/cm5. m2 (Figure 1). y Results This study included 389 patients with 6,624 patient-days. The median age of patients was 69 years and 224 (58%) were male. Among these 389 patients, 31 patients had 46 operation-related MEs during their hospital stay and the incidence of operation-related MEs was 12 per 100 patients. Operation-related AEs occurred in 29 patients with 43 events. The most frequent symptoms for operation-related MEs were skin (26%), bleeding (21%), and central nervous system (14%). Among 46 operation-related MEs, 43 (93%) were not intercepted, and they resulted in operation-related AEs that were considered as preventable operation-related AEs. Nine of preventable operation-related AEs (21%) were fatal or life-threatening: fi ve were nerve injury during operation and stroke after neurosurgical operation, and one biliary peritonitis after gastrectomy and cholecystectomy, and tension pneumothorax after lung lobectomy, and two unexpected massive bleeding due to vessels injury. Conclusion Etomidate use is associated with lower postoperative SVRI which is increased in the presence of G homozygosity for TNFβ polymorphism. P498 Epidemiology of operation-related medical errors in inpatients in Japan: the JET study y References 1. Iribarren JL, et al. J Cardiothorac Surg. 2010;5:26. 1. Iribarren JL, et al. J Cardiothorac Surg. 2010;5:26. 2. Iribarren et al. Interact Cardiovasc Thorac Surg. 2008;7:1071-4. 2. Iribarren et al. Interact Cardiovasc Thorac Surg. 2008;7:1071-4. Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study y M Soares1, JM Kahn2, FA Bozza1, T Lisboa3, LP Azevedo4, W Viana5, L Brauer6, PE Brasil1, DC Angus2, JI Salluh1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Sta. Casa de Porto Alegre, Brazil; 4Hospital Sírio Libanês, São Paulo, Brazil; 5Hospital Copa DOr, Rio de Janeiro, Brazil; 6Hospital Sao Luiz Itaim, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P500 (doi: 10.1186/cc14580) Figure 1 (abstract P501). HEWS prior to critical event. Methods We conducted a prospectively identifi ed, retrospectively gathered cohort study at two hospitals of consecutively admitted medical and surgical patients over a 6-month period. One hospital had a rapid response team (RRT) and used HEWS with a trigger of 5 while the other was undergoing implementation of the HEWS without a RRT. HEWS was calculated for each patient on the fi rst day of admission and for the 3 days prior to inpatient arrest or death. A study investigator reviewed all events for accuracy. Our outcome of interest was a composite of inpatient cardiac arrest and hospital mortality. Introduction The aim was to investigate the impact of organizational factors on patient outcomes in a large sample of Brazilian ICUs. Introduction The aim was to investigate the impact of organizational factors on patient outcomes in a large sample of Brazilian ICUs. Methods A retrospective cohort study of 59,483 patients admitted to 78 ICUs in 51 hospitals during 2013. We retrieved demographic, clinical and outcome data from an electronic ICU quality registry (Epimed Monitor System). We surveyed ICUs using a standardized questionnaire regarding hospital and ICU structure, organization, staffi ng patterns, process of care, and family care policies. We used multilevel logistic regression analysis to identify characteristics associated with hospital mortality. y Results There were 7,138 patients admitted over 6 months. We found 0.5% of patients suff ered an inpatient arrest and 3.6% of patients died. Moreover, 66% of patients who died or arrested were admitted to the hospital without a RRT. Patients who arrested or died had more comorbidities defi ned by the Charlson Comorbidity Index (CCI) of 6.0 and 8.2 respectively compared with the general population, which had a CCI of 5.2. Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Results A total of 6,084 patients were included in the analyses, adding up to more than 12 million data points. Using this multidimensional dataset, a 3D representation of the clusters of survivors and nonsurvivors was built, showing how their trajectories diverge through time. Patterns in the evolution of individuals or subgroups of patients can be identifi ed using this approach. For example, the evolution of a new patient can be visualised, progressing through the clusters as his severity changes. His expected mortality can be predicted at any point in time, with an AUC ROC constantly above 0.85. Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) p y Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) y Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) Introduction Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be Introduction Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be S175 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P501). HEWS prior to critical event. Conclusion Machine learning tools off er an appealing mathematical framework for modelling complex medical situations. This proof of concept demonstrates that the application of computational sciences to high-quality data such as the MIMIC-II database has the potential to lead to the development of meaningful tools which will ultimately be capable of assisting physicians in making the right decision at the right time for an individual patient. P500 P500 Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study M Soares1, JM Kahn2, FA Bozza1, T Lisboa3, LP Azevedo4, W Viana5, L Brauer6, PE Brasil1, DC Angus2, JI Salluh1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Sta. Casa de Porto Alegre, Brazil; 4Hospital Sírio Libanês, São Paulo, Brazil; 5Hospital Copa DOr, Rio de Janeiro, Brazil; 6Hospital Sao Luiz Itaim, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P500 (doi: 10.1186/cc14580) Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Only tight cooperation between clinicians and data scientists can help close the gap that currently separates these two worlds, for the ultimate benefi t of patients. Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study The median and mean HEWS at time of admission was 1 and 1.7 for the general population, 2 and 2.4 for patients who suff ered an inpatient arrest and fi nally 3 and 3.8 for those who died. There was a rise in median HEWS from 2 to 5 in the 24 hours prior to in patient arrest or death. See Figure 1. y Results ICUs were mostly medical or medical–surgical (62.79%) and located in private hospitals (67.86%). Approximately half (40.51%) had critical care training programs. Median physician and nurse staff – bed ratios were 0.15 (IQR, 0.12 to 0.19) and 0.71 (0.61 to 0.84); board- certifi ed intensivists were present 24/7 in 16 (21%) of ICUs. Routine clinical rounds occurred in 67 (86%) and daily clinical checklists were used in 36 (46%) ICUs. Most frequently implemented protocols focused on sepsis management and VAP and CLABSI prevention. Median number of patients per center was 898 (IQR 585 to 1,715) and there were 67% medical admissions; 18% patients received mechanical ventilation (MV). Median SAPS 3 score was 41 (33 to 52) points. ICU and hospital mortality rates were 9.6% and 14.3%, respectively. Adjusting for relevant patients’ characteristics (SAPS 3 score, diagnostic admission category, chronic health status, comorbidities, MV use), case-volume and type of ICU, the ICU size (OR = 1.50 (95% CI, 1.45 to 1.95), for 11 to 20 beds; OR = 2.02 (1.40 to 2.92), for >20 beds) and ≥2 clinical protocols (OR = 0.65 (0.42 to 0.99)) were the organizational characteristics associated with mortality. g Conclusion We found that a 2.5-fold increase in HEWS occurred 24 hours prior to critical events. Similar to previous studies, a RRT in conjunction with HEWS is the best system to reduce unanticipated adverse events. An absolute HEWS of 5 and/or a rapidly increasing HEWS should trigger rapid assessment and treatment to reduce preventable inpatient deaths and arrests. Reference 1. McNeill, Bryden. Resuscitation. 2013;84:1652-67. 1. McNeill, Bryden. Resuscitation. 2013;84:1652-67. Conclusion In a large sample of Brazilian ICUs, the implementation of clinical protocols was associated with better outcomes. Conversely, mortality was higher in larger ICUs. Ethnicity and trial recruitment h y g g Acknowledgements Funded by IDOR, CNPq and FAPERJ. Endorsed by BRICNet. HL Cronshaw, SW Scott, S Bowrey, JP Thompsoni HL Cronshaw, SW Scott, S Bowrey, JP Thompson Leicester Royal Infi rmary, Leicester, UK , , y, Leicester Royal Infi rmary, Leicester, UK yi y Critical Care 2015, 19(Suppl 1):P502 (doi: 10.1186/cc14582 Introduction Surrogate consent and time-sensitive recruitment in critical care research is challenging, yet low enrolment numbers or omitting ethnic groups skew results and conclusions. Patients from diff erent ethnic groups may respond diff erently to therapeutics [1]. There are few data about the eff ect of ethnicity on recruitment into ICU trials. Our ICU recruits to national trials and serves an increasingly non- White British population (24% of population). We undertook this study to determine whether ethnicity aff ects ICU consent rates. Validation of an electronic early warning score using decision tree analysis: proposal M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) Introduction Decision tree analysis uses an algorithm to classify data items by recursively posing a series of questions about items within a dataset. Each question leads to another node and potentially more questions until a predefi ned end condition is reached or no more questions can be asked (Figure 1). We hypothesize that scores generated using the decision tree method will improve upon our existing Hamilton Early Warning Score (HEWS) for a composite endpoint of cardiac arrest, unplanned ICU admission or death. Introduction Timely availability of a kidney specialist poses a formidable challenge in ICUs located in tier II and tier III cities of the developing world. Renal replacement therapy (RRT) is often required in the ICU for acute renal failure patients but availability of a nephrologist/ specialist is scarce, leading to unnecessary and risky transfer to higher centers in metropolitans or even worse to death. We explored whether a remotely monitored ICU – an electronic ICU (eICU) – would help mitigate this demand–supply gap. Methods A database of 156,642 electronically captured vital signs from 6,757 consecutively admitted patients to eight medical and surgical wards will be used to train and test the decision tree early warning score. One-third of the data will be withheld from the algorithm for use as a testing set. The algorithm will look for signifi cant changes in vitals 72 hours prior to an outcome and develop the score based upon the resulting relative risk of the composite endpoint happening given a certain vital sign. The scores and predictions generated by the decision tree analysis will then be compared with that of the inception HEWS cohort. y Methods This retrospective study was conducted at four Critinext affi liates where the eICU was being used to provide 24 × 7 support on 89 ICU beds from a remote command center with intensivist and other requisite staff . The eICU had complete access to the patient’s real-time vitals, hemodynamic parameters, imaging, laboratory values, audiovisuals and appropriately engineered smart alerts. The eICU model was further extended in initiating and getting RRT done in patients whenever deemed necessary in times of unavailability of a specialist at the same site. P501 Hamilton Early Warning Score: predict, prevent and protect B Tam, M Xu, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P501 (doi: 10.1186/cc14581) Introduction This study determined the pattern of decline prior to an inpatient arrest. We implemented the Hamilton Early Warning Score (HEWS) within our electronic vital signs documentation to track and trigger care for deteriorating patients. Other EWS have been described in the literature with varying success [1]. In contrast to previous observational studies, we chose to implement a score modifi ed from published EWS using the consensus opinion of a steering committee and evaluate the score in real time. f Methods We performed a retrospective review of screening logs from three national UK trials (PROMISE, BALTI-P, GAiNS) and one local trial (Nociceptin in Sepsis). We analysed consent rates of eligible patients by ethnicity, age, sex, interventional or observational trial, and ethnicity of the researcher seeking consent. We performed chi-squared analysis, and entered signifi cant values into a logistic regression model using SPSS v22. S176 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results The planned analysis for determining the discriminatory and predictive ability of the decision tree HEWS will be conducted with area under the receiver operating characteristic curves. We will test whether the current HEWS has the appropriate sensitivity and specifi city when compared with that of the decision tree score. The AUROC will be calculated for both the training set of data as well as the separate population of additional medical and surgical patients. The two scores will also be plotted along an effi ciency curve, comparing the percentage of vitals that precede a critical event with the percentage of vitals that produce a EWS value greater than or equal to a given EWS value. Results We identifi ed 332 eligible patients across all trials, of whom 37 (11%) were not White British (nWB). Analysis demonstrated consent/assent refusal being signifi cantly associated with: nWB (14, 38%, P <0.001), interventional trial (21, 25%, P = 0.003) and diff erent researcher–patient ethnicities (P <0.001). P504 P503 Validation of an electronic early warning score using decision tree analysis: proposal M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) P504 Can an electronic ICU support timely renal replacement therapy in resource-limited areas of the developing world S Gupta1, A Kaushal1, S Dewan2, A Varma1 1Fortis Escorts Heart Institute, New Delhi, India; 2Fortis Memorial Research Institute, Gurgaon, India Critical Care 2015, 19(Suppl 1):P504 (doi: 10.1186/cc14584) . Lilly CM, et al. Chest. 2014;145:500-7. 1. Bjornsson TD, et al. J Clin Pharmacol. 2003;43:943-67. P501 Logistic regression analysis confi rmed these as independent factors (nWB OR = 4.5, 95% CI = 2.1 to 9.8, P <0.001; interventional trial OR = 2.7, 95% CI = 1.4 to 5.2, P = 0.003; data points missing for researcher–patient ethnicity so variable excluded).f Conclusion This initial study suggests that ethnicity may aff ect assent/ consent to ICU research, with patients from diff erent ethnicities being four times less likely to be recruited. Whilst data are incomplete for researcher–patient ethnicity, our data suggest that this may be an important factor and may infl uence future consent processes. We believe that the role of ethnicity warrants further investigation, not only in clinical trials but also in areas such as organ donation. Reference Conclusion Decision tree analysis methodology with real-life vital signs can produce an EWS superior to previous observational studies. Using a decision tree, especially one that composites all vitals, may show that certain vitals are more predictive of critical events than others. Data will be used to further improve our current HEWS score. 1. Bjornsson TD, et al. J Clin Pharmacol. 2003;43:943-67. Validation of an electronic early warning score using decision tree analysis: proposal Patient baseline demographics, including risk factors, severity score, all-cause mortality at 30 days, transfers to higher center for RRT and its prevention were recorded. Descriptive analysis was performed. Between-group comparison was performed by applying the chi-squared statistic, signifi cance was assumed at a value of P <0.05. Out of a total of 5,146 admissions, 752 inpatient fi les with acute kidney injury/acute renal failure were reviewed, January to July 2013 (n = 373) and July 2013 to January 2014 (n = 379) pre and post eICU implementation respectively.i Figure 1 (abstract P503). Sample heart rate decision tree. p p y Results While baseline demographics and the patient profi le in the two groups did not show statistically signifi cant diff erence, mean APACHE II score was 14.25 ± 1.94 and 14.65 ± 1.76 pre and post eICU respectively; there was a statistically signifi cant diff erence in all-cause mortality at 30 days which decreased from 31 (8.3%) to 16 (4.2%) pre and post eICU respectively, a reduction of >49% (P = 0.030) and transfer out for RRT came down by >77%, from 15 (4%) to two (0.5%) post eICU implementation (P <0.002). Conclusion Over the years there is now broad consensus over the benefi ts of eICU intervention in deprived areas [1]. There is now a need for a paradigm shift to elevate specialized care to improve outcomes. Our small study has clearly indicated the benefi ts in outcome and economics even while providing intervention in such remote areas. An eICU as a bridge to the demand–supply gap needs to be explored and utilized further to its full potential in the emerging world. Reference Reference Figure 1 (abstract P503). Sample heart rate decision tree. Figure 1 (abstract P503). Sample heart rate decision tree. 1. Lilly CM, et al. Chest. 2014;145:500-7. S177 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P506). Mean change in systolic and diastolic BP and HR in relation to ICU work experience Table 1 (abstract P506). Mean change in systolic and diastolic BP and HR in relation to ICU work experience P505 Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study M Soares1, DC Angus2, JI Salluh1, AB Cavalcanti3, F Colombari4, R Costa5, E Silva6, A Japiassu7, JM Kahn2, FA Bozza1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Hospital do Coracao, São Paulo, Brazil; 4Hospital Alemao Oswaldo Cruz, São Paulo, Brazil; 5Hospital Quinta DOr, Rio de Janeiro, Brazil; 6Hospital Israelita Albert Einstein, São Paulo, Brazil; 7Rede Amil de Hospitais, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P505 (doi: 10.1186/cc14585) Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study Group 1 Group 2 P value SBP (mmHg) 3.22 –0.40 0.053 DBP (mmHg) 2.08 –0.27 0.061 HR (bpm) 4.46 1.16 0.048 M Soares1, DC Angus2, JI Salluh1, AB Cavalcanti3, F Colombari4, R Costa5, E Silva6, A Japiassu7, JM Kahn2, FA Bozza1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Hospital do Coracao, São Paulo, Brazil; 4Hospital Alemao Oswaldo Cruz, São Paulo, Brazil; 5Hospital Quinta DOr, Rio de Janeiro, Brazil; 6Hospital Israelita Albert Einstein, São Paulo, Brazil; 7Rede Amil de Hospitais, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P505 (doi: 10.1186/cc14585) Results The mean duration of working in an ICU was 7.3 ± 5.32 years (from 2 to 18 years). The nurses were grouped according to experience – Group A: 17 nurses with <10 years of experience (mean: 4.4 years), Group B: six nurses with >10 years (mean: 15.6 years). There were 640 BP–HR measurements. The mean systolic BP, diastolic BP and HR did not diff er between the two groups (systolic BP: 111.2 ± 10.9 vs. 113.7 ± 14.1 mmHg, P = 0.654; diastolic BP: 72.8 ± 8.2 vs. 71.9 ± 8.1 mmHg, P = 0.835; HR: 81.4 ± 7.2 vs. 78.1 ± 8.4 bpm, P = 0.365). Nevertheless, the mean change in BP and HR during the shift did diff er between the two groups, with the more experienced nurses showing a trivial reduction in systolic and diastolic BP and minor increase in HR whereas the less experienced ones showed slight increase in both BP and HR measurements (Table 1), reaching almost statistical signifi cance. For the less experienced nurses in Group 1, it was noted that the mean changes were bigger in night shifts although the limited number of measurements did not allow robust statistical analysis. Introduction The aim was to evaluate outcomes and resource use and to investigate the association between organizational factors and effi cient resource use in a large sample of Brazilian ICUs. Methods A retrospective cohort study in 59,483 patients (medical admissions: 39,734 (67%)) admitted to 78 ICUs (private hospitals, n = 67 (86%); medical or medical–surgical, n = 62 (79%)) during 2013. We retrieved demographic, clinical and outcome data from an electronic ICU quality registry (Epimed Monitor System). P506 Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) y y Methods We included all adult patients (≥18 years) with a fi rst-time ambulance transport to the ED at Odense University Hospital in the period 1 April 2012 to 30 September 2013. Ambulance personnel recorded vital signs and other clinical fi ndings on a structured form on paper during the ambulance transport. Each contact was linked to information from population-based healthcare registers in order to identify comorbid conditions and information on mortality. Demographic factors and fi rst registered vital sign were analysed by univariate logistic regression analysis, with 7-day mortality as outcome. Results In total, 18,572 fi rst-time ambulance contacts were identifi ed in the period of inclusion. Overall 7-day mortality was 4.3% (95% CI = 4.0 to 4.6). Univariate analysis showed increasing age, Charlson Comorbidity Index ≥2, vital parameters outside the normal reference range and summoned physician-assisted mobile emergency care units to be associated with 7-day mortality. Further analyses are currently being carried out. Prehospital transported patients: a resource for accessing prognostic risk factors p g C Bech1, M Brabrand2, A Lassen1 1Od U i it H it l Od C Bech1, M Brabrand2, A Lassen1 1Odense University Hospital, Odense, Denmark; 2Sydvestjysk Sygehus Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P507 (doi: 10.1186/cc14587) Conclusion We observed a great variability in outcome and resources in a large sample of Brazilian ICUs. Implementation of clinical protocols and nursing staffi ng patterns can be targets to improve the effi ciency in resource use in emerging countries such as Brazil. Critical Care 2015, 19(Suppl 1):P507 (doi: 10.1186/cc14587) Introduction The survival of patients transported by ambulance to the emergency department (ED) depends on clinical conditions, patient-related factors and organisational prehospital set up. Data and information concerning patients in the prehospital system could form a valuable resource for assessing potential risk factors associated with adverse outcomes and mortality. Our aim was to describe ambulance transports to the ED and identify prognostic factors accessible in the prehospital phase and associated with 7-day mortality.i Acknowledgements Funded by IDOR, CNPq and FAPERJ. Endorsed by BRICNet. Reference 1. Rothen et al. Intensive Care Med. 2007;33:1329-36. 1. Rothen et al. Intensive Care Med. 2007;33:1329-36. Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study We surveyed ICUs using a standardized questionnaire regarding hospital and ICU structure, organization, staffi ng patterns, process of care and family care policies. Effi cient resource use was assessed by estimating standardized mortality rates (SMR) and standardized resource use (SRU) adjusted for the severity of illness according to the SAPS 3 score, as proposed by Rothen and colleagues [1]. y Conclusion The less experienced ICU nurses, with <10 years of ICU work experience, tended to increase their BP and HR levels during the shift, a fi nding probably heightened during night shifts. Further research, including not only cardiovascular parameters, is warranted to uncover the eff ects of shift-work pattern in ICU nurses, taking into account this specifi cally stressful work environment. Results The median admissions per center was 898 (IQR 585 to 1,715) and SAPS 3 score was 41 (33 to 52) points. Median ICU length of stay was 2 (1 to 5) days, and ICU and hospital mortality rates were 9.6% and 14.3%, respectively. Estimated SMR and SRU were 0.97 (0.72 to 1.15) and 1.06 (0.89 to 1.37), respectively. There were 28 (36%) most effi cient (ICUs with both SMR and SRU <median), 28 (36%) least effi cient (ICUs with both SMR and SRU >median), 11 (14%) overachieving (ICUs with low SMR and high SRU) and 11 (14%) underachieving (ICUs with high SMR and low SRU) ICUs. Most effi cient ICUs were usually located in private and accredited hospitals, with step-down units and training programs in critical care. In univariate analyses comparing most and least effi cient ICUs, ≥2 clinical protocols (OR = 7.22 (95% CI, 1.41 to 36.97)) and graduated nurse/bed ratio >0.25 (OR = 4.40 (1.04 to 18.60)) were associated with effi cient resource use. Daily checklists also tended to be associated with effi cient resource use (OR = 2.89 (0.95 to 8.72), P = 0.057). P508 Contribution of medical senior house offi cers to a medical referral in the emergency department i g Conclusion In 2011 the Royal College of Physicians emphasized the impact that the quality of the care provided within the fi rst 48 to 72 hours had on clinical outcomes. An evaluation of consultant input into acute admissions management revealed that hospitals in which two or more ward rounds of all acute medical unit patients were performed daily had a lower adjusted case fatality rate for patients with hospital stays over 7 days. Despite twice-daily consultant ward rounds of all new acute admissions and the addition of a third 4:00 pm round from Monday to Friday, only 62% of patients were seen by a consultant within 12 hours. With 67% of patients being admitted between the hours of 12:00 am and 12:00 pm, it is possible that the substitution of an evening round for one of the afternoon rounds would help increase the number of patients seen within the target time frame. This would require a change in the working pattern of the acute medicine consultants. f Introduction In Irish hospitals, the medical senior house offi cer (SHO) is the most junior fully qualifi ed doctor on the medical on-call team. After a patient has been seen by an emergency department doctor of any level, they are almost always referred directly to the medical SHO. This process has been shown to delay a patient’s ward admission by 3 hours 30 minutes [1]. We attempted to quantify the additional benefi t for the patient of being seen by the on-call medical SHO, in terms of patients discharged, new diagnoses reached, and new treatments initiated. g g Methods The emergency department notes and clinical charts of 182 patients were assessed. This constituted a random sample of patients referred by emergency department doctors to the medical team on call over a 2-month period (November to December 2011). Results Discharged: 3/182 (1.6%) of patients referred to the medical team were discharged directly by the medical SHO. Diagnosed: medical SHOs suggested a diagnosis which was diff erent from, or additional to, the ED doctor, in 52/182 cases (28.6%). However, the medical consultant only agreed with this diagnosis in 25 cases (13.7%). This means an incorrect new diagnosis was reached more often than not (14.9%). Treatment: the majority of cases (116/182 (63.7%)) saw no new treatment initiated by the medical SHO. P508 Contribution of medical senior house offi cers to a medical referral in the emergency department Of the rest, only 31 (17%) had a new treatment initiated by the medical SHO which was continued on by the medical consultant through the admission. P510 Interhospital critical care transfer delays result from organisational not geographical factors: secondary analysis of deteriorating ward patients in 49 UK hospitals DJ Wong1, SK Harris2 1King’s College Hospital, London, UK; 2University College London, UK Critical Care 2015, 19(Suppl 1):P510 (doi: 10.1186/cc14590) Conclusion Few direct discharges, new diagnoses, or key new treatments were initiated by the medical SHO in the emergency department. A paper from our hospital shows that more patients referred in by GPs to ED are admitted compared with those referred in to the acute medical assessment unit, with comparable disease severity (43% vs. 12.5) [2]. That paper highlighted the fact that the junior level of the medical NCHDs who see patients in the ED may contribute to their lack of discharging/decision-making zeal. Our survey further illustrated this feature. Our study provided no evidence that a formal medical assessment should delay a patient progressing to the medical ward. Additional genuine urgent OPD appointment slots could be another benefi cial measure. Introduction Critically ill patients may require interhospital transfer for specialist care or because of a lack of local ICU capacity. Harm is assumed from delays that result, but it is not clear whether these delays are due to transfer distances or defi ciencies in the organisation of care. Methods In total, 151 of 15,602 deteriorating ward patients in the (SPOT)light study [1] were transferred rather than admitted locally. We defi ned delay as the time from critical care assessment in the fi rst hospital to arrival in critical care in the second hospital. We used hospital postcodes to derive latitude and longitude, and calculated both geodesic (straight-line) distances (Figure 1) and road distances between the sites using R version 3.1.1 [2]. We compared daytime versus overnight (7:00 pm to 7:00 am) transfer durations assuming traffi c would contribute less to delay overnight. Mapping and visualisation was performed on Quantum GIS version 2.4 [3]. i References 1. Gilligan P, et al. The referral and complete evaluation time study. Eur J Emerg Med. 2010;17:349-53. 1. Gilligan P, et al. The referral and complete evaluation time study. Eur J Emerg Med. 2010;17:349-53. 2. Watts M, et al. Acute medical assessment units: an effi cient alternative to in-hospital acute medical care. Ir Med J. 2011;104:47-9. 2. Watts M, et al. Acute medical assessment units: an effi cient alternative to in-hospital acute medical care. Ir Med J. 2011;104:47-9. Results The median delay to admission was 22 hours (range 41 to 167 hours). The median geodesic distance was 18 km (range 1 to 141 km), and road distance was 24 km (range 2 to 180 km). Correlations between time delay and geodesic/road distances were weak (Figure 2, Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) Introduction The aim of the study was to assess the blood pressure (BP) and heart rate (HR) changes during shifts in ICU nurses in relation to their work experience. Our hypothesis was that less experienced nurses, in comparison with more experienced ones, would be subjected to more work stress and this could be demonstrated by higher changes in BP and HR during shifts. Methods We enrolled 23 nurses working in an 8-hour shift schedule at a general adult ICU. Demographic and clinical data were obtained by completing a short questionnaire. The nurses were invited to measure their BP and HR at the beginning, in the middle and at the end of their shift. An ESH/BSH-certifi ed automatic device was used for the BP and HR measurements. Conclusion We found that several prehospital-registered vital signs recorded by ambulance personnel at fi rst contact with the patient were prognostic factors of 7-day mortality. S178 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P508 P508 Contribution of medical senior house offi cers to a medical referral in the emergency department GF Fitzpatrick University Hospital, Limerick, Ireland Critical Care 2015, 19(Suppl 1):P508 (doi: 10.1186/cc14588) of patients were fi rst seen by a consultant within 12 hours of admission, with a range from 23 minutes to 26 hours. When looking at patients admitted during the weekdays, 63% of them were seen within 12 hours; for those admitted at the weekend the fi gure was 57%. Reference 1. Royal College of Physicians. Acute medical care: the right person in the right setting-fi rst time. Report of the Acute Medicine Task Force. London: RCP; 2007. References 1. Gantner D, et al, Intensive Care Med. 2014;40:1528-35. 2. Flower M, et al, Crit Care. 2011;15:P464. R2 = 0.015 and 0.011, respectively). Transfer delays in the daytime and overnight were similar (Wilcoxon rank sum, P = 0.6).i P512 g Conclusion Interhospital transfers are subject to clinically signifi cant delays, and substantial travel distances. Delays are only weakly correlated to distances travelled and may refl ect delays resulting from organisational ineffi ciencies. We infer that eff orts to improve the effi ciency of transfer should focus on local organisational issues. There was no diff erence in the duration taken for overnight versus daytime transfers. R f Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients y B Greatorex, EC Colley B Greatorex, EC Colley Figure 1 (abstract P510). Map of transfers. y Great Western Hospital, Swindon, UK p , , Critical Care 2015, 19(Suppl 1):P509 (doi: 10.1186/cc14589) Critical Care 2015, 19(Suppl 1):P509 (doi: 10.1186/cc14589) Introduction In 2007, the Acute Medicine Task Force made recommendations about the operation and staffi ng of acute medical units (AMU). Consultant-led care was seen as critical to ensuring high standards of care for patients and maintaining effi cient patient fl ow [1]. It also recommended that during the hours when the AMU is staff ed by a consultant, all new patients should be seen within 6 to 8 hours. Patients admitted overnight should have a consultant review within 12 to 14 hours. Following the introduction of a 4:00 pm consultant ward round of newly admitted acute medical patients to the existing 8:00 am and 2:00 pm rounds, it was our intention to establish whether our trust was meeting those recommendations. Methods We conducted a prospective survey of all new acute medical admissions over a 2-week period. Data collected included date and time of admission to the hospital, location on arrival, time of fi rst medical clerking, and time of fi rst consultant review. i Results Data were collected for 420 admissions. Sixty-seven percent of patients were admitted to the hospital between 12:00 am and 12:00 pm with a peak occurring between 4:00 pm and 6:00 pm. Sixty-two percent Figure 1 (abstract P510). Map of transfers. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S179 R2 = 0.015 and 0.011, respectively). Transfer delays in the daytime and overnight were similar (Wilcoxon rank sum, P = 0.6). Conclusion Interhospital transfers are subject to clinically signifi cant delays, and substantial travel distances. Delays are only weakly correlated to distances travelled and may refl ect delays resulting from organisational ineffi ciencies. We infer that eff orts to improve the effi ciency of transfer should focus on local organisational issues. There was no diff erence in the duration taken for overnight versus daytime transfers. References 1. Harris SK, et al. Intensive Care Med. 2014;40 Suppl 1:127. 2. www.r-project.org. 3 i Figure 2 (abstract P510). (a) Time duration versus geodesic distance (R2 = 0.015). (b) Time duration versus road distance (R2 = 0.011). in those patients who are discharged out of hours is nearly twice that of those discharged during the day [1]. Evaluation of patients with wild mushroom poisoning in the emergency department Introduction Wild mushroom poisoning (MP) is an important medical emergency that may have bad clinical outcome. We aimed to evaluate the clinical and laboratory features of patients with wild MP admitted to our emergency department in the Central Black Sea Region and to inform the emergency department physicians about early diagnosis and management of wild MP in the light of obtained data.i Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients These results have been replicated in our institution with a mortality of 8.7% (discharged 22:00 to 06:59) versus 4.8% (discharged 07:00 to 21:59). In the UK, NICE CG50 advised that transfer from critical care to the ward out of hours should be avoided and documented as an adverse event. We postulated that one important factor in our hospital is the decreased medical and nursing cover overnight and so looked at the delay from discharge to fi rst medical review and to outreach review. in those patients who are discharged out of hours is nearly twice that of those discharged during the day [1]. These results have been replicated in our institution with a mortality of 8.7% (discharged 22:00 to 06:59) versus 4.8% (discharged 07:00 to 21:59). In the UK, NICE CG50 advised that transfer from critical care to the ward out of hours should be avoided and documented as an adverse event. We postulated that one important factor in our hospital is the decreased medical and nursing cover overnight and so looked at the delay from discharge to fi rst medical review and to outreach review. Methods The case notes of 100 consecutive patients discharged to the ward between September 2013 and October 2013 were examined to identify the time of discharge from the ICU and the subsequent fi rst review by the receiving medical team and the Critical Care Outreach team. The grade of the doctor reviewing the patient was recorded. Results Of these 100 patients, 22 were discharged between 22:00 and 07:59. From the 100 case notes requested, only 50 were available for examination. Forty patients were discharged to the wards, with only 37 having further documented medical reviews in the notes. Only 62% of patients were reviewed by a consultant following intensive care, with over 20% of patients waiting more than 24 hours for any medical review. During this time 18% of patients received a review by the nurse- led outreach team. See Figure 1. Conclusion It is clear that a highly vulnerable group of patients who are recovering from critical illness [2] receive inadequate early follow-up within the hospital. We postulate that the delay in medical review and the lack of senior review may be caused by over 40% being discharged overnight and contribute to the increased mortality seen in our institution and the ANZICS study [1] with nighttime discharges. References Delayed ICU discharges and medical follow-up: a cause of increased mortality? The BASIC Patient Safety course was only administered to staff from ICU1, and safety culture was assessed in both units before and after, using a survey based on the Hospital Survey on Patient Safety Culture [2]. Relative risk (95% CI) of improvement: baseline to follow-up in hospitals in patient safety domains, adjusted for duration of work in the unit (≤10 years vs. >10 years), was calculated. Responses were coded according to the Survey User’s Guide, and positive response percentages for each patient safety domain were compared with the 2012 Agency for Healthcare Research and Quality (AHRQ) ICU sample of 36,120 respondents. organizational culture to improve patient safety culture is considered important. We conducted a prospective, controlled study to assess the impact of a standardized patient safety course on an ICU’s patient safety culture, using a validated patient safety culture assessment tool. Methods Staff from two ICUs – ICU1 (tertiary referral hospital) and ICU2 (district hospital) – in Hong Kong were recruited to compare changes in the measured safety culture before and after a patient safety course. The BASIC Patient Safety course was only administered to staff from ICU1, and safety culture was assessed in both units before and after, using a survey based on the Hospital Survey on Patient Safety Culture [2]. Relative risk (95% CI) of improvement: baseline to follow-up in hospitals in patient safety domains, adjusted for duration of work in the unit (≤10 years vs. >10 years), was calculated. Responses were coded according to the Survey User’s Guide, and positive response percentages for each patient safety domain were compared with the 2012 Agency for Healthcare Research and Quality (AHRQ) ICU sample of 36,120 respondents. Conclusion Wild MP can cause bad clinical outcome. The public should be informed about the probable hazards of wild mushroom ingestion because collection and consumption of wild mushrooms from nature is common. Public health units should take protective precautions against wild MP. Education of health personals regarding MP will lead to successful results in patient management. Results Preintervention and postintervention period response rates for ICU1 were 88.1% (37/42) and 79.3% (23/29); and for ICU2 63% (20/32) and 63% (15/24). Delayed ICU discharges and medical follow-up: a cause of increased mortality? Methods This study was designed retrospectively by examining fi les of the patients with wild MP who were admitted to Ondokuz Mayis University Emergency Department, between January 2008 and December 2012. Patients older than 18 years were included in the study. Patients were evaluated according to gender, age, location, duration between mushroom intake and the start of clinical symptoms, time of application to hospital, clinical features and fi ndings and treatment method. The number of patients has been compared with the regional distribution of population, monthly temperature and average annual rainfall. y p , , Critical Care 2015, 19(Suppl 1):P511 (doi: 10.1186/cc14591) Introduction Discharge from intensive care is a potentially vulnerable time for patients who are recovering from critical illness. Recent data from the ANZICS group have highlighted that the mortality diff erence Figure 1 (abstract P511). S180 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results A total of 420 patients poisoned by wild mushrooms were studied. The male/female ratio was 1/1.5. The age of patients changed from 18 to 92 and mean age was 46 years. MP constituted 13.3% of all intoxication cases. The time when the fi rst symptom occurred after mushroom intake was a mean 2 (0.17 to 2.15) hours. Of the patients, 47.6% lived in villages, 38.6% in towns and 13.8% in city centers. Admissions were mostly made in autumn, with 57.6%. Eighty-six percent of intoxications happened because of wild mushrooms collected in nature. The most frequent symptoms were nausea (93.8%), and vomiting (87.1%). Increase in liver function tests in 47 patients was observed. Two of these patients died while 10 patients were transferred to further centers for liver transplantation. The remaining patients were discharged from the hospital. organizational culture to improve patient safety culture is considered important. We conducted a prospective, controlled study to assess the impact of a standardized patient safety course on an ICU’s patient safety culture, using a validated patient safety culture assessment tool. Methods Staff from two ICUs – ICU1 (tertiary referral hospital) and ICU2 (district hospital) – in Hong Kong were recruited to compare changes in the measured safety culture before and after a patient safety course. P513 Triage after drug overdose: eff ect of the introduction of a medical psychiatry unit on the allocation of ICU beds D Kleinloog, F Braam Houckgeest, D Sierink Tergooiziekenhuizen, Hilversum, the Netherlands Critical Care 2015, 19(Suppl 1):P513 (doi: 10.1186/cc14593) y y D Kleinloog, F Braam Houckgeest, D Sierink y y D Kleinloog, F Braam Houckgeest, D Sierink Tergooiziekenhuizen, Hilversum, the Netherlands g Critical Care 2015, 19(Suppl 1):P513 (doi: 10.1186/cc14593) Introduction Many patients with drug overdose are sedated, but do not have medical reasons to warrant ICU admission. Historically, monitoring behavior and suicide risk was done in the ICU, until the patient was awake enough for psychiatric consultation. Conclusion The study provides supportive evidence that a structured, reproducible short course on patient safety is associated with a general improvement in the ICU’s patient safety culture, measured with a validated safety culture assessment tool. Methods A medical psychiatry unit (MPU) was instituted as part of the Department of Clinical Psychiatry. For all patients with drug overdose in the emergency department, a risk assessment was made by the intensivist. Those without ICU indication (such as cardiac or respiratory monitoring) were admitted to the MPU. Alternatively, when awake enough, they were seen by the psychiatrist immediately. We performed an analysis of all patients with drug overdose, admitted to our ICU (before MPU n = 88, after MPU n = 191). We used the Welch t test for comparisons. Delayed ICU discharges and medical follow-up: a cause of increased mortality? Post intervention, compared with ICU2, ICU1 showed signifi cantly improved perceptions of teamwork within the hospital unit, RR (95% CI for diff erence between ICUs) 1.55 (1.10 to 2.19, P = 0.01); and overall perception of safety, 1.94 (1.11 to 3.37, P = 0.02); but not increased frequency of reporting mistakes, 0.90 (0.33 to 2.49, P = 0.84). Overall, ICU1 demonstrated a greater improvement in positive responses in fi ve safety culture domains than staff from ICU2. Patient safety culture indices were generally poorer in the two ICUs than the average ICU in the AHRQ database. Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Before MPU Since MPU (18 months) (51 months) Diff erence 95% CI P value Admissions per month 4.9 3.5 –1.4 –2.7 to –0.1 0.04 APACHE II score 8.4 12.5 +4.0 +1.8 to +6.2 0.0004 APACHE III score 30.9 40.3 + 9.4 +2.6 to +16.2 0.0069 Length of stay 0.8 1.3 + 0.5 –0.0 to +1.0 0.05 Conclusion Introduction of an MPU was associated with reduced numbers of patients with drug overdose admitted to the ICU. Those admitted to the ICU after the institution of the MPU were sicker, probably indicating more appropriate use of ICU beds. P514 Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Ling, G Joynt, A Lee, W Samy, H Fung, CD Gomersall The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) Introduction It is estimated that about one in 10 patients may be harmed by adverse events during their hospital stay [1]. Transforming Before MPU Since MPU (18 months) (51 months) Diff erence 95% CI P value Admissions per month 4.9 3.5 –1.4 –2.7 to –0.1 0.04 APACHE II score 8.4 12.5 +4.0 +1.8 to +6.2 0.0004 APACHE III score 30.9 40.3 + 9.4 +2.6 to +16.2 0.0069 Length of stay 0.8 1.3 + 0.5 –0.0 to +1.0 0.05 Methods Patients admitted to the ICU between 21 September 2014 and 2 October 2014 were included. If an investigation did not involve ionising radiation or was not requested by intensive care clinicians it was excluded. The indication for imaging was noted, and patient notes were analysed no less than 48 hours after the imaging was reported. Figure 1 (abstract P515). Conclusion Introduction of an MPU was associated with reduced numbers of patients with drug overdose admitted to the ICU. Those admitted to the ICU after the institution of the MPU were sicker, probably indicating more appropriate use of ICU beds. References 1. de Vries EN, et al. Qual Saf Health Care. 2008;17;216-23. 2. Nieva VF, et al. Qual Saf Health Care. 2003;12 Suppl 2:ii17-23. Audit of imaging documentation on an ICU l N Arora, SJ Glover p Results After institution of the MPU, there was a 28% reduction in the number of patients with drug overdose per month, admitted to the ICU. Also, patients admitted to the ICU were sicker and stayed longer (see Table 1). There were no patients admitted to the ICU after initial MPU admission. p pi Critical Care 2015, 19(Suppl 1):P515 (doi: 10.1186/cc14595) Introduction The Ionising Radiation (Medical Exposure) Regulations 2001 recommend to ‘ensure that a clinical evaluation of the outcome of each medical exposure is recorded’ [1]. This audit looked at whether ICU documentation of investigations involving ionising radiation could be improved. Anticipated benefi ts would be improved communication between the multidisciplinary team and better- informed decision-making. Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU P514 P514 Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Ling, G Joynt, A Lee, W Samy, H Fung, CD Gomersall The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) P516 Barriers to the implementation of checklists in the ICU: a survey on the perceptions of 314 Brazilian critical care nurses and physicians J Salluh1, W Viana2, F Machado3, A Cavalvanti4, F Bozza1, M Soares1 1IDOR, Rio de Janeiro, Brazil; 2Hospital Copa D’OR, Rio de Janeiro, Brazil; 3UNIFESP, São Paulo, Brazil; 4Hcor, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P516 (doi: 10.1186/cc14596) Introduction Checklists have been used increasingly in the ICU aiming at mitigating avoidable adverse events, but their content is variable and little is known about barriers to their full implementation. This survey reported practices on the use of checklists and perceptions regarding barriers on its implementation. Methods A web-based survey was conducted among a convenience sample of Brazilian ICU nurses and physicians between August and October 2014. Standard descriptive statistics were used. Results A total of 314 professionals, 104 nurses (33.1%) and 210 physicians (66.9%), responded. The majority (82.4%) had more than 5 years of experience in intensive care. Checklists were applied every day, including weekends, in only 49.8% (n = 227). When we compared the barriers perceived by those working in smaller versus larger ICUs (<10 beds vs. >20 beds), the absence of a more comprehensive checklist content (93.6% vs. 81.9%, P = 0.026), the absence of specialized software or app (80.8% vs. 63.8%, P = 0.014), low availability of mobile devices (87.2% vs. 72.4%, P = 0.019), Internet unavailability (83.3% vs. 67.6%, P = 0.017), and the limited number of computers (88.5% vs. 76.2%, P = 0.0366) were the most often barriers to implementation. Checklists were applied with similar frequencies (<3 times a week, three to fi ve times a week, and every day, including on weekends, P >0.05) regardless of the ICU size. When the type of tool used (paper vs. electronic) was considered, the main barrier highlighted was the lack of 100% Internet availability in the ICU (64.8% vs. 100%, P = 0.009). Users of paper form had higher demands for more comprehensive checklist content (84.3% vs. 63.6%, respectively, P = 0.037) and experienced more barriers to team adherence (98.1% vs. 86.4%, respectively, P = 0.034) as compared with those using specialized software. Conclusion Although checklists are recognized as valuable tools for the adherence to best practice in the ICU, it is diffi cult to ensure the uniformity of their daily use. Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Li G J A L W S H F CD G ll g y y g The Chinese University of Hong Kong, Shatin, Hong Kong y g g g g Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) Introduction It is estimated that about one in 10 patients may be harmed by adverse events during their hospital stay [1]. Transforming Figure 1 (abstract P515). S181 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 to concentrate, multitasking and the need to frequently change priorities in patient care-related procedures. It is therefore a continuous challenge to communicate and implement plans in an early, effi cient and reliable way within the team and to constantly keep track about tasks that are completed, postponed or that are about to be forgotten. To facilitate every caregiver’s workfl ow and to early and effi ciently communicate, we implemented the After Round Comprehensive Plan Summary (ARCoPS) tool. to concentrate, multitasking and the need to frequently change priorities in patient care-related procedures. It is therefore a continuous challenge to communicate and implement plans in an early, effi cient and reliable way within the team and to constantly keep track about tasks that are completed, postponed or that are about to be forgotten. To facilitate every caregiver’s workfl ow and to early and effi ciently communicate, we implemented the After Round Comprehensive Plan Summary (ARCoPS) tool. Results As shown in Figure 1, imaging requests were generally poorly documented (61%). In total, 17/26 (65%) chest X-rays (CXRs) were documented. A total of 0/2 CT scans were documented, despite one showing acute changes. In total, 17/20 (85%) CXRs requested following procedures carried out on ITU (such as insertion of central venous catheters) were documented, and the three not documented had no signifi cant fi ndings. The six other CXRs were requested to investigate worsening respiratory function. None were documented. Five had signifi cant fi ndings. y Methods Most of our patient care-related therapeutic and diagnostic decisions are made during the morning round consisting of intensivists, nursing team, surgeons, respiratory therapists, dieticians, clinical pharmacists and physiotherapists. P516 Resource limitations in smaller ICUs and the absence of comprehensive digital tools, mobile devices and Internet availability preclude full compliance at the bedside. P518 P518 Multicenter Thai university-based surgical ICU study (Thai-SICU study): adverse events and outcome in the SICU S Kongsayreepong, K Chittawatanarat Siriraj Hospital, Mahidol University, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P518 (doi: 10.1186/cc14598) Introduction The aim of this Thai-SICU was to study the incidence and outcome of adverse events in nine SICU university-based hospitals in Thailand. Introduction The aim of this Thai-SICU was to study the incidence and outcome of adverse events in nine SICU university-based hospitals in Thailand. Methods This multicenter prospective observational study was done in >18 years old, admission >6 hours surgical patients admitted to the large, postgraduate medical training university-based SICU during April 2011 to January 2012. Patient data were divided into three main phases as admission, discharge data and daily CRFs during the ICU stay. The patients were followed until they were discharged from the ICU or up to 28 days of their ICU admission and up to 28 days following discharge from the ICU if they survived. Results Following a 19.7-month recruitment period, a total of 4,654 patients (17,579 ICU-days) were included in the analysis process. Admitted patients had the median age of 64 years. Most of the patients were admitted directly from the OR for postoperative monitoring with median APACHE II score 10, 23% were admitted with priority I who needed aggressive hemodynamic resuscitation and respiratory support. ICU mortality and 28-day mortality were 9.61% and 13.80%. Each day of ICU increment was associated with a 1.38-day increase of hospital stay (95% CI, 1.24 to 1.53; P <0.01). On the surveillance periods, the six most common adverse events were sepsis (19.5%), AKI (16.9%), new cardiac arrhythmias (6.17%), respiratory failure (5.83%), cardiac arrest (4.86%) and delirium (3.5%) respectively. The other events including reintubation within 72 hours, intraabdominal hypertension, acute MI, unplanned extubation, upper GI hemorrhage, pneumohemothorax, seizure, drug error and pulmonary aspiration were <3% each. The risk Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Li G J A L W S H F CD G ll Immediately thereafter, all plans for the next 24-hour period are again discussed, summarized, confi rmed, prioritized and organized by the multidisciplinary team under aspects of optimal patient care and workfl ow effi ciency by entering the plan data into a fl at screen visualized template connected to our intranet immediately accessible at every physician and nursing caregiver workstation. ii Conclusion Investigations following procedures were generally well documented, but investigations seeking pathology were not documented at all, regardless of the fi ndings. This may have infl uenced the management of the patient and compromised patient safety. As such, the audit was presented at a departmental meeting to emphasise the importance of imaging documentation. A place for investigations was added to the ICU patient list to improve communication between the team, and a second audit is planned to assess the impact of this. Reference Results Twelve months after implementation of the ARCoPS tool we conducted caregiver interviews and identifi ed the following eff ects: increase in treatment confi dence through standardized multidisciplinary decision-making; reduced loss of information through immediate plan summarization by the whole team; reduction of ambiguity and misunderstanding about care plans through early written documentation; higher level of security not to forget procedures or tasks; positive acceptance of the tool to fl exibly change priorities of care procedures; positive acceptance of the tool to mark accomplished tasks providing visual feedback about the care plan status; individual caregiver workfl ow economization through permanent treatment plan availability; and higher job satisfaction throughout the caregiver team. Conclusion ARCoPS tool utilization has become a daily routine in our ICU. It functions as an eff ective communication and workfl ow tool and has helped us to reduce patient care-related misunderstandings and delays. It also enhanced the economics of our work sequence, which also highly contributes to a better level of patient safety. Furthermore, it has markedly contributed to an improved level of quality of work for caregivers. 1. The ionising radiation (medical exposure) regulations 2000. London: Stationery Offi ce; 2000. 1. The ionising radiation (medical exposure) regulations 2000. London: Stationery Offi ce; 2000. P517 After Round Comprehensive Plan Summary tool: an effi ciency approach for the ICU R Marktanner, M Mostafa, A Shafei, H Hon, R Ashraf, P Sreedhara, N Syed, D Gharaibeh, A Taha Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P517 (doi: 10.1186/cc14597) Introduction ICU workfl ow for physicians, nurses and other healthcare providers is often complicated by distractions, interruptions, diffi culties Introduction ICU workfl ow for physicians, nurses and other healthcare providers is often complicated by distractions, interruptions, diffi culties S182 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We performed an observational study in a 17-bed, mixed clinical and surgical emergency department observation. Before the year 2012, the staff were composed of fi rst-year internal medicine and surgical residents. There were no senior physicians specifi cally assigned to this unit, and residents were supervised by the members of the emergency department team, who changed shifts on a daily basis. In February 2012, two senior physicians (one cardiologist and one intensive care doctor) were specifi cally assigned to supervise the internal medicine residents and provide horizontal care for medical patients during the day, from Monday to Friday. There was no change in assistance for surgical patients. The schedule for nights and weekends remained unchanged. Mortality and length of stay for medical and surgical patients were measured in 2011, 2012 and 2013.i of adverse events on 28-day mortality were signifi cant on cardiac arrest (RR = 9.45; P <0.01), ARDS (RR = 4.58; P <0.01), AKI (RR = 4.18; P <0.01), sepsis (RR = 3.62; P <0.01), iatrogenic pneumohemothorax (RR = 3.23; P <0.01), seizure (RR = 3.12; P <0.01), upper GI hemorrhage (RR = 2.97; P <0.01), cardiac arrhythmia (RR = 2.91; P <0.01), ALI (RR = 2.71; P <0.01), delirium (RR = 2.13; P <0.01), MI (RR = 2.12; P <0.01), unplanned extubation (RR = 2.06; P <0.01), abdominal hypertension (RR = 1.75; P <0.01) and reintubation within 72 hours (RR = 1.51; P = 0.02). Conclusion This is the largest systemic surveillance observation in the SICU. The study results are the reference for future research and also provide information for patient and relative advice when confronted with adverse events during SICU admission. P521 Results There were 6,203 surgical admissions during the 8-year study period. For both ICU and in-hospital mortality; the median LOS in days for survivors was 2.2 (IQR: 1.2 to 4.9) and that of nonsurvivors was 3.2 (IQR: 1.5 to 7.9). At 28 days, 1 year, and 2 years, the respective values were 2.2 (1.2 to 4.9) and 3.3 (1.5 to 6.7); 2.1 (1.2 to 4.7) and 3.1 (1.5 to 7.3); and 2.1 (1.2 to 4.6) and 3.0 (1.5 to 7.0), all P <0.0001. The greatest median LOS was found in neurosurgery and cardiothoracic surgery; 3.3 days (IQR: 1.7 to 9.5) and 3.1 days (IQR: 1.5 to 8.0) respectively. They corresponded to the specialities with the greatest percentage ICU (9.7% and 10.2%) and 2-year mortality (27.9%, and 35%). The greatest mortality and median LOS was found in ventriculostomy cases; 40.8% at 2 years and 10.6 days (IQR: 4.8 to 18.2). Retrospective observational cohort study of mortality and length of stay for surgical ICU admissions Introduction A signifi cant number require a postoperative ICU stay which may be prolonged [1]. Very limited data exist to characterise mortality and ICU length of stay (LOS) in diff erent surgical specialties [2,3]. We aim to describe this relationship in a large cohort of surgical patients. Conclusion Emergency department observation units are an alternative to alleviate emergency room overcrowding when there are no intensive care beds available. However, patients end up staying in those units for days and horizontal care by senior doctors may improve outcomes. p Methods We performed a retrospective observational cohort study of adult surgical admissions to the ICU of a large academic tertiary medical centre. The primary endpoint was 28-day mortality. We used simple descriptive statistics to characterise the population. The Wilcoxon rank- sum test or Kruskall–Wallis test was used, as appropriate, for tests of continuous data. Rejection for ICU admission: analysis and results of this modality of limitation of therapeutic eff ort Williams TA, et al. Br J Anaesth. 2010:104:459-64. P517 Results In the fi rst year after the implementation of the clinic intensive care team, mortality in internal medicine patients decreased from 47% to 34% in 2012 and 33% in 2013. Although other changes happened in this period (the number of beds decreased from 24 to 17, nurses and physical therapists were hired and trained specifi cally for this unit), we believe the horizontal staff was critical, because mortality between surgical patients remained almost unchanged in the same period of time (23% in 2011, 22% in 2012 and 23% in 2013), in spite of the structural improvements that equally aff ected those patients. Length of stay decreased from 6.35 days in 2011 to 3.43 in 2012 and 3.15 in 2013 in medical patients and from 3.9 days on average in 2011 to 3.2 in 2012 and 2.8 in 2013 in surgical patients. Rejection for ICU admission: analysis and results of this modality of limitation of therapeutic eff ort pf F Acosta, O Moreno, M Muñoz, R Fernandez, M Colmenero Hospital Universitario San Cecilio, Granada, Spain Critical Care 2015, 19(Suppl 1):P521 (doi: 10.1186/cc14601) Critical Care 2015, 19(Suppl 1):P521 (doi: 10.1186/cc14601) Introduction The aim was to know the frequency, criteria and implications of rejection for ICU admission to our ICU unit, a second- level hospital (18 beds). Introduction The aim was to know the frequency, criteria and implications of rejection for ICU admission to our ICU unit, a second- level hospital (18 beds). Methods An observational retrospective study in a time interval of 6 months (January to June 2013). We retrospectively registered all patients rejected for admission to our unit, analyzing the clinical rejection report used in our hospital. From this report we extracted diff erent variables: demographical (age, sex), provenance (emergency room, hospital), clinical (comorbidity, functional situation, diagnosis, reason of requesting admission), rejection motive (‘too good’, ‘too bad’, futility, lack of beds, patient rejection), whether it was defi nitive or conditional, whether the patient was admitted afterwards, and the state at hospital discharge. We realized a descriptive analysis (frequencies) and multivariant analysis of the factors related to futility rejection. Conclusion There is an association between postoperative ICU LOS and mortality that persists for at least 2 years after admission. Neurosurgery and cardiothoracic surgery patients appear to have a worse prognosis and also a more prolonged LOS. Our results may provide a more objective basis for clinical decisions, the use of limited resources, and inform on appropriate expectations of treatment. 1. Pearse RM, et al. Lancet. 2012:380:1059-65. y y j Results There were 165 rejections, which represents 25% of total ICU patients evaluated for admission. A total of 59.4% were male. Mean age was 69 ± 7 years (19 to 98). In total, 53.9% had more than two comorbidities (pluripathological) and 31.5% moderate to severe functional disability. The cause of rejection was in 55.2% of situations that the patient was ‘too good’, 37.6% related to qualitative futility, 4.8% was ‘too bad’ and in 1.2% a mix of lack of space (beds) and patient rejection. In the multivariant analysis the signifi cant variables related to futility rejection were age (by years) with an OR of 1.05 (1.02 to 1.08), severe functional disability, OR of 4.35 (2.09 to 9.06), and the hospital provenance with an OR of 2.82 (1.1 to 7.2). 2. Timmers TK, et al. Ann Surg. 2011:253:151-7. 3. References 1. Komindr A, et al. Int J Emerg Med. 2014;5:1-6. 1. Komindr A, et al. Int J Emerg Med. 2014;5:1-6. 2. Baugh C, et al. Health Care Manag Rev. 2011;36:28-37. 2. Baugh C, et al. Health Care Manag Rev. 2011;36:28-37. P519 Retrospective observational cohort study of mortality and length of stay for surgical ICU admissions M Hameed1, M Maruthappu2, D Marshall3, M Pimentel1, L Celi4, J Salciccioli3, J Shalhoub3 1University of Oxford, UK; 2National Institute for Health and Care Excellence, London, UK; 3Imperial College London, UK; 4Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2015, 19(Suppl 1):P519 (doi: 10.1186/cc14599) P522 Impact of age, physiological status and APACHE score on acceptance of patients to the ICU Introduction Evidence suggests that age, chronic health status and acute illness severity aff ect the decision-making of clinicians regarding admission to the ICU (ITU) [1-3]. This prospective service review assesses the impact of age, APACHE II score and WHO functional score towards admission acceptance or refusal to ITU in a tertiary-level facility. p Results Following implementation of the new transfer process, the average wait time decreased by 58 minutes, process time decreased by 9 minutes, and lead time decreased by 66.5 minutes. The handoff error rate decreased by 1.3 errors/patient and fi rst-time quality rate increased by 67%. Staff satisfaction improved from 48% to 73%. By elimination of the PACU stay, the costs involved in admission to the PACU were deferred. admission acceptance or refusal to ITU in a tertiary-level facility. Methods Design: a planned prospective review of all referrals over a 14- day period. Data collection: review (LT, DP, SP) of case notes of patients referred to ITU with the following variables collected: age, sex, APACHE II scores, WHO functional status score, grade of referrer and source of referral. Data were collected on 37 patients: 22 accepted to ITU and 15 refused admission. Statistics: data were analyzed using GraphPad 6.05. Categorical variables were expressed as mean and standard error of mean. For unpaired variables, statistical signifi cance is determined using unpaired t test. P <0.05 is considered statistically signifi cant. Results The WHO functional status was the most signifi cant variable aff ecting admission (P <0.001). The APACHE score of patients admitted to ITU was signifi cantly lower than refused patients (P = 0.039). Patient age did not aff ect admission status (P = 0.15). See Table 1. Methods Design: a planned prospective review of all referrals over a 14- day period. Data collection: review (LT, DP, SP) of case notes of patients referred to ITU with the following variables collected: age, sex, APACHE II scores, WHO functional status score, grade of referrer and source of referral. Data were collected on 37 patients: 22 accepted to ITU and 15 refused admission. Statistics: data were analyzed using GraphPad 6.05. Categorical variables were expressed as mean and standard error of mean. For unpaired variables, statistical signifi cance is determined using unpaired t test. P <0.05 is considered statistically signifi cant.i Conclusion A single, multidisciplinary bedside handoff process between the OR and ICU leads to cost and time savings. Improved interprofessional communication, handover and ward rounds in critical care (ICARUS) Introduction There is growing evidence that optimising communication and patient assessment practices including ward rounds and handoff s can improve clinical, safety and operational outcomes, particularly in the critical care setting [1,2]. Here we describe the design and baseline phases of a 5-year project utilising improvement sciences to optimise the quality of interprofessional communication, handoff s and rounding in one of the largest critical care units in the UK. Conclusion This study was performed to assess decision-making for admission to the ITU in times of increased demand and limited availability. We want to validate our fi ndings from this short pilot study in a larger prospective study. Multivariate regression analysis will be used to identify signifi cant features that aff ect clinician decision- making in critical care admission. Methods We obtained institutional ethical and research approvals. We used mixed methods including interviews of opinion leaders and a representative cross-section of staff , roundtables, a survey targeting the whole critical care team (n = 546) and a Delphi exercise to generate a baseline consensus for the need to improve and a set of novel quality improvement interventions and tools. We tested two of these in a pilot plan–do–study–act (PDSA) cycle. g References References 1. Reignier J, et al. Crit Care Med. 2008;36:2076-83. 2. Garrouste-Orgeas M, et al. Crit Care Med. 2005;33:750-5. 3. Azoulay E, et al. Crit Care Med. 2001;29:2132-6. References 1. Reignier J, et al. Crit Care Med. 2008;36:2076-83. 2. Garrouste-Orgeas M, et al. Crit Care Med. 2005;33:750-5. 3. Azoulay E, et al. Crit Care Med. 2001;29:2132-6. Results Baseline consensus for the need and potential to improve was obtained (97.4% and 94.5%). Despite a large degree of heterogeneity of perceptions around current communication and rounding practices, it was possible to develop a set of interventions based on consensus that could be applied in this complex setting. These included a handoff bundle, an operational touch-base, a unit-level safety briefi ng, a unit- level safety check, a lean rounding bundle and board rounds. These core interventions were supported by several more detailed resources describing the evidence base around best handoff and rounding practices; and a feedback document that described all outputs and recommendations from the ICARUS project. A pilot PDSA cycle demonstrated a 55.3% and 76.3% improvement in key information transfer using a safety briefi ng and board round summary. P522 Impact of age, physiological status and APACHE score on acceptance of patients to the ICU By elimination of redundant, nonvalue-added processes, less opportunity for medical errors occurred, with substantial improvements in fi rst-time quality. Such a process can be successfully attained while aff ecting staff satisfaction positively. g p y gi Results The WHO functional status was the most signifi cant variable aff ecting admission (P <0.001). The APACHE score of patients admitted to ITU was signifi cantly lower than refused patients (P = 0.039). Patient age did not aff ect admission status (P = 0.15). See Table 1. Table 1 (abstract P522) Accepted Refused P value Mean age 56.5 ± 4.4 65.3 ± 3.6 0.15 Sex 73% male 56% male N/A WHO 0.45 ± 0.2 2.21 ± 0.3 <0.001 APACHE 13.5 ± 1.6 18.8 ± 2.0 0.039 5 Improved interprofessional communication, handover and ward rounds in critical care (ICARUS) P Hopkins, A Chan, S Rasoli, C Bell, K Peters, A Feehan, J Dawson King’s Health Partners AHSC, London, UK Critical Care 2015, 19(Suppl 1):P524 (doi: 10.1186/cc14604) Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness f SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) Introduction This Six Sigma project was initiated to evaluate and improve the transfer of care of patients from the OR to the ICU. Medical errors are responsible for billions of dollars in increased healthcare spending. Miscommunication among healthcare providers is a major contributor to these errors, with handoff s a particularly vulnerable period in the care process. At our institution, surgical patients with scheduled admissions to the ICU are fi rst recovered in the postanesthesia care unit (PACU). With this process, multiple, unstructured, and individual handoff s occur in parallel between providers, which may lead to communication errors, diff erential information sharing, content variability, care delays, and ineffi ciency. Conclusion Despite wide heterogeneity in baseline beliefs, opinions and perceptions around inter-professional communication, rounding and handoff s, we were able to develop a novel set of feasible quality improvement interventions, targeting these areas, that can be applied in a large complex critical care setting. Furthermore, they can be driven by improvement science methodology and tested for eff ectiveness using qualitative and quantitative measures. We now plan to use these interventions to deliver quality improvements in communication practices in parallel with the planning and implementation phases of a new critical care facility and electronic clinical information system. R f Implementation of a horizontal intensive care team can impact effi ciency in an emergency observation unit in Brazil S Ribeiro, C Petrini, L Marino, R Brandao-Neto Hospital das Clinicas School of Medicine, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P520 (doi: 10.1186/cc14600) fi y g y S Ribeiro, C Petrini, L Marino, R Brandao-Neto Hospital das Clinicas School of Medicine, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P520 (doi: 10.1186/cc14600) Introduction Emergency department observation units are often used to monitor critical patients in a situation of constant emergency department overcrowding and lack of intensive care beds. However, those units are often understaff ed and might not have enough personnel and equipment to provide the same quality of care as a conventional ICU. Conclusion Rejection for admission to ICU units is a frequent medical activity in our day-to-day job. The type of patient most rejected is cardiologic, mostly evaluated for thoracic pain probably ischemic but with low risk. In second place we found patients for which we decide rejection based on subjective qualitative futility, related mostly to age, prior functional disability and provenance. S183 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P522 were conducted, which began by defi ning the current process via direct observation and value stream mapping of orthopedic and neurosurgical patients. A new process was then introduced, including direct transfer of the patient to the ICU and a single, structured, bedside report between all care providers. A standardized handoff tool was implemented. We used process times, wait times and information content as process measures and handoff errors as outcome measures. A 10-point satisfaction score was also measured. were conducted, which began by defi ning the current process via direct observation and value stream mapping of orthopedic and neurosurgical patients. A new process was then introduced, including direct transfer of the patient to the ICU and a single, structured, bedside report between all care providers. A standardized handoff tool was implemented. We used process times, wait times and information content as process measures and handoff errors as outcome measures. A 10-point satisfaction score was also measured. P522 Impact of age, physiological status and APACHE score on acceptance of patients to the ICU L Terry, S Passey, D Porter, F Clark, R Matsa Royal Stoke University Hospital, Stoke on Trent, UK Critical Care 2015, 19(Suppl 1):P522 (doi: 10.1186/cc14602) P523 P523 Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) P523 Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) P526 Out-of-hours discharge from critical care: does it matter? M Ahmed, G Lumley, S Nourse, A Hurding, A Thomas, M Healy The Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P526 (doi: 10.1186/cc14606) Introduction The aim of this audit was to assess the clinical impact of out-of-hours (OOH) discharge from the adult critical care unit (ACCU) in a tertiary care hospital. Discharging patients from critical care OOH has been associated with both higher mortality and increased rate of readmissions [1,2]. As a result, national guidance stipulates that OOH discharges from critical care should be avoided [3]. Results A total of 2,248 emergency admissions were analysed. The majority of patients were admitted within 1 week of hospital admission (n = 1,897). They were younger and had lower APACHE II scores (15 vs. 19; P <0.001). APACHE II scores were the same in all other groups (groups 2 to 5). Patients admitted to the ICU 3 weeks following hospital admission had signifi cantly higher hospital mortality (up to 50%; P <0.001) and ICU length of stay (12 ± 15 vs. 8 ± 10 days; P <0.001). ICU mortality remained the same in all groups (20 to 28%). ICNARC and SOFA scores were equal between the groups. The post-ICU lengths of stay were signifi cantly longer in groups 3 to 5. In-hospital CPR prior to admission to ICU was lower in patients from groups 4 and 5, probably signifying appropriate DNAR decisions made on the ward. Methods A retrospective snapshot analysis of patients was conducted at least 48 hours after discharge from the ACCU in October 2014. Patients discharged OOH (20:00 to 07:59) were compared with those discharged in hours (IH; 08:00 to 19:59). Analysis included: patient demographics, APACHE II score, handover procedure prior to discharge, follow-up by the receiving team, appropriate and timely drug prescribing, recognising and acting upon clinical deterioration and readmission rates. Conclusion Prolonged pre-ICU hospital admission is associated with higher hospital but not ICU mortality. Commonly measured scores do not refl ect this higher mortality in patients admitted after prolonged stay in hospital. Further research into parameters that may refl ect changes in physiological reserves may strengthen these scores for such patients. Results A total of 161 patients were discharged from the ACCU in October 2014. Of these, 46% of the patients were discharged OOH. P526 Forty-one (of 74) OOH and 19 (of 87) IH discharges were sampled for further analysis. The baseline demographics and APACHE II score were similar between both groups. The majority of discharges were delayed (>4 hours, 90% IH vs. 93% OOH). More patients had nursing handover completed if discharged IH (88% IH vs. 61% OOH) and doctors’ summaries were less frequently completed OOH (83% OOH vs. 94.4% IH). A management plan for the ward was outlined in 94% Pre-ICU length of hospital stay is a predictor of hospital but not ICU mortality y K Flavin, D Hall, G Marshall, P Zolfaghari g Royal London Hospital, London, UK Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P527 (doi: 10.1186/cc14607) Introduction Prolonged hospital stay prior to admission to the ICU was previously shown to be independently associated with poorer outcome [1,2]. This is probably due to slow deterioration of physiological function in hospital and infl uenced by processes leading to critical care admission [2]. We investigated whether commonly measured severity scoring systems (Acute Physiology and Chronic Health Evaluation (APACHE) II, Intensive Care National Audit and Research Centre (ICNARC) and Sequential Organ Failure Assessment (SOFA) scores) are signifi cantly diff erent in patients admitted with prolonged pre-ICU hospital length of stay, and describe mortality and hospital length of stay. Conclusion There is a higher prevalence of adverse events in readmitted patients with similarity in the type of adverse eff ects despite readmission. g References 1. Priestap F, et al. Crit Care Med. 2006;34:2946-51. p 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 3. Acutely ill patients in hospital. NICE; 2007 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 3 Acutely ill patients in hospital NICE; 2007 gii Results The readmission rate was 15% (n = 392), particularly among males (55% (n = 214)). A 14.3% adverse event rate was observed among the readmitted patients (9.5% among those who were not readmitted). The readmitted patients were older (median (md): 68.0 (95% CI: 65.7 to 67.3) vs. md: 71.0 (95% CI: 67.1 to 71.3); P <0.05) and remained hospitalized for a longer period of time (md: 5.0 (95% CI: 13.2 to 20.7) vs. md: 11.0 (95% CI: 17.0 to 33.2); P <0.05), but not necessarily in the CICU (P = 106).The most prevalent adverse events in readmitted patients were pressure ulcers (n = 16 (4.1%)), drug administration error (n = 13 (3.3%)) and enteral feeding tube (n = 10 (2.6%)). Meanwhile, among the nonreadmitted, phlebitis due to peripheral vein access and pressure ulcers (n = 42 (1.9%)), drug administration error (n = 41 (1.9%)) and enteral feeding tube (n = 31 (1.4%)). There was a tendency (P = 0.71) that readmitted patients were presented with higher prevalence of pressure ulcers (n = 16 (4.1%) vs. n = 42 (1.9%)). 3. Acutely ill patients in hospital. NICE; 2007 Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) Introduction Security policies for the patient are of interest to any health professional. The rates of adverse events in hospitals reach values ranging between 3.7 and 16.6%, being the highest range (40 to 70%) considered preventable or avoidable [1]. The objective of this study is to assess the prevalence and types of adverse eff ects in intensive care patients according to readmission. g p Conclusion This audit compares with current evidence suggesting harm from OOH discharge despite most discharges being delayed. Discharging patients is a complex hospital process, but focus needs to be on discharging patients IH. Therefore, areas for improvement include targeting forward fl ow of patients throughout the hospital, completion of handover documents IH and publication of guidance for receiving teams. Methods During 4 years the data from 2,582 patients admitted to the coronary ICU (CICU) were analyzed in the coronary care unit of the city of Presidente Prudente, Brazil. We analyzed the rate of readmissions, length of stay and the mainly detected adverse events. We considered signifi cant P <0.05 two-tailed and confi dence intervals at 95% (CI). p Reference 1. Forster AJ, et al. CMAJ. 2004;170:345-9. 1. Forster AJ, et al. CMAJ. 2004;170:345-9. y Methods A retrospective analysis of prospectively collected data of all emergency admissions in the ICNARC database of a 44-bed adult critical care unit within a major trauma centre over a 2-year period. Demographic data, APACHE II score, SOFA score, ICNARC model score, mortality, and length of hospital stay prior to and after ICU admission were collected. Five groups of patients were defi ned as follows: those admitted to ICU within 1 week of hospitalization (group 1), within 8 to 14 days (group 2), within 15 to 21 days (group 3), within 22 to 28 days (group 4), and more than 28 days (group 5). Chi-squared and ANOVA tests were performed using the SOFA statistics package. References Methods A multidisciplinary QI project was initiated with input from the ICU, anesthesia and surgical services. A series of PDSA cycles Lane D. Crit Care Med. 2013;41:2015. Starmer AJ. N Engl J Med. 2014;371:1803. S184 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of IH versus 65.% of OOH discharges. Seventy-eight per cent OOH versus 95% IH patients discharged were reviewed by a doctor within 24 hours. Twenty-nine per cent OOH versus 67% IH patients discharged were reviewed by a consultant within 24 hours. Following discharge a management plan was followed in 94% of IH patients versus 44% OOH, patients had drug charts correctly charted in 100% of IH cases versus 66% OOH and missed/delayed doses were documented in 11.1% of IH cases versus 61% OOH. There was no diff erence between the groups in incidence of clinical deterioration and recognition and follow-up of clinical deterioration. Two patients were readmitted within 48 hours from the OOH group. of IH versus 65.% of OOH discharges. Seventy-eight per cent OOH versus 95% IH patients discharged were reviewed by a doctor within 24 hours. Twenty-nine per cent OOH versus 67% IH patients discharged were reviewed by a consultant within 24 hours. Following discharge a management plan was followed in 94% of IH patients versus 44% OOH, patients had drug charts correctly charted in 100% of IH cases versus 66% OOH and missed/delayed doses were documented in 11.1% of IH cases versus 61% OOH. There was no diff erence between the groups in incidence of clinical deterioration and recognition and follow-up of clinical deterioration. Two patients were readmitted within 48 hours from the OOH group. P525 Adverse events in patients in a Brazilian intensive care unit according to readmission GM Plantier1, CE Bosso1, AC Correa2, BN Azevedo3, JS Alves2, CD Monteiro2, SV Ferreira3, GE Valerio3, JV Moraes3, V Raso3 1Instituto do Coração de Presidente Prudente, Brazil; 2Santa Casa de Presidente Prudente, Brazil; 3Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) Higgins TL, et al. Crit Care Med. 2003;31:45-51. Goldhill DR, et al. Intensive Care Med. 2004;30:1908-13. P525 Adverse events in patients in a Brazilian intensive care unit according to readmission Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) F Kavanagh, I Browne g National Maternity Hospital, Dublin, Ireland g National Maternity Hospital, Dublin, Ireland y p Critical Care 2015, 19(Suppl 1):P530 (doi: 10.1186/cc14610) y p Critical Care 2015, 19(Suppl 1):P530 (doi: 10.1186/cc14610) Introduction Pregnancy and labour usually progress uneventfully; however, serious complications can occur and develop rapidly, necessitating critical care admission and support. Successive confi dential enquires have highlighted defi ciencies in this area and suboptimal care leading to increased morbidity and mortality [1]. The National Maternity Hospital is the largest maternity hospital in the Republic of Ireland where a total of 8,954 babies were delivered in 2013. It is a stand-alone institution and onsite facilities include a two- bed dedicated anaesthesia lead high-dependency unit. Introduction Hospital costs are a constant concern within health, especially in the ICU. Hospital admissions and average life expectancy have been growing gradually mainly in older and critical patients. This study is aimed to observe the direct costs of patients admitted to an ICU and their relation to the SAPS 3, length of stay in the ICU and fi nal outcome. Methods A retrospective observational study in which the direct costs were studied (materials, medicines, oxygen therapy and hospital fees) for 1,790 ICU patients from November 2013 to November 2014. The readmissions within 48 hours were excluded and also 10% of patients who had the highest and lowest costs. The remaining 1,401 patients were divided by age groups. Methods A retrospective observational study was carried out from January 2011 to January 2014 especially looking at the following parameters: admitting diagnosis, demographics, length of stay and the number of admissions requiring transfer for tertiary-level care. y g g Results Of the patients studied, 54.6% were male. Average age was 57.8 years (18 to 105 years). The biggest ICU average cost was in the group of patients 81 to 90 years old (US$793.00), as well as longest ICU stay (9.25 days), highest SAPS 3 (53.96) and higher ICU and in-hospital mortality (14.29% and 19.25% respectively). This study shows that the direct cost of the ICU stay for older patients was higher than for younger patients. The diff erence was explained by the higher severity measured by SAPS 3 in the older age groups (Figure 1), and the required greater length of stay in the ICU (Figure 2). As might be expected, the mortality in the group of older patients was also signifi cantly higher. References Higgins TL, et al. Crit Care Med. 2003;31:45-51. Goldhill DR, et al. Intensive Care Med. 2004;30:1908-13. S185 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P528 Reference 1. Special issue: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–2008. The Eighth Report of the Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG. 2011;118 Suppl S1:1-203. 1. Special issue: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–2008. The Eighth Report of the Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG. 2011;118 Suppl S1:1-203. P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) q g y Results In total, 29,344 deliveries occurred. A total of 376 HDU admissions were recorded in this period, representing 1.28% of all admissions. The average age of patients admitted to the HDU was 34 years. The predominant reasons for admission were hypertensive disease of pregnancy (49.7%), haemorrhage (antepartum/postpartum) (36.4%), sepsis (4.2%) and other reasons (11.1%) including cardiac rhythm disturbances, neurological complications and pre-existing medical disease. In 2013 the average length of stay was 2 days. A total 6.1% of those admitted to HDU required transfer for tertiary-level ICU care in other centres during the study period, this represented 0.07% of all deliveries.i Conclusion This study showed that greater age is associated with higher severity measured by SAPS 3, higher direct costs, and higher mortality both in the ICU and in-hospital environment. Conclusion There is signifi cant demand within our institution for HDU care for our patients, with the number of admissions increasing in 2013. The main admitting diagnoses are hypertensive disease of pregnancy and haemorrhage with an increase in the number of patients being admitted for management of sepsis in 2013. This highlights the increasing awareness, recognition and management of this condition in pregnancy. The increased number of HDU admissions in 2013 could also be explained by the recent introduction of an early warning score for the deteriorating patient in our hospital but this would require further evaluation. The low number of transfers of patients to other tertiary centres underpins the importance of an anaesthesia lead service. Figure 1 (abstract P528). Figure 2 (abstract P528). Figure 1 (abstract P528). Figure 1 (abstract P528). Reference P530 Critical care in a maternity hospital: a retrospective observational study P530 Critical care in a maternity hospital: a retrospective observational study P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) P531 Post-traumatic stress symptoms in intensive care staff working in adult and paediatric settings G Colville1, J Hammond1, L Perkins-Porras2 1St George’s Hospital, London, UK; 2St George’s University of London, UK Critical Care 2015, 19(Suppl 1):P531 (doi: 10.1186/cc14611) P534 P534 Mortality in a French military burn centre: a 12-year retrospective study analysing seasonal variations M Boutonnet1, C Dussault2, N Donat1, P Laitselart1, A Cirodde1, J Schaal1, C Hoff mann1, P Jault1, T Leclerc1 1HIA Percy, Clamart, France; 2IRBA, Brétigny sur Orge, France Critical Care 2015, 19(Suppl 1):P534 (doi: 10.1186/cc14614) f Methods After local ethics committee approval, a total of 1,004 patients (2012, n = 492; 2013, n = 454; 2014, n = 58) treated in the ICU in a 2-year period were reviewed. This study included for evaluation 135 patients determined with traumatic or nontraumatic brain damage, with a more than 24-hour stay in the ICU. Reasons for brain damage were determined as brain damage associated with head trauma (Group HT), head trauma accompanying general body trauma (Group HT + GBT) and spontaneous haemorrhage (Group SH). The type of brain damage was defi ned from the radiological diagnosis (CT and/or MRI) as subarachnoid haemorrhage, intracranial haemorrhage (ICH), subdural haematoma (SDH), epidural haematoma (EDH), skull fracture, brain contusion or a combination of these (COM). Operations were evaluated as performed by the brain surgery department. Introduction Factors associated with mortality in severely burned patients are well known. We aimed to assess the seasonal variations of the mortality of patients admitted to the main French military intensive care burn unit (ICBU) and to determine their relations to seasonal variations of severity or other factors (for example, staffi ng). Methods We performed a retrospective study analysing the medical records of all patients admitted to the ICBU of Percy Military Hospital (France) between January 2000 and December 2011. Statistical analysis was performed with R version 3.1.2. We fi rst conducted univariate analyses with simple logistic regression, then a simple periodic regression for seasonal variations (with and without harmonics for robustness), and fi nally a multivariate logistic regression with periodic terms in order to adjust seasonal variations for known severity factors. Results A total of 1,913 patients were admitted for acute burn injury during the study period, with a male to female ratio of 2.34. Mean total body surface area burned (TBSA) was 23.2% (IQR: 10 to 30) and mean full thickness total body surface area burned (FTBSA) was 13.4% (IQR: 0 to 15). Association with amount of registration and outcome in pediatric severe trauma patients S Ohnishi, N Saito, T Yagi, Y Konda, Y Hara, H Matsumoto Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan Critical Care 2015, 19(Suppl 1):P533 (doi: 10.1186/cc14613) Figure 1 (abstract P531). Main themes of staff descriptions of traumatic events on ICU. Introduction Unexpected trauma is a one of the most major causes of death for children in the world. However, pediatric severe trauma patients are rare and scattered. Although there is a strong association between patient’s volume of trauma center volume and outcomes in adults, such a report is less in children. In this study, we aimed to clarify the relationship of the amount of registration and outcome in pediatric trauma patients. most commonly endorsed: patient death (and particularly untimely deaths on adult units); handling distressed families; and concerns about the quality of care and dealing with staff confl ict (see Figure 1). Conclusion A signifi cant minority of staff reported clinically signifi cant levels of post-traumatic stress related to their work. The facts that post- traumatic stress levels were higher on paediatric units despite their lower rates of mortality and that untimely deaths were frequently mentioned by adult unit staff suggest it may be that untimely deaths are particularly hard to deal with. More research is needed on the prevalence of distress in staff working in these settings. Methods This retrospective study analyzed data of the Japan Trauma Data Bank from 2004 to 2012. We registered pediatric patients aged younger than 12 years with severe multiple or torso trauma (maximum Abbreviated Injury Scale score ≥3 or Injury Severity Score ≥9, and excluded patients with cardiopulmonary arrest on arrival, burn, isolated head or limb injury, lack of data). We divided the facilities into six groups according to every 10 patients registered and compared mortality between each group. Results A total of 1,015 patients from 105 facilities were included in the study. Number of registrations: 0 to 10 patients: 673 patients/68 facilities, 11 to 20: 381/28, 21 to 30: 141/6, 31 to 40: 101/3, 41 to 50: 45/1, 51 to 60: 58/1. Victims of blunt trauma accounted for 98.1%. Median age was 7 (interquartile range: 5 to 10) years, median injury severity score (ISS) was 17 (10 to 26). P533 P533 Association with amount of registration and outcome in pediatric severe trauma patients S Ohnishi, N Saito, T Yagi, Y Konda, Y Hara, H Matsumoto Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan Critical Care 2015, 19(Suppl 1):P533 (doi: 10.1186/cc14613) Distribution and mortality factors of cases with traumatic and nontraumatic brain damage treated in ICU S G k T k N B k G K ö E Ak S K ki Distribution and mortality factors of cases with traumatic and nontraumatic brain damage treated in ICU S Goksu Tomruk, N Bakan, G Karaören, E Akcay, S Keskin Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P532 (doi: 10.1186/cc14612) S Goksu Tomruk, N Bakan, G Karaören, E Akcay, S Keskin Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P532 (doi: 10.1186/cc14612) Introduction Cases of traumatic and nontraumatic brain damage have high rates of morbidity and mortality. In this study of cases being treated in the ICU for a diagnosis of brain damage, it was aimed to evaluate the relationship between mortality and the distribution of reason for and resulting type of brain damage and to determine other factors aff ecting mortality. i Conclusion There was a strong association between amount of registration and outcome. Post-traumatic stress symptoms in intensive care staff working in adult and paediatric settings Figure 2 (abstract P528). Figure 2 (abstract P528). Introduction The objectives of this survey were to establish the prevalence of symptoms of post-traumatic stress in mixed staff groups working in adult and paediatric intensive care settings and to examine the main themes in staff descriptions of the most traumatic event they had experienced at work. p Methods A total of 355 health professionals working on three adult and four paediatric units at two centres were asked to rate their current level of post-traumatic stress symptoms on the Trauma Screening Questionnaire (TSQ).f Results Paediatric/neonatal intensive care staff were more likely to score above the clinical cutoff point for post-traumatic stress symptoms on the TSQ in relation to an incident at work than adult intensive care staff in this sample (PICU n = 33/193 (17%) vs. AICU n = 13/162 (8%), P <0.001). For the 172 staff who provided a description of the most traumatic event they had experienced, the following themes were S186 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P531). Main themes of staff descriptions of traumatic events on ICU. (P <0.01). No statistical diff erence was determined in mortality in terms of age, gender, duration in ICU, surgical treatment or not, or reasons for brain damage (P >0.05). Conclusion There is considerable variation in causes of head injury. From this retrospective study it can be suggested that mortality of neurological/neurosurgical patients, regardless of management method, depends on APACHE II, arrival GCS, number of ventilator-days and type of brain damage. Association with amount of registration and outcome in pediatric severe trauma patients Multivariate analysis adjusted for age, revised trauma score and ISS revealed that the amount of registration (every 10 patients) was independently associated with survival (odds ratio: 1.55, 95% confi dence interval: 1.06 to 2.26, P = 0.023). P534 Simple periodic regression showed a biannual seasonal eff ect on mortality, documented with a 1-year periodic (P <0.01) and a 6-month periodic (P = 0.01) dependency. Multivariate analysis with or without periodic terms identifi ed age, past medical history, TBSA, FTBSA, inhalation, intoxication and admission date as the only factors independently associated with mortality. Conclusion Predictive factors for mortality in our ICBU are in line with the literature. The documented seasonal variations in mortality are fully explained by variations in these severity factors. Complementary analyses are under way to further study a nonlinear age eff ect. Results Baseline liver function was assessed in 1,565 patients. Of those, 522 (33%) exhibited liver dysfunction at baseline. No relationship was found with mortality according to baseline liver dysfunction status at day 28 (HR, 0.847 (95% CI, 0.647 to 1.109); P = 0.2267), day 90 (HR, 0.883 (95% CI, 0.701 to 1.112); P = 0.2892) and day 180 (HR, 0.885 (95% CI, 0.710 to 1.103); P = 0.2761). A total of 403 (26%) patients developed a new liver dysfunction (257/1,043, 25%) or worsened liver dysfunction (146/522, 28%) in the 28-day post-baseline period. The overall median time to develop or to worsen liver dysfunction was 2 (1 to 4) days. Signifi cant relationships between the new onset or the worsening of liver dysfunction post baseline and mortality at day 28 (HR, 1.67 (95% CI, 1.26 to 2.21); P = 0.0004), day 90 (HR, 1.65 (95% CI, 1.30 to 2.09); P <0.0001) and day 180 (HR, 1.57 (95% CI, 1.26 to 1.97); P <0.0001) were found. P534 Inhalation injury was present in 441 (23.1%) cases, intoxication (CO, CN) was present in 88 (4.6%) cases, and associated traumatisms also y g y Results The patients comprised 73.3% males and 26.7% females with a mean age of 40.58 ± 24.14 years (range, 1 to 87 years), mean APACHE II score of 19.07 ± 10.19 (range, 2 to 41), mean GCS of 9.17 ± 4.42 (range, 3 to 15) and 68.1% of whom were discharged and 31.9% were exitus. When the causes of brain damage were evaluated according to the type, the most frequently seen in the HT and HT + GBT groups were COM (37.3%, 42.9%), followed by EDH (13.6%, 28.6%). In the SH group, the most common reason was ICH (43.9%) followed by SDH (24.4%). Directly proportionally, only an increase in APACHE II score increased the mortality risk 1.3-fold (logistic regression analyses, coeffi cient 0.658) but the duration of intubation and ICH ratio was statistically signifi cantly high and GCS was low in the exitus cases, and rates of EDH were determined as high in discharged cases compared with exitus S187 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods All of the patients enrolled in the PROWESS-SHOCK were included. Liver dysfunction at baseline was defi ned by a liver Sequential Organ Function Assessment (SOFA) score >0. The onset of a liver dysfunction post baseline was defi ned as any post-baseline increase of the hepatic SOFA score from 0. The worsening of liver dysfunction post baseline was defi ned as any increase of the hepatic SOFA score from the baseline assessment. The post-baseline period studied extended from study drug infusion to day 28. The main outcome was the mortality at day 180, and the secondary outcomes were the mortality at day 28 and at day 90. Cox proportional hazard models were used to estimate the hazard ratio of death. in 88 (4.6%) cases. The mortality rate was 10.9%. The following factors were associated with mortality in univariate logistic regression: age, body mass index, past medical history, TBSA, FTBSA, intoxication (CO, CN), inhalation injury, fl ame burns, self-infl icted burns (all P <0.0001), sex (P <0.001), and admission date (P <0.01). Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Results One patient was admitted twice during this period. The sample comprised 11 males and 11 females (50%, respectively). Featuring an average age of 52 years, 78% of them were admitted to the ICU because of respiratory failure, 50% were due to diff use alveoli hemorrhage, 36% due to sepsis, 4% hypovolemic shock and fi nally 4% because of tuberculosis. According to the BVAS/WG, 20 patients corresponded to severe disease, one to limited diseases and one to persistent disease. The average ICU length of stay was 20.6 days and as inpatients 43 days. While comparing the SOFA score between alive and deceased patients there was a 0.5-point diff erence (P = 0.077), 63% of the alive patients were diagnosed while they were in the ICU. Plasmapheresis was found to be a protector factor (P <0.05).if Introduction In cancer patients, sepsis is the main cause of admission to the ICU and is associated with elevated mortality rates and healthcare costs. In this population, specifi c factors such as poor functional status and immunosuppression secondary to malignancy and/or antineoplastic treatment contribute to decreased survival. The aim of this study is to identify predictors of mortality in cancer patients admitted to the ICU with septic shock. Methods This is a retrospective study that analyzed predictors of 30- day mortality in 269 patients admitted to the ICU of the Institute of Cancer of State of São Paulo, Brazil, from February 2012 to November 2014. Conclusion The BVAS/WG score was signifi cantly diff erent between alive and deceased patients. Plasmapheresis was found to be a survival predictor. This study has shown that both SOFA and APACHE II scores have no prognostic value in these patients. Results From 1,250 patients admitted to the ICU, 269 patients had the admission diagnosis of septic shock and were analyzed. The mean age was 62 ± 14 years and 152 patients (56.5%) were male. Most patients had solid cancer (93.6%), and 87 patients (34.5%) had gastrointestinal neoplasm. Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Upon admission, the median SOFA score was 4 (IQR: 2 to 7), median SAPS 3 score was 55 (IQR: 48 to 68) and median lactate level was 1.88 mmol/l (IQR: 1.22 to 3.21). After 48 hours of ICU admission, acute kidney injury (AKI) was diagnosed according to AKIN classifi cation as follows: 202 patients (75.1%) had grade 0, 49 (18.2%) grade 1, seven (2.6%) grade 2 and 11 (4.1%) had grade 3. The 30-day mortality rate was 24.9%. In the univariate analysis, associated variables with 30-day mortality were age, urea and creatinine levels at admission, SOFA score at admission, SAPS 3 score and 48-hour AKI. In multivariate analysis, the predictive factors for 30-day mortality were SOFA score at admission (OR = 1.12; 95% CI: 1.04 to 1.21, P = 0.002) and 48-hour AKI defi ned as AKIN grades 1, 2 and 3 (OR = 2.69; 95% CI: 1.45 to 4.97, P = 0.002). P534 P535 ICU outcomes in patients suff ering granulomatosis with polyangitis C Hernandez-Cardenas1, GL Lugo-Goytia1, MS Sierra Beltran2, G Dominguez Cherit3 1Instituo Nacional de Enfermedades Respiratorias, Mexico DF, Mexico; 2Medica Sur, Mexico DF, Mexico; 3Instituo Nacional de Ciencias Medicas y Nutrición Salvador Zubirán, Mexico DF, Mexico Critical Care 2015, 19(Suppl 1):P535 (doi: 10.1186/cc14615) Introduction This study aims to describe both the clinical course and prognostic factors of patients suff ering granulomatosis with polyangitis (Wegener granulomatosis) (GP) who were admitted to the Salvador Zubirán National Medical Sciences and Nutrition ICU.f Introduction This study aims to describe both the clinical course and prognostic factors of patients suff ering granulomatosis with polyangitis (Wegener granulomatosis) (GP) who were admitted to the Salvador Zubirán National Medical Sciences and Nutrition ICU.f Conclusion The new onset or the worsening of liver dysfunction during the course of septic shock impacts strongly on long-term mortality. Septic patients with liver dysfunction need long-term follow-up. Future research should focus on developing specifi c septic liver therapeutics and new tools for earlier detection of septic liver dysfunction. Reference Methods Twenty-two patients suff ering GP admitted to the ICU, between January 2002 and December 2012, were included. The Acute Physiology and Chronic Health Evaluation (APACHE) II prognostic score scale was used in order to assess the severity of illness on the fi rst ICU day. The Sequential Organ Failure Assessment (SOFA) score was used to measure organ dysfunction, and the Birmingham vasculitis activity score for Wegener granulomatosis (BVAS/WG) was used to assess vasculitis activity. The outcome measurements taken into account were ICU mortality and ICU length of stay. 1. Ranieri et al. N Engl J Med. 2012;366;2055-64. Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Introduction Knowledge of the impact of liver dysfunction on mortality during septic shock is limited. However, the liver appears to play a key role during septic illness. To better explore this issue, we investigated the data collected during the Prospective Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis and Septic Shock (PROWESS-SHOCK) trial in which a cohort of 1,697 septic shock patients were constituted [1]. The study aimed to assess the relationship of liver dysfunction at the onset and during the course of septic shock on short-term and long-term mortality. Conclusion In cancer patients with septic shock, SOFA score at admission and acute kidney injury at 48 hours of admission were predictors of 30-day mortality. These fi ndings reinforce the needing of early strategies of diagnosis and therapy in this subset of patients. S188 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 disease remission and 31 (11.2%) had newly diagnosed disease. The ICU mortality rate was 26%, hospital mortality was 35.7% and 6-month mortality was 55.2%. The median number of organ dysfunction was 3 (IQR 2 to 4) and respiratory failure was the leading dysfunction, being present in 209 patients (75.5%). During the ICU stay, 21 patients needed hemodialysis (8%), 69 (25%) needed mechanical ventilation, 162 (58%) used vasoactive agents and 22 (8%) had a decision for limitation of medical treatment. On univariate analysis, risk factors for hospital mortality were acute myeloid leukemia, hospital stay prior to ICU admission >4 days, number of organ dysfunction ≥2, colonization and infection by a multidrug-resistant (MDR) agent, use of mechanical ventilation, use of vasoactive agents and renal replacement therapy. I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar Institute of Cancer of State of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P539 (doi: 10.1186/cc14619) Introduction In recent decades, therapeutic advances resulted in increased survival of patients with hematologic malignancies. These patients are increasingly admitted to the ICU due to infections, treatment toxicity and decompensation of chronic diseases. The aim of this study is to evaluate ICU, hospital and 6-month mortality in patients with hematological malignancies admitted to the ICU and to identify predictors of ICU mortality. y g Conclusion In our study, ICU and hospital mortality were lower than the pooled mortalities seen in a recent large systematic review [3] – despite our study excluding planned postoperative admissions (who are known to have better outcomes). The 180-day mortality was signifi cantly lower than in a previous study at our own institution [4]. Our study looked at a number of factors that might reasonably be expected to be associated with short-term and long-term outcomes and identifi ed several that were independent predictors of death by 180 days. Methods We performed a retrospective study of 277 consecutive patients with hematological malignancies admitted to the ICU of the Institute of Cancer of State of São Paulo, Brazil, from January 2010 to December 2013. Patient clinical and laboratory characteristics, evaluation of organ dysfunctions and need for hemodialysis, mechanical ventilation and vasoactive agents in the ICU were collected. The primary outcome was ICU mortality. Data were analyzed with univariate and multivariate logistic regression. Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 Multivariate analysis revealed that renal replacement therapy (OR = 6.35 (95% CI: 1.5 to 25.92), P = 0.010), SOFA score (OR = 1.69 (95% CI: 1.38 to 2.06), P <0.001), RDW (OR = 1.27 (95% CI: 1.11 to 1.46), P = 0.001), lactate (OR = 1.04 (95% CI: 1.02 to 1.06), P <0.001), colonization of MDR agent (OR = 10.73 (95% CI: 2.13 to 53.96), P = 0.004) and hospital stay prior to ICU admission >4 days (OR = 4.72 (95% CI: 1.8 to 12.3), P = 0.002) were predictive factors of ICU mortality. Factors associated with short-term and long-term mortality in solid cancer patients admitted to the ICU R Fi h 1 C D i 1 S C i h 2 S Sl 1 L S 1 Factors associated with short-term and long-term mortality in solid cancer patients admitted to the ICU R Fisher1, C Dangoisse1, S Crichton2, S Slanova1, L Starsmore1, T Manickavasagar1, C Whiteley1, M Ostermann1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2King’s College London, UK Critical Care 2015, 19(Suppl 1):P540 (doi: 10.1186/cc14620) Introduction Despite multiple reports demonstrating an improvement in outcomes of critically ill cancer patients admitted to ICUs over the last two decades [1], there is concern that admission policies for patients with cancer are overly restrictive [2]. The purpose of this study was to identify factors associated with mortality in the 180 days following unplanned ICU admission in patients with nonhaematological malignancy. Introduction Despite multiple reports demonstrating an improvement in outcomes of critically ill cancer patients admitted to ICUs over the last two decades [1], there is concern that admission policies for patients with cancer are overly restrictive [2]. The purpose of this study was to identify factors associated with mortality in the 180 days following unplanned ICU admission in patients with nonhaematological malignancy. Conclusion The ICU mortality of critically ill patients with HM is high, particularly in the group on MV. Diff erent factors were independent predictors of mortality, but 46.4% of admitted patients survived because of adequate support possibilities and were transferred back to hematology ward. The ICU admission with organ support is according results important for life saving in this extremely high-risk patient group. g p References Methods We carried out a retrospective analysis of all patients with solid tumours admitted to the Guy’s Critical Care Unit (13-bed level 3 ICU) as an emergency between August 2008 and July 2012. Data were collected regarding patient demographics, type of cancer, reason for referral and organ support required during the ICU stay. References 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 2. Grgic Medic M, et al. Hematologic malignancies in the medical intensive care unit – outcomes and prognostic factors. Hematology. 2014 [Epub ahead of print]. y Results During the 4-year study period there were 356 unplanned admissions of patients with solid cancer (8.3% of all admissions). Three hundred individual patients were admitted and 180-day survival data were available for 293 of these. Mean age at fi rst admission was 65.2 years, 115 (38.3%) were female. The most frequently present malignancies were lung (42.7%), head and neck (17.3%) and renal (6.7%). ICU, hospital and 180-day mortality were 19.1%, 31.0% and 52.2% respectively. Those factors found to be independently associated (in multivariate analysis) with increased risk of 180-day mortality include: metastases (OR = 4.0, 95% CI = 2.1 to 7.6); sepsis on admission (OR = 2.2, 95% CI = 1.2 to 4.1); APACHE II score on admission (OR = 1.06, 95% CI = 1.004 to 1.12); need for inotropes/vasopressors during admission (OR = 2.3, 95% CI = 1.05 to 4.8); and need for renal replacement therapy during admission (OR = 4.65, 1.7 to 12.8). References 1. Mokart D, et al. Intensive Care Med. 2014;40:1570-2. 2. Azoulay E, et al. Ann Intensive Care. 2011;1:5. 3. Puxty K, et al. Intensive Care Med. 2014;40:1409-28. 4. McGrath S, et al. QJM. 2010;103:397-403. P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) Introduction The admission of malignant hematology patients to the ICU is combined with poor prognosis [1]. A young population on one side and serious prognosis on the other are the main characteristics. Do we help? We are analyzing continuously clinical characteristics, treatment, and outcomes of critically ill patients with hematologic malignancies admitted to the medical ICU to identify predictors of adverse outcome [2]. Methods Demographic characteristics, hematologic diagnosis, reasons for ICU admission, transplant status, the presence of neutropenia, and APACHE II and SOFA scores were analyzed. Predictors of ICU mortality were evaluated using univariate analysis. g y Results There was a total of 194 patients (103 male), APACHE II score by admission was 27 ± 8, SOFA 9 ± 3. Acute leukemia (L) in 81 patients (41.8%), chronic L in 19 patients (9.8%), lymphoma in 58 patients (29.9%), and multiple myeloma in 28 patients (14.4%) were the etiology. Respiratory insuffi ciency, hemodynamic instability, AKI and CNS disturbances were responsible for the admission of 169 patients (87.1%) from the hematology ward to the ICU. In total, 127 patients (59.7%) were mechanically ventilated; 93 required invasive mechanical ventilation (MV). Non-invasive ventilation started in 34 patients and was successful in 14 (6.5%). The ICU mortality rate was 104 patients (53.6%), and the mortality of MV patients was 98 (77.2%). Need for vasopressors at admission, MV, neutropenia, and APACHE II and SOFA scores were identifi ed as independent predictors of fatal outcome. Overall mortality of admitted patients was 53.6% (104 patients), and in ventilated patients was 77.2% (98 patients). Conclusion Patients from our institution have survival rates comparable with data from the literature. Our study suggests that mortality is associated with late ICU admission and colonization of MDR bacteria. P540 P540 Outcomes of patients with hematologic malignancies admitted to the ICU I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar Institute of Cancer of State of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P539 (doi: 10.1186/cc14619) y References References 1. Mokart D, et al. Intensive Care Med. 2014;40:1570-2. 2. Azoulay E, et al. Ann Intensive Care. 2011;1:5. 3. Puxty K, et al. Intensive Care Med. 2014;40:1409-28. 4. McGrath S, et al. QJM. 2010;103:397-403. Results The median age of the population was 57 years and 144 patients (52%) were male. Upon admission, 15 patients (5.4%) had S189 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P541 Characteristics and outcomes of lung cancer patients requiring ventilatory support: results from a multinational study AC Toff art1, JF Timsit1, J Salluh2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, B Almeida7, E Azoulay8, M Soares9 1Hopital A. Michalon CHU Grenoble, France; 2Post-graduation Program – Inst. Nacional de Cancer, Rio de Janeiro, Brazil; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton Hospital, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Hopital Saint Louis, Paris, France; 9DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P541 (doi: 10.1186/cc14621) P541 using Hosmer–Lemeshow goodness of fi t test. Data will be reported as median (range) or proportions (95% confi dence interval). P541 Characteristics and outcomes of lung cancer patients requiring ventilatory support: results from a multinational study AC Toff art1, JF Timsit1, J Salluh2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, B Almeida7, E Azoulay8, M Soares9 1Hopital A. Michalon CHU Grenoble, France; 2Post-graduation Program – Inst. Nacional de Cancer, Rio de Janeiro, Brazil; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton Hospital, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Hopital Saint Louis, Paris, France; 9DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil Critical Care 2015 19(S ppl 1) P541 (doi 10 1186/cc14621) Results A total of 2,769 patients were included, median age 65 (18 to 100) years and 52% female. Thirty-day mortality was 4.5% and 2.2% were admitted to ICU. Median time to ICU admission was 1 (0 to 70) day. Nursing staff assessed 65% and physicians 26% of admissions. NEWS could be calculated for 85%. Nursing staff had a discriminatory power of 0.865 (0.786 to 0.944) with little variation with experience. Calibration was acceptable, except for the least experienced nurses (<5 years). References 1. Fuchs L, et al. Intensive Care Med. 2012;38:1654-61. 2. Nielsson MS, et al. Acta Anaesthesiol Scand. 2014;58:19-26. Hospital of South West Jutland, Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P542 (doi: 10.1186/cc14622) Introduction Not all patients in need of critical care arrive in clinical distress and some deteriorate after arrival. Identifying these patients early in their clinical course could potentially improve outcome. The present study was performed with the aim of assessing whether nursing and physician staff were able to identify patients in need of critical care using only clinical judgment and to compare this with the National Early Warning Score (NEWS). y References Physicians had a discriminatory power of 0.789 (0.641 to 0.937), with little variation with experience. Calibration was very good, regardless of experience. NEWS had a discriminatory power of 0.809 (0.727 to 0.891) and poor calibration. There was no signifi cant diff erence in discriminatory power between the three assessments.f Critical Care 2015, 19(Suppl 1):P541 (doi: 10.1186/cc14621) Introduction The aim was to evaluate clinical characteristics and outcomes of patients with lung cancer requiring ventilatory support. Methods Secondary analysis of a prospective multicenter study including patients requiring either invasive (IMV) or non-invasive (NIV) mechanical ventilation for >24 hours admitted to 22 ICUs in six countries from Europe and South America during 2011. We used shared frailty models to identify factors associated with 6-month survival. f y p Conclusion Both nursing staff and physicians were as good as NEWS at identifying patients at increased risk of ICU admission after admission to a medical admission unit. However, both nursing staff and physicians had better calibration (accuracy) than NEWS. P543 5 3 Clinical characteristics in surviving and nonsurviving older patients admitted to the ICU Results Out of 449 patients admitted to the ICUs, 239 (small-cell (SCLC) = 34; non-SCLC = 205) required ventilatory support (NIV = 104; IMV = 135). Out of NIV patients, 31 (30%) were subsequently intubated for IMV. Main reasons for ventilatory support were sepsis (n = 119; among them, 102 patients had pneumonia), airway involvement by tumor (n = 79), ARDS (n = 47) and coma (n = 18). Mean SAPS II score was 54 ± 20 and median SOFA score was 7 (4 to 12) points. Hospital and 6-month mortality rates were 55% and 67%; 94 (39%) patients received treatment limitations in the ICU. Mortality according to ventilatory strategy was 56% for NIV only, 77% for NIV followed by IMV, and 70% for IMV only. In the multilevel model, adjusting for the hospital size, presence of step-down units, type of admission and treatment limitation, performance status (PS) 3 to 4 (HR = 2.25 (95% CI, 1.52 to 3.34)), metastasis (HR = 1.66 (1.18 to 2.33)) and the ventilatory strategy compared with NIV only (HR = 1.73 (1.02 to 2.92), for NIV followed by IMV; HR = 2.25 (1.51 to 3.35), for IMV only) were associated with increased mortality. Conversely, patients with sepsis had higher survival (HR = 0.67 (0.46 to 0.96)). Introduction In recent years the proportion of older people admitted to the ICU has increased. A variety of clinical and physiological factors are associated with outcome in these patients. The aim of this study is to determine the clinical characteristics associated with survival of ICU mechanically ventilated older patients. Introduction In recent years the proportion of older people admitted to the ICU has increased. A variety of clinical and physiological factors are associated with outcome in these patients. The aim of this study is to determine the clinical characteristics associated with survival of ICU mechanically ventilated older patients. Methods We retrospectively studied 74 patients, aged >65 years, admitted to the ICU who underwent mechanical ventilation. Standard demographic, clinical, and physiologic data were recorded. We examined the signifi cant diff erences in clinical characteristics between survivors and nonsurvivors using the Student t and chi-square tests. Methods We retrospectively studied 74 patients, aged >65 years, admitted to the ICU who underwent mechanical ventilation. Standard demographic, clinical, and physiologic data were recorded. Admission to intensive care can be reliably predicted using only clinical judgment Admission to intensive care can be reliably predicted using only clinical judgment M Brabrand Hospital of South West Jutland, Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P542 (doi: 10.1186/cc14622) M Brabrand 5 3 Clinical characteristics in surviving and nonsurviving older patients admitted to the ICU We examined the signifi cant diff erences in clinical characteristics between survivors and nonsurvivors using the Student t and chi-square tests. Results The mean age of patients studied (43 men and 31 women) was 79 ± 6.4 years. The type of admission was surgical 18%, trauma 26% and medical 57%. The ICU mortality of these patients was 57% and it was not associated with gender and cause of admission to ICU. Patients who survived had lower Charlson Comorbidity Index (P <0.05) and shorter duration of mechanical ventilation (P <0.01). The episodes of ventilation-associated pneumonia, sepsis and renal failure were less frequently in survivors (P <0.05). Also, in addition serum iron and cholesterol levels were signifi cantly lower in nonsurvivors (P <0.01). Conclusion The mortality of ICU older patients is high. VAP, sepsis and renal failure are frequent complications in nonsurvivors. Pre-existing comorbidities considerably aff ect mortality. R f su o s a d o su o s us g t e Stude t t a d c squa e tests. Results The mean age of patients studied (43 men and 31 women) was 79 ± 6.4 years. The type of admission was surgical 18%, trauma 26% and medical 57%. The ICU mortality of these patients was 57% and it was not associated with gender and cause of admission to ICU. Patients who survived had lower Charlson Comorbidity Index (P <0.05) and shorter duration of mechanical ventilation (P <0.01). The episodes of ventilation-associated pneumonia, sepsis and renal failure were less frequently in survivors (P <0.05). Also, in addition serum iron and cholesterol levels were significantly lower in nonsurvivors (P <0 01) Conclusion In a multinational study, 6-month survival in lung cancer patients requiring ventilatory support is better than perceived a priori. Palliative care should be preferred in patients with poor PS. p p p Acknowledgements Funded by INCA, CNPq and FAPERJ. cholesterol levels were signifi cantly lower in nonsurvivors (P <0.01). Conclusion The mortality of ICU older patients is high. VAP, sepsis and renal failure are frequent complications in nonsurvivors. Pre-existing comorbidities considerably aff ect mortality. References Frailty predicts increased resource use and postoperative care requirements after revision hip surgery P546 Emergency laparotomy clinical outcome according to patient characteristics, level of postoperative care and time of surgery T Banerjee1, M Templeton2, C Gore2 1St Mary´s Hospital, London, UK; 2Imperial College Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P546 (doi: 10.1186/cc14626) Critical Care 2015, 19(Suppl 1):P546 (doi: 10.1186/cc14626) Introduction Emergency laparotomies have poor outcomes with variable postoperative critical care provision [1-3]. All patients requiring an emergency laparotomy with an estimated risk of death of >10% should go to critical care. Time of surgery should not aff ect standard of care [3,4]. In advance of the National Emergency Laparotomy Audit (NELA) results [2], our objective was to see whether the level of postoperative care and time of surgery aff ect outcome. Results A total of 389 patients with a mean age of 68.7 years were identifi ed. Frail patients were signifi cantly more likely to need vasopressors postoperatively (P = 0.012). Each increase in frailty score was associated with 0.16 increase in length of stay on the HDU (P = 0.025). Analysis of patient location at 30 days shows that frail patients stay in hospital longer (P = 0.00). Frail patients also bleed more intraoperatively (P = 0.00 with a coeffi cient value of 239; that is, for every point increase in frailty, average blood loss increases by 239 ml). For each increase by unit of blood transfused, the length of stay increased by 5.3 days (P = 0.000). The use of epidural is not associated with increased need for postoperative vasopressors (P = 0.598). See Figure 1. g yf Methods Retrospective data were collected across the Imperial NHS trust for all emergency laparotomies over 3 months in 2014: length of stay in days (LOS); mortality; age; ASA; surgery time and postoperative care level, that is ward (L1), high dependency (L2), or ICU (L3). Statistical tests: Mann–Whitney, Pearson correlation (PCC) and multilinear regression analysis. g y Results Seventy-one patients underwent surgery. Overall mortality was 13% and 70% of patients went to a L2/3 bed. More ASA 1/2 patients went to L1 and all ASA 4/5 went to L2/3. Median (IQR) for age was 61 (44 to 67) for L1, 65 (48 to 73) for L2/3 (P = 0.11), LOS was 10 (7 to 16) for L1, 19 (12 to 57) for L2/3 (P = 0.002), and mortality (%) was 0 for L1 and 18 for L2/3. g p References 1. Saunders D, et al. Br J Anaesth. 2012;109:368-75. 1. Saunders D, et al. Br J Anaesth. 2012;109:368-75. 1. Saunders D, et al. Br J Anaesth. 2012;109:368 75. 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. , ; 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. 2. NELA project team. Executive summary, fi rst organisational report of the N ti l E L t A dit L d RC A 2014 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. Methods We prospectively collected data on 339 adult patients admitted to two ICUs in Rwanda between August 2013 and July 2014. We described demographic and presenting characteristics and outcomes. We assessed the discrimination and calibration of the MPMo-III model. Using stepwise selection, we then developed a new logistic model for mortality prediction, the R-MPM. Frailty predicts increased resource use and postoperative care requirements after revision hip surgery A Panickar1, N Singatullina1, J Stubbs1, C Johnson1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Leicester Hospitals, Leicester, UK Critical Care 2015, 19(Suppl 1):P544 (doi: 10.1186/cc14624) A Panickar1, N Singatullina1, J Stubbs1, C Johnson1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Leicester Hospitals, Leicester, UK Critical Care 2015, 19(Suppl 1):P544 (doi: 10.1186/cc14624) y Methods This was a prospective cohort study of all adult patients with a fi rst-time admission to a medical admission unit at a 450-bed regional teaching hospital over a 3-month period in 2010. All subspecialties of internal medicine are present as well as a level 2 ICU. Upon fi rst contact with the patient after arrival, nursing staff and physicians were asked to report their estimation of the probability of ICU admission (0 to 100%). Survival status was extracted from the Danish Civil Registry. All administrative details (including transfers to other hospitals, wards and ICUs) were extracted from the National Patient Registry, both ensuring complete follow-up. The discriminatory power (ability to identify patients at increased risk) was estimated using area under the receiver- operating characteristics curve. Calibration (accuracy) was assessed Introduction There is increasing demand for revision hip surgery in older patients with poor frailty. Our previously submitted work demonstrated that frailty predicts the need for medical review [1]. We reviewed patients for a further 16 months to see whether frailty impacts on care [2]. This is the largest reported study reviewing frailty and the need for organ supports and outcomes in complex orthopaedic surgery. Methods A retrospective note review of all patients from January 2012 to April 2014 undergoing revision hip surgery. Data collected included frailty, comorbidities, operative blood loss, anaesthetic technique and level of organ supports and patient location at 30 days. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S190 Figure 1 (abstract P544). Frailty versus length of stay. of 8.881. The MPMo-III predicted mortality with area under the curve of 0.720 and Hosmer–Lemeshow chi-square statistic of 16.391, indicating that the predictive risk scores of the MPMo-III were not well calibrated to the Rwandan data. Figure 1 (abstract P544). Frailty versus length of stay. Conclusion The MPMo-III had modest risk prediction capacity in a population of Rwandan ICU patients. The R-MPM is an alternative severity score with fewer and more accessible variables that provides better predictive power. This model needs to be validated in other ICUs. References References 1. Singatullina et al. Abstract. Crit Care. 2014;18 Suppl 1:P49N. 2. Rockwood K, et al. Can Med J. 2005;173:489-95. 1. Singatullina et al. Abstract. Crit Care. 2014;18 Suppl 1:P49N. 2. Rockwood K, et al. Can Med J. 2005;173:489-95. Frailty predicts increased resource use and postoperative care requirements after revision hip surgery For surgery between 08:00 and 17:59, 14 went to L1, 28 to L2/3. Mortality was 5% and LOS 15 (9 to 24). Between 18:00 and 21:59, two went to L1, 10 to L2/3. Mortality was 17% and LOS 22 (8 to 34). Between 22:00 and 07:59, fi ve went to L1, 12 to L2/3. Mortality was 29% and LOS 15 (9 to 36). ASA strongly predicted mortality (P = 0.006, PCC 0.32). There was a negative correlation between postoperative destination and mortality with all deaths happening in those who went to L2/3 (P = 0.038 and PCC –0.25); however, sicker patients may have gone here. There was a strong correlation between mortality and time of surgery, night surgery being a strong predictor of mortality (PCC 0.31, P = 0.008). LOS can be predicted by a combination of ASA, age and care level (P = 0.027); the postoperative care regression coeffi cient was negative (–18.04, SE 10.41) with prolonged LOS in patients admitted to L2/3, which could also be explained by illness severity. g Conclusion Frailty is associated with increased intraoperative resource use and postoperative care requirements independent of choice of anaesthetic technique. This type of surgery should be subject to health economic analysis as demand amongst the frailer surgical population increases. P545 P545 Predicting outcomes in critically ill patients in a resource-poor setting: the Rwanda Mortality Probability Model T Twagirumugabe1, E Riviello2, R Fowler3, V Novack4, A Mueller2, W Kiviri1, V Banner-Goodspeed2, D Talmor2 1University of Rwanda, Kigali, Rwanda; 2Beth Israel Deaconess Medical Center, Boston, MA, USA; 3University of Toronto, ON, Canada; 4Ben-Gurion University of the Negev, Beer Sheva, Israel Critical Care 2015, 19(Suppl 1):P545 (doi: 10.1186/cc14625) Predicting outcomes in critically ill patients in a resource-poor setting: the Rwanda Mortality Probability Model g y y T Twagirumugabe1, E Riviello2, R Fowler3, V Novack4, A Mueller2, W Kiviri1, V Banner-Goodspeed2, D Talmor2 p y y Conclusion Trust mortality is similar to that in the Emergency Laparotomy Network audit [1]. Higher ASA patients are appropriately going to L2/3 care. Baseline health status and time of surgery are the strongest predictors of mortality in emergency laparotomy patients. References Introduction ICU mortality prediction models provide robust tools for research and benchmarking in the developed world, but an ICU mortality prediction model has not been validated in a resource-poor setting. We sought to validate the Mortality Probability Admission Model, version III (MPMo-III) in two public ICUs in Rwanda and to develop a simplifi ed Rwanda Mortality Probability Model (R-MPM) for use in developing countries. P549 P549 Disturbed circadian rhythm in ICU patients as indicated by melatonin levels: a prospective pilot study K Kiss1, I Földesi1, L Kemény1, V Csernus2, Z Molnár1, J Singer3 1University of Szeged, Hungary; 2University of Pécs, Hungary; 3Hungarian Society for Clinical Biostatistics, Hungary Critical Care 2015, 19(Suppl 1):P549 (doi: 10.1186/cc14629) pi y Results Age, platelets, ALT and APACHE II were selected to be included in the new laboratory-based score. The AUC for the score was 0.714, which was higher than each of the individual laboratory parameters. The AUC was increased further to 0.781 by including all 14 variables (age, lactate, FiO2, urea, creatinine, ALT, APACHE II, platelet, bicarbonate, haemoglobin, pH, ionised Ca, carboxyhaemoglobin and albumin), although this improvement was not considered signifi cant as the confi dence intervals of the two scores (4 and 14 variables) overlapped. Conclusion A laboratory-based score was successfully established in ICU patients, revealing an AUC of 0.714 which is comparable with established scores in a similar population. The compilation of the variables to produce a laboratory-based score showed greater prognostic power than individual variables. Model developers require an AUC of >0.7 to be termed useful; however, in order to be used in a clinical setting the AUC must be at least 0.75. Further research including internal and external validation studies must be performed to optimise the model before clinical implementation. Introduction The aim of this prospective pilot study was to test how melatonin levels follow the circadian cycle in ICU patients. There is strong evidence that changes of circadian rhythm are refl ected in melatonin levels with peak levels at dawn and low levels during daytime [1]. The ICU stay is accompanied by disturbed circadian rhythm [2], which could potentially be the result of artifi cial lighting conditions. Methods Melatonin levels were determined in eight medical ICU patients on mechanical ventilation, without brain injury or infection. Arterial blood samples were taken on the day of admission at 18:00 (TE0) and 03:00 in the morning (TM0), then at the same time points 48 hours later (TE1, TM1). Blood samples were centrifuged at 3,000 rpm for 8 minutes, and then serum samples were stored at –80°C. Measurements were performed using a US-CEA908Ge melatonin ELISA kit. For statistical analysis, a binary (yes/no) variable was created from the pairs for each day, assigning ‘Yes’ if the TE values were greater than TM (melatonin peak reversion). Developing a laboratory-based score to predict mortality in patients admitted to the ICU A Iqbal1, I Welters2, R Kolamunnage-Dona3, C Toh3, C Downey2 1Institute of Ageing and Chronic Disease, Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P547 (doi: 10.1186/cc14627) A Iqbal1, I Welters2, R Kolamunnage-Dona3, C Toh3, C Downey2 1Institute of Ageing and Chronic Disease, Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P547 (doi: 10.1186/cc14627) g y p Results Patient median age was 34 (IQR 26 to 49) years; 48.7% were male. Mortality was 50.3%. The variables most predictive of mortality in univariate analyses were: age, sepsis within 24 hours of ICU admission, hypotension or shock at ICU admission, Glasgow Coma Scale score at ICU admission, and heart rate (beats per minute) at ICU admission. Using these fi ve variables, the R-MPM predicted mortality with area under the curve of 0.829 and Hosmer–Lemeshow chi-square statistic Introduction Scoring systems can be used to predict mortality in patients admitted to the ICU. They are produced using variables that are associated with an increased risk of mortality such as patient Introduction Scoring systems can be used to predict mortality in patients admitted to the ICU. They are produced using variables that are associated with an increased risk of mortality such as patient S191 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The citizens surveyed valued outcomes associated with quality of life and intact neurological function. Mortality and other indices of duration of critical illness were valued less. These preferences need to be confi rmed in larger groups with greater diversity. Future research also needs to further understand these outcome preferences in survivors of critical illness, clinicians, decision-makers, and researchers. Understanding the outcome preferences of pertinent stakeholders and whether these preferences are congruent is imperative to inform the design of future critical care trials. Conclusion The citizens surveyed valued outcomes associated with quality of life and intact neurological function. Mortality and other indices of duration of critical illness were valued less. These preferences need to be confi rmed in larger groups with greater diversity. Future research also needs to further understand these outcome preferences in survivors of critical illness, clinicians, decision-makers, and researchers. Understanding the outcome preferences of pertinent stakeholders and whether these preferences are congruent is imperative to inform the design of future critical care trials. Patient preferences for outcomes in critical care trials (OPTICS): preliminary resultsf p y J Muscedere1, F Lamontagne2, G Boyd1, M Herridge3, S Fleury1, T Sinuff 3 1Queen’s University, Kingston, ON, Canada; 2University of Sherbrooke, QC, Canada; 3University of Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P548 (doi: 10.1186/cc14628) Results Melatonin levels were not normally distributed and were ranging between 9.2 and 23.7 pg/ml at T0 and 11.3 and 17.9 pg/ ml at T1. The median diff erences of the morning and evening serum melatonin levels were: TM0 – TE0 = –2.8 (–3.8 – (–0.2)); TM1 – TE1 = –1.2 (–2.5 – (–0.4)) presented as median (IQR). The proportion of subjects with peak reversals was 92.9% (80.8 to 100.0%). Introduction Although patient-centered outcomes are important to inform therapeutic choices for critically ill patients, patient preferences for outcomes in critical care studies are unknown. It is also unknown whether outcome preferences diff er between researchers, decision- makers and those who have never been critically ill (citizens). The aim of the OPTICS Program is to investigate these preferences. Herein we report the preliminary results for outcomes preferences in citizens. Conclusion Our preliminary data suggest that circadian rhythm disturbances may occur in critically ill patients within 48 hours after admission, and can be detected by inversion of melatonin peaks. Despite the limitations of this study, it may justify the need for larger observational and randomized trials on the eff ect of light on melatonin levels and on outcomes in ICU patients. Methods We recruited and surveyed lay public members without a history of critical illness as to their preferences for outcomes in critical care trials. After an in-person educational session, citizens were asked to rank 11 potential critical care trial outcomes in order of personal preference. Each outcome was also rated for importance on a 7-point Likert scale. Participants were then asked to indicate their agreement with potential tradeoff s between potential outcomes. References 1. Pevet P, et al. J Physiol. 2011;105:170-82. 1. Pevet P, et al. J Physiol. 2011;105:170-82. 2. Castro R, et al. Annual update in Intensive Care and Emergency Medicine. J.-L. Vincent, editor. Berlin: Springer-Verlag; 2011. p. 766-80. 2. Castro R, et al. Annual update in Intensive Care and Emergency Medicine. J.-L. Vincent, editor. Berlin: Springer-Verlag; 2011. p. 766-80. pf p Results The in-person session was attended by 31 citizens whose had a mean age (SD) of 71.6 (5.9) years and 1.5 (1.4) chronic health conditions; 25 (81%) had partially or fully completed post-secondary school education. Of the 11 potential outcomes, the three outcomes ranked of highest importance were: permanent brain dysfunction, quality of life, and requirement for long-term institutional care. The three outcomes ranked of least importance were: duration of hospitalization, death after a prolonged illness, and occurrence of delirium. When rated on a 7-point Likert scale the results were similar. Of the participants, 24/27 (89%) indicated that they would be willing to receive a therapy which was associated with a higher mortality rate but resulted in an improved quality of life in survivors. Conversely, 16/27 (59%) indicated that they would not be willing to receive a therapy which increased the chances of survival but was associated with a reduced quality of life in the survivors. Developing a laboratory-based score to predict mortality in patients admitted to the ICU demographics, physiological measurements and coexisting conditions and can be used to evaluate ICU performance, to stratify patients in clinical trials and to assist in-hospital and healthcare decisions such as resource allocation. The aim of the project was to determine whether a general score derived from routine laboratory parameters could be used to predict mortality rates in patients admitted to the ICU in the UK. Methods P values were calculated using the t test, Mann–Whitney U test and chi-squared test, depending on distribution of data, in order to determine which variables were signifi cantly diff erent in the survivors and nonsurvivors of critical illness. Signifi cant variables were categorised into subgroups according to medically relevant landmarks and univariately analysed by assessing the correlation with mortality. Forward logistic regression models were used to choose the parameters to include in our score. ROC curves illustrated the sensitivity and specifi city of selected variables via their AUC. P549 The proportion of reversals and their 95% confi dence intervals were estimated using a GEE model for repeated binary data, assuming a binomial distribution and log link, and accounting for subject as a repetition factor. All calculations were done in SAS 9.4. P548 Patient preferences for outcomes in critical care trials (OPTICS): preliminary results J Muscedere1, F Lamontagne2, G Boyd1, M Herridge3, S Fleury1, T Sinuff 3 1Queen’s University, Kingston, ON, Canada; 2University of Sherbrooke, QC, Canada; 3University of Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P548 (doi: 10.1186/cc14628) Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay Both devices can be used to eff ectively monitor and characterize sleep in the ICU environment. g y Methods In this multicenter follow-up study of the Dexamethasone for Cardiac Surgery (DECS) trial, validated self-report questionnaires were sent 1.5 to 4 years after cardiac surgery and ICU treatment to assess symptoms of PTSD and depression, in relation to cumulative life stress (that is, childhood trauma, major stressful life events) and trait anxiety as determinants of psychopathology. Data were available for 1,125 out of 1,244 (90.4%) eligible participants. Mediating and moderating analyses were performed with multivariable linear regression to assess the eff ect of trait anxiety. Subgroup analyses were performed for both sexes. P552 P552 Hospital anxiety and depression after ICU survival: results of a post-ICU aftercare program D Ramnarain, C Slobbe, W Schapendonk, J Van Gorp, I Gnirrip, S Voermans, A Rutten, G Van der Nat, N Van der Lely St.Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P552 (doi: 10.1186/cc14632) Hospital anxiety and depression after ICU survival: results of a post-ICU aftercare program Introduction Although the ICU survival rate has increased in the last decade, the negative eff ects on mental health and related quality of life become more clear. In the literature the prevalence of anxiety and depressive symptoms post ICU ranges from 10 to 43% [1]. Early recognition and treatment of anxiety and depressive symptoms is important because depression caries a risk for suicide, limited quality of life, and delayed return to work. We studied hospital anxiety and depression (HAD) symptoms after ICU discharge. Results Trait anxiety partially mediates the relationship between cumulative life stress and PTSD (β-value reduction from 0.325 to 0.068; P = 0.000 to P = 0.003) and fully mediates the association between cumulative life stress and depression (β-value reduction from 0.282 to 0.015; P = 0.000 to P = 0.507). Trait anxiety was not a moderating factor between cumulative life stress and psychopathology. Full mediation of trait anxiety was found in female patients (n = 247), whereas only partial mediation was seen in male patients (n = 878) with regard to PTSD symptoms. As for depression, full mediation was present in both female and male patients. p y p g Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013 for more than 5 days were invited to visit our post-ICU aftercare clinic. Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay L Kok1, M Sep1, D Veldhuijzen2, S Cornelisse1, A Nierich3, J Van der Maaten4, P Rosseel5, J Hofl and6, J Dieleman1, C Vinkers1, L Peelen1, M Joëls1, D Van Dijk1, M Hillegers1 L Kok1, M Sep1, D Veldhuijzen2, S Cornelisse1, A Nierich3, J Van der Maaten4, P Rosseel5, J Hofl and6, J Dieleman1, C Vinkers1, L Peelen1, M Joëls1, D Van Dijk1, M Hillegers1 1University Medical Center Utrecht, the Netherlands; 2Leiden University, the Netherlands; 3Isala Clinics, Zwolle, the Netherlands; 4University Medical Center Groningen, the Netherlands; 5Amphia Hospital, Breda, the Netherlands; 6Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P550 (doi: 10.1186/cc14630) Introduction ICU survivors are at risk for post-traumatic stress disorder (PTSD) and depression. The development of psychopathology depends partially on stable personality factors such as trait anxiety. Introduction ICU survivors are at risk for post-traumatic stress disorder (PTSD) and depression. The development of psychopathology depends partially on stable personality factors such as trait anxiety. S192 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 distribution was light sleep 62.2% (PAT) and 74.1% (PSG); REM 13.0% (PAT) and 10.7% (PSG); deep sleep 14.5% (PAT) and 7.9% (PSG). Conclusion Both wristwatch-style PAT and miniature PSG devices successfully recorded sleep in ICU patients. Although the simple PAT device overestimated TST, sleep stage times were generally in agreement, especially REM time which correlated strongly. Both devices can be used to eff ectively monitor and characterize sleep in the ICU environment. distribution was light sleep 62.2% (PAT) and 74.1% (PSG); REM 13.0% (PAT) and 10.7% (PSG); deep sleep 14.5% (PAT) and 7.9% (PSG). Among ICU patients a high level of trait anxiety is relatively common and associated with intrusions, a symptom of PTSD. Independently, childhood trauma and stress exposure throughout life have been associated with depression. In cardiac surgery patients admitted to the ICU postoperatively, the eff ect of trait anxiety on the relationship between cumulative life stress and stress-related psychopathology remains unknown. Therefore we aimed to assess the mediating or moderating role of trait anxiety in this at-risk patient population. Conclusion Both wristwatch-style PAT and miniature PSG devices successfully recorded sleep in ICU patients. Although the simple PAT device overestimated TST, sleep stage times were generally in agreement, especially REM time which correlated strongly. Sleep monitoring in ICU patients Sleep monitoring in ICU patients S Namba1, K Hayashi1, T Hirayama1, T Hirayama1, Y Namba1, M Terado1, P Easton2, Y Ujike1 1Okayama University Hospital, Okayama, Japan; 2University of Calgary, AB, Canada Critical Care 2015, 19(Suppl 1):P551 (doi: 10.1186/cc14631) Sleep monitoring in ICU patients S Namba1, K Hayashi1, T Hirayama1, T Hirayama1, Y Namba1, M Terado1, P Easton2, Y Ujike1 1Okayama University Hospital, Okayama, Japan; 2University of Calgary, AB, Canada Critical Care 2015, 19(Suppl 1):P551 (doi: 10.1186/cc14631) Introduction Sleep disruption and deprivation is a continuing problem in the ICU. Strategies to improve sleep are confounded by diffi culties in monitoring and measuring sleep in the ICU; traditional polysomnography cannot be utilized. Practical, non-intrusive diagnostic monitoring of sleep is required. The aims were to test two new ambulatory sleep diagnostic devices to monitor sleep in the ICU, compare sleep data generated by the diff erent devices, and characterize sleep in the ICU. p Methods The devices were: Watch PAT 200 (Itamar), simple wristwatch style, employing peripheral arterial tone and actigraphy to evaluate sleep time and sleep stage by an automatic algorithm (PAT device); and ALICE PDx (Respironics Philips), miniature polysomnographic device utilizing EEG and EMG recordings, requiring post-study sleep technician scoring (PSG device). Nineteen ICU patients provided informed consent (mean age 37 years, two female). Diagnosis of most patients was trauma. Device technical problems terminated one ALICE PDx study and three Watch PAT study; one patient revoked consent. Therefore, 14 patients were recorded successfully in a private room in the ICU, while simultaneously wearing both devices, from 20:00 to 06:00. No patient received sedation. Subjective sleep quality was estimated by the visual analog scale. i References 1. Myhren H, et al. Crit Care. 2010;14:R14. 1. Myhren H, et al. Crit Care. 2010;14:R14. 2. Zimond M, et al. Behav Res Ther. 2003;41:1489-96. 2. Zimond M, et al. Behav Res Ther. 2003;41:1489-96. Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay Six weeks after discharge they received a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale (HADS) questionnaire [2]. Patients were asked to return the questionnaire prior to their visit. All data were analyzed and if the HADS score indicated a clinically signifi cant anxiety or depression, patients were referred to a psychologist for further analyses and treatment. All patient data were analyzed retrospectively. Results Seventy-nine patients, 54 men and 43 women, mean age 57 years. Median APACHE II and IV was 18 and 60 respectively. Median ICU and hospitals days were 9 and 20 respectively. Seventy-six percent were mechanically ventilated with a median of 5 days. Median time after ICU discharge to aftercare visit was 165 days. Patients were divided into three categories: 1, no HAD (45.4%); 2, possible HAD (9.3%); and 3, clinically signifi cant HAD (45.4%). Women compared with men showed signifi cantly more HAD symptoms (26.8% vs. 18.6%, P <0.05). Patients with subarachnoid hemorrhage, neurotrauma and multitrauma patients showed more HAD symptoms. Pain, fatigue, muscle weakness, impairment of daily activity dyspnea, and hoarseness were signifi cantly associated with clinically signifi cant HAD. No association between age and HAD was found. Diagnosis at ICU admission, length of stay, severity of illness, delirium and use of sedatives were not associated with HAD. Conclusion Prevalence of clinically signifi cant post-ICU HAD was 45.4%. Female sex and post-ICU physical complaints – pain, fatigue, muscle weakness, impairment in daily activities, hoarseness and dyspnea – were signifi cantly associated with HAD. Conclusion In cardiac ICU patients, trait anxiety mediates the infl uence of cumulative life stress on the occurrence of PTSD and depression symptoms. Further prospective research is necessary to establish these factors as reliable measures for the early identifi cation of ICU patients at risk for stress-related psychopathology. Somatic complaints after ICU survival: results of a post-ICU aftercare program p g D Ramnarain, W Schapendonk, I Gnirrip, G Van der Nat, A Rutten, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P554 (doi: 10.1186/cc14634) p g D Ramnarain, W Schapendonk, I Gnirrip, G Van der Nat, A Rutten, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P554 (doi: 10.1186/cc14634) Introduction Critical illness today is well recognized as being associated with new or worsening physical impairment, diminished mental health and cognitive dysfunction. We studied the scope of somatic complaints in ICU survivors 4 to 6 months after ICU treatment. Introduction Critical illness today is well recognized as being associated with new or worsening physical impairment, diminished mental health and cognitive dysfunction. We studied the scope of somatic complaints in ICU survivors 4 to 6 months after ICU treatment. yp yp p Conclusion Irrespective of defi nition using PCL-S or DSM-IV mapping, PTSD was identifi ed in no more than one in 10 survivors of critical illness at either 3 or 12 months post ICU, which is still nearly double the US population past-year PTSD prevalence. In ICU survivors with moderate probability PTSD by PCL-S, the CAPS gold-standard interview is challenging to complete and adds only a small number of diagnoses. However, two in fi ve ICU survivors will develop PTSD subtypes of avoidance or hyperarousal, which both occur twice as frequently as the intrusion subtype. Targeting predominant PTSD subtypes may help optimize treatment strategies for the ICU survivor, such as prolonged exposure and eye movement desensitization and reprocessing for those with the avoidance subtype, and pharmacologic antidepressants targeting the sympathetic nervous system to produce anxiolysis for those with the hyperarousal subtype. Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013, for 5 or more days, were invited to visit our ICU aftercare clinic. Six weeks after ICU discharge a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale questionnaire [1] and Impact of Event Scale Revised questionnaire [2], was sent. Patients were asked to return the questionnaires before visiting our clinic. The main purpose of the post- ICU aftercare was to screen for somatic complaints, mental health and cognitive dysfunction. If necessary, further examination or treatment was advised. All data were retrospectively analyzed. y y Results Ninety-seven patients visited our aftercare program in 2013. P553 Post-traumatic stress disorder after ICU discharge: results of a post-ICU aftercare program D Ramnarain, I Gnirrip, W Schapendonk, A Rutten, G Van der Nat, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P553 (doi: 10.1186/cc14633) Results Both devices calculated total sleep time (TST), but the results were signifi cantly diff erent (P <0.05), with mean TST reported as 443.07 and 270.8 minutes for PAT and PSG devices. VAS correlated tightly with TST calculated by the PSG device (r = 0.559, P <0.05). Both devices were able to successfully discern diff erent sleep stages, summarized as light sleep, deep sleep, and REM. Measurements of sleep stage were generally in agreement between the two devices; REM sleep time correlated strongly between PAT and PSG devices (P <0.05). Sleep stage Introduction Patients who survive ICU treatment may experience psychological distress for some time after discharge from the ICU. In the literature the reported prevalence of post-traumatic stress disorder (PTSD) ranges from 5 to 64% [1]. We studied PTSD symptoms in relation to ICU factors, demographic data and physical complaints reported by patients 4 to 6 months after ICU discharge. Introduction Patients who survive ICU treatment may experience psychological distress for some time after discharge from the ICU. In the literature the reported prevalence of post-traumatic stress disorder (PTSD) ranges from 5 to 64% [1]. We studied PTSD symptoms in relation to ICU factors, demographic data and physical complaints reported by patients 4 to 6 months after ICU discharge. S193 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013 for more than 5 days were invited to visit our ICU aftercare clinic. Six weeks after discharge a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale questionnaire and Impact of Event Scale (IES-R) questionnaire, was sent to the patient. Patients were asked to return the questionnaires prior to visiting the aftercare clinic. All data were analyzed and if the IES-R score indicated a possible PTSD, patients were referred to a psychologist for further analyses and treatment. All patient data were analyzed retrospectively. y Referencefi Referencefi 1. Griffi ths J, et al. Intensive Care Med. 2007;33:1506-18. , yp g Results Of the 180 eligible participants at 3 months, PTSD was identifi ed in 10 (6%) using PCL-S scores and 15 (8%) using DSM-IV mapping of the PCL-S. Of the 160 eligible participants at 12 months, PTSD was identifi ed in two (1%) using PCL-S scores and 10 (6%) using DSM-IV mapping of the PCL-S. Of those eligible for CAPS assessments, at 3 months only 13 of 24 (54%) interviews were completed resulting in three extra PTSD diagnoses, and at 12 months only six of 22 (27%) interviews were completed resulting in two extra PTSD diagnoses. At 3 and 12 months, the intrusion subtype was present in 25 (14%) and in 25 (16%), the avoidance subtype was present in 78 (43%) and 60 (38%), and the hyperarousal subtype was present in 82 (46%) and 71 (44%). Conclusion Irrespective of defi nition using PCL-S or DSM-IV mapping, PTSD was identifi ed in no more than one in 10 survivors of critical illness at either 3 or 12 months post ICU, which is still nearly double the US population past-year PTSD prevalence. In ICU survivors with moderate probability PTSD by PCL-S, the CAPS gold-standard interview is challenging to complete and adds only a small number of diagnoses. However, two in fi ve ICU survivors will develop PTSD subtypes of avoidance or hyperarousal, which both occur twice as frequently as the intrusion subtype. Targeting predominant PTSD subtypes may help optimize treatment strategies for the ICU survivor, such as prolonged exposure and eye movement desensitization and reprocessing for those with the avoidance subtype, and pharmacologic antidepressants targeting the sympathetic nervous system to produce anxiolysis for those with the hyperarousal subtype. Results Of the 180 eligible participants at 3 months, PTSD was identifi ed in 10 (6%) using PCL-S scores and 15 (8%) using DSM-IV mapping of the PCL-S. Of the 160 eligible participants at 12 months, PTSD was identifi ed in two (1%) using PCL-S scores and 10 (6%) using DSM-IV mapping of the PCL-S. Of those eligible for CAPS assessments, at 3 months only 13 of 24 (54%) interviews were completed resulting in three extra PTSD diagnoses, and at 12 months only six of 22 (27%) interviews were completed resulting in two extra PTSD diagnoses. y Referencefi At 3 and 12 months, the intrusion subtype was present in 25 (14%) and in 25 (16%), the avoidance subtype was present in 78 (43%) and 60 (38%), and the hyperarousal subtype was present in 82 (46%) and 71 (44%).i P553 The Pearson chi-squared test was used to compare groups and Cramer’s V analyses was used to examine strength of the association between groups. activity, pain and hoarseness were associated signifi cantly with PTSD and HAD. There was no signifi cant diff erence in somatic complaints between men and women. Conclusion Somatic complaints after ICU discharge are frequently reported in our post-ICU aftercare patients, infl uencing daily performance and quality of life. Patient-centered research and treatment focusing on somatic complaints is of great importance. References 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. Results Seventy-nine patients, 54 male and 43 women, with mean age 57 years. Median APACHE II and APACHE IV were 18 and 60 respectively. Median ICU days and hospital days were 9 and 20 respectively. Seventy- six percent of patients were mechanically ventilated with a median of 5 days. Median time of ICU discharge to aftercare visit was 165 days. Delirium occurred in 22 (22.7%) patients during ICU treatment. The prevalence of PTSD was 43.3% and was most seen in patients after subarachnoid hemorrhage (SAH) (28.6%). Pain, muscle weakness, fatigue, impairment in daily activity, dyspnea, and hoarseness reported during the ICU aftercare clinic visit were signifi cantly associated with PTSD. There was no signifi cant diff erence in men and women. Sedation, opiates, benzodiazepine, inotropic medication and delirium during ICU treatment were not associated with higher prevalence of PTSD. None of the other demographic data analyzed were signifi cantly associated with PTSD. Post-traumatic stress disorder prevalence and subtypes among survivors of critical illness Post-traumatic stress disorder prevalence and subtypes among survivors of critical illness M Patel1, J Jackson1, A Morandi2, T Girard1, C Hughes1, A Kiehl1, J Thompson1, R Chandrasekhar1, E Ely1, P Pandharipande1 1Vanderbilt University, Nashville, TN, USA; 2Ancelle Hospital, Cremona, Italy Critical Care 2015, 19(Suppl 1):P555 (doi: 10.1186/cc14635) M Patel1, J Jackson1, A Morandi2, T Girard1, C Hughes1, A Kiehl1, J Thompson1, R Chandrasekhar1, E Ely1, P Pandharipande1 1Vanderbilt University, Nashville, TN, USA; 2Ancelle Hospital, Cremona, Italy Critical Care 2015, 19(Suppl 1):P555 (doi: 10.1186/cc14635) Introduction Among North American survivors of critical illness, we aim to describe the prevalence of post-traumatic stress disorder (PTSD), and its subtypes of intrusion, avoidance, and hyperarousal. Methods In this prospective, observational, multicenter cohort study from 2009 to 2010, we screened adults (age ≥18 years) with new- onset respiratory failure, cardiogenic shock, or septic shock, who were admitted to medical and surgical ICUs in four facilities. At 3-month and 12-month follow-ups, high probability of PTSD was defi ned by 17-symptom PTSD Checklist – Event Specifi c Version (PCL-S) score ≥50. Also, PCL-S responses were mapped onto DSM-IV criteria for PTSD. To augment PTSD identifi cation, those with a moderate probability of post-ICU PTSD (PCL-S score ≥35) were further confi rmed with the Clinician Administered PTSD Scale (CAPS) structured interview. Moderate or greater symptoms for each PTSD subtype of intrusion, avoidance, and hyperarousal were categorized. Conclusion Prevalence of PTSD was 43.3% and most seen in patients after SAH, refl ecting the majority of patients treated in our ICU. PTSD was associated signifi cantly with pain, muscle weakness, fatigue, dyspnea, hoarseness and impairment of daily activity after a median 165 days post ICU treatment. Somatic complaints after ICU survival: results of a post-ICU aftercare program Median time after ICU discharge and visit to our after care clinic was 165 days. Twenty-fi ve patients died after ICU discharge. Fifty-four patients were excluded because of various reasons; that is, language barrier, psychiatric illness, mental handicap, hospital admittance elsewhere, great distance. Seventy patients (81.4%) had somatic complaints infl uencing daily performance and quality of life. Fatigue (74.4%), muscle weakness (48.8%), dyspnea (34.9%), impairment of daily activity (81.4%), pain (38.4%) and weight loss (33.3%) were the most frequently reported complaints. Pain was most reported in patients with subarachnoid hemorrhage (27.3%), multitrauma (15.2%) and pneumonia (12.1%). Pain was most localized in the head (15.6%), one or both legs (15.6%), back (10.9%), shoulder (9.3%), hip (9.3%) and thorax (6.3%). Muscle weakness, fatigue, dyspnea, impairment of daily Physiotherapy in the ICU: an evidence-based, expert-driven, practical statement J Sommers1, R Engelbert2, D Dettling1, R Gosselink3, P Spronk4, J Horn 1, F Nollet1, M Van der Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2School of Health, Amsterdam, the Netherlands; 3KU Leuven, Belgium; 4Gelre Hospital, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P558 (doi: 10.1186/cc14638) J Sommers1, R Engelbert2, D Dettling1, R Gosselink3, P Spronk4, J Horn 1, F Nollet1, M Van der Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2School of Health, Amsterdam, the Netherlands; 3KU Leuven, Belgium; 4Gelre Hospital, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P558 (doi: 10.1186/cc14638) g q y Results A total of 1,743 papers were retrieved, of which 18 studies were eligible for inclusion in the review. Studies had a combined population of 1,970 patients admitted to 38 ICUs from Europe, Asia and North America. Eleven studies were randomized controlled trials (RCTs). Interventions were classifi ed as four groups – music; therapeutic touch; diary and psychotherapeutic interventions. Ten studies found that music interventions were eff ective in the short term; however, follow- up results were limited and some studies were low quality. There was moderate quality evidence from three studies for the eff ectiveness of diary interventions, with medium-term follow-up results. There was mixed-quality evidence for therapeutic touch interventions in the short term from three studies. The two psychotherapeutic interventions studied were of moderate quality, and one showed promising results at 12-month follow-up.fi Introduction Evidence-based, expert-driven, practical statements improve quality and eff ectiveness of the diagnostic and therapeutic process of patient care. Although the eff ectiveness of physiotherapy treatment strategies in ICU patients has been described, statements or guidelines of physiotherapy for ICU patients are not available [1]. Guidelines on safety management and on the diagnostic and therapeutic process may support and guide clinical decision-making leading towards evidence-based tailored care. The aim of this study was to develop an evidence-based statement for the physiotherapy treatment of ICU patients with recommendations for eff ective and safe diagnostic assessment and intervention strategies. Conclusion The evidence for the effi cacy of nonpharmacological interventions to reduce short-term or long-term stress in intensive care patients was of low to moderate quality. Studies included mainly short- term and medium-term follow-up. This highlights the need for larger- scale, better-quality RCTs with longer-term outcome measurement. However, the results indicate that nonpharmacological, including psychological, approaches are likely to be benefi cial for reducing short- term or long-term stress in intensive care patients. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 particularly those of working age. More work is required to understand optimal rehabilitation pathways in this patient group. References particularly those of working age. More work is required to understand optimal rehabilitation pathways in this patient group. References effi cacy of such interventions. Previous work has shown that intensive care patients undergo many stressful experiences, which can aff ect their long-term psychological well-being. Studies have demonstrated a high prevalence of depression, anxiety or post-traumatic stress disorder after intensive care admissions. Methods A systematic review was carried out according to the Prisma statement. A search was conducted of Medline, Embase and Psychinfo databases. Inclusion criteria included studies of populations of adult patients in mixed or general ICUs. No study designs were excluded, but studies that focused on specifi c disease states were excluded. Included studies were assessed for risk of bias, using a quality checklist. P557 P557 Utilisation of existing community rehabilitation services by critical care survivors CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) Utilisation of existing community rehabilitation services by critical care survivors CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) l Results Three expert-based relevant clinical questions were formulated within the physiotherapy clinical reasoning process and were classifi ed according to the International Classifi cation of Functioning, Disability and Health. In a systematic literature search, 129 studies were identifi ed and assessed for methodological quality and classifi ed according to the level of evidence. The fi nal Evidence Statement consisted of recommendations for physiotherapy in ICU patients including safety criteria, a core set of instruments to assess impairments and activity restrictions and eff ective interventions. CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) Introduction Patients recovering from critical illness suff er many physical and psychological problems during their recovery, including muscle weakness, fatigue, signs and symptoms of PTSD, anxiety and depression [1]. At present, specialist intensive care follow-up and rehabilitation is inconsistent and in many geographical areas is nonexistent. As a result, many survivors of critical illness will require using existing community rehabilitation services [2]. The aim of this present service evaluation was to understand the utilisation of community rehabilitation services by critical care survivors. f Conclusion The Evidence Statement for physiotherapeutic diagnostics and intervention in ICU patients will contribute to the quality of clinical practice by supporting the clinical decision-making process. References 1. Kayambu G, Boots R, Paratz J. Physical therapy for the critically ill in the ICU: a systematic review and meta-analysis. Crit Care Med. 2013;41:1543-54. 2. Burgers JS, van Everdingen JJE. Evidence-based richtlijnontwikkeling in Nederland: the EBRO-platform. Ned Tijdschr Geneeskd. 2004;148:2057-9. Methods A database of acute referrals to community rehabilitation services was retrospectively analysed from 1 May 2014 to 31 October 2014. Age, referring specialty and reason for referral for rehabilitation were documented. This database was cross-checked with the critical care database in Glasgow Royal Infi rmary to identify which individuals had been admitted to critical care during their admission. Physiotherapy in the ICU: an evidence-based, expert-driven, practical statement Methods For the development of this evidence statement, we used the EBRO method, as recommended by the Dutch Evidence Based Guideline Development Platform [2]. This method consists of the identifi cation of clinically relevant research questions, followed by a systematic literature search, quality assessment, and summary of the evidence eventually leading to establishing of concept and fi nal recommendations based on feedback from experts. The fi nal recommendations were prepared according to this methodical approach and summarized in fi gures, fl owcharts and appendices. Nonpharmacological interventions to reduce short-term or long-term psychological stress in ICU patients: a systematic review D Wade1, Z Moon2, S Windgassen2, J Weinman2 1University College Hospital, London, UK; 2Kings College London, UK Critical Care 2015, 19(Suppl 1):P556 (doi: 10.1186/cc14636) Introduction A systematic review was performed of studies of nonpharmacological interventions aiming to reduce short-term or long-term stress in intensive care patients, as little is known about the S194 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) i Results A total of 697 hospitals records were analyzed. Patients had on average (SD) 67.8 (13.1) years and the majority of them were males (57%). Our results revealed that 65% of patients in the CICU received phase 4 and 43% of patients in the CICU received phase 5 of the early mobilization protocol. No diff erences in the proportion of patients receiving phase 4 or 5 were found among arrhythmia, coronariopathies and congestive heart failure groups. The only diff erence found was between congestive heart failure group and other cardiovascular pathologies (P <0.001). The congestive heart failure group was mobilized 5.6 times (95% CI: 2.7 to 11.5) and 3.2 times (95% CI: 1.7 to 5.7) more than the other cardiovascular pathologies group in phase 4 and 5, respectively. Introduction Increasing cost is an important issue in critical care medicine. We tried to analyze in a level 3 care ICU in Kolkata of a tertiary care hospital whether the diff erent payment options (self-paying vs. insurance/corporate paying) do affect the outcome in the critically ill insurance/corporate paying) do aff ect the outcome in the critically ill. Methods Our prospective study included 1,520 patients admitted consecutively to a level 3 care ICU for a period of 20 months. Readmitted patients during the same period were excluded. Payment method was documented for all and divided into two groups as self-paying and insurance/corporate paying. Outcome assessment was done using the APACHE IV model for all cases. Demographic data, number of observed deaths, predicted mortality rate (PMR), standardized mortality ratio (SMR), average length of stay (ALOS), predicted length of stay, and number of discharge against medical advice (DAMA) were documented for each group. Statistical analysis was carried out using unpaired Student t test and P <0.05 was considered signifi cant. Conclusion A considerable proportion of patients was mobilized without any serious complications in the CICU. Our fi ndings suggest that patients diagnosed with arrhythmia, coronariopathies and congestive heart failure can be equally mobilized in an ICU. p gi Results Of 1,520 patients, 995 (65.46%) cases were self-paying while 525 (34.54%) cases were insurance/corporate paying. P559 E l Early mobilization according to diagnosis in a Brazilian coronary ICU GS Zavanelli1, SA Padulla1, MR Franco1, RZ Pinto1, LL Faccioli1, DN Barbosa1, DT Neves2, CE Bosso2 1Universidade Estadual Paulista – UNESP, Presidente Prudente, Brazil; 2Instituto do Coração de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P559 (doi: 10.1186/cc14639) g Results Over this 6-month period 769 patients were referred from their parent specialty for community rehabilitation in North East Glasgow. Thirty-three of the 769 patients (4.3%) referred had a critical care stay during their admission. Of these, eight patients were referred for rehabilitation by orthopaedics, eight by medicine for the older patients, 11 from acute medicine and the remaining six from other specialties. Six of the 769 patients who had a critical care admission were of working age (<1%). Two individuals were admitted to critical care following trauma whilst four had complex social needs prior to their critical care admission. This included an individual with a high body mass index. None of the individuals of working age were referred as a consequence of their critical care stay. Introduction Early mobilization has been advocated to improve muscle function and, consequently, the patient quality of life after discharge. Nevertheless, few studies have explored it in a coronary ICU (CICU). The aims of the present study were to describe the use of an early mobilization protocol in a CICU and to investigate whether diff erent groups of diagnoses respond similarly to this protocol. Introduction Early mobilization has been advocated to improve muscle function and, consequently, the patient quality of life after discharge. Nevertheless, few studies have explored it in a coronary ICU (CICU). The aims of the present study were to describe the use of an early mobilization protocol in a CICU and to investigate whether diff erent groups of diagnoses respond similarly to this protocol. Conclusion This service evaluation demonstrates that very few critical care survivors are referred to community rehabilitation services, Methods This is a retrospective observational study conducted in a medium-sized hospital located in the city of Presidente Prudente, S195 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Brazil. P559 E l The early mobilization protocol consists of fi ve phases: 1 – passive exercises for the unconscious patient; 2 – active exercises associated with respiratory exercises (patient lying on the bed); 3 – phase 2 exercises with the patient sitting on the bed; 4 – phase 2 exercises with the patient sitting on a chair or in a standing position; 5 – phase 4 exercises plus walking. All hospital records from patients, between September 2013 and August 2014, were included in this study. Data extracted from hospital records were: age, gender, diagnosis (arrhythmia, coronariopathies, congestive heart failure and other pathologies), length of stay, number of discharge and number in each phase of the early mobilization protocol. Pearson chi-square test was used to compare the number of mobilizations (phase 4 and 5) per group of diagnoses. Odds ratios were calculated for those comparisons found to be statistically signifi cant (P <0.05). with mortality for pulmonary artery catheter, platelet transfusion and vasoactive drug infusion (one drug) (P >0.05). Conclusion In this large retrospective multicenter study, the TISS item associated with the highest risk of death was cardiac arrest and/or countershock. Unexpectedly, the independent eff ect of emergency admission was of comparable magnitude in terms of impact on hospital mortality. Of these, in-ICU cardiac arrest might be amenable to preventive measures and should be studied further. P561 P560 Need for therapeutic interventions as a predictor of mortality in intensive care Need for therapeutic interventions as a predictor of mortality in intensive care I Ef dij 1 R R j1 S H 2 MB Sk if 1 M R i ik i 3 Need for therapeutic interventions as a predictor of mortality in intensive care I Efendijev1, R Raj1, S Hoppu2, MB Skrifvars1, M Reinikainen3 1HUS, Helsinki, Finland; 2TAYS, Tampere, Finland; 3PKKS, Joensuu, Finland Critical Care 2015, 19(Suppl 1):P560 (doi: 10.1186/cc14640) I Efendijev1, R Raj1, S Hoppu2, MB Skrifvars1, M Reinikainen3 1HUS, Helsinki, Finland; 2TAYS, Tampere, Finland; 3PKKS, Joensuu, Finland Critical Care 2015, 19(Suppl 1):P560 (doi: 10.1186/cc14640) Introduction Various therapeutic interventions needed in critical care may refl ect a high risk of death. We evaluated associations between commonly used interventions and hospital mortality in Finnish ICU patients. Methods We retrieved data on adult patients treated in Finnish ICUs between 2003 and 2013 from the Finnish Intensive Care Consortium database. We used the Therapeutic Intervention Scoring System (TISS-76) for categorizing ICU interventions and the Simplifi ed Acute Physiology Score (SAPS II) for quantifying severity of illness. We excluded readmissions, patients with missing outcome, SAPS II and TISS data. We also excluded very common interventions (arterial line, bolus intravenous medication), very rare ones (prevalence <1%), and interventions only applicable in specifi c populations (intracranial pressure monitoring, intra-aortic balloon assist). We grouped several TISS categories when applicable. We performed a backward stepwise binary logistic regression analysis with TISS items to assess the impact of each intervention on hospital mortality (expressed as odds ratio (OR) with 95% confi dence intervals (CIs)). Age, admission type, and SAPS score (minus age and admission type scores) were adjusted for in the multivariate analysis. P562 P561 Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) Payment options: do they aff ect outcome in the critically ill A Kar, A Datta In the self-paying group, mean age was 59.65 years ± 17.26 SD (median 62), APACHE IV score mean was 62.50 ± 33.61 SD (median 57), average LOS 4.67 days ± 4.29 SD (median 3), PMR was 22.71, 226 observed deaths, 85 cases of DAMA, and SMR was 1.00 (CI = 0.87 to 1.14). In the insurance/ corporate-paying group, mean age was 61.75 years ± 17.19 SD (median 65), APACHE IV score mean was 58.53 ± 32.94 SD (median 54), average LOS was 5.64 days ± 5.61 SD (median 4), PMR was 21.26, 113 observed deaths, six cases of DAMA, and SMR was 1.01 (CI = 0.83 to 1.21). In the two compared groups, predicted mortality and SMR were not statistically signifi cant (P = 0.2808); however, ALOS in the insurance/ corporate paying group was signifi cantly higher than the self-paying group (P = 0.0002), mean age of the insurance/corporate paying group was signifi cantly higher than the self-paying group (P = 0.02), and incidence of DAMA is signifi cantly higher in the self-paying group (8.54%) as compared with insurance/corporate paying group (1.14%). Root-cause analysis showed DAMA cases are mostly fi nancial (>95%). Conclusion Statistically signifi cant diff erences in ALOS and DAMA in the two groups are probably due to cost of healthcare not aff ordable to all. Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P562 (doi: 10.1186/cc14642) Introduction Source of admission to the ICU is of importance. We tried to identify the diff erent sources of ICU admission to a level 3 ICU of a tertiary care hospital in Kolkata and analyze whether the overall patient outcome is aff ected by the diff erent sources of admission. Results We identifi ed 161,134 patients eligible for analysis. The multivariate analysis showed that the highest risk for hospital mortality in all patients was associated with cardiac arrest and/or countershock, OR 2.58 (95% CI = 2.43 to 2.73), SAPS II emergency admission, OR 2.52 (95% CI = 2.32 to 2.74), vasoactive drug infusion (>1 drug), OR 1.66 (95% CI = 1.59 to 1.73) and blood transfusion (a combined TISS item), OR 1.53 (95% CI = 1.44 to 1.63). TISS items associated with the lowest risk of mortality in general population were: active anticoagulation, OR 0.51 (95% CI = 0.49 to 0.53), induced hypothermia, OR 0.68 (95% CI = 0.62 to 0.74) and measurement of cardiac output by any method, OR 0.87 (95% CI = 0.83 to 0.91). All aforementioned associations were statistically signifi cant (P <0.001). There was no notable association ff Methods Our prospective study included 2,056 patients admitted to a level 3 care ICU over a period of 2 years. Numbers of readmissions were not considered. ICU outcome was analyzed using the APACHE IV model and source of admission to the ICU was documented as either from emergency (ER), from the fl oor or from other hospital. Analysis was carried out between diff erent groups based on admission using unpaired Student t test and P <0.05 was considered signifi cant. Number of ventilations and the mortality rate in each group were also documented. S196 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Of 2,056 admissions, 1,223 cases (59.48%) were from ER, 809 cases (39.35%) were from fl oor and 24 cases (1.16%) were from other hospitals. Association between out-of-hours discharge and mortality in adult patients leaving critical care Association between out-of-hours discharge and mortality in adult patients leaving critical care S Edie, K Burt, J Paddle Royal Cornwall Hospital, Truro, UK Critical Care 2015, 19(Suppl 1):P564 (doi: 10.1186/cc14644) Introduction Out-of-hours (OOH) discharge from critical care is associated with a signifi cantly increased mortality rate in Australasia [1]. In the UK, daytime discharges from critical care are considered a core standard [2]. We sought to assess the impact of OOH discharge from critical care on mortality in a large general ICU, where operational pressures appear to have led to a high rate of OOH discharges. Methods Retrospective data for all patients admitted to our ICU from April 2007 to September 2014 were recorded, using routinely collected data from our databases. Adult patients (>15 years) discharged from their fi rst ICU admission during each hospital stay (episode) were included. Patients that died on the unit and those discharged for palliative care were excluded. Patients transferred to other centres were no longer subject to discharge within our control and were therefore also excluded. Patients discharged directly home from ICU were excluded. We defi ned OOH discharges as those occurring between 22:00 and 06:59, a standard defi nition in UK practice. Mortality status at the time of hospital discharge for each episode was used. We also recorded the readmission rate to ICU. The relative risk (RR) for OOH mortality and readmission was calculated. Statistical signifi cance was accepted at P <0.05. Conclusion The severity of illness index in patients admitted to the ICU from fl oors is signifi cantly higher than emergency admissions. Overall outcome for patients transferred to the ICU from the fl oor is worse based on mortality rate, SMR, and ALOS when compared with the emergency group. Readmission to the ICU: is it a big concern? An analysis A Ahmed, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P563 (doi: 10.1186/cc14643) Readmission to the ICU: is it a big concern? An analysis A Ahmed, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P563 (doi: 10.1186/cc14643) Results Of 4,476 index cases, 714 died on the unit and 80 were discharged for palliative care. A total of 490 patients were excluded for transfer to other centres and discharge directly home. Data were missing for three patients, which left 3,189 records for analysis. In total, 2,711 patients were discharged during daytime hours, of which 145 (5.35%) died. A total of 478 patients were discharged at night, 40 died (8.37%). The RR for OOH mortality was 1.56 (95% CI = 1.12 to 2.19, P = 0.0091). Readmission rate was 5.2% by day, 6.1% at night. The RR for readmission was 1.17 (95% CI = 0.79 to 1.72, P = 0.436). Introduction Readmission to the ICU is an important quality indicator of ICU care. We conducted a prospective study in a level 3 care ICU in Kolkata of a tertiary care hospital to analyze whether there are overall outcome diff erences when comparing the readmission group with the entire group. Conclusion Our data demonstrate an association between critical care discharge time and mortality, to a statistically signifi cant level. Due to the retrospective observational nature of the study, causation cannot be assumed; however, a number of factors may contribute to the increased risk of harm to patients discharged from the ICU at night. Further work will focus on annual OOH mortality trends, thereby gaining an insight into whether bed occupancy demands impact on the necessity for nighttime discharges. Methods Our prospective study included 2,140 patients admitted to a level 3 care ICU over a period of 1 year. The number of readmissions (n = 85) during the same period was also documented. Readmission was defi ned as all patients who were transferred back to the ICU prior to hospital death/discharge during the above period. ICU outcome was calculated using the predictive APACHE IV model. Payment methods were documented as either self-paying or corporate/insurance paying. A comparison analysis between the entire group with the readmission group was done using unpaired Student t test and P <0.05 was considered statistically signifi cant. Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed In the ER group, mean APACHE IV was 55.03 ± 31.49 SD (median 50), PMR 16.26, observed deaths 198, ALOS 4.78 days ± 4.83 SD (median 3), SMR 0.995 (CI = 0.86 to 1.14), mean age 60.52 years ± 17.63 SD (median 63), 323 ventilations. In the fl oor group, mean APACHE IV was 65.17 ± 34.40 SD (median 60), PMR 27.03, observed deaths 234, ALOS 5.23 days ± 5.22 SD (median 3), SMR 1.07 (CI = 0.94 to 1.21), mean age 61.38 years ± 15.72 SD (median 64), 302 ventilations. In the other hospital group, mean APACHE IV was 55.29 ± 29.82 SD (median 50), PMR 18.0, observed deaths 2, ALOS 6 days ± 5.85 SD (median 3), SMR 0.46 (CI: 0.23 to 0.88), mean age 56.08 years ± 17.79 SD (median 56.5), six ventilations. During analysis, the other hospital group was omitted because of inadequate sample size. There was statistically signifi cant diff erences in APACHE IV (fl oor >ER, P <0.0001), PMR (fl oor >ER, P <0.0001), ALOS (fl oor >ER, P = 0.04) noted between the fl oor and ER groups. Number of ventilations (37.33% vs. 26.4%), SMR (1.07 vs. 0.995), and mortality rate (28.92% vs. 16.19%) were also signifi cantly higher for patients admitted from the fl oor. No signifi cant statistical diff erence was observed in age between two groups (P = 0.26). y References 1. Gantner D, et al. Int Care Med. 2014;40:1528-35. 1. Gantner D, et al. Int Care Med. 2014;40:1528-35. 2. Faculty of Intensive Care Medicine and Intensive Care Society. Core standards for intensive care units (2013). http://www.fi cm.ac.uk/standards. 2. Faculty of Intensive Care Medicine and Intensive Care Society. Core standards for intensive care units (2013). http://www.fi cm.ac.uk/standards. y gi Results In the entire group (n = 2,140), mean APACHE IV was 50.34 ± 31.54 SD (median 42), PMR 15.49, observed deaths 327, ALOS 4.05 days ± 4.55 SD (median 3), SMR 0.99 (CI = 0.88 to 1.1), mean age 60.55 years ± 15.68 SD (median 63), 490 ventilations, 72.71% of patients were self-paying while 27.29% of patients were corporate/insurance paying. In the readmission group (n = 85), mean APACHE IV was 77.16 ± 33.72 SD (median 73), PMR 38.89, observed deaths 42, ALOS 5.23 days ± 4.18 SD (median 4), SMR 1.27 (CI = 0.95 to 1.67), mean age 64.79 years ± 14.40 SD (median 67), 43 ventilations, 75.3% of patients were self- paying while 24.7% of patients were corporate/insurance paying. During comparison between the two groups there were statistically signifi cant diff erences, with the readmission group having signifi cantly higher APACHE IV (P <0.0001), PMR (P <0.0001), ALOS (P = 0.002), age (P = 0.005), and SMR (1.27 vs. 0.99) compared with the entire group. Percentage of patients requiring ventilation (50.59% vs. 22.90%) and mortality rate (49.11% vs. 15.28%) were also signifi cantly higher in the readmission group. Readmission was signifi cantly higher in the self- paying group. Root-cause analysis showed most readmissions were due to deteriorating conditions (desaturation, hypotension, sepsis, arrhythmias); however, it was also associated with cases where transfer policy from the ICU was not followed by stakeholders and fi nancial issues were a cause of early transfer. P566 Determination of brain death for adult patients with ECMO I Ceylan, R Iscimen, E Cizmeci, N Kelebek Girgin, F Kahveci Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P566 (doi: 10.1186/cc14646) f Results The CFA showed a good fi t indicating factorial validity (CFI: 0.97), reliabilities were from α 0.79 to 0.93 and ICCs were signifi cant (~0.20, P <0.001). HLM revealed that unit-level IL of nurses and residents was positively related to PS (b = 0.34, P <0.001). Being a resident and working in a smaller unit also predicted PS. As expected, unit-level PS was negatively related to individual PFC (b = –0.38, P = 0.025). Further predictors of higher PFC were: being a nurse, having more than 5 years of job experience and higher workload. PS mediated the relationship between unit-level IL and individual PFC (indirect eff ect: –0.13, P <0.001). Additional analyses revealed that attendings’ PFC was negatively related to their perception of residents PS (b = –0.44, P = 0.019). Introduction ECMO support in ARDS is an emerging strategy when conventional treatment modalities fail. ECMO has advantages on oxygenation and circulation but also it has some unfavorable eff ects. The most serious complication is brain death due to cerebrovascular hemorrhage. An apnea test is the most important component in confi rming brain death. For patients supported by ECMO, apnea testing remains challenging. Brain-death diagnosis is often made without an apnea test. p Methods We present two cases who receive V-V ECMO support after progression to ARDS. After initiation of ECMO we used sedation to prevent movement and improve adaptation to mechanical ventilation. Also we used anticoagulation with heparin to prevent thromboembolic events and ECMO circuit occlusion. On daily follow-up we noticed that patients had lost their pupil reactions to light. Their sedation was ceased and a computed brain tomography was performed. Both patients had intracerebral hemorrhage. We decided to determine brain death with apnea tests. We increased ECMO blood fl ow and fi O2 and then decreased sweep gas fl ow and disconnected the patient from mechanical ventilation respectively. In one patient we did not see any spontaneous breathing eff orts after carbon dioxide retention. We concluded that the apnea test was successful and confi rmed brain death. P566 On the other hand, we confi rmed the brain death of the other patient with cerebral angiography due to the occurrence of hypoxia and hypotension during apnea testing. p p Conclusion A sense of PS in an ICU team might reduce futile care by increasing the safety of speaking-up behavior of nurses and residents. PS can be enhanced by attending physicians who practice inclusive leadership behavior to foster autonomy and participation of residents and nurses. References 1. Manthous CA, et al. Am J Respir Crit Care Med. 2011;184:17-25. 2. Nembhard IM, et al. J Organ Behav. 2006;27:941-66. End-of-life decisions: how do patients die in the ICU? Introduction One of the main goals of intensive care medicine is to reduce the mortality of critically ill patients. However, it is essential to recognize end-of-life care as an integral component of critical care. Besides survival, the success of intensive care should also include the quality of lives preserved and the quality of dying. The objective of this study was to evaluate the incidence and type of end-of-life decisions (ELD) in critical patients that died in an ICU. y Results We experienced some challenges while determining brain death in patients under ECMO support for ARDS. It is challenging to conduct the apnea testing during ECMO support. Auxiliary tests are required for patients who cannot tolerate the changes needed to conduct the apnea test. With increasing use of ECMO therapies, clinicians may come face to face with more complicated life-ending decisions. p Methods Analysis of all patients included in an ICU running database and who died from 1 November 2013 to 31 October 2014. The following variables were evaluated: age, gender, reason for admission, SAPS II, length of ICU stay and type of ELD. To classify ELD, four concepts were defi ned: ‘Comfort care’, a change from curative therapy to comfort care therapy; ‘Limited therapy’, maintenance of curative therapy but without escalating it (for example, no renal substitution); ‘Decision not to resuscitate’, not to perform advanced life support if cardiac arrest occurs; and ‘Without previous end-of-life decisions’, when there was no prior decision regarding the ELD. Conclusion Current guidelines do not include brain death criteria using supportive therapies such as ECMO and therefore should be updated. References 1. Goswami S, et al. J Cardiothor Vasc Anesth 2013;27:312-4. 2. Jarrah RJ, et al. Pediatr Crit Care Med. 2014;15:38-43. 3. Marasco SF, et al. Heart Lung Circ 2008;17 Suppl:S41–7. 4. Farrah JM, et al. Arch Neurol. 2011;68:1543-9. P567 Results A total of 507 patients were admitted to the ICU and 132 died (26%). Reasons for admission in those who died were septic shock (47%), post cardiac arrest (13%), cardiogenic shock (8%), and nontraumatic brain bleeding (8%). Fifty-three patients (40%) died after a ‘Comfort care’ decision, 28 patients (21%) after ‘Decision not to resuscitate’ and 14 (11%) after a ‘Limited therapy’ decision. Thirty-seven patients died ‘Without previous end-of-life decisions’. However, specifi cally in this group, when looking for individual records, 32 patients died (86%) in the fi rst 48 hours after the admission and four (11%) had evidence of brain death and were organ donors, which leaves one patient (3%) in whom there was no ELD. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU D Schwarzkopf1, J Felfe2, CS Hartog1, F Bloos1 1Jena University Hospital, Jena, Germany; 2Helmut Schmidt University, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P567 (doi: 10.1186/cc14647) Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) , Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) Introduction Severe critical illness requiring treatment in the ICU may have a serious impact on patients and their families. However, optimal follow-up periods are not defi ned and data on health-related quality of life (HRQOL) before ICU admission as well as those beyond 2-year follow-up are limited. The aim of our study was to assess the impact of ICU stay up to 5 years after ICU discharge. Methods We performed a long-term prospective cohort study in patients admitted >48 hours to a medical–surgical ICU. The Short-Form 36 was used to evaluate HRQOL before admission (by proxy within 48 hours after admission of the patient), at ICU discharge and at 1, 2 and 5 years following ICU discharge (all by patients). Changes in HRQOL were assessed using linear mixed modeling. Conclusion Readmission to the ICU was associated with worse outcome in our study group. Lack of adherence to transfer policy by concerned stakeholders was a concern as well as increasing cost of healthcare. Results We included a total of 749 patients (from 2000 to 2007). At 5 years after ICU discharge, 234 patients could be evaluated. After S197 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 correction for natural decline in HRQOL, the mean scores of four dimensions – physical functioning (P <0.001), physical role (P <0.001), general health (P <0.001) and social functioning (P = 0.003) – were still signifi cantly lower 5 years after ICU discharge compared with their preadmission levels, although eff ect sizes were small (<0.5). Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study Conclusion Five years after ICU discharge, survivors still perceived a signifi cantly lower HRQOL than their preadmission HRQOL (by proxies), and that of an age-matched general population. Importantly however, after correction for natural decline, the eff ect sizes were small suggesting that patients regain their age-specifi c HRQOL 5 years after their ICU stay. correction for natural decline in HRQOL, the mean scores of four dimensions – physical functioning (P <0.001), physical role (P <0.001), general health (P <0.001) and social functioning (P = 0.003) – were still signifi cantly lower 5 years after ICU discharge compared with their preadmission levels, although eff ect sizes were small (<0.5). report less perceived futile care (PFC). We also expected that attending physicians’ inclusive leadership (IL), which invites nurses’ and residents’ participation [2], would decrease PFC and that PS mediates this relationship. Methods The hypotheses were tested in a cross-sectional, multicenter paper-and-pencil survey addressing medical staff on participating ICUs. A total of 22 ICUs and four intermediate care units were included in the sample and 73 attendings, 147 residents and 659 nurses participated in the study (52% participation). Psychometric properties were tested by confi rmatory factor analysis (CFA), Cronbach’s α and intraclass correlations (ICC). A series of hierarchical linear models (HLM) were conducted to test the study hypotheses separately among nurses/residents and attendings. IL and PS were entered as unit-level predictors (mean values per unit). Covariates were demographics, working hours per week, workload and unit size (number of staff ). Mediation eff ects were tested.i gf Conclusion Five years after ICU discharge, survivors still perceived a signifi cantly lower HRQOL than their preadmission HRQOL (by proxies), and that of an age-matched general population. Importantly however, after correction for natural decline, the eff ect sizes were small suggesting that patients regain their age-specifi c HRQOL 5 years after their ICU stay. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU D Schwarzkopf1, J Felfe2, CS Hartog1, F Bloos1 1Jena University Hospital, Jena, Germany; 2Helmut Schmidt University, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P567 (doi: 10.1186/cc14647) Introduction Psychological safety (PS), for example safety of speaking up, fosters team learning and prevents treatment errors on the ICU [1]. Since speaking up might also prevent excessive and inappropriate (futile) care for patients, we hypothesized that teams with higher PS Conclusion In this study, ‘Comfort care’ was the main ELD, which is in line with the concept that ELD are essential to ensure that care provided is S198 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 examine the association of palliative care screening criteria with adverse patient outcomes. consistent with quality of life and death. The apparent large proportion of patients ‘Without previous end-of-life decisions’ was due to patients who died in the fi rst 48 hours after ICU admission corresponding to conditions refractory to treatment. Additionally, this study also draws our attention to better plan ICU admissions and hospital outreach in order to reduce early ICU mortality. consistent with quality of life and death. The apparent large proportion of patients ‘Without previous end-of-life decisions’ was due to patients who died in the fi rst 48 hours after ICU admission corresponding to conditions refractory to treatment. Additionally, this study also draws our attention to better plan ICU admissions and hospital outreach in order to reduce early ICU mortality. Methods We performed an observational unicentric study on a 12-bed, medical emergency department intensive care unit (EDICU). A three- item palliative care screen was developed from consensus reports. A senior critical care physician screened patients upon admission using these questions during a 10-week period. The questions were: does this patient suff er from a life-limiting disease (end-stage lung, liver, heart or kidney disease, severe neurological disability, extreme frailty, locally advanced or metastatic cancer, advanced-stage AIDS). If the answer to the fi rst question is yes, we proceed to the next one: do you believe this patient will survive to hospital discharge? Answers to those questions were recorded, SAPS III was calculated and all patients were followed until death, discharge or transfer to another center. Diff erences in mortality and SAPS III score between groups were examined using a Student’s t test. Proportions were compared using chi-square test. References Table 1 (abstract P569). Respondents advocating withdrawal for the patient Withdrawal self (%) Withdrawal family (%) Day 3 71 67 Day 7 83 76 Day 28 96 88 Day 42 98 97 Table 1 (abstract P569). Respondents advocating withdrawal for the patient 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. Parents’ return to the hospital after the death of their children: importance of palliative care after death p p G Halal, PL Lago, J Piva, M Halal p g Critical Care 2015, 19(Suppl 1):P572 (doi: 10.1186/cc14652 Introduction To analyze the perception of parents regarding their return to the hospital where their children died to participate in a conversation with doctors and to analyze the feelings of parents about their participation in a study evaluating the care provided in the moments leading up to the death of children. p Withdrawal self (%) Withdrawal family (%) Day 3 16 10 Day 7 23 15 Day 14 25 19 Day 42 42 29 Methods A descriptive exploratory qualitative study. The study sites were the pediatric ICUs of the Hospital São Lucas and Hospital de Clinicas de Porto Alegre. Fifteen parents of children who died in the PICUs studied participated in the study. Data collection occurred in 2010 and was conducted through semistructured interviews. Data were analyzed using thematic content analysis. The research was approved by the research ethics committees of both hospitals. Conclusion Of the ICU physicians who would withdraw care for their patient, the majority would also want the same for themselves. The disparity between decision to continue to treat the patients versus treating self or family increased with increasing length of stay. Reference Results The ability to return to the hospital and talk to medical assistants was considered by parents as a positive and enlightening opportunity. Parents who participated in the study understood this moment as an opportunity to be heard and demonstrated the intention to contribute with their experiences in order to improve care in the hospitals studied. Conclusion We conclude that there is a need to implement measures to provide palliative care to parents after the death of their children. It is necessary to consider the possibility of providing families with follow-up meetings with the multidisciplinary team after the death of children. 1. Korones DN. What would you do if it were your kid? N Engl J Med. 2013;369:1291-3. 1. Piva J, Lago P, Othero J, Garcia PC, Fiori R, Fiori H, et al. Evaluating end of life practices in ten Brazilian paediatric and adult intensive care units. J Med Ethics. 2010;36:344-8. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU P <0.05 was considered statistically signifi cant. P569 Do intensivists prognosticate patients diff erently from themselves or their loved ones? Do intensivists prognosticate patients diff erently from themselves or their loved ones? S Gupta, C Green, R Tiruvoipati, J Botha S Gupta, C Green, R Tiruvoipati, J Botha Peninsula Health, Frankston, Australia Critical Care 2015, 19(Suppl 1):P569 (doi: 10.1186/cc14649) Introduction There is a paucity of data about whether our treatment philosophy is diff erent for our patients as compared with what we would have wanted for ourselves, or while acting as surrogate decision- makers for our loved ones. y gi Results During the period, 191 patients were admitted to the EDICU, from which 151 had complete data and follow-up. A total of 63 patients (41.7%) suff ered from a life-limiting disease and were evaluated as having a high probability of death in 1 year. This group was further divided between 35 patients who in the moment of initial screening were expected to die in this hospital admission and 28 patients who were believed to survive to discharge. Comparison between these two groups showed patients believed to die at this hospital admission had higher SAPS III scores (66.9 vs. 59, P = 0.010) and hospital mortality (48.6% vs. 10.7%, P = 0.001). Methods An anonymous survey was sent to all the members of Australia and New Zealand Intensive Care Society and the College of Intensive Care Medicine (CICM). The fi rst section comprised a hypothetical case scenario spanning over 6 weeks of ICU stay for a patient. At four diff erent stages of the ICU stay, responders were requested to answer multiple- choice questions regarding the philosophy of treatment, based on their perceived prognosis of the patient at that particular time. The following two sections contained the same set of questions with the hypothetical scenario of responders acting as surrogate decision-makers for the patient and that of responders being patients themselves, in the same situation. The responses were compared amongst three sections at each stage using the chi-square test. Conclusion A high percentage of patients admitted to our EDICU have life-limiting disease and might benefi t from palliative care. These patients can be identifi ed using simple screening questions at admission and positive answers to those questions can be associated with worse outcomes. Results A total of 115 responses were received from the fellows of CICM. The results are presented in Tables 1 and 2. P573 How readable are our Patient Information Sheets? L Strachan, M Booth Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2015, 19(Suppl 1):P573 (doi: 10.1186/cc14653) Results Sixty-eight knowledge to care gaps were proposed, rated and revised by the committee over three rounds of review, resulting in 13 priorities for improvement. Then, 1,103 providers (62% response rate) representing nurses, respiratory therapists, allied health professionals and physicians evaluated the priorities and rated nine as necessary. In multivariable logistic regression analyses, provider (profession, experience and teaching status of ICU) and knowledge to care gap characteristics (strength of supporting evidence, potential to benefi t the patient, potential to improve patient/family experience, and potential to decrease costs) were associated with priorities rated as necessary. After disseminating the results to all network members, 627 responded (35% response rate) and indicated that the priorities were reasonable choices for quality improvement initiatives (87%), that they were highly supportive of working on initiatives targeting the priorities (61%) and would be willing to act as local champions for the initiatives (n = 92 individuals). Introduction We often need to obtain consent for clinical studies in the ICU. Participant Information Sheets (PIS) can be diffi cult to understand. A recent French publication [1] supports our hypothesis that PIS have poor readability scores. Methods Protocols submitted for ethics approval between 2008 and 2009 were obtained with permission from the Scotland A Ethics Research Committee. Ethical approval was not required for this observational study. All header, footers, diagrams and tables were removed. Readability scoring was performed using the Flesch Reading Ease and Flesch–Kincaid (FK) grades. Statistical analysis using Excel and MiniTab was then performed. The readability of these documents was compared with everyday documents – newspaper articles, politicians’ speeches [2] and standard contract agreements. p g Results A total of 104 protocols containing 209 PIS were reviewed. Of these, 99 (47%) were written for patients, 56 (27%) for GPs, 26 (12%) for relatives, 17 (8%) for carers, fi ve (2%) for legal representatives and six (3%) were summary sheets only. Sixty-seven (64%) of these protocols were submitted by academic institutions (for example, university or health boards) and 37 (36%) by pharmaceutical companies. Results are expressed as the median and 25th and 75th percentiles. P573 The word count and number of pages were higher for those PIS submitted by pharmaceutical companies compared with academic institutions: 1,561 (471; 5,167) versus 1,177 (626.5; 1,559.8) with P <0.05 and 4 (2; 10) versus 3 (2; 4) with P <0.05 respectively. The Flesch Reading Ease (63 (56; 69) vs. 60 (52.6; 65.4)) and FK grades (3 (5.4; 7.2) vs. 6.8 (6; 7.6)) were similar for both groups. Further subanalysis demonstrated that PIS designed for GPs had a lower word count, lower Flesch and higher FK grade compared with those for patients – the diff erence in Flesch and FK grade were compared using a Mann–Whitney test and were statistically signifi cant. Conclusion Our research approach engaged a diverse group of stakeholders to identify nine priorities for improving the quality and value of care provided to critically ill patients. This methodology can be used to engage stakeholders and identify priorities for quality improvement in other healthcare systems and domains. Additional work is required to reconcile provider/decision-maker and patient/ family priorities. p H Stelfox1, E McKenzie1, S Bagshaw2, M Gill1, P Oxland1, D Boulton1, D Oswell1, M Potestio1, D Niven1 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P575 (doi: 10.1186/cc14655) y gi Conclusion The FK grade is equivalent to US school grade level. The US government advises all policies produced should have a FK grade of <9. Our study suggests that protocols submitted to the ethics committee are easy to read, comparing favourably with broadsheet journalism and standard contract, for example loan contract. However, the average reading age in the UK is 9 years [3], suggesting participants may struggle with the information provided. References Introduction With increasing emphasis on patient and family-centred care, it follows that patients and their family members should be included when priorities for improving care are established. We therefore used a novel methodology that employs former patients and family members as researchers to describe the experiences of critically ill patients and their families with ICUs and to identify opportunities for improvement. journalism and standard contract, for example loan contract. However, the average reading age in the UK is 9 years [3], suggesting participants may struggle with the information provided. References p Methods Using the patient engagement framework developed by Marlett and Emes, we engaged four former patients and family members trained in qualitative research methods to conduct and analyse semistructured focus groups and interviews with adult patients who had recovered from critical illness and family members of both surviving and deceased patients. Participants were recruited from 13 ICUs in Alberta, Canada. Focus groups and interviews were recorded, transcribed and analysed using phenomenological reduction. Data collection continued until thematic saturation was reached. 1. Menoni V. The readability of information and consent forms in cl research in France. PLos One. 2010;5:e10576. Menoni V. The readability of information and consent forms in clinic research in France. PLos One. 2010;5:e10576. 2. http://www.britishpoliticalspeech.org/speech-archive.l 3. Gillies K. Patient information leafl ets for UK randomised controls. Trial. 2014;15:62. References 1. Piva J, Lago P, Othero J, Garcia PC, Fiori R, Fiori H, et al. Evaluating end of life practices in ten Brazilian paediatric and adult intensive care units. J Med Ethics. 2010;36:344-8. Introduction A high percentage of patients admitted to ICUs fulfi ll one or more criteria for palliative care. There are currently few comprehensive studies in critical care settings that have set out to 2. Lago PM, Nilson C, Pedro Piva J, Vieira AC, Halal MG, de Carvalho Abib GM, et al. Nurses’ participation in the end-of-life process in two paediatric intensive 2. Lago PM, Nilson C, Pedro Piva J, Vieira AC, Halal MG, de Carvalho Abib GM, et al. Nurses’ participation in the end-of-life process in two paediatric intensive S199 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 care units in Brazil. Int J Palliat Nurs. 2011;17:264, 267-70. 3. Abib El Halal GM, Piva JP, Lago PM, El Halal MG, Cabral FC, Nilson C, et al. Parents’ perspectives on the deaths of their children in two Brazilian paediatric intensive care units. Int J Palliat Nurs. 2013;19:495-502. Methods Using a modifi ed RAND/UCLA Appropriateness Methodology, a committee of 38 providers and decision-makers representing a population-based clinical network of adult (n = 14) and pediatric (n = 2) medical–surgical ICUs in Alberta, Canada (population 4 million) serially proposed, rated and revised potential knowledge to care gaps as priorities for improvement. The priorities developed by the committee were sent to the network’s 1,790 frontline providers to rate their importance. The fi nal list of priorities that were rated as important was disseminated to all network members for feedback. Using patient researchers to understand patient and family experiences in ICUs p H Stelfox1, E McKenzie1, S Bagshaw2, M Gill1, P Oxland1, D Boulton1, D Oswell1, M Potestio1, D Niven1 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P575 (doi: 10.1186/cc14655) Survey of visiting hours in critical care units in English trauma centres One hundred per cent were able have questions answered satisfactorily. Linked to the FS-ICU, we have seen marked improvements in decision-making and satisfaction. Methods A telephone survey on visiting times was conducted in 53 adult critical care units in trauma centres in England. A list of trauma centres was obtained from the NHS England website. All critical care units (other than obstetric high dependency units and coronary care units, unless part of a cardiothoracic critical care unit) within each hospital were surveyed. Each respondent was asked about the visiting hours, whether children were allowed to visit and how many visitors to a bed space. Conclusion We have shown progressive improvement over 3 years across all domains. Marked improvement in information provision and decision-making support from 53% to 96% over 3 years since introducing the FWR correlates with the improved overall satisfaction (Figure 1). Interestingly FWR is more helpful than relatives anticipated. The FWR was very well received and our results suggest an unrecognised need is being met. Because this was a pragmatic study, we feel this is a true representation of family satisfaction. It is encouraging that communication, information and decision-making support continue to improve. They have become embedded in the fabric of our critical care practice and lead to marked improvement in satisfaction for families. Reference Results Fifty-three units with between four and 75 beds and covering the whole of England were surveyed: there was a 100% response rate. Visiting hours varied between hospitals and between units within the same hospital. Nine units (17%) had open visiting hours, although most gave advice on times to avoid such as nursing handover. The majority of units (44.83%) operated restricted visiting with a median (range) of 6 (2 to 9) hours. All units allowed a maximum of two visitors to the bedspace. Children were allowed in nine units without restriction, the remaining units advised that it may not be appropriate for children to visit and it was at the discretion of the parents and medical staff . 1. Wall et al. Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med. 2007;35:271-9. f Conclusion The majority of adult critical care units in England, including our own, have restricted visiting policies. Survey of visiting hours in critical care units in English trauma centres Figure 1 (abstract P577). Trends in family satisfaction. E Taylor, N Bunker E Taylor, N Bunker The Royal London Hospital, London, UK round (FWR), to enhance and standardise communication and improve satisfaction. Following introduction of the FWR we have audited family satisfaction using the validated FS-ICU questionnaire [1]. round (FWR), to enhance and standardise communication and improve satisfaction. Following introduction of the FWR we have audited family satisfaction using the validated FS-ICU questionnaire [1]. Methods This was a prospective study of relatives’ satisfaction for patients completing their critical care episode. The questionnaire was completed anonymously and data collected. This was a pragmatic study, no changes were made to communication strategies. Results There is a high degree of satisfaction across all domains of the FS-ICU including treatment of family and provision of information (Figure 1). One hundred per cent found FWR to be helpful, only 55% had anticipated this. Fifteen per cent changed their perception of critical care. It enabled 15% to raise new concerns. One hundred per cent were able have questions answered satisfactorily. Linked to the FS-ICU, we have seen marked improvements in decision-making and satisfaction. Introduction The purpose of this study was to assess the visiting restrictions placed on families visiting adult patients on critical care units within trauma hospitals in England. Whilst it is well recognised that high-quality care for patients is of paramount importance, we should also be aware that supporting patients’ families off ers long- term benefi ts for patient, family and hospital. In our own unit we are reviewing whether we could adopt a more fl exible attitude to visiting times and assessing how to provide a more welcoming environment to relatives. To inform our own review and in order to develop a best practise approach, we surveyed all of the major trauma centres in England. Methods This was a prospective study of relatives’ satisfaction for patients completing their critical care episode. The questionnaire was completed anonymously and data collected. This was a pragmatic study, no changes were made to communication strategies. Results There is a high degree of satisfaction across all domains of the FS-ICU including treatment of family and provision of information (Figure 1). One hundred per cent found FWR to be helpful, only 55% had anticipated this. Fifteen per cent changed their perception of critical care. It enabled 15% to raise new concerns. Survey of visiting hours in critical care units in English trauma centres Visiting policies are a source of debate amongst staff in intensive care with concerns about open visiting including increased workload and interruptions to normal routine [1]. This is consistent with the views of staff at our own unit who, in appreciative enquiry, have expressed mixed opinions about extending visiting times. Extending visiting times is only part of a wider project to improve the way relatives experience intensive care whilst ensuring both medical and nursing staff feel supported, creating an environment for optimal communication. P578 Bereavement care in UK ICUs: a national survey M Berry1, E Brink2, V Metaxa2 1Imperial Healthcare Trust, London, UK; 2King’s College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P578 (doi: 10.1186/cc14658) Introduction For the families of critically ill patients, the death of a loved one in the ICU is often an unexpected and traumatic event, characterised by diffi cult decisions regarding withholding or withdrawing life-sustaining therapy. Increasingly the importance of bereavement care (BC) in the ICU is being acknowledged, although reports continue to highlight the inadequacies around end-of life care in the critical care environment. In 1998, the Intensive Care Society (ICS) published guidelines mapping out BC in the ICU [1]. We aimed to compare BC in ICUs across England against the recommendations set out by the ICS. 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. P574 Stakeholder engagement to identify priorities for improving the quality and value of care provided to critically ill patients H Stelfox1, D Niven1, S Bagshaw2, E McKenzie1, M Potestio1, F Clement1, D Zygun2 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P574 (doi: 10.1186/cc14654) Results Thirty-two participants including patients (n = 11) and family members (n = 21) participated in fi ve focus groups (n = 23 participants) and eight interviews (n = 9 participants). Participants articulated themes refl ecting important components of care organised across three phases of the ICU experience; admission to ICU, daily care in ICU and after ICU discharge. Admission to ICU comprised three themes: patient and family transition into ICU, patient and family disorientation upon admission to ICU and preferred staff actions to help patients/ family adapt to the ICU. The daily care phase of ICU consisted of fi ve themes: honouring patient’s voices, needing to know, making decisions, culture in ICU and medical care. The experience after ICU discharge comprised two themes: transition from ICU to a hospital ward and long-term eff ects of critical illness. Participants identifi ed fi ve Critical Care 2015, 19(Suppl 1):P574 (doi: 10.1186/cc14654) Introduction Healthcare systems do not make optimal use of evidence, which results in suboptimal patient care. Large amounts of scientifi c evidence are generated but not implemented into patient care (knowledge to care gap). We sought to identify and prioritize knowledge to care gaps in critical care medicine as opportunities to improve quality and value in care. S200 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P577). Trends in family satisfaction. priorities for improvement: provide families with a guide/navigator; educate providers about the fragility of family trust; improve provider communication skills; inform patients about the long-term eff ects of critical illness; and develop strategies to facilitate continuity of care between providers. p Conclusion Patients and family members are an untapped resource and engaging them as researchers is a viable strategy to identify opportunities for quality improvement that are patient and family centred. P577 y Methods All adult ICUs in England were contacted over a 2-week period, using a standardised questionnaire based on the nine domains identifi ed by the ICS. All answers were collected anonymously using SurveyMonkey®. An 80% compliance rate was deemed acceptable.i y Methods All adult ICUs in England were contacted over a 2-week period, using a standardised questionnaire based on the nine domains identifi ed by the ICS. All answers were collected anonymously using SurveyMonkey®. An 80% compliance rate was deemed acceptable. Results From the 148 ICUs identifi ed, 113 answered the questionnaire (76%). Forty-three per cent of the responders had access to training in BC and in communication skills, and 54% had a named member of staff responsible for training, writing, auditing and developing the R Handslip, A Molokhia Introduction Patient satisfaction is a crucial part of clinical care and there is now increasing recognition of the importance of family involvement and satisfaction in the provision of care for the critically ill. Since 2012 our unit has introduced a consultant-led family ward Results From the 148 ICUs identifi ed, 113 answered the questionnaire (76%). Forty-three per cent of the responders had access to training in BC and in communication skills, and 54% had a named member of staff responsible for training, writing, auditing and developing the S201 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion This is the fi rst national audit of BC in the ICU since the initial ICS guideline publication. Even though most ICUs provided relatives with information around the time of death, training, auditing and adequate facilities do not meet the recommended standards. The lack of adherence is defi nitely multifactorial and requires further research. In the meantime, vigorous implementation of these guidelines is warranted in order to ensure optimal care for the bereaved families. Reference BC policy. When asked about the presence of a written BC policy only 45% responded positively, and even less (19%) had provisions for audit and development of the service. Information to staff about cultural and religious rites around the time of death, and to relatives on what to do after a death was available in 81% and 96% respectively. The general practitioner was informed of the deaths taking place in the ICU in 77% of the cases. . Intensive Care Society (1998). http://www.ics.ac.uk/ics-homepage/ guidelines-and-standards/. P577 In more than 70% of the participating ICUs, eff orts were made to ensure privacy of the grieving relatives and to have dedicated follow-up facilities for the bereaved. Even though staff support programmes were recognised as paramount, only 54% of the ICUs had formal ones set up.
https://openalex.org/W3196925190	https://www.journal.acorn.org.au/cgi/viewcontent.cgi?article=1149&context=jpn	English	null	Prevention is better than cure: Understanding metabolic syndrome (MetS) and the occupational risks for perioperative nurses	Journal of perioperative nursing	2,021	cc-by	3,249	Prevention is better than cure: Understanding metabolic Prevention is better than cure: Understanding metabolic syndrome (MetS) and the occupational risks for perioperative syndrome (MetS) and the occupational risks for perioperative nurses nurses Follow this and additional works at: https://www.journal.acorn.org.au/jpn Part of the Perioperative, Operating Room and Surgical Nursing Commons This work is licensed under a Creative Commons Attribution 4.0 License. Follow this and additional works at: https://www.journal.acorn.org.au/jpn Part of the Perioperative, Operating Room and Surgical Nursing Commons This work is licensed under a Creative Commons Attribution 4.0 License. Recommended Citation Recommended Citation Turnbull, Daniel and Foran, Paula (2021) "Prevention is better than cure: Understanding metabolic syndrome (MetS) and the occupational risks for perioperative nurses," Journal of Perioperative Nursing: Vol. 34 : Iss. 3 , Article 7. Available at: https://doi.org/10.26550/2209-1092.1149 https://www.journal.acorn.org.au/jpn/vol34/iss3/7 This Discussion paper is brought to you for free and open access by Journal of Perioperative Nursing. It has been accepted for inclusion in Journal of Perioperative Nursing by an authorized editor of Journal of Perioperative Nursing. Recommended Citation Recommended Citation Turnbull, Daniel and Foran, Paula (2021) "Prevention is better than cure: Understanding metabolic syndrome (MetS) and the occupational risks for perioperative nurses," Journal of Perioperative Nursing: Vol. 34 : Iss. 3 , Article 7. Available at: https://doi.org/10.26550/2209-1092.1149 https://www journal acorn org au/jpn/vol34/iss3/7 Volume 34 Issue 3 Volume 34 Issue 3 Article 7 Recommended Citation Recommended Citation Recommended Citation Recommended Citation Turnbull, Daniel and Foran, Paula (2021) "Prevention is better than cure: Understanding metabolic syndrome (MetS) and the occupational risks for perioperative nurses," Journal of Perioperative Nursing: Vol. 34 : Iss. 3 , Article 7. Available at: https://doi.org/10.26550/2209-1092.1149 https://www.journal.acorn.org.au/jpn/vol34/iss3/7 This Discussion paper is brought to you for free and open access by Journal of Perioperative Nursing. It has been accepted for inclusion in Journal of Perioperative Nursing by an authorized editor of Journal of Perioperative Nursing. This discussion paper is available in Journal of Perioperative Nursing: https://www.journal.acorn.org.au/jpn/vol34/ iss3/7 This discussion paper is available in Journal of Perioperative Nursing: https://www.journal.acorn.org.au/jpn/vol34/ iss3/7 Cover Page Footnote Cover Page Footnote Metabolic syndrome (MetS) is a condition with interconnected abnormalities of the metabolic system that has been labelled by the World Health Organization as a main cause of death worldwide. Risk factors for MetS include occupational stress, disturbances to circadian rhythms, sleep disorders and changed eating habits, which are all associated with shift work. As shift work and occupational stress are common in perioperative nursing, the risk of developing MetS is increased for perioperative nurses. This discussion paper aims to bring an awareness and understanding of MetS to perioperative nurses and identifies the occupational risks in the perioperative environment that may lead to its development. It also presents some possible strategies to mitigate the risk factors or prevent this condition for perioperative nurses in the future. Emerging scholar article Authors Daniel Turnbull GradDipNsg (Periop), BN, RN MACN Dr Paula Foran PhD, RN, FACORN, FACPAN, MACN Abstract Metabolic syndrome (MetS) is a condition with interconnected abnormalities of the metabolic system that has been labelled by the World Health Organization as a main cause of death worldwide. Risk factors for MetS include occupational stress, disturbances to circadian rhythms, sleep disorders and changed eating habits, which are all associated with shift work. As shift work and occupational stress are common in perioperative nursing, the risk of developing MetS is increased for perioperative nurses. This discussion paper aims to bring an awareness and understanding of MetS to perioperative nurses and identifies the occupational risks in the perioperative environment that may lead to its development. It also presents some possible strategies to mitigate the risk factors or prevent this condition for perioperative nurses in the future. Keywords: metabolic syndrome, circadian misalignment, shift work, occupational stress and breast cancer1,5,6,7. Research has also theorised that MetS affects approximately 30 per cent of the adult population world-wide8. Introduction Metabolic syndrome (MetS) was previously known as ‘syndrome x’ and was first recognised by Gerald M Raevan in the 1980’s1. The pathophysiology of MetS is complex and comprises interconnected abnormalities of the metabolic system including lipid and glucose metabolism1. MetS is diagnosed by the simultaneous presence of three or more of the following factors: hypertension, dyslipidaemia, central obesity and hypertriglyceridemia1–4. The syndrome has been linked to co-morbidities of the liver and reproductive system, thrombotic states and inflammatory diseases. It has been identified as having an increased risk of mortality, with the World Health Organization labelling it as a main cause of death worldwide alongside cardiovascular disease, type 2 diabetes mellitus A systematic review of literature by Ranasinghe et al. in 2017 provided an alternative estimate of the prevalence of MetS, estimating that 20 to 25 per cent of the adult population may be suffering from the disorder1. This review was the first to complete a comprehensive systematic evaluation of literature regarding prevalence of MetS in the Asia–Pacific region; however, from a possible 51 countries, only 15 studies were found revealing the importance of further data collection1. In Ranasinghe’s study several variables were considered, including age, occupation and gender1. Females generally had a higher prevalence of MetS except in some specific areas, one of which was rural Australia1. Journal of Perioperative Nursing Volume 34 Number 3 Spring 2021 acorn.org.au e-46 More recent studies found similar findings where MetS had an increased prevalence in women; however, data around prevalence in shift workers varied from nine per cent to 30 per cent, with some studies suggesting a two per cent prevalence in nurses specifically3,6,8,9. While most studies found age to be a factor in prevalence – the under 40 age group were at high risk – other studies acknowledged the presence of MetS in all age groups3,4,6,9. Despite the variation in data and research on the prevalence of MetS, which may be due to different study designs, it is important for the health of all nurses to investigate the occupational risks of shift work and the potential to develop MetS with its associated risk of mortality10,11. identified a further increase of risk in rotating shift workers12, and this was also highlighted by Khosravipour et al. in their similar systematic review of 38 observational studies10. Introduction The increased risk of developing MetS in shift workers has been attributed to the desynchronisation, or misalignment, of the circadian rhythm and workers’ disrupted sleep– wake cycles5,6,7,12. MetS and periperative nursing Perioperative nursing often involves shift work, being on-call and changing rosters. Meal breaks can be short, requiring nurses to alter their dietary habits and eat quickly while at work. Perioperative nurses may experience occupational stress with long work hours and physically demanding tasks such as lifting heavy instrument trays, moving and positioning patients and wearing lead aprons. Thus there are a number of occupational risks for MetS associated with working in the operating room. Circadian misalignment Circadian rhythm influences temperature and the sleep–wake cycle and is synchronised by the retina’s exposure to light which stimulates photosensitive cells connected to the suprachiasmatic nucleus in the hypothalamus gland in the brain14,15. The suprachiasmatic nucleus then innervates the sympathetic nervous system which regulates humoral, neural and endocrine signals that lead to predictable behaviours of metabolism and physical cellular performance14,15. Extended exposure to light disrupts this circadian rhythm, especially in night shift workers, and may result in insulin resistance and glucose intolerance from altered hormonal secretions, potentially leading to the development of MetS3,14. A systematic review of metabolic and cardiovascular consequences of shift work by Kervezee et al. identified evidence that disruption to circadian rhythms, or circadian misalignment, is linked to elevated glucose levels and insulin resistance in varying degrees, particularly in the majority of night shift workers14. The review also found that shift workers were at a greater risk of developing metabolic disorders from short-term circadian rhythm misalignment14. The risk of developing MetS from circadian misalignment is compounded by disrupted sleep and altered eating regimes7 both of which are common with shift work. Sleep deprivation and sleep disorders Sleep deprivation has been theorised to affect the endoplasmic reticulum within cellular structures. These organelles are responsible for processing secretory and membrane proteins which have a link to insulin functionality, lipidystrophy, obesity and type 2 diabetes7. Shift work, quick turn-around times between shifts, duration of shifts and night shift may all contribute to the development of sleep disorders, which are known risk factors for MetS6,7,15. A small cross-sectional study on MetS in night shift workers (n=60), revealed that insomnia symptoms were found in 40 per cent of the participants9. Rosa et al. also identified in their systematic review of randomised control trials and observation studies (n=24) of shift work and nurses’ health, that sleep disorders were more prevalent for staff rotating onto morning shift from night shifts when following a 3 x 8-hour rotation schedule6. Other studies have suggested night shift workers sleep less than day workers, and some studies show workers on a rapidly changing shift rotation sleep fewer hours than workers permanently on night shift6,14. Rosa et al. also noted that all shift-work hours interfere with at least one main meal and that appetite levels are affected by short sleep cycles6. Recommendations Managers need to have an awareness of the risk factors for MetS and put strategies in place to mitigate the occupational risks that could result in nurses developing MetS3,6,13. Strategies include raising awareness of MetS, education about diet and the importance of physical exercise in avoiding metabolic disorders, and promotion of healthy behaviours2,3,6. Benefit could be gained by introducing a health care program for staff with increased risk of developing MetS and implementing a food diary to help control calorie consumption2,16. It is known that perioperative nurses suffer fatigue due to emotionally and physically demanding work and staffing practices19. Thus, managers also need to critically think about shift scheduling and rotations, and Shift work Shift work has been identified as increasing the risk of developing MetS3,4,6,10,12,13. A systematic review and meta-analysis by Wang et al. looked at the risk of MetS associated with shift work. The review of 36 studies, with a combined total of 216 527 participants, revealed that shift workers had an increased risk of developing MetS compared to regular day workers (OR = 1.35, 95%CI: 1.24–1.48; I2 = 74.6%)12. Wang et al. also Increased risk of MetS may come from appetite changes in shift workers due to unregulated meal times, sleep disturbances, changes in lifestyle and demands of shift work6. Changes to eating regimes can lead to altered insulin responses, and disrupted secretion of appetite hormones may lead to higher caloric consumption6,14. A small study by Molzof et al. comparing female Journal of Perioperative Nursing Volume 34 Number 3 Spring 2021 acorn.org.au e-47 day- and night-shift workers (n=17) and the impact of meal timing on cardiometabolic syndrome indicators, revealed that irregular eating patterns were observed more in shift workers, and that night shift workers’ food intake was associated with multiple risk factors specific for MetS5. This study also found that increased lipid levels and weight gain was associated with increased food consumption in the evening5. While the research by Molzof et al. had a small sample size and was restricted to female shift workers, other studies have also shown that high calorie consumption by shift workers and calorie consumption in the evening increases the risk of developing MetS2,7. Another interesting finding, from a cross-sectional data analysis of nurses (n=1638) by Jung et al., is that faster eating speeds have been associated with weight gain and MetS2. Jung et al. also found that nurses have meal breaks, on average, between six and 28 minutes long2. Higher caloric consumption by nurses during these meal breaks could also be attributed to occupational stressors16. of stress, leading to increased energy from gluconeogenesis and lipid mobilisation in adipose tissue7,17. A cross-sectional analysis of a multicentre cohort of civil servants (n=15 105) by Santos et al. observed that higher levels of cortisol were noted in irregular shift workers on their rostered days off, thereby resulting in prolonged stress responses13. Although this analysis did not focus on the nursing profession, the analysis revealed that shift work contributed independently to the risk of developing MetS13. Conclusion MetS is a complex and potentially fatal condition believed to affect nearly a third of the world’s population. Shift work and the associated disturbances to circadian rhythms, sleep and eating habits, as well as occupational stress, are recognised as risk factors for developing MetS and are occupational risks for perioperative nurses. Further research into and reporting about MetS will improve the understanding of the physiological mechanisms underlying MetS and the risk factors that contribute to its development. Increasing awareness among the perioperative nursing profession will enable the development and implementation of strategies that may mitigate the risks factors of MetS and reduce its prevalence thus improving the overall health of the perioperative nursing workforce. Shift work Chronic stress can also cause burnout with some studies suggesting a link between burnout and MetS17. Others studies suggest there has been no definite association between MetS and burnout, although association with components of MetS were found18. Due to the inconsistency of evidence from multiple studies, more research is required on stress-related burnout and MetS18. day- and night-shift workers (n=17) and the impact of meal timing on cardiometabolic syndrome indicators, revealed that irregular eating patterns were observed more in shift workers, and that night shift workers’ food intake was associated with multiple risk factors specific for MetS5. This study also found that increased lipid levels and weight gain was associated with increased food consumption in the evening5. While the research by Molzof et al. had a small sample size and was restricted to female shift workers, other studies have also shown that high calorie consumption by shift workers and calorie consumption in the evening increases the risk of developing MetS2,7. Another interesting finding, from a cross-sectional data analysis of nurses (n=1638) by Jung et al., is that faster eating speeds have been associated with weight gain and MetS2. Jung et al. also found that nurses have meal breaks, on average, between six and 28 minutes long2. Higher caloric consumption by nurses during these meal breaks could also be attributed to occupational stressors16. frequency and length of shifts, as well as ensuring adequate rest periods to reduce detrimental effects on staff and increase wellbeing6,15. As the risk of developing MetS is ever present in the perioperative environment, it is important for organisations to screen workers who are exposed to shift work, identify those at high risk of developing this dangerous disorder and provide education and resources to reduce the risk. Finally, as research into MetS in the perioperative domain is limited, it is recommended that organisations facilitate research in operating suites across Australia to explore the prevalence of and prevention strategies for this potentially dangerous disorder to improve the health of nurses6. Occupational stress Research has shown that occupational stress may increase the risk of MetS8. Increased stress has been associated with shift work, specifically rotating shift work, with research theorising that one in every four nurses are being affected6. In addition, the perioperative environment can be a stressful environment. Excess stress during work can increase the production of cortisol which could potentially lead to insulin resistance and development of MetS17,18. Cortisol production by the adrenal gland is stimulated by adrenocorticotropic hormone from the hypothalamic– pituitary–adrenal axis in times Journal of Perioperative Nursing Volume 34 Number 3 Spring 2021 acorn.org.au e-48 5. Molzof HE, Wirth MD, Burch JB, Shivappa N, Hebert JR, Johnson RL et al. The impact of meal timing on cardiometabolic syndrome indicators in shift workers. Chronobiol Int 2017;34(3):337–348. 12. Wang Y, Yu L, Gao Y, Jiang L, Yuan L, Wang P et al. Association between shift work or long working hours with metabolic syndrome: A systematic review and dose-response meta-analysis of observational studies. Chronobiol Int 2021;38(3):318–333. Acknowledgment This paper was submitted to the University of Tasmania as part fulfilment of subject CNA803, Advanced Clinical Nursing Practice, for the Master of Clinical Nursing (Perioperative Nursing). The author sincerely wishes to thank Dr Paula Foran, unit coordinator, for her guidance throughout the master course and work in preparing this paper for publication. 6. Rosa D, Terzoni S, Dellafiore F, Destrebecq A. Systematic review of shift work and nurses’ health. Occup Med 2019;69(4):237–243. 13. Santos AE, Araújo LF, Griep RH, Castro Moreno CR, Chor D, Barreto SM et al. Shift work, job strain, and metabolic syndrome: Cross-sectional analysis of ELSA-Brasil. Am J Ind Med 2018;61(11):911–918. 7. Ferraz-Bannitz R, Beraldo RA, Coelho PO, Moreira AC, Castro M, Foss-Freitas MC. Circadian misalignment induced by chronic night shift work promotes endoplasmic reticulum stress activation impacting directly on human metabolism. Biology 2021;10(197):1–13. 14. Kervezee L, Kosmadopoulos A, Boivin DB. Metabolic and cardiovascular consequences of shift work: The role of circadian disruption and sleep disturbances. Eur J Neurosci 2020;51(1):396–412. 8. Kuo W, Bratzke LC, Oakley LD, Kuo F, Wang H, Brown RL. The association between psychological stress and metabolic syndrome: A systematic review and meta- analysis. Obes Rev 2019;20(11):1651–1664. 15. Garde AH, Begtrup L, Bjorvatn B, Bonde JP, Hansen J, Hansen ÅM et al. How to schedule night shift work in order to reduce health and safety risks. Scand J Work Environ Health 2020;46(6):557–569. References 1. Ranasinghe P, Mathangasinghe Y, Jayawardena R, Hills A, Mirsa A. Prevalence and trends of metabolic syndrome among adults in the Asia-Pacific region: A systematic review. BioMed Central 2017;17(101):1–9. 9. Holanda NCP, Castro Moreno CR, Marqueze EC. Metabolic syndrome components: Is there a difference according to exposure to night work? Chronobiol Int 2018;35(6):801– 810. 16. Bigand T, Cason M, Diede T, Wilson M. Pilot testing an electronic food diary among registered nurses working night shifts. Chronobiol Int 2020;37(9/10):1377–1383. 2. Jung H, Dan H, Pang Y, Kim B, Jeong H, Lee JE et al. Association between dietary habits, shift work and the metabolic syndrome: The Korea nurses’ health study. Int J Environ Res Public Health 2020;17(20). 17. Merces MC, Gomes AMT, Coelho JMF, Servo MLS, Marques SC, Júnior AD. Scientific evidence on the association between burnout and metabolic syndrome: Integrative review. Acta Paul Enferm 2019;32(4):470–476. 10. Khosravipour M, Khanlari P, Khazaie S, Khosravipour H, Khazaie H. A systematic review and meta-analysis of the association between shift work and metabolic syndrome: The roles of sleep, gender,and type of shift work. Sleep Med Rev 2021;57(101427):1–14. DOI: 10.1016/j. smrv.2021.101427. 3. Nikpour M, Tirgar A, Hajiahmadi M, Hosseini A, Heidari B, Ghaffari F et al. Shift work and metabolic syndrome: A multi-center cross- sectional study on females of reproductive age. Biomedical Reports 2019;10(5):311–317. 18. Chico-Barba G, Jiménez-Limas K, Sánchez- Jiménez B, Sámano R, Rodriguez-Ventura AL, Castillo-Pérez,et al. Burnout and metabolic syndrome in female nurses: An observational study. Int J Environ Res Public Health 2019;16(11):1–11. 11. Loef B, Baarle D, van der Beek AJ, Beekhof PK, van Kerkhof LW, Proper KI. The association between exposure to different aspects of shift work and metabolic risk factors in health care workers, and the role of chronotype. Plos One 2019;14(2):1–14. 4. Rosa D, Villa G, Montanari E, Destrebecq A, Terzoni S. The relationship between urolithiasis, metabolic syndrome and nurse shift work: A literature review. Int J Urol Nurs 2020;14(2):57–66. 4. Rosa D, Villa G, Montanari E, Destrebecq A, Terzoni S. The relationship between urolithiasis, metabolic syndrome and nurse shift work: A literature review. Int J Urol Nurs 2020;14(2):57–66. 19. Nijkamp N, Foran P. The effects of staffing practices on safety and quality of perioperative nursing care – an integrative review. JPN 2021;34(1):e15–e22. Journal of Perioperative Nursing Volume 34 Number 3 Spring 2021 acorn.org.au Journal of Perioperative Nursing Volume 34 Number 3 Spring 2021 acorn.org.au e-49
https://openalex.org/W2121157972	https://www.frontiersin.org/articles/10.3389/fnsys.2014.00164/pdf	English	null	Circadian gating of neuronal functionality: a basis for iterative metaplasticity1	Frontiers in systems neuroscience	2,014	cc-by	15,714	†Present address: Tongfei A. Wang, Department of Physiology, University of California, San Francisco, San Francisco, USA Keywords: circadian rhythms, gating, plasticity, iterative metaplasticity, hippocampus, suprachiasmatic nucleus, glutamatergic, signaling INTRODUCTION Although observed throughout the brain, plasticity is best studied in the hippocampus, a site critical to establishing and recalling new memories. Cellular and molecular mechanisms by which stimuli generate synaptic changes in hippocampal sub-regions fall into two classes: those that cause long-term potentiation (LTP) vs. long-term depression (LTD). Plastic changes in the hippocampus develop along linear timelines, initiated by the stimulus, fol- lowed by multiple sequential steps that are necessary to establish long-term, persistent functional changes. The ability of salient stimuli to induce persistent changes in the structure and function of neurons is a fundamental modulatory process that confers the ability to modify physiology and behav- ior, learning from experience (Markham and Greenough, 2004). Abbreviations: AVP, arginine vasopressin; BDNF, brain-derived neurotrophic fac- tor; CaMKII, calcium-calmodulin dependent protein kinase II; cAMP, cyclic adenosine triphosphate; cGMP, cyclic guanine monophosphate; CICR, calcium- induced calcium release; CREB, cAMP response element-binding protein; DHA, dehydroascorbic acid; ERK, extracellular signal-regulated kinase; GABA, gamma- amino butyric acid; GAD, glutamic acid decarboxylase, GC, guanylate cyclase; GRP, gastrin-releasing peptide; GSH, glutathione; GSSH, glutathione disulphide; ipRGCs, intrinsically photoreceptive retinal ganglion cells; JNK, c-jun kinase; K2P, two-pore domain potassium channel; KCNK, potassium channel subfamily K; LTD, long-term depression; LTP, long-term potentiation; MAPKK/MEK, MAPK kinase; MAPKs, mitogen activated protein kinases; NMDA/ NMDA•R, N-methyl D-aspartate/ N-methyl D-aspartate receptor; NO, nitric oxide; NOS, nitric oxide synthase; NPAS2, neuronal PAS-domain protein 2; ODNs, oligodeoxynucleotides; PACAP, pituitary adenylate cyclase-activating peptide; PKA, cAMP-dependent pro- tein kinase; PKG, cGMP-dependent protein kinase; PVN, paraventricular nucleus; RHT, retinohypothalamic tract; ROR, retinoic acid-related orphan receptor; RXO, reduction-oxidation oscillator; RyR, ryanodine receptor; SCN, suprachiasmatic nucleus; TASK, TWIK-related acid-sensitive K+ channel; tPA, tissue-type plas- minogen activator; TREK, TWIK-related K+ channel; TrkB, tropomyosin-related receptor kinase; TTO, transcription-translation oscillator; TTX, tetrodotoxin; TWIK, Tandem of P domains in a Weak Inward rectifying K+ channel; VIP, vasoactive intestinal peptide; VIP•R (also VPAC•R), vasoactive intestinal peptide receptor; Vm, membrane potential. Among the most salient stimuli for life on Earth are the alter- nating environmental states of day and night. The cycle of day and night partitions the availability of energy, both thermal and nutri- ent. Alternating activity vs. rest behaviors align with these envi- ronmental states. 1This paper is dedicated to the memory of our friend and colleague, William T. Greenough, a pioneer in the discovery of brain plasticity. REVIEW ARTICLE published: 19 September 2014 doi: 10.3389/fnsys.2014.00164 REVIEW ARTICLE published: 19 September 2014 doi: 10.3389/fnsys.2014.00164 REVIEW ARTICLE Rajashekar Iyer 1, Tongfei A. Wang 2† and Martha U. Gillette 1,2* 1 Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA 2 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Rajashekar Iyer 1, Tongfei A. Wang 2† and Martha U. Gillette 1,2* 1 Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA 2 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Brain plasticity, the ability of the nervous system to encode experience, is a modulatory process leading to long-lasting structural and functional changes. Salient experiences induce plastic changes in neurons of the hippocampus, the basis of memory formation and recall. In the suprachiasmatic nucleus (SCN), the central circadian (∼24-h) clock, experience with light at night induces changes in neuronal state, leading to circadian plasticity. The SCN’s endogenous ∼24-h time-generator comprises a dynamic series of functional states, which gate plastic responses. This restricts light-induced alteration in SCN state-dynamics and outputs to the nighttime. Endogenously generated circadian oscillators coordinate the cyclic states of excitability and intracellular signaling molecules that prime SCN receptivity to plasticity signals, generating nightly windows of susceptibility. We propose that this constitutes a paradigm of ∼24-h iterative metaplasticity, the repeated, patterned occurrence of susceptibility to induction of neuronal plasticity. We detail effectors permissive for the cyclic susceptibility to plasticity. We consider similarities of intracellular and membrane mechanisms underlying plasticity in SCN circadian plasticity and in hippocampal long-term potentiation (LTP). The emerging prominence of the hippocampal circadian clock points to iterative metaplasticity in that tissue as well. Exploring these links holds great promise for understanding circadian shaping of synaptic plasticity, learning, and memory. SYSTEMS NEUROSCIENCE published: 19 September 2014 doi: 10.3389/fnsys.2014.00164 Keywords: circadian rhythms, gating, plasticity, iterative metaplasticity, hippocampus, suprachiasmatic nucleus, glutamatergic, signaling Edited by: Jason Robert Gerstner, Washington State University, USA Jason Robert Gerstner, Washington State University, USA Reviewed by: Robert N. S. Sachdev, Yale University, USA Jason Robert Gerstner, Washington State University, USA Correspondence: Martha U. Gillette, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S Goodwin Ave., Urbana, IL 61801, USA e-mail: mgillett@illinois.edu †Present address: Tongfei A. Wang, Department of Physiology, University of California, San Francisco, San Francisco, USA Reviewed by: Robert N. S. Sachdev, Yale University, USA Jason Robert Gerstner, Washington State University, USA Correspondence: Martha U. Gillette, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S Goodwin Ave., Urbana, IL 61801, USA e-mail: mgillett@illinois.edu †Present address: Tongfei A. Wang, Department of Physiology, University of California, San Francisco, San Francisco, USA Reviewed by: Robert N. S. Sachdev, Yale University, USA Jason Robert Gerstner, Washington State University, USA Reviewed by: Robert N. S. Sachdev, Yale University, USA Reviewed by: Robert N. S. Sachdev, Yale University, USA Jason Robert Gerstner, Washington State University, USA Correspondence: Martha U. Gillette, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S Goodwin Ave., Urbana, IL 61801, USA e-mail: mgillett@illinois.edu †Present address: Tongfei A. Wang, Department of Physiology, University of California, San Francisco, San Francisco, USA Jason Robert Gerstner, Washington State University, USA Correspondence: Martha U. Gillette, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S Goodwin Ave., Urbana, IL 61801, USA il ill @illi i d *Correspondence: Martha U. Gillette, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S Goodwin Ave., Urbana, IL 61801, USA †Present address: Internally, day-night changes in metabolism, physiology, and behavior are organized by an endogenous time- keeping system that oscillates with a circadian (circa, about, and dian, a day) period. The multitude of interacting elements that generate and coordinate circadian rhythms throughout the body constitute the circadian timing system. Endogenous circadian rhythms are fundamental properties of all cells. They emerge from September 2014 \| Volume 8 \| Article 164 \| 1 Frontiers in Systems Neuroscience www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. adapt to these changes would have held a competitive advantage (Ouyang et al., 1998; Woelﬂe et al., 2004; Gerstner, 2012). Indeed, selection for mechanisms that internalized timekeeping embedded circadian clocks within genomes and basal metabolism (Lowrey and Takahashi, 2011; O’Neill and Reddy, 2011; O’Neill et al., 2011; Gillette and Wang, 2014). This encoded the pat- terning of environmental state within genes of some prokaryotes and all eukaryotes, enabling them to organize day vs. night- time processes internally and predict these environmental state changes. self-regenerative oscillations in transcription-translation of core timing (clock) genes, gene products, and their post-translational modiﬁcation and in cellular metabolism of reduction-oxidation (redox) states. Cellular circadian rhythms of various tissues, e.g., heart, lung, blood, and brain, are synchronized by the hypothala- mic suprachiasmatic nucleus (SCN) (Lehman et al., 1987; Ralph et al., 1990; Yoo et al., 2004). The SCN coordinates the myriad body clocks via diverse output signals and adjusts its circadian state via inputs that communicate desynchronization with envi- ronmental and internal conditions. Light-stimulated resetting of SCN circadian phase is a form of neuronal plasticity, circadian plasticity, mediated by cellular processes similar to those that cause long-term changes in hippocampal state, but susceptibility is temporally restricted to night. Rhythmic behaviors that anticipate rather than merely react to environmental changes would offer signiﬁcant beneﬁts. Functional and physiological changes can be initiated in advance of environmental changes, optimizing behavior and aligning it more closely to daily environmental cycles. Further, because day-night durations undergo seasonal variations, an anticipa- tory, endogenous rhythm generator would adapt and entrain to changing ratios of light: dark periods, generating seasonal plas- ticity in behaviors (Bartness et al., 1993; Ebling et al., 1995; Nuesslein-Hildesheim et al., 2000; Meijer et al., 2010). †Present address: As understanding of mechanisms that lead to memory for- mation in the hippocampus has grown, questions have arisen about how such plasticity is regulated to prevent network hyper- stimulation, saturation, and impaired recall and cognition. This led to the prediction and substantiation of metaplastic regula- tory mechanisms. Metaplasticity is a persistent change in the state of synapses or neurons due to past activity or modulation that alters responses to subsequent plasticity-inducing stimuli. Consequently, responses may be altered in amplitude or duration compared with the basal response (Abraham and Bear, 1996). Metaplastic modulation can be local, involving single or nearby synapses, or extend throughout the neuron or network (Hulme et al., 2014). Behavioral expression of metaplasticity includes responses to environmental stimuli (enrichment or stress), devel- opmental deprivation (visual or tactile stimulation), and changes due to associative learning (Abraham, 2008). Anticipatory, near-24-h circadian rhythms are generated by molecular and cellular processes, and result in appropriately timed cycles of physiology, metabolism, and behavior. They per- sist in constant darkness. The endogenous period of circadian clocks under these aperiodic conditions is close-to, but not exactly, 24-h (King and Takahashi, 2000; Okamura et al., 2002). Consequently, free-running rhythms drift out of correspondence with day and night of the solar cycle. They re-adjust in response to light signals that occur during the subjective night of circa- dian clock processes. Thus, when animals are exposed to regular 24-h cycles of light and dark or light presented brieﬂy at regular 24-h intervals, the rhythms undergo entrainment, aligning with the time-schedule dictated by light (Golombek and Rosenstein, 2010). Because light can communicate environmental timing to entrain the circadian system, it is a zeitgeiber (time-giver). g In this review, we consider the commonality of mediators of plasticity effectors and mechanisms between LTP in the hip- pocampus and circadian plasticity in the SCN. Once initiated, these feed-forward processes involving excitability and intra- cellular networks transform these systems into new, persistent states. On a higher-order level, the ability of the SCN to express plasticity-induced state changes is dependent upon the context that is modulated by the cycling circadian clock and the associated state changes in SCN neurons. This susceptibility to light-induced plasticity returns each night, as the circadian clock repeats its daily cycle, a phenomenon we term iterative metaplasticity. We pro- pose that the hippocampal circadian clock similarly modulates the potential for LTP and other forms of synaptic plasticity by iterative metaplasticity. CIRCADIAN RHYTHMS ARE GENERATED BY MOLECULAR AND METABOLIC OSCILLATORS How do molecules and metabolism generate self-sustaining near 24-h oscillators? The endogenous timing mechanism consists of a transcription-translation oscillator (TTO) with negative feedback (Lowrey and Takahashi, 2011) that interacts with a reduction- oxidation oscillator (RXO) (Gillette and Wang, 2014). In the TTO, heterodimers of positive transcription factors CLOCK/BMAL (or CLOCK/NPAS2 in some brain regions) bind to E-box motifs in the promoters of Per 1/2/3, Cry 1/2, and Rev-Erbα, activat- ing transcription. The protein products undergo complex post- translational modiﬁcations. Under appropriate conditions, PER and CRY proteins heterodimerize, translocate to the nucleus, and repress transcriptional activation by CLOCK/BMAL. An addi- tional regulatory feedback loop involves the negative regulator, REV-ERBα (a nuclear heme receptor) and the positive regula- tor, retinoic acid-related orphan receptor (ROR) (Lowrey and Takahashi, 2004; Yin et al., 2007, 2010). These antagonistic reg- ulators compete for binding to ROR elements (ROREs) within the promoters of Bmal1 and CLOCK. REV-ERBα also can mod- ulate transcription by binding to ROREs in the Per and Cry Frontiers in Systems Neuroscience WHY ARE THERE INTERNAL CIRCADIAN SYSTEMS? cAMP was the ﬁrst conﬁrmed non- transcriptional cytosolic oscillator in SCN neurons (O’Neill et al., 2008), followed by Ca2+ (Harrisingh et al., 2007), PKC (Robles et al., 2010), small G protein (Brancaccio et al., 2013), and other small molecules (Dodd et al., 2007). p The RXO emerges from robust, near-24-h rhythms of cel- lular metabolic state. Redox state oscillates in SCN samples ex vivo, as well as brain slices in vitro. This RXO modulates the membrane potential (Vm) of SCN neurons and thus the state of excitability (see further). Drivers of the RXO are presently unknown, however, redox oscillations are wide spread in cir- cadian systems (O’Neill and Reddy, 2011; O’Neill et al., 2011). Transcriptional regulation via REV-ERBα, an endogenous heme receptor, is exquisitely sensitive to cellular redox state. This is one of several nodes of interaction between the TTO and the RXO (Gillette and Wang, 2014). The interacting oscillators gen- erate rhythms in the synthesis of key cellular proteins, ﬁring rate of neurons, and release of neuropeptides (Hatcher et al., 2008; Wang et al., 2012). These clock-controlled rhythms can have dis- tinct phase-relationships, patterns, and amplitudes. Time-of-day restrictions on susceptibility to phase-resetting signals emerge from the complexity of theses dynamic systems. ( ) Beyond VIP, numerous neuropeptides have been found to be important to regulating clock function. Examples include the fol- lowing. (1) Mice lacking arginine vasopressin (AVP) receptors are more resistant to jet-lag and their recovery period is more rapid than wildtypes (Yamaguchi et al., 2013). This feature results from the looser coupling between cells in AVP receptor-deﬁcient SCN, which maintains the basic circadian rhythm under steady state, but is more responsive to zeitgebers for extreme phase-shift. (2) Gastrin-releasing peptide (GRP) is another major neuropeptide in the brain clock. Activation of GRP-receptors produces long- lasting excitation in SCN neurons (Gamble et al., 2007). This response is stronger in subjective night than daytime. The under- lying mechanism involves inhibition of resting K+ current and subsequent membrane depolarization (Gamble et al., 2011). (3) Pituitary adenylate cyclase-activating peptide (PACAP) is present in retinohypothalamic tract (RHT) terminals that invest SCN tissue. PACAP alters the excitability of SCN neurons in a bi- modal manner: it suppresses the discharge of some of the neurons via VIP•R2 receptors, while excites others via VIP•R1 receptors (Reed et al., 2002). WHY ARE THERE INTERNAL CIRCADIAN SYSTEMS? The internal milieu is not resting in a basal state when stimuli are encountered. Rather, the internal environment—from the level of cells to tissues, organs, and brain and body systems–oscillates with a major regular periodicity near 24 h, as well as various ultradian rhythms. Consequently, patterning of behavioral outputs changes signiﬁcantly over this period, so that some behaviors occur in the day, others at night, and some are expressed at dawn and dusk. Daily internal oscillations are a result of adaptation to a major environmental variable over the course of evolution: the ever- changing cycle of day and night generated by the Earth’s rotation on its axis. Early organisms that optimized cycles of behavior to September 2014 \| Volume 8 \| Article 164 \| 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. promoters. Transcription-factor binding is a dynamic process, which permits regulation based on relative amounts and states. Rates of synthesis and proteasomal degradation of clock pro- teins are important to rhythm generation (Gillette and Mitchell, 2002; Nitabach et al., 2002; Lundkvist et al., 2005; Golombek and Rosenstein, 2010; Van Ooijen et al., 2011). When PER and CRY are ubiquitinated and degraded, the cycle of Per and Cry transcription-translation repeats. The TTO takes ∼24-h to complete one cycle, as does an accessory loop comprising the transcription factor REV-ERBα. REV-ERBα binds to ROREs in promoter regions of the Clock and Bmal1 genes, initiating their transcription. the free-running circadian clock, resulting in arrhythmic behavior (Nitabach et al., 2002). While links between oscillations in Vm and circadian plasticity have not been fully established, recent studies suggest important roles for the neuropeptides released in the SCN. Their circa- dian rhythm is driven by oscillation in Vm, which ﬁne-tunes the neuronal responses to input signals. Among the more than 200 neuropeptides identiﬁed in SCN, vasoactive intestinal peptide (VIP) is the most characterized and well studied. VIP is an essen- tial neuropeptide for the synchrony of the brain clock (Aton et al., 2005). VIP binds to a G-protein-coupled receptor (VIP•R 2), activating cAMP and PKA pathways (Hao et al., 2006). cAMP is essential for the maintenance of intrinsic circadian rhythmicity; it is also important in information processing within the brain clock (Hastings et al., 2008). CIRCADIAN OSCILLATION IN SCN EXCITABILITY CIRCADIAN OSCILLATION IN SCN EXCITABILITY The daily rhythm of electrical activity in SCN neurons is essen- tial for the central pacemaker to synchronize circadian clocks throughout the body to each other and to changing environ- mental time cues (Brown and Piggins, 2007; Colwell, 2011). SCN neurons exhibit a daily ﬂuctuation of spontaneous action poten- tials (SAP), with higher frequency during the daytime than the night. The SAPs are autonomously generated by the SCN (Brown and Piggins, 2007), and their patterned behavior can be detected both in vivo and in vitro, by single- or multi-unit recording (Inouye and Kawamura, 1979; Green and Gillette, 1982; Welsh et al., 1995). Dissection of underlying ionic mechanisms by patch- clamp recordings of membrane properties of SCN neurons from mouse (Belle et al., 2009) and rat (Wang et al., 2012) reveals at least three ionic factors, K+ and Ca2+ currents and [Ca2+ i ], underlie rhythmic oscillating membrane potential (Vm) (Belle et al., 2009). These ionic features represent three functional cate- gories (Colwell, 2011): (1) currents providing the excitatory drive that elevates Vm to the threshold of action-potential generation; (2) currents responding to the excitatory drive and generating action potentials; and (3) currents contributing to the nightly silencing of ﬁring through hyperpolarization of the membrane. Modulation of these currents could be on the levels of channel expression, localization, post-translational modiﬁcation of con- ductance, and/or gating properties. Circadian oscillation in Vm is necessary for timekeeping. Electrical silencing of pacemaker neu- rons in Drosophila by genetic manipulation of K+ channels stops The SCN releases these, and many other neuropeptides, with levels of release following a circadian pattern (Hatcher et al., 2008). It is thought that these neuropeptides also play a role in synchronizing clocks in other brain regions by carrying time-of- day information from the SCN (Lee et al., 2010). It follows that neuropeptides may contribute to clock-based metaplasticity. Frontiers in Systems Neuroscience WHY ARE THERE INTERNAL CIRCADIAN SYSTEMS? PACAP also modulates light-induced phase- shift in complex ways: it facilitates phase-delay but attenuates phase-advance; the underlying mechanism has yet to be revealed (Chen et al., 1999). CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY The SCN processes information about signiﬁcant variations in availability of light or nutrients that necessitate adjustment of circadian timing. It dynamically responds to cues that communi- cate mismatch between internal and environmental time. Light, the signal of day-length, alters clock gene expression, as well as phases of the circadian oscillations in heart rate, ingestion, and wheel-running behavior. Whereas the rest-activity cycles of nocturnal and diurnal ani- mals are in anti-phase, SCN electrical activity in nocturnal and diurnal animals alike peaks during the light phase (Fuller et al., 2006; Brown and Piggins, 2007). This indicates the switch in peak activity in other brain regions that drive the behavioral differences between nocturnal and diurnal animals lies down- stream of the SCN, and afﬁrms the autonomy of SCN electrical activity rhythms. Extra-SCN brain tissue, such as the paraven- tricular nucleus (PVN), drifts out of synchrony in vitro but rapidly re-synchronizes to the rhythm of the SCN in co-culture (Tousson and Meissl, 2004). Mediators of this inter-region syn- chronization are subjects of current study. Multiple modes of communication of phase have been identiﬁed, including the electrical communication via neuronal circuits, as well as via diffusible signals (Silver et al., 1996; Tousson and Meissl, 2004; Guo et al., 2005; Kalsbeek et al., 2006; Welsh et al., 2010). Thus, in each cell of the body an endogenous circadian clock con- trols the daily timing of cell-speciﬁc transcription, cell dynamics, and signaling, but it relies on information from the SCN for coordination. g The SCN has the unusual property of responding to light dif- ferently at different points in the circadian cycle (Figure 1). The phase of rhythms in clock gene expression, neuronal ﬁring, and locomotor activity are unaffected by light in the daytime, but change signiﬁcantly when light is encountered during nighttime. The response to nocturnal light is bifurcated (Ding et al., 1994). Light in the early night (the period after the normal light-off) sig- nals a prolonged day; the clock responds by delaying its phase. In the late night (the period before lights-on), however, light signals an early dawn, advancing clock phase prematurely to a daytime state. The SCN generates such differential responses by selectively gating its susceptibility to inputs (see further). Gating is regu- lated at multiple levels, including neurotransmitter receptors and effectors of intracellular signaling pathways (Gillette and Mitchell, 2002; Golombek and Rosenstein, 2010). CIRCADIAN CLOCKS IN THE BRAIN Circadian rhythms of clock genes have been reported in sev- eral brain regions, including the prefrontal cortex, olfactory bulb, and hippocampus (Abe et al., 2002; Granados-Fuentes et al., 2006; Li et al., 2013). The hippocampus exhibits circadian oscil- lation in the expression of Per2, a hallmark of the TTO. The amplitude and persistence of LTP in the CA1 region varies in a cir- cadian manner (Chaudhury and Colwell, 2002; Chaudhury et al., 2005). Mutations in Per 2 that impair the circadian clock result in abnormal hippocampal LTP (Wang et al., 2009). This supports a necessary role for the circadian clock in permitting and enabling hippocampal plasticity. The SCN is acknowledged to be the central circadian clock in mammals (Lehman et al., 1987; Ralph et al., 1990). The SCN maintains its autonomous circadian rhythm when surgically iso- lated from the rest of the brain in vivo (Inouye and Kawamura, 1979) and in vitro, where synchronized near-24-h rhythms of electrical activity, metabolism, and clock-gene expression persist (Schwartz et al., 1980; Gillette and Prosser, 1988; Prosser et al., 1989; Yamazaki et al., 1998; Wang et al., 2012). Whereas all cells of the body possess intrinsic clocks, in the absence of the SCN the myriad cellular TTOs drift out of phase with one another. This was demonstrated elegantly in a mouse bearing a transgene reporter, PER 2::LUCIFERASE, where circadian rhythms in this clock protein are expressed as bioluminescence and can be mea- sured non-invasively in all cells and tissues (Yoo et al., 2004). When tissues were assessed in vivo after SCN lesion or cultured in isolation in vitro, individual cells from all tissues examined con- tinued to exhibit circadian oscillations, but with a range of phases that appeared arrhythmic when averaged (Welsh et al., 1995; Yoo et al., 2004). THE MANY BRAIN CONNECTIONS OF THE SCN The SCN has direct and indirect connections with many brain sites. Recent assessments identify 35 brain regions projecting directly to the SCN, and if multi-synaptic inputs are included, this number increases to 85 projecting regions (Morin et al., 2006; Morin, 2013). Input from the retina via the retinohypothalamic tract (RHT), the thalamic intergeniculate nucleus via the genicu- lohypothalamic tract (GHT) and the median raphae nucleus are September 2014 \| Volume 8 \| Article 164 \| 3 www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. considered critical to the “circadian visual system” (Morin and Allen, 2006). A study of the various primary and secondary inputs conducted using retrograde tracers (Krout et al., 2002) found many sources for SCN afferents, including regions in the hip- pocampal formation. Many brain regions are targets for efferent projections from the SCN (Abrahamson and Moore, 2001; Morin, 2013). Of particular interest is the indirect connection of the SCN, via the dorsomedial hypothalamus, to the locus coeruleus (Aston- Jones et al., 2001; Markov and Goldman, 2006), a region known to mediate cortical and hippocampal activation (Berridge and Foote, 1991). Intrinsically photoreceptive retinal ganglion cells (ipRGCs) act as photon-counters, marking the presence, duration, and inten- sity of light (Berson et al., 2002; Hattar et al., 2002). Their axons innervate the SCN and communicate the presence of light by releasing glutamate and the co-localized peptide, PACAP, onto the ventral SCN (Chen et al., 1999). The SCN integrates these signals and transmits information about the environmental light proﬁle to all other cellular circadian clocks in the brain and body. The SCN also receives timing signals from other, less potent, zeitgeibers including locomotor activity, sleep/wake states and nutritional status (Stephan, 1989; Lamont et al., 2005; Fuller et al., 2006), resulting in feedback control of the circadian timing system. Frontiers in Systems Neuroscience CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY Underlying differences in susceptibility are cyclic states of excitability and intracellular signaling elements that prime SCN receptivity to plasticity signals. We propose that this gating of light-signaling responsiveness, which cycles over the night and day, is a paradigm of iterative metaplasticity, the repeated, anticipatory susceptibility to induction of neuronal plasticity. FIGURE 1 \| The cyclic series of dynamic cellular states of the endogenous circadian clock is characterized by changing susceptibility that recurs every ∼24 h to signals that alter clock state, a paradigm of FIGURE 2 \| Signal transduction at the suprachiasmatic nucleus (SCN) in response to light activates divergent pathways in early vs. late night. Light experienced at night transmits signals via the FIGURE 2 \| Signal transduction at the suprachiasmatic nucleus (SCN) in response to light activates divergent pathways in early vs. late night. Light experienced at night transmits signals via the retinohypothalamic tract (RHT) to the SCN causing glutamate release. Glutamatergic activation of the NMDA receptor is necessary and sufﬁcient for initiating state changes, and leads to inﬂux of extracellular Ca2+. Ca2+/calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase (NOS) are activated, increasing levels of nitric oxide (NO). In the early night, the rise of NO activates ryanodine receptors (RyR) on the intracellular endoplasmic reticulum where Ca2+ is stored. Intracellular Ca2+ (Ca2+ i ) is released via the activated RyR and, through a mechanism yet to be elucidated, leads to phosphorylation of cAMP response element-binding protein (pCREB) and subsequent increased expression of clock genes. During the late night, however, NO activates guanylyl cyclase (GC), cGMP synthesis, and increased activity of cGMP-dependent protein kinase (PKG). Activation of this and other kinases again leads to increased pCREB and transcription of key clock genes. This simpliﬁed model includes only those elements necessary and sufﬁcient to stimulate state changes similar in amplitude and timing to light-induced responses. synthase (nNOS) (Agostino et al., 2004), triggering nitric oxide (NO) production. NOS activity is necessary for light-induced plasticity in the SCN (Ding et al., 1994, 1997; Melo et al., 1997), and the response to light or glutamate increases in the presence of an NO donor (Melo et al., 1997). Guido et al., 1999) and the clock gene Per 1 (Moriya et al., 2000). NMDA receptor antagonist application effectively blocks light- induced changes. In subjective daytime, glutamate exposure does not activate these signaling pathways, nor does it cause a change in phase. CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY The periodic recurrence of night and day provides an oscillatory environmental context for life on Earth. A near-24-h dynamic series of functional states organizes differential responses to light that depend upon time-of-day. Light-driven glutamatergic signals (lightning bolts) at night (blue) alter suprachiasmatic nucleus (SCN) state-dynamics and outputs; daytime (yellow) stimulation is without effect. Circadian timekeeping mechanisms generate these windows of susceptibility, poising the SCN to respond appropriately to a temporal error signal- in early night moving clock state back to an earlier time or in late night advancing it prematurely toward morning. These long-lasting changes in clock state express the hallmarks of neuronal plasticity. The gate to light-signaling is open transiently during nighttime (green arrow), but closed in daytime (red arrow). The gating mechanisms permissive for state changes are clock-driven, preceding the light signal. Thus, light-induced plasticity occurs only if the functional state of cells is permissive at that time. Underlying differences in susceptibility are cyclic states of excitability and intracellular signaling elements that prime SCN receptivity to plasticity signals. We propose that this gating of light-signaling responsiveness, which cycles over the night and day, is a paradigm of iterative metaplasticity, the repeated, anticipatory susceptibility to induction of neuronal plasticity. FIGURE 1 \| The cyclic series of dynamic cellular states of the endogenous circadian clock is characterized by changing susceptibility that recurs every ∼24 h to signals that alter clock state, a paradigm of iterative metaplasticity. The periodic recurrence of night and day provides an oscillatory environmental context for life on Earth. A near-24-h dynamic series of functional states organizes differential responses to light that depend upon time-of-day. Light-driven glutamatergic signals (lightning bolts) at night (blue) alter suprachiasmatic nucleus (SCN) state-dynamics and outputs; daytime (yellow) stimulation is without effect. Circadian timekeeping mechanisms generate these windows of susceptibility, poising the SCN to respond appropriately to a temporal error signal- in early night moving clock state back to an earlier time or in late night advancing it prematurely toward morning. These long-lasting changes in clock state express the hallmarks of neuronal plasticity. The gate to light-signaling is open transiently during nighttime (green arrow), but closed in daytime (red arrow). The gating mechanisms permissive for state changes are clock-driven, preceding the light signal. Thus, light-induced plasticity occurs only if the functional state of cells is permissive at that time. Frontiers in Systems Neuroscience CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY Light is communicated to the SCN from the retina by glu- tamatergic neurotransmission from the RHT (Ding et al., 1994; Welsh et al., 2010). During subjective nighttime, glutamate or the agonist N-methyl D-aspartate (NMDA), are sufﬁcient to activate light signaling in vivo (Colwell and Menaker, 1992; Vindlacheruvu et al., 1992; Gannon and Rea, 1993, 1994) or the SCN brain slice in vitro (Ding et al., 1994; Shirakawa and Moore, 1994). They mimic the effects of light: changing circadian phas- ing, inducing c-fos and other immediate early genes (Ebling, 1996; In addition to its autonomous ∼24-h rhythmicity, the SCN holds a privileged position among circadian clocks at the cell and systems levels in receiving direct information about the presence and intensity of environmental light from the retina via the RHT. It is then able to transmit this information to peripheral clocks. September 2014 \| Volume 8 \| Article 164 \| 4 www.frontiersin.org www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. FIGURE 2 \| Signal transduction at the suprachiasmatic nucleus (SCN) in response to light activates divergent pathways in early vs. late night. Light experienced at night transmits signals via the retinohypothalamic tract (RHT) to the SCN causing glutamate release. Glutamatergic activation of the NMDA receptor is necessary and sufﬁcient for initiating state changes, and leads to inﬂux of extracellular Ca2+. Ca2+/calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase (NOS) are activated, increasing levels of nitric oxide (NO). In the early night, the rise of NO activates ryanodine receptors (RyR) on the intracellular endoplasmic reticulum where Ca2+ is stored. Intracellular Ca2+ (Ca2+ i ) is released via the activated RyR and, through a mechanism yet to be elucidated, leads to phosphorylation of cAMP response element-binding protein (pCREB) and subsequent increased expression of clock genes. During the late night, however, NO activates guanylyl cyclase (GC), cGMP synthesis, and increased activity of cGMP-dependent protein kinase (PKG). Activation of this and other kinases again leads to increased pCREB and transcription of key clock genes. This simpliﬁed model includes only those elements necessary and sufﬁcient to stimulate state changes similar in amplitude and timing to light-induced responses. FIGURE 1 \| The cyclic series of dynamic cellular states of the endogenous circadian clock is characterized by changing susceptibility that recurs every ∼24 h to signals that alter clock state, a paradigm of iterative metaplasticity. CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY The light signaling pathway bifurcates in early vs. late night, downstream of NO (Figure 2). During the early night, glutamate- induced plasticity requires Ca2+-induced-Ca2+ release (CICR) from neuronal ryanodine receptors (RyRs). When RyRs are phar- macologically activated, the effects of light/glutamate in the early night are reproduced. The same agents have no effect in late night or daytime. Further, inhibition of RyRs blocks light-/glutamate- induced phase delay in the early night both in vivo and in vitro, but has no effect in late night or daytime (Ding et al., 1998). Initial steps of glutamate signaling include NMDA recep- tor (NMDA•R) activation, Ca2+ inﬂux into cells in the SCN (Ding et al., 1998; Obrietan et al., 1998; Colwell, 2000, 2001), and stimulation of downstream kinases (Figure 2). Among the earliest changes is in Ca2+/calmodulin-dependent kinase Type II (CaMKII), which is activated by auto-phosphorylation. Inhibition of CaMKII in early night blocks light-induced phase delays and changes in c-fos and Per 1/Per 2 expression in the SCN (Golombek and Ralph, 1995; Fukushima et al., 1997; Yokota et al., 2001). Active pCaMKII phosphorylates neuronal nitric oxide During late night, on the other hand, NO activates guanylyl cyclase (GC), which increases cGMP levels. This in turn leads to the activation of cGMP-dependent kinase (PKG) (Weber et al., September 2014 \| Volume 8 \| Article 164 \| 5 www.frontiersin.org www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. Time-of-day-dependent neurotransmission via the inhibitory neurotransmitter gamma-amino butyric acid (GABA) pro- foundly affects SCN responses to light. GABA and its biosyn- thetic enzyme, glutamatic acid decarboxylate (GAD), are widely expressed in SCN neurons (Card and Moore, 1984; van den Pol, 1986; Okamura et al., 1989; Moore and Speh, 1993). Both GABAAand GABAB receptors have been localized within the SCN by binding and functional studies (Francois-Bellan et al., 1989; Liou et al., 1990; Mason et al., 1991). Further, both GAD activ- ity and GABA levels undergo circadian oscillations in the rat SCN (Aguilar-Roblero et al., 1993). Modulatory effects of GABA sig- naling on light-induced plasticity are complex, and consistent with a model wherein GABAB receptors regulate photic signals via presynaptically modulating glutamatergic input from the RHT, whereas GABAA receptors are proposed to act in the photic signal transduction cascade in local circuits downstream of glutamater- gic inputs (Ralph and Menaker, 1989; Gillespie et al., 1997). CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY GABAergic activation of GABAB receptors diminishes release of labeled glutamate at the SCN in response to optic nerve stimula- tion (Liou et al., 1986). Consistent with this observation, GABAB receptor agonists reduce light-induced phase delays in early night; an antagoinist of GABAB receptors enhances this phase delay (Gillespie et al., 1997). With regard to light-effects in late night, microinjections of agonists of either GABAAand GABAB recep- tor agonists into the SCN region signiﬁcantly diminish phase- advances. Thus, GABAergic modulation of glutamatergic and downstream synapses contributes to gating and amplitude of the light-response in the SCN. Inhibition of the phase shift has been proposed to be mediated by the opposing effects of light and GABA on Period gene expression (Ehlen et al., 2008). Additionally, GABA modulates light-induced plasticity by induc- ing divergent responses in intracellular Ca2+ mobilization in SCN neurons (Irwin and Allen, 2009). 1995; Ding et al., 1998; Tischkau et al., 2003b). Phase advance caused by light/glutamate in the late night is mimicked by cGMP analogs (Prosser et al., 1989) and blocked by pharmacological inhibition of PKG (Weber et al., 1995; Mathur et al., 1996; Ding et al., 1998; Ferreyra and Golombek, 2001). The same inhibition does not impact the phase delay induced in early night. In both pathways, downstream from the steps described, there is transient and rapid phosphorylation of the Ca2+-cAMP response element-binding protein (CREB), leading to transcrip- tion of Per 1, as well as other CRE-mediated genes (Ginty et al., 1993; Ding et al., 1997; Gau et al., 2002). Phase advance during late night is blocked by antisense oligodeoxynucleotides (αODNs) of CRE sequences. These Ca2+/cAMP response element decoys (CRE-decoys) sequester pCREB and selectively block the advance in clock phase (Tischkau et al., 2003a). Other factors downstream of glutamate-induced signaling in the SCN are the mitogen-activated protein kinases (MAPKs) and cAMP-dependent kinase (PKA). Both light-induced phase delays and advances are partially blocked by inhibitors of p44 MAPK/ERK1 and MAPK kinase (MAPKK/MEK) transcription (Obrietan et al., 1998; Tischkau et al., 2000; Butcher et al., 2002; Antoun et al., 2012). Light pulses during the subjective night induce activation of members of all three MAP kinase pathways: ERK, JNK, and p38 (Butcher et al., 2003; Pizzio et al., 2003). Signaling via p44/42 MAPK (ERK1/ERK2) is necessary for Ca2+- induced CRE-mediated gene transcription (Obrietan et al., 1998; Butcher et al., 2002; Antoun et al., 2012). CIRCADIAN OSCILLATION IN SUSCEPTIBILITY TO LIGHT-INDUCED PLASTICITY PKA activation by light and glutamate has differential effects on SCN state at early vs. late night. Upon light or glutamate stimulation, PACAP and PKA enhance the amplitude of light-/glutamate-induced phase delay in early night, and diminish the effect in late night (Tischkau et al., 2000). These effects are like those of PACAP, which were described earlier. Although the complete signaling cascade from light to clock gene expression remains unresolved, the roles of these well- studied effectors downstream of light/glutamate provide insights into the most completely studied form of circadian plasticity in the SCN (Figure 2). The elucidation of the roles of several of these effectors in SCN plasticity, including pCREB and RyRs, was inﬂuenced by their role in plasticity signaling in the hippocampus (Ding et al., 1994, 1997, 1998). Various regulators of light-induced plasticity affect the net- work properties of SCN neurons. Brain-derived neurotrophic factor (BDNF) is expressed in the SCN. The cognate recep- tor of BDNF, tropomyosin-related receptor kinase B (TrkB), is expressed in the ﬁbers of the RHT (Allen and Earnest, 2005). BDNF expression and release are rapidly increased by neuronal activity, which regulates presynaptic release and direct activation of membrane channels (Rose et al., 2004; Blum and Konnerth, 2005). This enhanced expression and response to optic nerve stimulation suggest a role for BDNF in modulating photic input to the SCN (Allen and Earnest, 2005). BDNF- and TrkB-deﬁcient mice exhibit a reduction in the phase-shifting effects of light on the circadian system (Liang et al., 2000). BDNF cannot shift the phase of the clock itself (Prosser et al., 2008; Mou et al., 2009), but interfering with BDNF signaling in the SCN blocks or strongly inhibits phase shifts induced by light and glutamate in the subjec- tive night (Liang et al., 2000; Michel et al., 2006). SCN treatment with either BDNF or tissue-type plasminogen activator (tPA) per- mits light and/or glutamate to induce phase shifts in the daytime (Liang et al., 2000; Mou et al., 2009). BDNF thus contributes to gating of SCN responsiveness to state-changing signals, a concept we will explore in the next section. Gating of the light-response in the SCN is conferred by regulaton of glutamatergic synaptic transmission in the SCN (Kim et al., 2006). CIRCADIAN GATING OF CLOCK FUNCTION: ITERATIVE METAPLASTICITY Because SCN responsiveness to light-induced state changes waxes and wanes with each daily circadian cycle, states of sensitivity to plastic change repeat each night, when light has the poten- tial to interrupt the anticipated darkness. Thus, the nocturnal state of SCN activity alters the magnitude and/or duration of plastic events compared with the daytime state. Dynamic link- age of temporal history and state with current responsiveness deﬁnes metaplasticity (Abraham and Bear, 1996). At night, the SCN responds to light as an error signal–it infers a mismatch between the timing of the clock and the environmental light cycle. The SCN must be able to distinguish nocturnal light as an error signal to respond with temporally appropriate phase-resetting. The restricted, time-of-day dependence of susceptibility of the Frontiers in Systems Neuroscience September 2014 \| Volume 8 \| Article 164 \| 6 www.frontiersin.org www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. SCN to light establishes conditions for the iterative metaplastic state. circadian variation in hamsters. NR2B mRNA level is high from late day through early night, with the phosphorylated protein peaking in the late night (Wang et al., 2008). Changes in phos- phorylation of NMDA•R subunits can correspond to changes in functional properties. An endogenous daily rhythm in the mag- nitude and duration of NMDA•R-induced Ca2+ transients in rat SCN neurons peaks during the night, as does the rhythm in NMDA•R-evoked currents (Pennartz et al., 2001). Thus, evidence indicates that peaks in mRNA abundance of certain subunits pre- cede peaks in NMDA•R phosphorylation. NMDA function peaks in the night in rats, anti-phase to mRNA abundance. Together, these observations indicate that the clock primes the SCN to respond to photic signals in anticipation of encountering light signals at night. During the night, the manner in which phase is affected varies depending on proximity of the signal to the subjective dusk or dawn encoded in the circadian clock. Dynamic gating of inputs to the SCN ensures that the same input (light) has restricted and dif- ferential effects on output that depend on the time of day during stimulation. Gating is achieved by clock-driven rhythms in Vm, expression of plasticity modulators, and expression/receptiveness of intracellular effectors of plasticity. This implies the presence of a reciprocal relationship wherein various inputs alter timekeep- ing elements and induce plasticity, and their oscillations are in turn controlled by the circadian clock. cAMP/PKA In constant conditions, the rat SCN exhibits spontaneous oscilla- tions in cAMP levels and cAMP-dependent protein kinase (PKA) activity. The endogenous levels of cAMP peak at the end of day and of night (Prosser and Gillette, 1991). While cAMP levels rise in response to light/glutamate stimulation, applica- tion of agonists to cAMP/PKA does not mimic the effect of light/glutamate. However, if cAMP/PKA is activated simulta- neously with light/glutamate stimulation, resultant plasticity is enhanced in early night, and diminished in late night. Thus, the cAMP/PKA system toggles responses, changing the state of sig- naling pathways based on time of activation (Tischkau et al., 2000). CaMKII Calmodulin kinase II (CaMKII), which is activated in neurons by elevated Ca2+ i , undergoes circadian changes in autophospho- rylation state in hamsters, with peak abundance of pCaMKII occurring during subjective day. Total CaMKII levels in the SCN do not vary. Light pulses during the subjective night rapidly phos- phorylate CaMKII, which is necessary for the signaling cascade resulting in circadian plasticity (Agostino et al., 2004). INTRACELLULAR Ca2+ cGMP levels and cGMP-dependent protein kinase (PKG) activity also display spontaneous oscillations in the SCN. Tissue cGMP levels remain relatively constant throughout subjective day and night, with a sharp peak appearing at the time corresponding to end of night. PKG activity is markedly higher at end of subjective night compared to the end of subjective day and the middle of subjective night. Sensitivity of the SCN to light-induced increase in cGMP levels and PKG activity occurs just before these val- ues peak endogenously (Tischkau et al., 2003b). This rise in cGMP/activation of PKG is required for clock dynamics to pro- ceed; if either is blocked, clock state reverts by 3.5 h and then recapitulates the intervening period (Tischkau et al., 2004). Peak PKG activity may signal the transition from night to the day- time state. A light-pulse at late subjective night may prematurely shift the clock to this state, thus advancing the clock phase to a point it normally transits at the end of the night state of the circadian cycle and that is required for entry into the daytime state. Membrane excitability inﬂuences neuronal plasticity through intracellular signal pathways where Ca2+ plays an essential role. SCN neurons exhibit spontaneous oscillation of intracellular Ca2+ (Ca2+ i ) concentration, with peak levels during the daytime (Ikeda et al., 2003). While Ca2+ i oscillation is not completely driven by membrane events, the action potential-induced open- ing of voltage-gated Ca2+ channels (L-type) is a proven source of Ca2+ i elevation during the daytime (Irwin and Allen, 2007). CIRCADIAN GATING OF CLOCK FUNCTION: ITERATIVE METAPLASTICITY Circadian control of input allows the clock to prime SCN neurons in anticipation of the acti- vation of speciﬁc signaling cascades at speciﬁc times of the day. Key signaling elements that are potential targets of metaplastic regulation follow. MEMBRANE EXCITABILITY The proposed model is that SCN neurons are highly electrically active during daytime, preventing additional excitatory input from the RHT from inducing further changes. At night, however, the neurons are hyperpolarized, thus excitatory input from the RHT elicits signiﬁcant responses. Supporting this hypothesis, up- regulation of K+ currents and the consequential hyperpolarized status of Vm during nighttime are essential for gating of phase- shift responses. In excitable cells, Vm is mainly set by a class of two-pore domain K+ channels (K2P, TASK, and TREK chan- nels) (Hallie, 2001). Gating of these channels is independent of voltage across the membrane; the current is termed leak or back- ground current. K2P channels are encoded by the KCNK gene family. Both Kcnk1 and Kcnk2 are expressed in the SCN (Lein et al., 2007); in particular, Kcnk1 exhibits a robust rhythm in the SCN (Panda et al., 2002). Interestingly, a functional study suggests that the K+ leak current is regulated by metabolic oscillation in SCN neurons, which provides a non-transcriptional pathway of the clockwork machinery to modulate the membrane excitability (Wang et al., 2012). Frontiers in Systems Neuroscience CREB Phosphorylation of the Ca2+-cAMP response element-binding protein (forming pCREB) links stimulation of these intracellular signaling pathways to transcriptional activation of genes whose promoters bear the Ca2+-cAMP response element (CRE). An endogenous oscillation in basal levels of pCREB occurs in rat and mouse SCN, with a corresponding oscillation in expression of CRE-mediated genes. pCREB levels peak from the middle to end of subjective night. This is followed by peak expression of CRE-mediated genes, which occurs from late-subjective night to mid-subjective day (Obrietan et al., 1999). Light induces 133Ser pCREB at levels proportionate to light intensity and expression of CRE-mediated genes only at subjective night (Ginty et al., 1993; Ding et al., 1997; Gau et al., 2002). This indicates that there is strict regulation of CRE-mediated induction of gene expression during the circadian cycle, with only the night being favorable for the CREB/CRE-transcription pathway (Ding et al., 1997). GABA There is a diurnal rhythm of activity of GAD, which synthesizes GABA (Aguilar-Roblero et al., 1993). GABA release within the SCN also oscillates (Aton et al., 2006), peaking at the early night (Itri and Colwell, 2003). The peak in GABA release coincides with the timing of the most hyperpolarized state of Vm in SCN neu- rons (Wang et al., 2012). This release pattern is modulated by VIP signaling (Itri and Colwell, 2003; Itri et al., 2004; Aton et al., 2006). Deeper understanding of the signaling mechanisms underly- ing synaptic plasticity has raised questions as to the manner in which these plasticity mechanisms are modulated. While a num- ber of factors can regulate plasticity at the time of occurrence, activity-dependent processes that modulate plasticity by altering the state of synapses or cells prior to plasticity-inducing events are metaplastic. That is, synaptic metaplasticity can be considered the plasticity of synaptic plasticity. It acts to enhance the salience of subsequent exposure to certain types of stimulation, and prevent saturation of LTP and LTD, which can have negative effects on learning and memory as well as neuronal health (Deisseroth et al., 1995; Philpot et al., 2007; Abraham, 2008; Mockett and Hulme, 2008). BDNF BDNF mRNA levels in the SCN peak during the early subjective day, and BDNF protein levels peak during the subjective night (Liang et al., 1998). The expression of this rhythm in BDNF is dependent on a tetrodotoxin (TTX)-sensitive neuronal circuit (Baba et al., 2008). These observations support a role for BDNF in gating circadian responses to nocturnal light. COMMONALITIES IN PLASTICITY PATHWAYS: SITES OF CLOCK-DRIVEN METAPLASTICITY IN THE HIPPOCAMPUS The best-characterized form of activity-dependent synaptic plas- ticity is long-term potentiation (LTP). Deﬁned as persistent enhancement in synaptic efﬁcacy due to repeated activation of the same synapses, it is widely considered, along with long-term depression (LTD), to be the physiological basis for acquiring new memories and enhancing nascent ones (Malenka and Nicoll, 1999; Lisman, 2003; Cooke and Bliss, 2006). While the molecu- lar basis of LTP is still incompletely determined, the roles of a large number of signaling mechanisms and molecular effectors have been elucidated. Most in vitro studies on LTP have been conducted by delivering high frequency stimulation to Schaffer collateral ﬁbers connecting the CA3 and CA1 pyramidal neurons. Glutamatergic synapses were the ﬁrst investigated for LTP and the well-studied synapses between Schaffer collateral ﬁbers and CA1 pyramidal neurons exhibit LTP mediated by NMDA•R activation (Bliss and Collingridge, 1993; Lisman, 2003). There is, however, synapse-to-synapse heterogeneity in the molecular mechanisms associated with LTP. NMDA RECEPTOR The SCN exhibits circadian expression of mRNAs for subunits of the NMDA receptor (NMDA•R) subunits ε3 and ζ1 in rats; mRNA levels are high during daytime and low during night- time, whereas protein levels change in anti-phase. Expression of these mRNAs increases in response to light stimulation in the subjective night (Ishida et al., 1994). Both expression and phosphorylation of the ε2/NR2B subunit of NMDA•R undergo September 2014 \| Volume 8 \| Article 164 \| 7 www.frontiersin.org www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. MAPK the changes in the state of synapses or neurons that impact the amplitude and persistence of subsequent instances of plasticity (Abraham, 2008). Circadian rhythms have been implicated previously as a form of metaplasticty (Gerstner and Yin, 2010; Gerstner, 2012). Therefore, we propose, that the alteration of sig- nal responsivity of SCN cells is a metaplastic form of modulation of plasticity. Three members of the mitogen-activated protein kinase (MAPK) family, ERK, JNK, and p38, undergo oscillations in phosphoryla- tion in hamsters, with peak phosphorylation levels during the day. All three are phosphorylated in response to light at mid-subjective night (Pizzio et al., 2003). In mice, ERK1 and ERK2 signaling is induced by light pulse in the subjective night. Levels of phospho- rylated ERK (pERK) show a circadian oscillation, with peak levels at mid-to late-subjective day (Obrietan et al., 1998). Three members of the mitogen-activated protein kinase (MAPK) family, ERK, JNK, and p38, undergo oscillations in phosphoryla- tion in hamsters, with peak phosphorylation levels during the day. All three are phosphorylated in response to light at mid-subjective night (Pizzio et al., 2003). In mice, ERK1 and ERK2 signaling is induced by light pulse in the subjective night. Levels of phospho- rylated ERK (pERK) show a circadian oscillation, with peak levels at mid-to late-subjective day (Obrietan et al., 1998). With the core clock driving SCN metaplasticity, SCN neu- rons can show cyclic variation in their metaplastic state. This is distinct from metaplastic regulation as it has been described pre- viously (Abraham and Bear, 1996; Abraham, 2008). Persistence of clock-driven metaplasticity is restricted to a regular, discrete period within the 24-h daily cycle. Metaplastic effects that have been studied previously persist from a few minutes to a few days (Abraham, 2008). Therefore, we propose that the repetitive pat- tern of circadian neuronal state-changes constitutes a paradigm of iterative metaplasticity (Figure 1). CONCLUSION Endogenous rhythms in expression/function of these effectors and modulators are timed to allow temporally speciﬁc signal- ing cascades. Functional states of SCN cells with respect to these molecules are varied in advance of the point in time when the plasticity-inducing event is anticipated, namely light in the early or late night (Gillette and Wang, 2014). The clock thus primes cells to respond to plasticity signals in a speciﬁc, time-of-day dependent manner. Key to the concept of metaplasticity is that any physiolog- ical or biochemical change in the state of the cell or synapse needs to persist beyond the initial activity that triggers the meta- plastic changes. This distinguishes the paradigm from direct synaptic regulation, which occurs concurrently with synaptic plasticity. The initial bout of activity, or priming, changes the These intracellular effectors overlap with some of the effectors that contribute to the standard paradigm for metaplasticity, September 2014 \| Volume 8 \| Article 164 \| 8 Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. functional state of the synapse, neuron, and network, and thus its susceptibility to future plasticity-inducing events (Abraham, 2008; Hulme et al., 2014). to facilitate LTP in the CA1 region of the rat hippocampus (Nakatsuka and Natsume, 2014). Further, the clock gene Per 2 oscillates in isolated hippocampal slices, indicating the presence of an endogenous clock in the hippocampus. In support of this link, mutations in Per2 cause abnormal LTP in the hippocampus, mediated by decreased phosphorylation of CREB (Wang et al., 2009). There is emerging evidence demonstrating that the time-of-day affects the magnitude and persistence of synap- tic plasticity. Several studies have shown circadian variation in the efﬁcacy of LTP (Chaudhury et al., 2005; Nakatsuka and Natsume, 2014). Time-of-day effects may be one mechanism of metaplasticity. How the brain clock interacts with synaptic plasticity to cause circadian variation is a question ripe for deeper investigation. Both direct and indirect interactions can be hypothesized. While the peripheral hippocampal clock may itself drive rhythms in levels/activity of speciﬁc molecules, there also may be signals from the core SCN clock that drive or synchronize these rhythms. Signals, such as neuropeptides, hormones, and small molecules, may reach the hippocampus directly, or regu- late hippocampal activity indirectly. For instance, signaling from the SCN is known to be critical for rhythmic variation in hor- mone levels. CONCLUSION Several hormones that are known to affect hip- pocampal LTP, including melatonin and cortisol, are released in circadian patterns controlled by the SCN (Reppert et al., 1981; Gillette and Mitchell, 2002; Chaudhury et al., 2005; Chan and Debono, 2010). This could be one mechanism by which the cycling of the clock can lead to time-of-day changes in LTP. In the SCN itself, glutamate application has been shown to induce LTP of ﬁeld potentials activated by RHT stimulation in a time-of -day dependent manner. These experiments were performed with SCN slices in vitro, indicating that the core clock contributes to these time-of-day dependent changes in LTP (Nisikawa et al., 2002). In this review, we have detailed effectors of SCN plasticity that are under circadian control. Several of these effectors are also involved in hippocampal LTP, including glutamate, NMDA•R, CaMKII, Ca2+, NO, RyR, cGMP/PKG, cAMP/PKA, MAPK, and CREB (Lu et al., 1999; Lu and Hawkins, 2002; Monfort et al., 2002; Cooke and Bliss, 2006; Irvine et al., 2006; Zorumski and Izumi, 2012). While a detailed description of the role of these molecules in LTP is outside the scope of this review, the common- ality of signaling molecules between plasticity-inducing events in the SCN and hippocampal LTP is striking. Further, signaling cascades involving some of these molecules are also common to both processes. The NO-GC-cGMP-PKG pathway, which is crit- ical in late night signaling in the SCN, has also been shown to contribute to late-phase LTP (L-LTP) (Lu et al., 1999; Lu and Hawkins, 2002; Ping and Schafe, 2010). The cAMP-MAPK-CREB pathway is also involved in both signaling processes (Gerstner and Yin, 2010). In both cases, this signaling cascade induces plastic- ity by targeting CREB. Glutamate-induced NMDA•R activation, Ca2+ inﬂux, and CaMKII phosphorylation are critical signals that mediate both hippocampal LTP and SCN state changes (Malenka and Bear, 2004). Further, several of the signaling ele- ments described, including NMDA•R, Ca2+ i , PKC, NO, CaMKII, and MAPK, previously have been hypothesized as sites for meta- plastic regulation of synaptic plasticity (Abraham, 2008; Lucchesi et al., 2011; Zorumski and Izumi, 2012). Lastly, hippocampal plasticity is modulated by molecules that are also modulators of SCN plasticity, like BDNF (Bramham and Messaoudi, 2005; Lu et al., 2008; Minichiello, 2009; Schildt et al., 2013) and GABA (Arima-Yoshida et al., 2011; Nakatsuka and Natsume, 2014). Frontiers in Systems Neuroscience CONCLUSION We propose that (1) the commonality of plasticity elements in the SCN and hippocampus, (2) the existence of a circadian clock in the hippocampus that modulates the ability to acquire LTP over ∼24-h, and (3) evidence for communication between the SCN and the hippocampus all point to clock-driven, iterative metaplasticity in the hippocampus. Further exploration around- the-day of hippocampal expression and activity of molecules, such as NO, PKG, GABA, that have been discussed in this review will shed more light onto clock driven iterative metaplasticity in the hippocampus. The 12-h-limited duration of the persis- tence of SCN metaplasticity each 24-h cycle makes it a valuable model to probe how such processes affect neurophysiology and the molecular effectors of synaptic plasticity. Such understanding is also critical to elucidating other, non-circadian mechanisms of metaplasticity. Cycling of the circadian clock results in iterative metaplasticity via regulation of the effectors and modulators of plasticity in the SCN. Might such a relationship also exist between clock cycling and hippocampal plasticity? One indication of such a paradigm comes from a study that showed that activity of MAPK as well as adenylyl cyclase, and levels of cAMP vary in a circadian fash- ion in the hippocampus. These oscillations parallel Ras activity and phosphorylation of MAPK kinase and CREB. The varia- tions persist under free-running conditions, indicating they are endogenous in nature. These oscillations were shown to impact long-term memory (Eckel-Mahan et al., 2008). A further study showed that oscillations of adenylyl cyclase and MAPK in the hippocampus are dependent upon an intact SCN (Phan et al., 2011). Endogenous circadian rhythms Autonomous, self-generated near-24-h rhythms at any level organization of life, such as expression/function of proteins, cellular physiology, neuropeptide release, and amplitude of behavior. Circadian rhythms are deﬁned by their ability to persist with near-24-h periods in the absence of exogenous temporal cues. For example, animals maintained in aperiodic environments such as constant darkness and SCN brain slices maintained in vitro exhibit various circadian rhythms, ranging from the rhythmic patterning of wakefulness and sleep, neuronal ﬁring rate, neuropeptide release to cellular metabolic state. They are driven at the cellular level by a transcription-translation oscillator (TTO) and a redox oscillator (RXO). Circadian plasticity p y A persistent change of circadian-clock state in response to signiﬁcant stimulation during a discrete phase of the ∼24-h cycle. When it occurs inappropriately at night, environmental light can permanently change, or reset, the state of the SCN clock. The clock dynamically controls its own susceptibility by circadian plasticity. AUTHOR CONTRIBUTIONS GABAergic system in the suprachiasmatic nuclei of the rat. Neurosci. Lett. 157, 199–202. Rajashekar Iyer, Tongfei Wang and Martha Gillette wrote the text; Martha Gillette revised the text, and provided expertise and funding. Allen, G. C., and Earnest, D. J. (2005). Overlap in the distribution of TrkB immunoreactivity and retinohypothalamic tract innervation of the rat suprachiasmatic nucleus. Neurosci. Lett. 376, 200–204. doi: 10.1016/j.neulet.2004.11.076 CONCLUSION Considering circadian gating of inputs in the context of hip- pocampal and SCN metaplasticity provides striking insights on the ways in which signaling mechanisms conserved between two brain regions can impact their functional states. As a well studied system with an iterative impact on plasticity, the circadian clock offers a compelling model system for the study of metaplastic- ity. Conversely, new insights on links between circadian rhythms and synaptic plasticity can positively impact the study of SCN and hippocampal function. Further understanding of the interactions between these two critical brain processes will require that future research in either ﬁeld is more deeply informed by the distinct methodological considerations of the other. The potential ben- eﬁts to understanding the substrates and dynamics of cognitive disorders in both cases can be enormous. A role for GABA in circadian rhythms of LTP has been demon- strated, with nighttime disinhibition of a GABAA network shown September 2014 \| Volume 8 \| Article 164 \| 9 Frontiers in Systems Neuroscience Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org Circadian gating: iterative metaplasticity Iyer et al. Metaplasticity A plasticity regulatory phenomenon where experience alters the ability of a system to respond to a subsequent plasticity-inducing stimulus. Metaplasticity was originally described as a mechanism for regulating and tuning synaptic plasticity, but also can alter cell or network state. A critical feature of metaplasticity is that once triggered, metaplastic change must persist long enough to impact a plasticity event occurring at a later time. A metaplastic state can either increase or decrease the amplitude or duration of responses to a later plasticity event. Iterative Metaplasticity Circadian gating Circadian gating Restricted sensitivity and response to plasticity signals that depends upon the time of day. Whether, and to what extent, phase is reset by neurotransmitter signals is under the control of the circadian clock, which is able to gate sensitivity by regulating membrane state and the expression and function of various signaling effectors of plasticity. Iterative Metaplasticity p y Gating of receptivity to subsequent signals that repeats on a cyclic timebase. An example is gating of susceptibility to light-induced plasticity by the dynamic of the circadian clock in the SCN. Gating of receptivity is achieved by clock-generated, ∼24-h rhythms in neuronal membrane state and/or expression or activation state of intracellular signaling pathways permissive for light/glutamate stimulation. ACKNOWLEDGMENTS Antoun, G., Bouchard-Cannon, P., and Cheng, H. Y. (2012). Regulation of MAPK/ERK signaling and photic entrainment of the suprachiasmatic nucleus circadian clock by Raf kinase inhibitor protein. J. Neurosci. 32, 4867–4877. doi: 10.1523/JNEUROSCI.5650-11.2012 We thank past and present members of the Gillette lab, especially Jennifer Mitchell, for insightful discussions. 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https://openalex.org/W2793549097	https://www.revista.classica.org.br/classica/article/download/477/449	Portuguese	null	Da filologia à iconografia. A permanência do arcaico nas imagens tríplices de Hécate	Classica	2,001	cc-by	2,742	Classica, Sao Paulo, v. 1 311 4, n. 1311 4, p. 1 01 -1 07,2000/2001. Classica, Sao Paulo, v. 1 311 4, n. 1311 4, p. 1 01 -1 07,2000/2001. Haiganuch Sarian: Da filologia h iconografia A permanencia do arcaico nas imagens tripiiis de Hecate. Os autores modernos impuseram o termo kicckzatov (ou ~ K ~ Z E L O V ) para designar as representacoes de Hecate triplice. Ora, as fontes literarias antigas parecem revelar que este termo nao se aplicava unicamente a Hecate triplice. A este respeito, o testemunho mais importante, muitas vezes citado mas pouco entendido, e o das Vespas de Aristofanes, 800- 804: os atenienses erigiram uma pequena tribuna "como um Hekateion, por toda parte diante de suas portas" (chnuq~ 'EK&ZELOV mwa~oU z6v Bupov). Porem, aqui o poeta refere-se a deusa "como um Hekateion" (v.804). que se pode interpretar como uma capela (iepov) ou uma estatua de Hecate conforme o escolio ao texto de Aristofanes. Com efeito, este escoliasta diz muito claramente que 'EK~ZELOV (OU 'E~&zaiov) e o kpbv ' E ~ & q g ou ' E ~ & q g &yahpa, tambem equivalente de E~amimov Por outro lado, a mesma palavra aparece em outra passagem de Aristofanes, as Ras 366 (z6v 'E~azaiov) e a explicacao do escoliasta para este verso nao deixa nenhuma duvida sobre o seu sentido: ~ a z a z6v ayahp&zov q g ' E ~ & q c ("das estatuas de Hecate"). O testemunho de outros comentadores tambem e revelador: Hesiquio, s.v. k ~ a z a w escreve com clareza z a npo ~ 6 . h Oqkijv ' E K & ~ $ ay&Xpaza, nVEc 6E za EV zpto60q ("estatuas de Hecate diante das portas, aquelas que se colocam nas encruzilhadas"); o mesmo sentido de "estatua de Hecate" e dado pelo lexicografo Suda: kicazatov: ' E K & ~ &yahpa. Podemos entao concluir que os termos &K&ZELOV, k~&zatov, k~amjmov podiam significar i&@v, capela, nicho ou simplesmente um local consagrado a Hecate, ou &yahpa, estatua da deusa sob sua forma triplice ou mesmo simples. Convem ressaltar que alguns documentos figurados confirmam a equivalencia dos termos &K&Z~LOV e &yahpa a proposito das imagens triformes e da deusa com um so corpo. Assim, as representacoes de Hecate guardia das portas - xpobpaia - sao indiferen- temente imagens desta divindade com um so corpo ou triplice: figura Unica diante do pala- cio de Hades (Sarian, "Hekate", 11-27.28 e 43), diante de uma casa (idem, n" 4546, 58 b-c), diante ou dentro de locais sagrados (idem, na 50.58'. 59-61,63 e 106). Da filologia a iconografia A permanencia do arcaico nas imagens triplices de Hecatel HAIGANUCH SARIAN Museu de Arqueologia e Etnologia Universidade de Sao Paulo RESUMO: Examinando alguns textos antigos que mencionam o termo d~6cta~ov com que os autores modernos designam as imagens triplices de Hecate, verifica-se que este termo nao se aplica unicamente a Hecate triplice mas significa tambem capela, nicho ou simplesmente um local consagrado a Hecate, ou ainda a estatua da deusa sob sua forma triplice ou simples. A partir desta constatacao, retoma-se a passagem de Pausanias 11, 30, 2 referente a Hecate de tr3s corpos do escultor Alcamenes, obra arcaizante que originou varias copias das epocas helenistica e sobretudo romana imperial, conservantismo que se fundamenta sobretudo no pro- fundo sentido religioso que a imagem expressava. PALAVRAS-CHAVE: Grecia; Hecate; religiao; arte; tradicao literaria; tradicao iconografica. Entre as varias figuracoes de Hecate, dois tipos principais foram frequentemente opostos pelos autores antigos: o da deusa com um so rosto ( p o v o ~ o q ) e o de Hecate de tres corpos e tres rostos (ou tres cabecas) que aparece nos textos literarios, epigraficos e papirologicos com os epitetos zpipopcpo~, ' ~ p t & o q , '~p~dcpolh~. E muito provavel que estes tipos tenham coexistido na imagistica, mas as representacoes de Hecate com um s6 corpo sao anteriores as de Hecate trimorfa: os dois tipos deviam seguir, contudo, de perto ou de longe, uma tradicao iconografica muito mais antiga, que nao temos a possibilidade de determinar com precisao. Os tipos de monumentos sobre os quais aparecem a imagem de Hecate sao muito variados: um lugar de destaque e reservado a escultura, plena ou em relevo; a pintura dos vasos aticos e italiotas privilegiou sobretudo as representacoes de Hecate com um so corpo em cenas mitologicas de carater ritual. Na pintura mural romana, Hecate e sempre figurada com o corpo Unico em espacos domesticos sagrados ou em paisagens bucolicas sacralizadas. Enfim, a imagistica de Hecate difundiu-se nas emissoes monetarias gregas e romanas e em um certo tipo de gliptica: as impressoes de selos e os entalhes magicos. Na categoria de relevos, e preciso assinalar tambem monumentos mais raros como os instrumentos divinatorios e as estelas funerarias. Haiganuch Sarian: Da filologia h iconografia A permanencia do arcaico nas imagens tripiiis de Hecate. Haiganuch Sarian: Da filologia h iconografia A permanencia do arcaico nas imagens tripiiis de Hecate. Hecate aparece tambem, cumprindo a mesma funcao, mas em sua forma triplice, diante de uma casa (idem n" 208-211) e diante ou dentro de santuarios (idem n"112, 207 e 212). Esta interpretacao e comprovada pela celebre passagem de Pausanias II,30,2 que se serve da expressao ayddpaza ' E K & ~ G z p h e nao a palavra k~&zatov para designar a Hecate triplice do escultor Alcamenes. Tal referencia de Pausanias e o mais antigo testemunho de uma Hecate do tipo zpipopcpoc, pois que seu autor, Alcamenes, e escultor contemporaneo de Fidias e, continua o periegeta, havia expressado, pela primeira vez, em tres corpos associados, a concepcao triplice da deusa, modificando deste modo o tipo com um s6 corpo ainda conservado em Egina pelo escultor Miron (aprox. 460 a.C.). Diz o texto mais explicitamente: Classica, Sao Paulo, v. 13/14, n. 13/14, p. 101-107,200012001. ("Parece-me que Alcgrnenes fez pela primeira vez tres estatuas de ("Parece-me que Alcgrnenes fez pela primeira vez tres estatuas de Hecate, associadas entre si; os atenienses chamam-na de Epipyrgidia; situava-se junto ao templo da Vitoria Apteram). Esta Hecate de Alcamenes, que os atenienses denominavam de Epipyrgidia (termo que significa "sobre a cidadela"), erigia-se portanto ao lado do templo de Atena Nike na Acropole. Fullerton (1986, p.669-675) propoe situa-la, de modo mais preciso, entre o angu- lo noroeste do bastiao e o muro norte do templo (fig. 1). A data de sua instalacao deve estar associada aos trabalhos de reconstrucao realiza- dos no templo de Atena Nfke, por volta de 430 a.C. Por outro lado, a base retangular de um monumento triplice conservado in situ proximo aos Propileus (fig. 2), justamente diante de sua entrada e datando da epoca das Guerras Medicas, poderia convir perfeitamente a uma estatua de Hecate triforme (Amandry, 1976, p.87-90; Linfert, 1978, p.25-34; Dinsmoor, 1980, p.31-34). Esta hipotese, apesar de sedutora, (ver Amandry, 1976, p.89-90), nao nos oferece nenhum indicio sobre o estilo desta Hecate triplice. No tocante a Hecate de Alcamenes, a maioria dos estudiosos acredita que se tratava de uma obra de estilo arcaizante (Kraus, p.84-112; Harrison, 1965). por varios motivos, dos quais um e bem evidente: a permanencia de um arcaismo explicado sobretudo pelo tradici- onalismo religioso mais do que por tendencias arcaizantes do escultor. Esta justa observa- cao de Harrison reveste-se de um valor suplementar quando se considera o enorme alcance da sobrevivencia deste estilo. Trata-se. Nota 1 - Este artigo e uma versao adaptada de um aspecto da iconografia de Hecate por mim publicada em "Hekate", Lexicon Iconographicum Mythologiae Classicae Vii, doravante mencionada como Sarian, "Hekate". 1 - Este artigo e uma versao adaptada de um aspecto da iconografia de Hecate por mim publicada em "Hekate", Lexicon Iconographicum Mythologiae Classicae Vii, doravante mencionada como Sarian, "Hekate". Haiganuch Sarian: Da filologi h iconografia A pemandncia do arcaico nas imagens tripiii de Hecate. Haiganuch Sarian: Da filologi h iconografia A pemandncia do arcaico nas imagens tripiii de Hecate. cintura alta em apoptygma, polos na cabeca. Hecate aqui se apresenta na forma simples e nao triplice, mas pode ser relacionada com os exemplares da Agora (fig. 3) e da Escola Britanica de Atenas (fig. 4) pelo desenho do seu drapejamento (Zagdoun, 1989, p.15 1). E de se admirar que estes elementos arcaizantes estejam presentes na quase totalida- de de exemplares representando Hecate, dentre os documentos conhecidos do sec. IJJ a.C. ate o periodo romano tardio, nao apenas na Atica como tambem em outras regioes do mun- do grego e romano. Este estilo se multiplica nas inumeras estatuetas de que o exemplar da Agora de Atenas S 852, datado dos sec. I-JJ d.C., e um excelente representante (Sarian, "Hekate", no 133) (fig. 6). no qual vemos as tres divindades sobre uma base quadrangular, ao redor de uma coluna, segurando uma longa tocha. O estilo arcaizante se reproduz tambem em outras categorias de documentos figura- dos, como, por exemplo, em relevos, moedas, e entalhes. Uma tao longa permanencia dos caracteres arcaizantes nas representacoes de Hecate triplice justifica-se plenamente na me- dida em que a imagem desta deusa com tres corpos nao podia estar dissociada do sentido profundamente religioso que ela expressava. E, pois, a religiao, mais do que a arte, que origina este consewantismo estilistico das Hecates triformes arcaizantes. Haiganuch Sarian: Da filologia h iconografia A permanencia do arcaico nas imagens tripiiis de Hecate. por certo, de uma invencao atica do ultimo quartel do sec. V a.C. e nao devemos ficar surpresos em encontra-la nas varias copias originarias de Atenas nas epocas helenistica e sobretudo romana imperial. Tal "estilo arcaizante" e uma expressao moderna criada artificialmente pelos arqueo- logos e designa hoje "tout art qui, en Grece et dans le monde romain, se refere volontairement et consciemment a l'art archaique grec et en reproduit les principes caracteristiques" (Zagdoun, 1989, p. 11). Seus elementos constitutivos foram largamente definidos por Harrison a partir das esculturas arcaizantes descobertas na Agora de Atenas (Harrison, 1965, p. 51- 61). Nesta caracterizacao incluem-se exemplares de Hecate triplice, as tres figuras estreita- mente encostadas uma a outra, vestidas com um longo quiton e um peplos marcado por cintura alta em apoptygma. Os autores antigos exprimiram a natureza triplice de Hecate conforme esquemas figurativos muito variados. Os exemplares mais proximos da Hecate de Alcamenes, do ponto de vista iconografico e estilistico, sao as estatuetas da Agora (Sarian, "Hekate", no 130) (fig. 3) e da Escola Britanica de Atenas (idem no 131) (fig. 4), as duas datadas provavelmente do inicio do sec. I d.C. Reproduzem certamente a obra de um escultor do sec. V a.C., dai tratar-se com grande probabilidade de copias da Hecate triplice de Alcame- nes: a cintura larga, nem muito alta nem muito baixa, a representacao da parte superior da vestimenta numa construcao livre do estilo do final do sec. V a.C., foram comparadas com inumeras obras desta epoca, particularmente o relevo votivo de Xenocrateia (idem no 106) (fig. 3, datado de aprox. 410-400 a.C.: a cena figurada mostra uma mortal e uma crianca que se apresentam a um grupo de divindades; na extremidade direita do relevo, temos a imagem de Hecate, em posicao hieratica e vestida com quiton longo, peplos marcado por Referencias bibliograficas: AMANDRY, P. Trepieds d' Athenes: I. Dionysies. Bulletin de Correspondance Hellenique, Paris, n. 100, p.15-93, 1976. CHARBONNEAUX, J.; MARTIN, R.; VILLARD, F. Grece Classique. Paris: Gallimard, 1969. ("L'Univers des Formes"). DINSMOOR, Jr., W. B. The propylaia to the Arhenian akropolis. I. The predecessors. Princeton, New Jersey: American School of Classical Studies at Athens, 1980. ECKSTEIN, F. Das Hekataion in der Btitish School zu Athen. Antike Plastik, Lief IV. Berlin: Verlag Gebr. Mann, 1965. FUCHS, W. Zur Hekate des Alkamenes. Boreas, Munstersche Beitrage zur Archaologie, Miinster, v. 1, p.32-35, 1978. FULLERTON, M. D. The location and archaism of the Hekate Epipyrgidia. Archaologischer Anzeiger, Berlin, p.669-675, 1986. . The archaistic style in Roman statuaty. Mnemosyne Suppl. 110. Leiden: E. J. Brill, 1990 e archaistic style in Roman statuaty. Mnemosyne Suppl. 110. Leiden: E. J. Brill, Classica, Sao Paulo, v. 13/14, n. 13/14, p. 101-107, 2000/2001. HARRISON, E. B. The Athenian agora, XI. Archaic and archaistic sculpture. Princeton: The American School of Classical Studies at Athens, 1965. KRAUS, T. Hekate. Studien zu Wesen und Bild der Gottin in Kleinasien und Griechenland. Heidelberg: C. Winter Universitats Verlag, 1960. LINFERT, A. Die Propylaen der Akropolis von Athen - ein Dach fUr viele. Mitteilungen des Deutschen Archaologischen Instituts. Athen Abt., Berlin, v.93, p.25-34, 1978. MARCADE, J. Problemes de la sculpture archaique et de la sculpture archaisante. In: MARCADE, J. Etudes de sculpture et d'iconographie antiques. Scripta Varia, 1941- 1991. Paris: Publications de la S O ~ ~ O M ~ , 1993. p.61-73. SARIAN, H. "Hekate". Lexicon Iconographicum Mythologiae Classicae, VI. Zuricht MUnchen: Arternis Verlag, 1992. vol. I: 985-1018; vol. 11: 654-673. VON RUDLOFF, R. Hekate in ancient Greek religion. Victoria: Homed Owl Publishing, 1999. WILLERS, D. Hekate Epipyrgidia. Zur der Anfangen der Archaistischen Plastik in Gnechenland. Mitteilungen des Deustschen Archaologischen Instituts. Athen. Abteilung, Berlin, Beiheft IV, 1975. ZAGDOUN, M.-A. Lu sculpture archai'sante dans l'art hellenistique et dans l'art romain du haut-empire. Paris: Ecole Francaise d9Athenes; De Boccard, 1989. (B.E.F.A.R., 269). SARIAN, H. De la philologie a l'iconographie. La permanente de l'archaique dans les images triples d'Hecate. Classica, Sao Paulo, 13/14, p. 101-107,2000/2001 SARIAN, H. De la philologie a l'iconographie. La permanente de l'archaique dans les images triples d'Hecate. Classica, Sao Paulo, 13/14, p. RESUME: En examinant quelques textes anciens ou apparait le mot ticcizaiov avec leque1 les auteurs modernes designent les images triples d'Hecate, on verifie que ce mot ne s'applique pas uniquement a Hecate triple mais a egalement le sens de chapelle, niche ou simplement un lieu consacre a Hecate, ou encore Ia statue de Ia deesse sous sa forme triple ou a un seu1 corps. On a repris alors le passage de Pausanias 11, 30, 2 ou il est question de I'Hecate a trois corps du sculpteur Alcamenes, oeuvre archaisante qui est I'origine de nombreuses copies a I'epoque hellenistique et a I'epoque romaine imperiale, conservatisme fonde surtout sur le profond sens religieux exprime par cette image. MOTS-CLES: Grece; Hbcate; religion; ar!; tradition litteraire; tradition iconographique. Referencias bibliograficas: 101-107,2000/2001 RESUME: En examinant quelques textes anciens ou apparait le mot ticcizaiov avec leque1 les auteurs modernes designent les images triples d'Hecate, on verifie que ce mot ne s'applique pas uniquement a Hecate triple mais a egalement le sens de chapelle, niche ou simplement un lieu consacre a Hecate, ou encore Ia statue de Ia deesse sous sa forme triple ou a un seu1 corps. On a repris alors le passage de Pausanias 11, 30, 2 ou il est question de I'Hecate a trois corps du sculpteur Alcamenes, oeuvre archaisante qui est I'origine de nombreuses copies a I'epoque hellenistique et a I'epoque romaine imperiale, conservatisme fonde surtout sur le profond sens religieux exprime par cette image. Haiganuch Sarian: Da filologia a iconografia g g g A permanhcia do arcaico nas imagens triplices de Hecate Fig. 1 - Reconstituicao da HCcate Epipyrgidia, situada no bastiao do templo de Atena Nike na Acropole de Atenas. Von Rudloff, R.. fig. 4. Fig. 2 - Base situada proxima aos Propileus de Athenas. Amandry, P., p.88. fig. 54. Fig. 1 - Reconstituicao da HCcate Epipyrgidia situada no bastiao do templo de Atena Nike na Acropole de Atenas. Von Rudloff, R.. fig. 4. Fig. 2 - Base situada proxima aos Propileus de Athenas. Amandry, P., p.88. fig. 54. Fig. 1 - Reconstituicao da HCcate Epipyrgidia, situada no bastiao do templo de Atena Nike na Acropole de Atenas. Von Rudloff, R.. fig. 4. Fig. 2 - Base situada proxima aos Propileus de Athenas. Amandry, P., p.88. fig. 54. Fig. 4 - Hecate triplice arcaizante. The British School at Athens. Eckstein. F,. pr. 12. Fig. 3 - Htcate triplice arcaizante. Agora de Atenas S 1277. Harrison, E.B., pr. 32. Fig. 4 - Hecate triplice arcaizante. The British School at Athens. Eckstein. F,. pr. 12. Fig. 3 - Htcate triplice arcaizante. Agora de Atenas S 1277. Harrison, E.B., pr. 32. Classiea, Sao Paulo, v. 13/14, n. 13/14, p. 101 -1 07,2000/2001. Fig. 5 - Relevo votivo de Xenocratkia. Atenas, Museu Nacional. Charbonneaux, J., Martin, R., Villard, E, fig. 195. Fig. 5 - Relevo votivo de Xenocratkia. Atenas, Museu Nacional. Charbonneaux, J., Martin, R., Villard, E, fig. 195. Fig. 6 - Hecate trfplice arcaizante. Agora de Atenas, S 852. Hanison, E. B., pr. 36, no 147. Fig. 6 - Hecate trfplice arcaizante. Agora de Atenas, S 852. Hanison, E. B., pr. 36, no 147.
https://openalex.org/W3024473630	https://europepmc.org/articles/pmc7291158?pdf=render	English	null	EPICANCER—Cancer Patients Presenting to the Emergency Departments in France: A Prospective Nationwide Study	Journal of clinical medicine	2,020	cc-by	8,138	EPICANCER—Cancer Patients Presenting to the Emergency Departments in France: A Prospective Nationwide Study Olivier Peyrony 1,* , Jean-Paul Fontaine 1, Sébastien Beaune 2,3,4, Abdo Khoury 4,5, Jennifer Truchot 4,6,7, Frédéric Balen 8,9, Rishad Vally 10, Jacques Schmitt 11, Kasarra Ben Hammouda 12, Mélanie Roussel 13, Céline Borzymowski 14, Cécile Vallot 15, Veronique Sanh 16, Elie Azoulay 17,18 and Sylvie Chevret 18,19 Olivier Peyrony 1,* , Jean-Paul Fontaine 1, Sébastien Beaune 2,3,4, Abdo Khoury 4,5, Jennifer Truchot 4,6,7, Frédéric Balen 8,9, Rishad Vally 10, Jacques Schmitt 11, Kasarra Ben Hammouda 12, Mélanie Roussel 13, Céline Borzymowski 14, Cécile Vallot 15, Veronique Sanh 16, Elie Azoulay 17,18 and Sylvie Chevret 18,19 Olivier Peyrony 1,* , Jean-Paul Fontaine 1, Sébastien Beaune 2,3,4, Abdo Khoury 4,5, Jennifer Truchot 4,6,7, Frédéric Balen 8,9, Rishad Vally 10, Jacques Schmitt 11, J y J q Kasarra Ben Hammouda 12, Mélanie Roussel 13, Céline Borzymowski 14, Cécile Vallot 15, Veronique Sanh 16, Elie Azoulay 17,18 and Sylvie Chevret 18,19 1 Department of Emergency Medicine, Saint-Louis University Hospital, Assistance Publique-Hôpitaux de Paris, 1 avenue Claude Vellefaux, 75010 Paris, France; jean-paul.fontaine@aphp.fr 1 Department of Emergency Medicine, Saint-Louis University Hospital, Assistance Publique-Hôpitaux d Paris, 1 avenue Claude Vellefaux, 75010 Paris, France; jean-paul.fontaine@aphp.fr 2 Department of Emergency Medicine, Ambroise Paré University Hospital, Assistance Publique-Hôpitau Paris, 92100 Boulogne-Billancourt, France; sebastien.beaune@aphp.fr g p p 3 INSERM UMRS 1144, Paris-Descartes University, 75006 Paris, France y 4 Initiatives de Recherche aux Urgences (IRU) Research Network, Société Française de Médecine d’Urgence (SFMU), 75010 Paris, France; akhoury@chu-besancon.fr (A.K.); jennifer.truchot@aphp.fr (J.T.) Department of Emergency Medicine & Critical Care, Besançon University Hospital, 25000 Besançon, France Department of Emergency Medicine SMUR Lariboisière University Hospital Assistance 5 Department of Emergency Medicine & Critical Care, Besançon University Hospital, 25000 Besançon, France 6 Department of Emergency Medicine, SMUR, Lariboisière University Hospital, Assistance Department of Emergency Medicine & Critical Care, Besançon University Hospital, 25000 Besançon, France 6 Department of Emergency Medicine, SMUR, Lariboisière University Hospital, Assistance 6 Department of Emergency Medicine, SMUR, Lariboisière University Hospital, Assistance P bli Hô it d P i 75010 P i F 6 Department of Emergency Medicine, SMUR, Lariboisi Publique-Hôpitaux de Paris, 75010 Paris, France 7 Faculty of Medicine, Paris Diderot University, 75010 Paris, France 8 Department of Emergency Medicine, Toulouse University Hospital, 31059 Toulouse, France; balen.f@chu-toulouse.fr 9 Faculty of Medicine, Toulouse III—Paul Sabatier University, 31330 Toulouse, France 10 Department of Emergency Medicine, SAMU 33, Pellegrin University Hospital, 33000 Bordeaux, France; rishad.vally@chu-bordeaux.fr 11 Department of Emergency Medicine, SAMU 68, Mulhouse Hospital, 68100 Mulhouse, France; jacques.schmitt@ghrmsa.fr 12 Department of Emergency Medicine, Colmar Hospital, 68000 Colmar, France; kasarra.benhammouda@ch-colmar.fr 12 Department of Emergency Medicine, Colmar Hospital, 68000 Colmar, France; 13 Department of Emergency Medicine, Rouen University Hospital, F-76031 Rouen, France; melanie.roussel@chu-rouen.fr 14 Department of Emergency Médicine, Jean Bernard Hospital, 59322 Valenciennes, France; borzymowski-c@ch-valenciennes.fr 15 Department of Emergency Medicine, Annecy Genevois Hospital, 74370 Annecy, France; cvallot@ch-annecygenevois.fr 16 Department of Emergency Medicine, SAMU 95, René Dubos Hospital, 95300 Pontoise, France; vsanh@hotmail.fr 17 Intensive Care Unit, Saint-Louis University Hospital, Assistance Publique-Hôpitaux de Paris, 75010 Paris, France; elie.azoulay@aphp.fr 18 Centre of Research in Epidemiology and StatisticS (CRESS), INSERM, UMR 1153, Epidemiology and Clinical Statistics for Tumor, Respiratory, and Resuscitation Assessments (ECSTRRA) Team. Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine www.mdpi.com/journal/jcm 1. Introduction Due to a growing number of new cases and decrease in mortality over recent years, the number of patients with malignancies is expected to increase over the next decade [1]. In France in 2018, there were 382,000 new cases of cancer and 157,400 cancer-related deaths, which represents approximately 29% of all-cause deaths. Currently, 3.7 million individuals (5% of the total French population) are cancer survivors (i.e., individuals who are living with cancer or have a past history of cancer) [2]. These patients are likely to use emergency care resources for medical complications that may reveal malignancy or be due to its treatments or cancer progression [3,4]. Complications may occur even after remission [5]. Descriptive data are therefore needed to both describe how emergency departments (EDs) are attended by cancer patients and report their particular outcomes, such as the need for hospitalization, Intensive Care Unit (ICU) admission and mortality [6]. The Comprehensive Oncologic Emergencies Research Network (CONCERN) [7] identiﬁed several research priorities, including the need to collect epidemiologic data. However, only retrospective data based on nationwide survey databases have described the characteristics of the ED visits of patients with cancer [8–10]. These studies provided a very comprehensive picture of the reasons why cancer patients visit EDs in the US, but no such results have been reported in Europe. As the number of patients living with cancer is expected to grow in following years, such data might be valuable to better address unmet needs and identify an optimal standard of care for this ED population. Therefore, in a large prospective nationwide study, we aimed to describe the use of French EDs by cancer patients, with a particular emphasis on patients’ characteristics and predictors of 30-day all-cause mortality following hospital admission. EPICANCER—Cancer Patients Presenting to the Emergency Departments in France: A Prospective Nationwide Study University of Paris, 75010 Paris, France; sylvie.chevret@u-paris.fr 19 Department of Biostatistics and Medical Information, Saint-Louis University Hospital, Assistance Publique-Hôpitaux de Paris, 75004 Paris, France * Correspondence: o.peyrony@hotmail.fr; Tel.: +33-1-42-49-84-04 ceived: 28 April 2020; Accepted: 14 May 2020; Published: 17 May 2020 Received: 28 April 2020; Accepted: 14 May 2020; Published: 17 May 2020 Received: 28 April 2020; Accepted: 14 May 2020; Published: 17 May 2020 Abstract: Background: We aimed to estimate the prevalence of cancer patients who presented to Emergency Departments (EDs), report their chief complaint and identify the predictors of 30-day all-cause mortality. Patients and methods: we undertook a prospective, cross-sectional study during three consecutive days in 138 EDs and performed a logistic regression to identify the predictors of J. Clin. Med. 2020, 9, 1505; doi:10.3390/jcm9051505 www.mdpi.com/journal/jcm 2 of 14 J. Clin. Med. 2020, 9, 1505 30-day mortality in hospitalized patients. Results: A total of 1380 cancer patients were included. The prevalence of cancer patients among ED patients was 2.8%. The most frequent reasons patients sought ED care were fatigue (16.6%), dyspnea (16.3%), gastro-intestinal disorders (15.1%), trauma (13.0%), fever (12.5%) and neurological disorders (12.5%). Patients were admitted to the hospital in 64.9% of cases, of which 13.4% died at day 30. Variables independently associated with a higher mortality at day 30 were male gender (Odds Ratio (OR), 1.63; 95% CI, 1.04–2.56), fatigue (OR, 1.65; 95% CI, 1.01–2.67), poor performance status (OR, 3.00; 95% CI, 1.87–4.80), solid malignancy (OR, 3.05; 95% CI, 1.26–7.40), uncontrolled malignancy (OR, 2.27; 95% CI, 1.36–3.80), ED attendance for a neurological disorder (OR, 2.38; 95% CI, 1.36–4.19), high shock-index (OR, 1.80; 95% CI, 1.03–3.13) and oxygen therapy (OR, 2.68; 95% CI, 1.68–4.29). Conclusion: Cancer patients showed heterogeneity among their reasons for ED attendance and a high need for hospitalization and case fatality. Malignancy and general health status played a major role in the patient outcomes. This study suggests that the emergency care of cancer patients may be complex. Thus, studies to assess the impact of a dedicated oncology curriculum for ED physicians are warranted. Keywords: cancer; emergency department; epidemiology 2.1. Objectives The main objectives of this study were to estimate the prevalence of cancer patients that presented to EDs in France, report their chief complaints and describe their characteristics. The secondary objective was to identify the predictors of mortality among the patients admitted to the hospital after ED presentation. 2.2. Patients and Study Design This was a cross-sectional national prevalence study. During three consecutive days, from Tuesday the 6th to Thursday the 8th of February 2018, all EDs in France that consented to participate prospectively included all the consecutive cancer patients they attended. J. Clin. Med. 2020, 9, 1505 3 of 14 In France, patients can present to the ED through self-referral or after having called the dispatch center “Services d’Aide Médicale Urgente” (SAMU), where an emergency physician decides the appropriate level of response by sending the patient either an ambulance; the ﬁre department; or a Mobile Intensive Care Unit staﬀed by an emergency physician, a nurse and a paramedic for pre-hospital medical assistance when a life-threatening condition is suspected. Medical advice can also be provided, or the patient can be referred to a general practitioner or to the ED. Overall, among all the 622 French EDs, 138 (22.2%) from the Initiative de Recherche aux Urgences (IRU) research network coordinated by the French Society for Emergency Medicine (SFMU) agreed to participate in the study (Table S1). After giving informed consent to participate, all consecutive patients with solid cancer or hematologic malignancy were included, whatever their reason for attending the ED. Exclusion criteria were an age below 18 years and the remission of malignancy for more than ﬁve years. 2.2. Patients and Study Design Attending emergency physicians collected standardized data, including demographic data (age, gender); ﬁrst medical contact prior to ED attendance (none; a non-emergency physician such as a general practitioner, referring oncologist or radiologist; or a SAMU medical dispatch center); reasons why patients were seeking emergency care; underlying malignancy (type and location of the malignancy, time since diagnosis, presence of metastases, disease status); performance status (i.e., a scale developed by the Eastern Cooperative Oncology Group that describes the patient’s ability to care for himself and perform daily activities, ranging from 0 “fully active” to 4 “completely disabled”); presence of life-threatening conditions (shock, acute respiratory or neurological failure according to the attending emergency physician); vital signs at triage, including numerical pain rating scale (NPRS) with a 0 to 10 range, triage level at ED presentation on a 1 to 5 scale (1 being the most severe) [11] and shock-index (heart rate/systolic blood pressure); access to the referring oncologist or the oncologic medical record; in case of a life-threatening condition, the presence of advanced directives (resuscitation or palliative status) in the patient’s medical record; the need to access the referring oncologist’s advice; investigations, interventions and treatments realized in the ED; and hospital admission or discharge after the ED visit. For patients admitted to the hospital from the ED, the assessment included whether they were admitted to ICU, the length of the hospital stay and the 30-day status (discharged, still hospitalized or death). The primary outcome was 30-day all-cause mortality following hospital admission. The study was registered on ClinicalTrials.gov (NCT03393260) and approved by the Institutional Review Board of the French Speaking Society for Respiratory Medicine—Société de Pneumologie de Langue Française (number CEPRO 2017-038). 2.3. Statistical Analysis Patient pathways before ED attendance were described for all patients. Further results only dealt with patients enrolled in the ED. When the same patient was attended by several EDs successively, only the data from the last admission was considered. Descriptive statistics were reported, namely median with interquartile range (IQR) for continuous variables and the number with percentage for binary and categorical variables with a comparison based on the Mann Whitney test or chi square test, respectively. In patients admitted to the hospital, we used logistic regression to identify the predictive factors of all-cause mortality at day 30. Univariable models were ﬁrst ﬁtted. Then, variables associated with the outcome on the basis of p-values less than 0.1 by univariable analyses were selected for a multivariable analysis. When several variables shared a close clinical signiﬁcance, we only selected variables that appeared more accurate and with the best metrological properties. Clinically relevant variables, such as age or gender, were forced into the model whatever their p-value. Then, a backward selection procedure was applied and variables with p-values of less than 0.05 were removed. Missing data were imputed, except for the outcome, using multiple imputation by chained equations [12,13]. The multivariable logistic model was applied to the imputed datasets and ﬁnal estimates were obtained according to Rubin’s rules. The primary analyses of mortality only dealt with patients with available outcomes. Then, sensitivity analyses were conducted; a simple imputation of the outcome (by dead 4 of 14 J. Clin. Med. 2020, 9, 1505 or alive) was performed, as well as simple stochastic imputation based on the observed mortality of 13.4%, assuming outcomes were missing completely at random. All the p-values were two-sided, with values of 0.05 or less considered as statistically signiﬁcant. Data were analyzed with R 3.5.0 software (the R Foundation for Statistical Computing, Vienna, Austria). 3. Results During the three-day study period, a total of 1380 cancer patients visited the ED in the participating centers. The number of cancer patients included by each center varied greatly from one center to another and ranged from 0 to 40 during these three days, with a median of eight patients per center. The median prevalence was 2.8% (IQR, 1.7%–4%; range, 0–11.1%) (Table 1). The patient trajectories before attending the ED are shown in Figure 1. Among these 1380 patients, 502 (36.4%) presented through self-referral to the ED, 395 (28.6%) were referred by a non-emergency physician to the ED, while 433 (31.4%) ﬁrst called an emergency physician in a SAMU dispatch center. Of all the patients referred to the ED by a non-emergency physician, 31 (7.8%) were sent by their referring oncologist. Among the 433 patients that ﬁrst called the SAMU dispatch center, 396 (91.4%) were referred to the ED without pre-hospital medical assistance and 37 (8.6%) with pre-hospital medical assistance. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 15 Figure 1. Flowchart of cancer patients attending emergency departments in France throughout the 3-day study period (ED—emergency department; SAMU—Service d’Aide Médicale Urgente). Medical contact prior to ED attendance No previous medical contact Non emergency physician Prior call to SAMU medical dispatch center Unknown 502 (36.4%) 395 (28.6%) 433 (31.4%) 50 (3.6%) Pre-hospital medical assistance 37 (8.5%) ED 1380 Deceased in the ED 6 (0.4%) Discharged home from the ED 478 (34.6%) Hospital admission 896 (65.0%) Status at day 30 Alive 745 (83.1%) Discharged home Still hospitalized Unknown Loss to follow up Deceased 558 (62.2%) 156 (17.4%) 31 (3.5%) 31 (3.5%) 120 (13.4%) Figure 1. Flowchart of cancer patients attending emergency departments in France throughout the 3-day study period (ED—emergency department; SAMU—Service d’Aide Médicale Urgente). Figure 1. Flowchart of cancer patients attending emergency departments in France throughout the 3-day study period (ED—emergency department; SAMU—Service d’Aide Médicale Urgente). Figure 1. Flowchart of cancer patients attending emergency departments in France throughout the 3-day study period (ED—emergency department; SAMU—Service d’Aide Médicale Urgente). 5 of 14 J. Clin. Med. 2020, 9, 1505 Table 1. Number of inclusions and prevalence of cancer patients in the participating 138 French emergency departments. Table 1. Number of inclusions and prevalence of cancer patients in the participating 138 French emergen departments. 3. Results Variable Missing Data ED, No 138 Patients Included, No 1380 No of Patients Included by Center 0 Mean 10 Median 8 IQR 4–14 Max 40 Prevalence, % 17 Mean 3.1 Median 2.8 IQR 1.7–4.0 Max 11.1 ED—emergency department; IQR—interquartile range. p p p p g g departments. Variable Missing Data ED, No 138 Patients Included, No 1380 No of Patients Included by Center 0 Mean 10 Median 8 IQR 4–14 Max 40 Prevalence, % 17 Mean 3.1 Median 2.8 IQR 1.7–4.0 Max 11.1 ED—emergency department; IQR—interquartile range. ED—emergency department; IQR—interquartile range. 3.1. General Characteristics and Reasons for Seeking Emergency Care The patient characteristics are summarized in Table 2. The patients had mostly solid malignancies (86.1%), were in complete or partial remission (57.0%) and had a good performance status (75.7%). The most frequent reasons why patients sought ED care were fatigue (16.6%); dyspnea (16.3%), gastro-intestinal disorders such as diarrhea, vomiting or abdominal pain (15.1%); trauma (13.0%); fever (12.5%); and neurological disorders (12.5%) (Figure 2). According to the emergency physician in charge of the patient, these reasons were related to the malignancy in 687 cases (52%) but varied depending on the chief complaint (Figure 2). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 7 of 15 Figure 2. Reasons for cancer patients’ attendance to emergency departments. Figure 2. Reasons for cancer patients’ attendance to emergency departments. Figure 2. Reasons for cancer patients’ attendance to emergency departments. Figure 2. Reasons for cancer patients’ attendance to emergency departments. 6 of 14 J. Clin. Med. 2020, 9, 1505 Table 2. Patient characteristics. Variable Missing Data (n) N 1380 Age (Years), Median (IQR) 71 (61–82) 30 Female Gender, n (%) 598 (43.6) 8 Underlying Malignancy, n (%) 4 Solid Malignancy 1185 (86.1) Digestive and pancreas 223 (18.8) Breast 187 (15.8) Prostate 175 (14.8) Lung 172 (14.5) Kidney and bladder 117 (9.9) Endometrium and ovary 61 (5.1) Head and neck 57 (4.8) Skin 55 (4.6) Other 138 (11.7) Hematologic Malignancy 191 (13.9) Lymphoma 73 (38.2) Chronic leukemia 38 (19.9) Myeloma 37 (19.4) Acute leukemia 22 (11.5) Other 21 (11.0) Time Since Diagnosis, n (%) 122 <6 months 236 (18.8) 6 months–5 years 753 (59.9) >5 years 269 (21.3) Disease Status, n (%) 285 Complete remission 270 (24.7) Partial remission 354 (32.3) Uncontrolled malignancy 471 (43.0) Metastatic, n (%) 380 (47.1) 573 Speciﬁc Treatments in the Last 3 Months, n (%) 134 Chemotherapy 401 (32.2) Hormonotherapy 127 (9.2) Radiotherapy 73 (5.9) Surgery 65 (5.2) Immunotherapy 47 (3.8) Other 17 (1.4) Poor Performance Status (>2), n (%) 283 (24.3) 213 Patient Alone at Home, n (%) 225 (27.7) 569 Home Nursing Service or Nursing Home Care, n (%) 483 (37.3) 85 IQR—inter-quartile range. 3.2. Presentation at Inclusion 3.4. Investigations, Interventions and Treatments Table 4 reports the investigations, interventions and treatments performed or initiated in the ED. Nearly all the patients (91.8%) had at least one investigation or intervention, such as a blood sample (76.6%) or an X-ray (43.6%). One quarter of the patients underwent a CT-scan. Analgesics (31.2%) and antibiotics (14%) were the most commonly administered treatments. Among the 213 patients with an NPRS ≥6, 33 (15.5%) received morphine. The patients received intensive care medications such as ﬂuid challenge, vasoactive agents, non-invasive ventilation, mechanical ventilation or cardiopulmonary resuscitation in 12.6% of the cases. The patients had oxygen therapy in 14.8% of the cases. Among the patients who carried a long-term central venous catheter (CVC) for their chemotherapies, it was used in 13.4% of the cases. 3.3. Access to the Oncologic Medical Record For 570 (42.7%) of the cases, the patients presented to an ED outside their referring oncology center. The emergency physician had no immediate access to the oncologic medical record of 519 (40.2%) patients. Immediate contact with the referring oncologist was considered unnecessary for 946 (71.7%) patients. Among the 102 patients critically ill at ED admission, no information in the medical record regarding resuscitation or palliative care status was recorded in 65 (69.9%) patients. Among those for whom this information was available, 24 (85.7%) had a palliative care status mentioned in their medical record. 3.2. Presentation at Inclusion 3.2. Presentation at Inclusion Table 3 shows the cancer patients’ severity, depending on the triage level, the shock-index, the emergency physician’s assessment and the pain assessment at ED arrival. 7 of 14 J. Clin. Med. 2020, 9, 1505 Table 3. Clinical severity of cancer patients upon emergency department presentation. Table 3. Clinical severity of cancer patients upon emergency department presentation. Variables at ED Presentation Missing Data (n) N 1380 Critically Ill According to the Emergency Physician, n (%) 102 (8.3) 144 Respiratory failure 44 (3.6) Shock 44 (3.6) Altered mental status 19 (1.5) High Shock-Index a (≥1), n (%) 130 (9.9) 71 Triage Level, n (%) 373 1 50 (5.0) 2 205 (20.3) 3 520 (51.6) 4 200 (19.9) 5 32 (3.2) Pain Assessment at ED Arrival 1029 (74.6) NPRS ≥6 213 (20.7) ED emergency department, NPRS numeric pain rating scale, a Heart rate/systolic arterial blood pressure. 3.5. Outcome after ED Visit and at Day 30 The median length of stay in the ED was 5 h and 50 min (IQR: 3 h and to 8 h and 51 min). When admitted to the hospital, the patients were admitted to their re oncologic department in 176 (19.6%) of the cases and to the ED short stay unit in 229 (25.6%) cases. Fifty-nine (6.6%) patients were admitted to the ICU, either (n = 38) directly from the during their hospitalization (n = 21), one half of whom were admitted during the first three d their hospital stay. At day 30 after hospital admission, 558 (62.2%) patients had been discharg (13.4%) had died and 156 (17.4%) were still in the hospital. The median length of hospital stay (3–12) days. Among the hospitalized patients, Figure 3 shows the rates of in-hospital mo according to the reasons why they attended EDs. Fatigue; neurological, metabolic and u disorders; and shock were the symptoms that carried the highest mortality rate (>20%). Table 4. Investigations, interventions and treatments performed in the EDs for cancer patients. Table 4. Investigations, interventions and treatments performed in the EDs for cancer patients. Variable Missing Data (n) N 1380 Exams, n (%) 0 None 113 (8.2) Blood sample 1057 (76.6) X-ray 602 (43.6) CT-scan 358 (25.8) ECG 158 (11.4) Ultrasound 68 (4.9) Other 36 (2.6) Interventions, n (%) 0 Peripheral catheter 1008 (73.0) Oxygen therapy 204 (14.8) Fluid challenge 164 (11.9) Puncture/drainage/catheterization 88 (6.4) Management of traumatism 43 (3.1) Non-invasive ventilation/mechanical ventilation 11 (0.8) Cardiopulmonary resuscitation 1 (0.1) Red Cells or Platelets Transfusion, n (%) 61 (4.4) Medications, n (%) 0 Analgesia 430 (31.2) Antibiotics 193 (14.0) Morphine 54 (3.9) Cardiovascular/coagulation 46 (3.3) Nebulization 25 (1.8) Sedation/neurological 12 (0.9) Vasoactive agents 7 (0.5) Other 31 (2.2) Long-Term Central Venous Catheter Carrier, n (%) 243 (23.0) 324 Use of Long-Term Central Venous Catheter, n (%) 28 (13.4) 34 ED—emergency department. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3.5. Outcome after ED Visit and at Day 30 As shown in the flowchart (Figure 1), 896 (64.9%) patients were admitted to the hospital a (0.4%) died in the ED. The median length of stay in the ED was 5 h and 50 min (IQR: 3 h and 4 to 8 h and 51 min). 3.5. Outcome after ED Visit and at Day 30 As shown in the ﬂowchart (Figure 1), 896 (64.9%) patients were admitted to the hospital and six (0.4%) died in the ED. The median length of stay in the ED was 5 h and 50 min (IQR: 3 h and 45 min to 8 h and 51 min). When admitted to the hospital, the patients were admitted to their referring oncologic department in 176 (19.6%) of the cases and to the ED short stay unit in 229 (25.6%) of the cases. Fifty-nine (6.6%) patients were admitted to the ICU, either (n = 38) directly from the ED or during their hospitalization (n = 21), one half of whom were admitted during the ﬁrst three days of their hospital stay. At day 30 after hospital admission, 558 (62.2%) patients had been discharged, 120 (13.4%) had died and 156 (17.4%) were still in the hospital. The median length of hospital stay was 6 (3–12) days. Among the hospitalized patients, Figure 3 shows the rates of in-hospital mortality according to the reasons why they attended EDs. Fatigue; neurological, metabolic and urologic disorders; and shock were the symptoms that carried the highest mortality rate (>20%). 8 of 14 J. Clin. Med. 2020, 9, 1505 Table 4. Investigations, interventions and treatments performed in the EDs for cancer patients. Variable Missing Data (n) N 1380 Exams, n (%) 0 None 113 (8.2) Blood sample 1057 (76.6) X-ray 602 (43.6) CT-scan 358 (25.8) ECG 158 (11.4) Ultrasound 68 (4.9) Other 36 (2.6) Interventions, n (%) 0 Peripheral catheter 1008 (73.0) Oxygen therapy 204 (14.8) Fluid challenge 164 (11.9) Puncture/drainage/catheterization 88 (6.4) Management of traumatism 43 (3.1) Non-invasive ventilation/mechanical ventilation 11 (0.8) Cardiopulmonary resuscitation 1 (0.1) Red Cells or Platelets Transfusion, n (%) 61 (4.4) Medications, n (%) 0 Analgesia 430 (31.2) Antibiotics 193 (14.0) Morphine 54 (3.9) Cardiovascular/coagulation 46 (3.3) Nebulization 25 (1.8) Sedation/neurological 12 (0.9) Vasoactive agents 7 (0.5) Other 31 (2.2) Long-Term Central Venous Catheter Carrier, n (%) 243 (23.0) 324 Use of Long-Term Central Venous Catheter, n (%) 28 (13.4) 34 ED—emergency department. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3.5. Outcome after ED Visit and at Day 30 As shown in the flowchart (Figure 1), 896 (64.9%) patients were admitted to the hospital a (0.4%) died in the ED. Figure 4. Effects on the 30-day mortality of covariates identified by multivariate logistic regression after imputation of the missing data. * Reference: hematological malignancy. Figure 4. Eﬀects on the 30-day mortality of covariates identiﬁed by multivariate logistic regression after imputation of the missing data. * Reference: hematological malignancy. Figure 4. Effects on the 30-day mortality of covariates identified by multivariate logistic regression after imputation of the missing data. * Reference: hematological malignancy. Figure 4. Eﬀects on the 30-day mortality of covariates identiﬁed by multivariate logistic regression after imputation of the missing data. * Reference: hematological malignancy. Table 5. Multivariable analysis. Variables independently associated with a 30-day mortality for patients hospitalized after visiting the ED (N = 896) before and after the imputation of missing data, except for the outcome that was not imputed (355 patients had at least one missing data, among which 31 concerned the outcome). Table 5. Multivariable analysis. Variables independently associated with a 30-day mortality for patients hospitalized after visiting the ED (N = 896) before and after the imputation of missing data, except for the outcome that was not imputed (355 patients had at least one missing data, among which 31 concerned the outcome). Complete Cases After Imputation N = 541—Deaths = 81 N = 865—Deaths = 120 OR (95% CI) p OR (95% CI) p Male Gender 1.96 (1.13–3.52) 0.02 1.63 (1.04–2.56) 0.03 Age (≥60 years) 1.90 (0.89–4.47) 0.11 1.82 (0.93–3.53) 0.08 Fatigue 1.75 (0.97–3.13) 0.06 1.64 (1.01–2.67) 0.049 Poor Performance Status (>2) 2.78 (1.63–4.76) 0.0002 3.00 (1.87–4.80) <0.00001 Solid Malignancy a 3.70 (1.41–12.81) 0.02 3.05 (1.26–7.40) 0.01 Uncontrolled Malignancy 2.00 (1.14–3.57) 0.02 2.27 (1.36–3.80) 0.002 Neurological Disorders 2.69 (1.34–5.28) 0.005 2.38 (1.36–4.19) 0.003 High Shock-Index b (≥1) 2.03 (1.03–3.94) 0.04 1.80 (1.03–3.13) 0.04 Oxygen Therapy 2.43 (1.34–4.37) 0.003 2.68 (1.68–4.29) <0.0001 ED—emergency department; OR—odds ratio; 95% CI—95% confidence interval. ED—emergency department; OR—odds ratio; 95% g y p ; ; malignancy; b heart rate/systolic arterial blood pressure. 3.5. Outcome after ED Visit and at Day 30 When admitted to the hospital, the patients were admitted to their ref oncologic department in 176 (19.6%) of the cases and to the ED short stay unit in 229 (25.6%) cases. Fifty-nine (6.6%) patients were admitted to the ICU, either (n = 38) directly from the during their hospitalization (n = 21), one half of whom were admitted during the first three d their hospital stay. At day 30 after hospital admission, 558 (62.2%) patients had been discharge (13.4%) had died and 156 (17.4%) were still in the hospital. The median length of hospital stay (3–12) days. Among the hospitalized patients, Figure 3 shows the rates of in-hospital mo according to the reasons why they attended EDs. Fatigue; neurological, metabolic and ur disorders; and shock were the symptoms that carried the highest mortality rate (>20%). Figure 3. In-hospital mortality rates depending on the reasons for attending emergency departmen in hospitalized cancer patients. Figure 3. In-hospital mortality rates depending on the reasons for attending emergency departments in hospitalized cancer patients. Figure 3. In-hospital mortality rates depending on the reasons for attending emergency departme in hospitalized cancer patients Figure 3. In-hospital mortality rates depending on the reasons for attending emergency departments in hospitalized cancer patients. J. Clin. Med. 2020, 9, 1505 9 of 14 In hospitalized patients, variables independently associated with a higher mortality at day 30 were male gender (OR, 1.63; 95% CI, 1.04–2.56), fatigue (OR, 1.65; 95% CI, 1.01–2.67), poor performance status (OR, 3.00; 95% CI, 1.87–4.80), solid malignancy (OR, 3.05; 95% CI, 1.26–7.40), uncontrolled malignancy (OR, 2.27; 95% CI, 1.36–3.80), ED attendance for a neurological disorder (OR, 2.38; 95% CI, 1.36–4.19), high shock-index (OR, 1.80; 95% CI, 1.03–3.13) and oxygen therapy (OR, 2.68; 95% CI, 1.68–4.29) (Figure 4). The results of the multivariable logistic model with and without multiple imputation are reported in Table 5. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 10 of 15 Figure 4. Effects on the 30-day mortality of covariates identified by multivariate logistic regression after imputation of the missing data. * Reference: hematological malignancy. Figure 4. Eﬀects on the 30-day mortality of covariates identiﬁed by multivariate logistic regression after imputation of the missing data. * Reference: hematological malignancy. 4. Discussion The ﬁrst objective of our study was to estimate prospectively the prevalence of cancer patients in French EDs; we found that it was, in median, 2.8%. Even if our study was conducted during a short period and did not concern all French EDs, it should be emphasized that almost one ED in four participated all around the country, providing the ﬁrst large prospective nationwide prevalence estimate of ED use by cancer patients. This number was close to the 3.4% prevalence reported in a single French ED prospective study in 2007 [14], as well as to those reported in other non-French nationwide retrospective studies [8,9,15]. Based on diagnosis codes, these studies reported a prevalence of ED cancer patients in the US of 4.2% for Rivera et al. [8] and 3.7% for Hsu et al. [9], whereas it was 2.4% for van der Meer et al. in Australia [15]. These nationwide retrospective studies have the beneﬁt of giving a large picture of emergency care use by cancer patients but could underestimate their prevalence. This may be the case when the reason for seeking care is not directly related to the malignancy and cancer is not taken into account when coding the diagnosis. Furthermore, the prevalence may also vary depending on the deﬁnition used for inclusion, and terms like “cancer patients”, “active cancer”, “remission” or “cancer-related visit” need to be more accurately deﬁned [6]. We observed that the prevalence varied from one center to another depending on their speciﬁcity in onco-hematology, but was somewhat lower than the 5% prevalence of cancer survivorship reported by the French National Institute of Cancer [2]. This could be due to the fact that we chose to include patients with active cancer or who had been in remission for less than ﬁve years. Our ﬁrst result reveals that the number of cancer patients attending EDs every day is substantial and that this requires speciﬁc attention from the emergency community with further research on this topic. The second objective of our study was to describe the reasons why cancer patients sought emergency care. We showed that the most frequent reason was fatigue, followed by potential life-threatening conditions such as dyspnea, neurological disorders and fever. a Reference: h t l i l li b h t t / t li t i l bl d Complete Cases After Imputation N = 541—Deaths = 81 N = 865—Deaths = 120 OR (95% CI) p OR (95% CI) p Male Gender 1.96 (1.13–3.52) 0.02 1.63 (1.04–2.56) 0.03 Age (≥60 years) 1.90 (0.89–4.47) 0.11 1.82 (0.93–3.53) 0.08 Fatigue 1.75 (0.97–3.13) 0.06 1.64 (1.01–2.67) 0.049 Poor Performance Status (>2) 2.78 (1.63–4.76) 0.0002 3.00 (1.87–4.80) <0.00001 Solid Malignancy a 3.70 (1.41–12.81) 0.02 3.05 (1.26–7.40) 0.01 Uncontrolled Malignancy 2.00 (1.14–3.57) 0.02 2.27 (1.36–3.80) 0.002 Neurological Disorders 2.69 (1.34–5.28) 0.005 2.38 (1.36–4.19) 0.003 High Shock-Index b (≥1) 2.03 (1.03–3.94) 0.04 1.80 (1.03–3.13) 0.04 Oxygen Therapy 2.43 (1.34–4.37) 0.003 2.68 (1.68–4.29) <0.0001 ED—emergency department; OR—odds ratio; 95% CI—95% conﬁdence interval. a Reference: hematological malignancy; b heart rate/systolic arterial blood pressure. J. Clin. Med. 2020, 9, 1505 10 of 14 10 of 14 3.6. Sensitivity Analyses The results of the multivariable logistic models after imputing the 31 missing outcomes were similar, though the inﬂuence of the shock-index was no longer signiﬁcant in the case of imputation by “death”. Fatigue was no longer signiﬁcant in the case of imputation by “alive” or by stochastic imputation (Table S2). 4. Discussion While symptoms like fatigue or gastro-intestinal disorders, which were also frequent, may seem less alarming to emergency physicians, these symptoms could also hamper discharge and participate in the high rate of hospitalization and death among cancer patients after an ED visit [10,16,17]. Indeed, fatigue had a high mortality rate in hospitalized patients and was associated with mortality in the multivariable analysis. In other studies, weakness was also found to be associated with mortality in non-cancer patients in the ED [18,19]. Contrary to other studies in all-comer ED patients [20,21], dyspnea was not associated with mortality in the univariable analysis, whereas this condition was one of the most frequent reasons for cancer patients to attend the ED and had a high mortality rate. More generally, the reasons why patients attended EDs in our study were similar to those found in other studies that focused on ED cancer patients [8–10,15,22–24], except for trauma, probably because of the inclusion period with snowfalls all across the country. These findings suggest that cancer patients may attend EDs for multiple reasons, either specific to malignancy progression or cancer treatment or due to other non-cancer-related causes. Thus, diagnostic workup can be challenging for a non-experienced emergency physician. Studies focusing on frequent and life-threatening complications in cancer patients, such as febrile neutropenia; dyspnea; or neurological disorders like spinal cord compression, confusion or seizures, are mandatory in the emergency setting [6]. We also sought to describe cancer patients’ pathways before ED arrival, their clinical severity, their management throughout ED attendance and their outcomes. Patients presented mostly through self-referral to the ED or after calling medical dispatch centers. Very few patients were referred by their referring oncologist, leading to the risk that emergency physicians missed out on important 11 of 14 J. Clin. Med. 2020, 9, 1505 information concerning malignancy status and patient prognosis. Moreover, nearly half of the patients attended an ED that was not located in their referring oncologic center, and their oncologic medical record was often not accessible. Surprisingly, emergency physicians declared that contact with the referring oncologist was not necessary in two-thirds of the cases. One patient in ten was described as critically ill and one in four had the highest triage levels at arrival. Very few patients did not have any exam or intervention; most had a blood sample, radiograph, CT-scan or ultrasound and were treated with analgesics, antibiotics or oxygen therapy. 4. Discussion A higher use of morphine in patients with high NPRS levels and long-term CVC should be promoted. This high burden of care among these patients illustrates their need for urgent care, consistent with the high rates of hospital admission of 64.9% and in-hospital mortality of 13.4%. This high rate of hospitalization among these patients is similar to those found in other studies that ranged from 29% to 90% [8–10,15,23,24]. The mortality rate of 7.9% reported by van der Meer et al. was lower than ours but concerned all ED visits, while ours concerned only hospitalized patients [15]. In this study, patients with cancer-related visits were eight times more likely to die in the hospital than those with visits unrelated to cancer. Vandyk et al. reported in a meta-analysis a median mortality rate of 13%, but this rate varied depending on the timeline, ranging from 1% for mortality in the ED to 56% for mortality at 3 months [24]. Karakoumis et al. prospectively enrolled more than 1200 patients who attended EDs for non-speciﬁc complaints during four years and showed that patients with cancer had the highest mortality rate at day 30 (31%), which was six times higher than the overall patient mortality [25]. Finally, our study identiﬁed that male gender, fatigue, poor performance status, solid and uncontrolled malignancy, ED attendance for a neurological disorder, high shock-index and oxygen therapy were predictors of 30-day all-cause mortality among hospitalized cancer patients. Mortality was associated with some characteristics inherent to malignancy, such as solid or uncontrolled malignancy, but also with the patient’s general health status such as fatigue as a reason for attending the ED and a dependency level assessed by the performance status. The impact of general health status upon cancer patients’ outcomes may seem obvious and has already been described by other authors [26–28]. Nonetheless, it is crucial to take into account these characteristics upon ED management, so as to establish conjointly the goals of care as soon as possible in order to determine the appropriate intensity of care, orientation and monitoring [4]. We also identiﬁed clinical prognostic factors for cancer patients, like shock-index or the need for oxygen therapy. 4. Discussion If these characteristics are non-speciﬁc of patients with malignancies, the use of shock-index at ED triage is very simple and has already proven its utility in recognizing patients at a high risk of complication [29–32], as is the case with the quick Sequential Organ Failure Assessment score [33]. Hypoxemia is a predicting factor of ICU admission among patients with malignancies, and the level of oxygen delivery has shown to be an early and independent predictor of 28-day mortality in cancer patients admitted to the ICU for acute respiratory failure [34,35]. It should alert emergency clinicians to admit these patients to departments with an adequate level of monitoring. Limitations In order to motivate the highest number of recruiting centers, the dedicated form was short and follow-up was conducted only for admitted patients. Therefore, details about malignancy status, speciﬁc treatments, symptoms, ED management and long-term follow-up for the whole cohort were not recorded. Additionally, inclusions took place for three consecutive working weekdays, but a seven-day collection could have given a more thorough picture. Nevertheless, this study gives a general picture of cancer patients visiting EDs in our country. Despite the simple format of the inclusion form, there was missing data, mostly regarding malignancy characteristics or patients’ general health status. Such missing data is consistent with the fact that clinicians lacked speciﬁc information concerning cancer status. Likewise, the surprisingly low number of speciﬁc treatments in the last three months may be due to missing information and highlights the need for more accessible data in the EDs on patients’ oncologic follow-up. Finally, we cannot exclude non-observed confounding factors. J. Clin. Med. 2020, 9, 1505 12 of 14 12 of 14 5. Conclusions This large prospective nationwide study about cancer patients seeking emergency care, who represent 3% of total ED attendance, showed some heterogeneity among reasons for ED attendance and a high need for hospitalization and case fatality. Mortality predictors among hospitalized cancer patients were male gender, fatigue, poor performance status, solid and uncontrolled malignancy, ED attendance for a neurological disorder, high shock-index and oxygen therapy. Malignancy and general health status, information about which is often not available in the emergency setting, both play a major role in patient outcomes, and should be taken into account by emergency physicians to adapt the level of care. This study suggests that the emergency care of cancer patients may be complex. Thus, studies to assess the impact of a dedicated oncology curriculum for ED physicians are warranted. Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/9/5/1505/s1. Table S1: collaborators and members of the Initiatives de Recherche aux Urgences (IRU) research network who participated in the study, Table S2: sensitivity multivariable analyses imputing the missing outcomes either all by death or alive status, or by simple stochastic imputation, assuming missing completely at random outcomes, based on the observed mortality of 13.4%. Author Contributions: Conceptualization, J.-P.F., E.A. and S.C.; data curation, O.P., S.B., A.K., J.T., F.B., R.V., J.S., K.B.H., M.R., C.B., C.V. and V.S.; formal analysis, O.P., J.-P.F., E.A. and S.C.; investigation, O.P.; methodology, O.P. and S.C.; project administration, O.P., S.B., A.K. and J.T.; resources, S.C.; supervision, O.P., J.-P.F., E.A. and S.C.; validation, O.P., J.-P.F., S.B., A.K., J.T., F.B., R.V., J.S., K.B.H., M.R., C.B., C.V., V.S., E.A. and S.C.; writing—original draft, O.P., J.-P.F., E.A. and S.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors thank the local investigators of the Initiatives de Recherche aux Urgences research network (Table S1), and the Société Française de Médecine d’Urgence (SFMU), Guillemette Leriche, for her valuable help, the staﬀfrom the department of biostatistics and medical information (Saint-Louis hospital) for their help in logistics and especially Martine Nyunga for data management. Conﬂicts of Interest: All authors declare no potential conﬂict of interest related to the study. References 1. Howlader, N.; Noone, A.M.; Krapcho, M.; Miller, D.; Bishop, K.; Kosary, C.L.; Yu, M.; Ruhl, J.; Tatalovich, Z.; Mariotto, A.; et al. SEER Cancer Statistics Review, 1975–2014; National Cancer Institute: Bethesda, MD, USA, 2014. 2. Defossez, G.; Le Guyader-Peyrou, S.; Uhry, Z.; Grosclaude, P.; Colonna, M.; Dantony, E.; Delafosse, P.; Molinié, F.; Woronoﬀ, A.S.; Bouvier, A.M.; et al. 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https://openalex.org/W4396525774	http://jtuh.org/index.php/jtuh/article/download/2594/2376	Arabic	null	قياس مستوى الكفاءة الذاتية الانفعالية لدى طلبة المرحلة الثانوية	Mağallaẗ ǧāmiʻaẗ tikrīt li-l-ʻulūm al-insāniyyaẗ/Journal of Tikrit university for humanities	2,023	cc-by	11,011	M.M Mouna Mohamad shehab College of Education for Human Sciences, Tikrit University Tikrit University for Humanities The current research aims to reveal sentimental self-efficacy among secondary school students according to the variable of gender. The research sample entails (300) students of the secondary stage. The researcher uses the measure of emotional self- efficacy which consists of (42) items. After analyzing the data, the results have shown that there is a statistically significant difference in the level of sentimental self- efficacy among students as well as between male and female variables. Tikrit University for Humanities The current research aims to reveal sentimental self-efficacy among secondary school students according to the variable of gender. The research sample entails (300) students of the secondary stage. The researcher uses the measure of emotional self- efficacy which consists of (42) items. After analyzing the data, the results have shown that there is a statistically significant difference in the level of sentimental self- efficacy among students as well as between male and female variables. Measuring the Level of Sentimental Self – efficacy among Secondary School Students A B S T R A C T 319 319 Journal of Tikrit University for Humanities (2023) 30 (6) 319-342 ikrit Univer © 2023 JTUH, College of Education for Human Sciences, Tikrit University M.M Mouna Mohamad shehab 319 UNDER THE CC BY LICENSE http://creativecommons.org/licenses/by/4.0/ Journal of Tikrit Univers © 2023 JTUH, College of Education for Human Sciences, Tikrit University DOI: http://doi.org/10.25130/jtuh.30.6.2.2023.17 قياس مستوى الكفاءة الذاتية االنفعالية لدى طلبة المرحلة الثانوية أ.م.د عامر مهدي صالح / جامعة تكريت/ كلية التربية للعلوم االنسانية م.م منى محمد شهاب/ جامعة تكريت/ كلية التربية للعلوم االنسانية الخالصة: يهدف البحث الحالي الكشف عن الكفاءة الذاتية االنفعالية لدى طلبة المرحلة الثانوية وفقا لمتغير ( الجنس وقد بلغت عينة البحث300 ) طالب وطالبة من طالب المرحلة الثانوية وقامت الباحثة ببناء ( مقياس الكفاءة الذاتية االنفعالية إذ تكون من42 )فقرة وقد أظهرت النتائج بعد تحليل البيانات ,وجود فرق دال إحصائيا في مستوى الكفاءة الذاتية االنفعالية لدى الطلبة ودلت النتائج ايضا وجود فرق دال إحصائيا .في مستوى الكفاءة الذاتية االنفعالية وفقا لمتغير الذكور/اإلناث ولصالح الذكور ikrit Univer © 2023 JTUH, College of Education for Human Sciences, Tikrit University DOI: http://doi.org/10.25130/jtuh.30.6.2.2023.17 قياس مستوى الكفاءة الذاتية االنفعالية لدى طلبة المرحلة الثانوية أ.م.د عامر مهدي صالح / جامعة تكريت/ كلية التربية للعلوم االنسانية م.م منى محمد شهاب/ جامعة تكريت/ كلية التربية للعلوم االنسانية الخالصة: ا قياس مستوى الكفاءة الذاتية االنفعالية لدى طلبة المرحلة الثانوية أ.م.د عامر مهدي صالح / جامعة تكريت/ كلية التربية للعلوم االنسانية م.م منى محمد شهاب/ جامعة تكريت/ كلية التربية للعلوم االنسانية الخالصة: يهدف البحث الحالي الكشف عن الكفاءة الذاتية االنفعالية لدى طلبة المرحلة الثانوية وفقا لمتغير ( الجنس وقد بلغت عينة البحث300 ) طالب وطالبة من طالب المرحلة الثانوية وقامت الباحثة ببناء ( مقياس الكفاءة الذاتية االنفعالية إذ تكون من42 )فقرة وقد أظهرت النتائج بعد تحليل البيانات ,وجود فرق دال إحصائيا في مستوى الكفاءة الذاتية االنفعالية لدى الطلبة ودلت النتائج ايضا وجود فرق دال إحصائيا .في مستوى الكفاءة الذاتية االنفعالية وفقا لمتغير الذكور/اإلناث ولصالح الذكور 319 319 319 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 :الكلمات المفتاحية.قياس, مستوى الكفاءة الذاتية االنفعالية , المرحلة الثانوية وال أ :مشكلة البحث :- تعد الكفاءة الذاتية االنفعالية من ا لمفاهيم النفسية التي تنبثق منن المكن ا االنفعنالي ال ننداني ل نذا ممنا يعني ون د عالقة طردية ارتباطية بين الس ك والعمل الكفاءة الذاتية االنفعالية وتعقد الذا,إ ا االمنر النذ س ف يتألف من خ يط وينعكس ايجابيا ع ى انتاج نمط شخصية مميزة في مجتمع مضنرر يعناني لعقن د من الضغ ط النفسية التي تك ا و تتر ب التكيف السريع لها قبل أا تنعكس ع ى شخصية الفرد بشنكل خنا , والتأثير ع ى صحته النفسية واالنتماعية وت افقه النفسي واالنتمناعي, وقند يننعكس ع نى المجتمنع بشنكل عننام مننن خننالر الهنن ر االمننراا النفسننية الع ننل واالمننراا التنني تننسد إلننى ننعف بنيننة ا نسنناا . وقنند وند( Nelis,2011, ) عالقة بين الكفاءة الذاتية االنفعالية طردية بين االشخا لدى الفنرد والصنحة النفسنية ,إو ا انخفاا ارتفاع مست ى الكفاءة الذاتية االنفعالية بين لندى الفنرد تحسنن منن السنعادة النفسنية والعامنة ع ى المست يين النفسي والجسمي تشكيل وتنظيم النذا , وفهنم اخخنرين بمروننة ومهنارة ومسنسولية ويقن العالقننة االنتماعيننة وهننذا مننا اشننار إليننه دراسننة أوسننكاا وايننس ( Ozcan,Essen,2016 ",) حينني يننرى (فرينندلماير1999 ) أا النمنن االنفعننالي لننيس مرعبننا منعننزال وانمننا يننرتبط مننع النمنن المعرفنني واالنتمنناعي ." ( Nelis,Kotso,2011:34 ) ثانيا :اهمية البحث :- تتفق الدراسات والنظريا السيك ل نية ع نى اا أعثنر مرح نة يكن ا فيهنا االنسناا متق باً ومتغيراً في نميع أونه ومجناال حياتنه النفسنية واالنفعالينة واالنتماعينة والعاطفينة وااليمانينة هني مرح ة المراهقة( الشبا ) وهي المرح ة التي تنتهني عنندها مرح نة الرف لنة ومنن هننا عناا البند ل مختصنين بتربيننننة هننننذا االنسنننناا وت نيهنننن ه وخاصننننة فنننني مجننننار المشنننناعر والع اطننننف واالنفعنننناال .( داود واخروا1990 )في( شاعر, 2018 : 3 ) ويسعد(سيد أحمد) إذ يقن ر بنأا ارسنرة هني الحصنن النذ تترن ر فينه الشخصية ا نسانية وت ع به أص ر التربيع االنتمناعي . (أحمند, 1998 : 6 )فني(فاطمنة, 2017 : 383 ) باتن المهارا االنفعالية تحتل مكانه متزايدة ارهمية فني المجتمعنا المخت فنة, ويكثنر التأعيند ع يهنا فني المراننع المتخصصة وغير المتخصصة , وينظر الي م ل نم االنفعنالي ل ينافعين ع نى اننه مكن ا اساسني منن مك ننا التربية , وقد أدعى ن لماا بأننه ال قيمنة ل نذعاء المرتفنع منن دوا الحيناة االنفعالينة السن يمة, فكالهمنا النذعاء 320 320 320 320 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 العق نننننننننني واالنفعننننننننننالي يتفنننننننننناعالا معننننننننننا بصنننننننننن رة معقنننننننننندة ننننننننننندا ومسننننننننننتمرة . ثالثا : إهداف البحث : يهدف البحث : 1 - التعرف ع ى مست ى الكفاءة الذاتية االنفعالية لدى ط بة المرح ة الثان ية . 1 - التعرف ع ى مست ى الكفاءة الذاتية االنفعالية لدى ط بة المرح ة الثان ية . 2 - التعرف ع ى مست ى الكفاءة الذاتية االنفعالية و فقا لمتغير( الذع ر / االناث .) رابعا : حدود البحث :- ( يتحدد البحي الحالي ع ى طال المرح ة الثان ية في محافظة صالح الدين ل عام الدراسني2020 / 2021 , و عال الجنسين ذع ر / اناث , الدراسة الصباحية فقط .) 2 - التعرف ع ى مست ى الكفاءة الذاتية االنفعالية و فقا لمتغير( الذع ر / االناث .) رابعا : حدود البحث :- ( يتحدد البحي الحالي ع ى طال المرح ة الثان ية في محافظة صالح الدين ل عام الدراسني2020 / 2021 , و عال الجنسين ذع ر / اناث , الدراسة الصباحية فقط .) وال أ :مشكلة البحث :- ( (Mitternuber,2008 ونتيجة التغيرا الحياتية واالعاديمية السريعة والمفانئة التي يمنر بهنا االفنراد والتني تنزداد ي منا بعند ين م تجع ه في انفعار دائم , وتنظيم هذه االنفعاال يتر ب الت افق والتأهيل حيار هذه االنفعاال والتصنرف ااائهنا بما ال يسثر ع ى عالقت ه باخخرين ويحافظ ع ى صحت ه ا لنفسية ,والتحديا العديندة التني ت اننه المجتمنع ار ا االنتماعيننة والمعرفيننة والتكن ل نيننة والصننحية والثقافيننة والبيئيننة تتر ننب مننن الفننرد أا يكنن ا ع ننى قنندرا وعفننننناءا انفعالينننننة عالينننننة ل تعامنننننل منننننع عنننننل ذلننننن والت اصنننننل بكفننننناءة منننننع اخخنننننرين . ( Dreher,1985:14 ) ثالثا : إهداف البحث : يهدف البحث : خامسا : تحديد المصطلحات :- خامسا : تحديد المصطلحات :- - الكفاءة الذاتية االنفعاليةEmotional competence - الكفاءة الذاتية االنفعاليةEmotional competence 2 - عرفه( Eisenbery1998 ) ( فهم الشخص النفعاالته و انفعاال االخرين و الميل الى االهار انفعاالته بارس االفضل من الناحية الم قفية و الثقافية و القدرة ع ى السيررة ع ى انفعاالته واختيار وتعديل الس عيا النانمة عن االنفعاال و االهارها بأس الئق انتماعيا بهدف تحقيق اهدافه .) ( Eisenbery 1998 ) عرفتها الباحثة نظريا : بأنها مجم عة من المهارا والقدرا الشخصية واالنتماعية التي تمكن من فهم وإدارة ومعالجة انفعاالته وتنظيمها والحكم ع يها والتعبير عنها ع ى نح مناسب لتحقيق أع ى درنا الت افق النفسي واالنتماعي ) 321 321 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 ال فصل الثاني اإلطار النظري -الكفاءة الذاتية االنفعالية :- ترى النظرية السلوكية إا الس ك ا نساني ه عبارة عنن مجم عنة منن العنادا المتع مة التي يكتسبها الفرد خالر مراحل حياته وترعز ع ى اهمية الحن ادث البيئينة والتفاعنل معهنا, وتنرى النظرية السن عية أا أعثنر السن ك يمكنن اعتسنابه عنن طرينق النتع م وأا السن ك قابنل ل تعنديل والتغيينر . (أب أسعد2010 ) في(أاهر, 2020 : 43 ) مصر ح تم اقتراحه من قبل سارني ويشمل س س ة من القدرا والمهارا التي تتداخل مع بعضنها إلنى حند ما ,وقد صمم ليس عقدرة عق ية مستق ة تأتي باختبار مالئنم ل نذعاء( Mental apfitude اسنتعداد عق ني ) إذ أا االنابة الصحيحة في م قف ينر ع ى تحد انفعالي ال يمكن معرفتها مسبقاً وانما الحقا عما أنه ال يقصد بالكفاءة االنفعالية الذاتية سنمة شخصنية تنربنع فني الفنرد وانمنا تنبثنق منن التفاعنل التعنام ي بنين االشنخا والسياق . ( Saarni,1999,p3 ) ًيفضل عثير من الباحثين استخدام مصر ح الكفاءة الذاتية االنفعالينة بندال( EI ) منن النذعاء االنفعنالي ويرننع ذل إلى سببين ارور : إا استخدام مصر ح الكفاءة الذاتية االنفعالية يسعد أا المهارا االنفعالية قاب ة ل تعنديل ومن ثم فإا الكفاءة الذاتية يمكن تر يرهنا ,والثناني : أا غالبينة مقناييس النذعاء االنفعنالي والتني تعتمند ع نى التقريننر الننذاتي ال تقننيس أقصننى أداء فنني السنن ك باعتبننار أا الننذعاء ينتمنني لمقنناييس ارداء ارقصننى . - الكفاءة الذاتية االنفعاليةEmotional competence 73) Zeidner,et al;2004; ) 73) Zeidner,et al;2004; ) يشير عل من( Wei,zhang:2006,2 ) إلى أا الكفاءة االنفعالية تعد بعندا شنديد ارهمينة فني ترن ير الفنرد حيننننني تنننننسثر فننننني ترننننن ر إدراعنننننا الفنننننرد ودافعيتنننننه ل نننننتع م وا بنننننداع , ويسعننننند عنننننل منننننن ( (Goleman:2001,Saarni:1999,Coetzee:2006 ع ى ارتباط الكفاء ة االنفعالية بالقدرة ع ى االبدا ع و االتقاا والت افق مع التغيرا االنتماعية و الثقافية وتقدير الذا . أبعاد الكفاءة الذاتية االنفعالية حسب سارني المك نة لها تتمثل في ما يأتي : 1 -- ال عي بالحالة االنفعالية يتضمن المعرفة بإمكانية خبرة االنفعاال المتناقضة في وق ما. 2 - تعني القدرة ع ى التعرف ع ى انفعاال االخرين وفهمها من من ما تعنيه القدرة ع ى تفسير وتقييم س ك التعبير ال فظي وغير ال فظي لألشخا اخخرين . أبعاد الكفاءة الذاتية االنفعالية حسب سارني المك نة لها تتمثل في ما يأتي : 1 -- ال عي بالحالة االنفعالية يتضمن المعرفة بإمكانية خبرة االنفعاال المتناقضة في وق ما. 2 - تعني القدرة ع ى التعرف ع ى انفعاال االخرين وفهمها من من ما تعنيه القدرة ع ى تفسير وتقييم س ك التعبير ال فظي وغير ال فظي لألشخا اخخرين . 322 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 3 -- ويقصد بالقدرة ع ى استخدام قام س االنفعاال المعرفة بالمشاعر المستخدمة عبر الت اصل في ثقافة معينة . 4 -تمكن مهارة المشارعة العاطفية من فهم الخبرة االنفعالية ل ناس اخخرين . 5 - تعد المعرفة باا المشاعر قد ال تترابق بالضرورة مع مشاعر الظاهرة وفني الخنارج منن نمن القندرة ع نى التميينز بنين الخبنرة االنفعاليننة الداخ ينة والتعبينر الخنارني عنن االنفعننار , وينربنق هنذا ع نى الخبننرة االنفعالية الذاتية وع ى الخبرة االنفعالية لألشخا اخخرين ع ى حد س اء , وتعد هذه القدرة ع نى درننة منن ار همية في العالقا االنتماعية بشكل خا . ( Saarni,2002:12 ) ا لدراسا السابقة :- دراسة عفاف( 2017 ) ( (القدرة التنبسية لبيئة الت اصل االسر والكفاءة الذاتية االنفعالية واالنتماعية واالعاديمية بالمرونة المعرفية لدى ط بة الصف العاشر في محافظة عج ا) 4 -تمكن مهارة المشارعة العاطفية من فهم الخبرة االنفعالية ل ناس اخخرين . - الكفاءة الذاتية االنفعاليةEmotional competence 5 - تعد المعرفة باا المشاعر قد ال تترابق بالضرورة مع مشاعر الظاهرة وفني الخنارج منن نمن القندرة ع نى التميينز بنين الخبنرة االنفعاليننة الداخ ينة والتعبينر الخنارني عنن االنفعننار , وينربنق هنذا ع نى الخبننرة االنفعالية الذاتية وع ى الخبرة االنفعالية لألشخا اخخرين ع ى حد س اء , وتعد هذه القدرة ع نى درننة منن ار همية في العالقا االنتماعية بشكل خا . ( Saarni,2002:12 ) ( (القدرة التنبسية لبيئة الت اصل االسر والكفاءة الذاتية االنفعالية واالنتماعية واالعاديمية بالمرونة المعرفية لدى ط بة الصف العاشر في محافظة عج ا) هدف الدراسة التعرف غ ى القدرة التنبسية لبيئة الت اصل االسر والكفناءة الذاتينة االنفعالينة واالنتماعينة واالعاديمية بالمرونة المعرفية لدى ط بة الصف العاشر في محافظة عج ن ا وتك نن العيننة منن400 ًطالبنا وطالبة وتم استخدام ثالثة مقاييس وهي مقيناس المروننة المعرفينة ومقيناس بيئنة الت اصنل االسنر ومقيناس الكفاءة الذاتية االنفعالية واالنتماعية واالعاديمية عما أالهر النتائج أا مست ى المرونة المعرفية لندى عيننة الدراسة ناء بدرنة مت سرة ع ى االداة عكل عما أا مست ى الكفاءة الذاتية االنفعالية واالنتماعية واالعاديمية لدى افراد عينة البحي ناء بدرنة مرتفعة ع ى االداة عكل وأشار النتائج أا نمط الت اصل االسر ارعثنر انتشاراً لدى أفراد عينة الدراسة واالهر النتائج ون د فروق ذا داللة احصائية في المرونة المعرفينة وفني نمط الت اصل ارسنر نمنط الحن ار ونمنط الراعنة وااللتنزام وفني الكفناءة الذاتينة االنفعالينة و االنتماعينة واالعاديمية تعزى لمتغير الجنس ا ناث وون د أثر مشترك ومقداره39 % لبيئنة الت اصنل ارسنر والكفناءة الذاتية االنفعالية ل تنبس بالمرونة المعرفية . (عفاف, 2017 ) الفصل الثالي الفصل الثالي ندور ( 1 ) توزيع طلبة المجتمع حسب الجنس والتخصص ت اسم المدرسة الصف المجموع علمي أدبي ذكور إناث ذكور إناث 1 ثانوية تكريت للمتميزين 18 ــــ ــــ ــــ 18 2 إعدادية المغيرة للبنين 50 ــــ ـــــــ ـــــ 50 3 ثانوية تكريت للمتميزات ـــ 10 ــــ ــــ 10 4 ثانوية المتفوقين للبنين ـــــ --- ---- ---- --- 5 إعدادية الخنساء للبنات --- 38 ---- 18 56 6 ثانوية المتفوقات للبنات --- --- --- --- --- تحدد مجتمع البحي الحالي بر بة المدارس الثان ية التابعة لمديرية تربية محافظة صالح الدين ل عام الدراسي( 2021 - 2022 ) ، والبالغ عددهم( 12929 ) طالباً وطالبة، إذ يب غ عدد الذع ر( 6698 ) طالب ا وعدد االناث( 6231 ) طالبة، وقد اقتصر البحي الحالي ع ى ط بة المدارس ا عدادية والثان ية التابعة لمديرية تربية محافظة صالح الدين– قسم تربية تكري ، والبالغ عددهم( 1231 ) طالباً وطالبة، م اعين ع ى( 8 ) مدرسة إعدادية، و( 14 ) مدرسة ثان ية وعاا ت ايعهم حسب الجدور( 1 ) ي ح ذل . ندور ( 1 ) توزيع طلبة المجتمع حسب الجنس والتخصص ت اسم المدرسة الصف المجموع علمي أدبي ذكور إناث ذكور إناث 1 ثانوية تكريت للمتميزين 18 ــــ ــــ ــــ 18 2 إعدادية المغيرة للبنين 50 ــــ ـــــــ ـــــ 50 3 ثانوية تكريت للمتميزات ـــ 10 ــــ ــــ 10 4 ثانوية المتفوقين للبنين ـــــ --- ---- ---- --- 5 إعدادية الخنساء للبنات --- 38 ---- 18 56 6 ثانوية المتفوقات للبنات --- --- --- --- --- تحدد مجتمع البحي الحالي بر بة المدارس الثان ية التابعة لمديرية تربية محافظة صالح الدين ل عام الدراسي( 2021 - 2022 ) ، والبالغ عددهم( 12929 ) طالباً وطالبة، إذ يب غ عدد الذع ر( 6698 ) طالب ا وعدد االناث( 6231 ) طالبة، وقد اقتصر البحي الحالي ع ى ط بة المدارس ا عدادية والثان ية التابعة لمديرية تربية محافظة صالح الدين– قسم تربية تكري ، والبالغ عددهم( 1231 ) طالباً وطالبة، م اعين ع ى( 8 ) مدرسة إعدادية، و( 14 ) مدرسة ثان ية وعاا ت ايعهم حسب الجدور( 1 ) ي ح ذل . الفصل الثالي إجراءات البحث : يتضمن هذا الفصل عر اً لإلنراءا التي اعتمدتها الباحثة لغرا تحقيق أهداف هذا البحي والتي البد من تحديد مجتمع البحي واختيار عينة ممث ة له وإعداد مقياس يتسم بالصدق والثبا ومن ثم استعمار ال سائل ا حصائية المناسبة لتح يل بيانا هذا البحي ومعالجتها، وس ف يتم في هذا الفصل استعراا هذه ا نراءا وعما يأتي : - 323 323 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 منهج البحث : اعتمد الباحثة المنهج ال صفي االرتباطي، وذل لمالئمة طبيعة وأهنداف البحني، ذلن را هذا المنهج ه استقصاء ينصب ع ى الاهرة من الظ اهر التع مية أو النفسية عما هي قائمة في الحا ر بقصد تشخيصها وعشف ن انبها وتحديد العالقا بين عناصرها أو بينها وبين ال اهر تع يمية أو نفسية أو انتماعية أخننننننننننرى مننننننننننع تفسننننننننننير ومقارنننننننننننة ل ت صننننننننننل إلننننننننننى تعميمننننننننننا ذا معنننننننننننى . (الزوبعي و اخروا، 1981 : 52 .) م جتمع البحي : يقصد بمجتمع البحي نميع أفراد الظاهرة قيد الدراسة وقد عرفه أب ح يج( 2002 ) بأنه ارفراد بعامة الذين لهم خصائص معينة يمكن مالحظتها، والمح ال حيد ل مجتمع ه ون د خاصية مشترعة بين ارفراد، وير ق ع ى خصائص المجتمع التي يمكن مالحظتها معالم المجتمع . ( ،أب ح يج2002 : 44 .) منهج البحث : اعتمد الباحثة المنهج ال صفي االرتباطي، وذل لمالئمة طبيعة وأهنداف البحني، ذلن را هذا المنهج ه استقصاء ينصب ع ى الاهرة من الظ اهر التع مية أو النفسية عما هي قائمة في الحا ر بقصد تشخيصها وعشف ن انبها وتحديد العالقا بين عناصرها أو بينها وبين ال اهر تع يمية أو نفسية أو انتماعية أخننننننننننرى مننننننننننع تفسننننننننننير ومقارنننننننننننة ل ت صننننننننننل إلننننننننننى تعميمننننننننننا ذا معنننننننننننى . (الزوبعي و اخروا، 1981 : 52 .) م جتمع البحي : يقصد بمجتمع البحي نميع أفراد الظاهرة قيد الدراسة وقد عرفه أب ح يج( 2002 ) بأنه ارفراد بعامة الذين لهم خصائص معينة يمكن مالحظتها، والمح ال حيد ل مجتمع ه ون د خاصية مشترعة بين ارفراد، وير ق ع ى خصائص المجتمع التي يمكن مالحظتها معالم المجتمع . ( ،أب ح يج2002 : 44 .) بين ارفراد، وير ق ع ى خصائص المجتمع التي يمكن مالحظتها معالم المجتمع . الفصل الثالي ( ،أب ح يج2002 : 44 .) تحدد مجتمع البحي الحالي بر بة المدارس الثان ية التابعة لمديرية تربية محافظة صالح الدين ل عام الدراسي( 2021 - 2022 ) ، والبالغ عددهم( 12929 ) طالباً وطالبة، إذ يب غ عدد الذع ر( 6698 ) طالب ا وعدد االناث( 6231 ) طالبة، وقد اقتصر البحي الحالي ع ى ط بة المدارس ا عدادية والثان ية التابعة لمديرية تربية محافظة صالح الدين– قسم تربية تكري ، والبالغ عددهم( 1231 ) طالباً وطالبة، م اعين ع ى( 8 ) مدرسة إعدادية، و( 14 ) مدرسة ثان ية وعاا ت ايعهم حسب الجدور( 1 ) ي ح ذل . ندور ( 1 ) توزيع طلبة المجتمع حسب الجنس والتخصص ت اسم المدرسة الصف المجموع علمي أدبي ذكور إناث ذكور إناث 1 ثانوية تكريت للمتميزين 18 ــــ ــــ ــــ 18 2 إعدادية المغيرة للبنين 50 ــــ ـــــــ ـــــ 50 3 ثانوية تكريت للمتميزات ـــ 10 ــــ ــــ 10 4 ثانوية المتفوقين للبنين ـــــ --- ---- ---- --- 5 إعدادية الخنساء للبنات --- 38 ---- 18 56 6 ثانوية المتفوقات للبنات --- --- --- --- --- تحدد مجتمع البحي الحالي بر بة المدارس الثان ية التابعة لمديرية تربية محافظة صالح الدين ل عام الدراسي( 2021 - 2022 ) ، والبالغ عددهم( 12929 ) طالباً وطالبة، إذ يب غ عدد الذع ر( 6698 ) طالب ا وعدد االناث( 6231 ) طالبة، وقد اقتصر البحي الحالي ع ى ط بة المدارس ا عدادية والثان ية التابعة لمديرية تربية محافظة صالح الدين– قسم تربية تكري ، والبالغ عددهم( 1231 ) طالباً وطالبة، م اعين ع ى( 8 ) مدرسة إعدادية، و( 14 ) مدرسة ثان ية وعاا ت ايعهم حسب الجدور( 1 ) ي ح ذل . الفصل الثالي ندور ( 1 ) توزيع طلبة المجتمع حسب الجنس والتخصص ع ع ت اسم المدرسة الصف المجموع علمي أدبي ذكور إناث ذكور إناث 1 ثانوية تكريت للمتميزين 18 ــــ ــــ ــــ 18 2 إعدادية المغيرة للبنين 50 ــــ ـــــــ ـــــ 50 3 ثانوية تكريت للمتميزات ـــ 10 ــــ ــــ 10 4 ثانوية المتفوقين للبنين ـــــ --- ---- ---- --- 5 إعدادية الخنساء للبنات --- 38 ---- 18 56 6 ثانوية المتفوقات للبنات --- --- --- --- --- 324 324 324 324 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 7 ثانوية العقيدة للبنات --- 67 --- --- 67 8 ثانوية عقبة بن نافع 16 --- 35 --- 51 9 ثانوية المستنصرية للبنات --- 43 ---- 32 75 10 ثانوية المرجان للبنات --- 18 --- --- 18 11 ثانوية الزهور للبنات --- 50 --- 37 87 12 إعدادية عمرو بن جندب الغفاري 64 --- 30 --- 94 13 ثانوية الجامعة المختلطة 23 3 4 2 32 14 إعدادية البيان للبنات --- 33 --- --- 33 15 ثانوية ام المؤمنين للبنات --- 50 --- 25 75 16 ثانوية ميسلون للبنات --- 95 --- 78 173 17 ثانوية تكريت المسائية للبنين --- --- 16 --- 16 18 ثانوية ميسلون المسائية للبنات --- 10 ---- 10 20 19 إعدادية ابن المعتم للبنين 82 --- 32 --- 114 20 إعدادية الفرقان للبنين 60 --- 43 --- 103 21 إعدادية البارودي للبنات --- 43 --- 25 68 22 إعدادية خالد بن الوليد للبنين 71 --- ---- ---- 71 المجموع 1231 ة 325 عينة البحث : العينة نزء من مجتمع البحي، تحمل خصائصه وتمث ه في الج انب ا حصائية لغرا تعميم النتائج ع يه، وقد عرفها الن ح( 2004 ":) بأنها ذل الجزء من مفردا الظاهرة الترب ية م ع البحي الذ تختاره الباحثة ع ى وفق شروط معينة، لتمثل المجتمع ارص ي ." ( ،الن ح2004 : 49 ) وفي ء تحديد مجتمع البحي وأهدافه اختار الباحثة عينة عش ائية من( 6 ) مدارس ب غ( 300 )طالب وطالبة، وفق متغير الجنس والتخصص الدراسي، والجدور( 2 ) ي ح ذل . الفصل الثالي جدول( 2 ) توزيع أفراد عينة البحث على المدارس مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 ت اسم المدرسة الصف الخامس العينة علمي أدبي ذكور إناث ذكور إناث 1 ثانوية البيان للبنات - 25 - 25 50 2 إعدادية الخنساء للبنات - 25 - 25 50 3 ثانوية ابن المعتم للبنين 25 - 25 - 50 4 ثانوية العقيدة للبنات - 25 - 25 50 5 إعدادية المغيرة للبنين 25 - 25 - 50 6 إعدادية الفرقان للبنين 25 - 25 - 50 مجموع الطلبة 150 150 300 ا البحي : تعرف أداة البحي بأنها طريقة م عية ومقننة لقياس الاهرة من الس ك( ،أب نادو2003 : 398 ) ، ورنل تحقيق أهداف البحي ا لحالي تر ب ت افر أدوا لقياس متغيرا البحي الثالث، وعاالتي : - األداة األولى : مقياس الكفاءة الذاتية االنفعالية من انل قياس متغير الكفاءة الذاتية االنفعالية اط ع الباحثة ع ى مجم عة من الدراسا واردبيا والمقاييس والمرانع الخاصة بم ع الكفاءة الذاتية االنفعالية، ومرانعة بعض المقاييس العربية وارننبية التي تخص هذا المتغير، وند الباحثة انه من ارفضل بناء أداة لقياس الكفاءة الذاتية االنفعالية لمحدودية المقاييس المح ية والعربية واعتمادها ع ى المقاييس ارننبية التي قد تك ا غير مناسبة الختالف الثقافا والبيئا التي أنري فيها عن ثقافة مجتمعنا والروفه . ا يعد م ع صياغة فقرا مقياس الكفاءة الذاتية االنفعالية من الم عا المهمة نداً، رنه ع ما نجح الباحثة في صياغة فقرا مقياسه بشكل ع مي ودقيق في قياس الظاهرة المراد قياسها حقق المقياس الغرا المعد من أن ه، وقد أعد الباحثة( 43 ) فقرة لقياس الكفاءة الذاتية االنفعالية ولكل فقرة أربعة بدائل هي( ًدائماً، غالباً، أحياناً، نادرا) ، عما راع الباحثة في إعداد المقياس : - 1 - أا تصاغ الفقرا ب غة مفه مة بالنسبة لعينة البحي . الفصل الثالي 326 مجاالت مقياس الكفاءة الذاتية االنفعالية وعدد فقراته مجاالت مقياس الكفاءة الذاتية االنفعالية وعدد فقراته ت المجال عدد الفقرات 1 المعرفة االنفعالية 11 2 تنظيم االنفعاال 10 3 إدارة االنفعاال 12 4 المهارا االنتماعية 10 لصدق الظاهري : Face validity الصدق الظاهري : Face validity رنل التحقق من صالحية مقياس الكفاءة الذاتية االنفعالية ول حكم ع ى صالحية عل فقرة من فقراته في قياس ما و ع رن ه، عر الباحثة ارداة بص رتها ارولية ع ى مجم عة من الخبراء والمختصين في الع م الترب ية والنفسية لألخذ بآرائهم وت نيهاتهم، وقد حص الفقرا ع ى نسبة اتفاق أعثر من( 80 )% ، عما اعتمد الباحثة ع ى مربع عا ل تأعد من مدى صالحية الفقرا ، واعتماداً ع ى رأ الخبراء والمحكمين قام الباحثة بتعديل صياغة بعض الفقرا ، وبذل ب غ عدد فقرا المقياس( 43 ) فقرة إذ عان قيمة مربع عا الجدولية( 3،84 ) عند مست ى داللة( 0،05 ) ، وعما مبين في الجدور( 4 .) 327 جد ول( 4 ) الموافقين وغير الموافقين وقيمة مربع كاي المحسوبة والجدولية لفقرات مقياس الكفاءة الذاتية االنفعالية مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 المجار الفقرا الم افق ا غير الم افقين قيمة مربع عا المحس بة قيمة مربع عا ندولية مست ى الداللة 0.05 العدد النسبة المئ ية العدد النسبة المئ ية المعرفة االنفعالية 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 ، 11 18 100 % 0 0 % 18 3.84 دالة تنظيم االنفعاال 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 16 88 % 2 22 % 8.8 دالة إدارة االنفعاال 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 ، 11 ، 12 15 83 % 3 17 % 8.4 دالة المهارا االنتماعية 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 18 100 % 0 0 % 18 دالة • عينة وضوح المقياس وتعليماته وتحديد الوقت إا الهدف من هذا التربيق ه لغرا التعرّف ع ى مدى و ح تع يما المقياس وفقراته ومالئمتها لمجتمع البحي بشكل أفضل، فضالً عن حسا الزمن الذ يتر به المختبرين لغرا إعمار إناباتهم ع ى المقياس وع يه طبق المقياس عن عينة تتألف من( 40 ) طالباً وطالبة من ط بة المرح ة الثان ية تّم اختيارهم برريقة عش ائية ومن عال التخصصين، وبناء ع ى نتائج التربيق فقد تبين أا فقرا المقياس وا حة ال ق ا ا ا ة ف ال ط ال ة ق ق قة مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 المجار الفقرا الم افق ا غير الم افقين قيمة مربع عا المحس بة قيمة مربع عا ندولية مست ى الداللة 0.05 العدد النسبة المئ ية العدد النسبة المئ ية المعرفة االنفعالية 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 ، 11 18 100 % 0 0 % 18 3.84 دالة تنظيم االنفعاال 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 16 88 % 2 22 % 8.8 دالة إدارة االنفعاال 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 ، 11 ، 12 15 83 % 3 17 % 8.4 دالة المهارا االنتماعية 1 ، 2 ، 3 ، 4 ، 5 ، 6 ، 7 ، 8 ، 9 ، 10 18 100 % 0 0 % 18 دالة • عينة وضوح المقياس وتعليماته وتحديد الوقت إا الهدف من هذا التربيق ه لغرا التعرّف ع ى مدى و ح تع يما المقياس وفقراته ومالئمتها لمجتمع البحي بشكل أفضل، فضالً عن حسا الزمن الذ يتر به المختبرين لغرا إعمار إناباتهم ع ى المقياس وع يه طبق المقياس عن عينة تتألف من( 40 ) طالباً وطالبة من ط بة المرح ة الثان ية تّم اختيارهم برريقة عش ائية ومن عال التخصصين، وبناء ع ى نتائج التربيق فقد تبين أا فقرا المقياس وا حة ومفه مة وقد ب غ مت سط الزمن المستغرق في ا نابة ع ى المقياس( 15 )دقيقة . مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 وبهذا يك ا قد ب غ عدد ارفراد في المجم عتين الع يا والدنيا ( 216 ) ًطالباً وطالبة ثّم وفقا لمست ى الق ة التمييزية ل فقرة ومعامل ارتباط الفقرة بالدرنة الك ية وع ى النح اختي : ولتحقيق ذل فقد تّم تربيق المقياس ع ى عينة عش ائية من ط بة المرح ة الثان ية ب غ ( 400 ) طالب وطالبة تم اختيارهم برريقة عش ائية من مجتمع البحي، وبعد تربيق المقياس ع ى العينة صحح إنابا ًالر بة ثّم استخرن الدرنة الك ية لكل فرد من أفراد عينة التح يل ا حصائي، ومن ثّم رتب الدرنا تنااليا ثم أخذ نسبة( 27 )%عمجم عة ع يا و ( 27 )% عمجم عة دنيا وقد أوصى عي ي(Kelly) عند تح يل مفردا االختبار االعتماد ع ى النسبة27 % من ارفراد في عل من المجم عتين الررفيتين واستبعد نسبة46 % ال سرى، ويشير إلى أا هذه النسبة تجعل المجم عتين في أفضل ما يك ا في الحجم والتباين(Kelly, 1955,p; 468) . وبهذا يك ا قد ب غ عدد ارفراد في المجم عتين الع يا والدنيا ( 216 ) ًطالباً وطالبة ثّم وفقا لمست ى الق ة التمييزية ل فقرة ومعامل ارتباط الفقرة بالدرنة الك ية وع ى النح اختي : القوة التمييزية للفقرات تم حسا التمييز(الفرق )بين المجم عتين المتررفتين باستعمار االختبار التائي لعينتين مستق تين، وبذل عان نميع الفقرا دالة إحصائياً عند م اانة القيم التائية المحس بة بالقيمة ندولية البالغة( 1،96 ) وعند مست ى داللة( 0،05 ) وبدرنة حرية( 214 ) ويدر ذل ع ى أا نميع الفقرا ذا تمييز نيد، ما عدا الفقرة ( 4 ) ، والجدور( 5 ) يبين ذل . تم حسا التمييز(الفرق )بين المجم عتين المتررفتين باستعمار االختبار التائي لعينتين مستق تين، وبذل عان نميع الفقرا دالة إحصائياً عند م اانة القيم التائية المحس بة بالقيمة ندولية البالغة( 1،96 ) وعند مست ى داللة( 0،05 ) وبدرنة حرية( 214 ) ويدر ذل ع ى أا نميع الفقرا ذا تمييز نيد، ما عدا الفقرة ( 4 ) ، والجدور( 5 ) يبين ذل . 327 جد ول( 4 ) الموافقين وغير الموافقين وقيمة مربع كاي المحسوبة والجدولية لفقرات مقياس الكفاءة الذاتية االنفعالية التحليل اإلحصائي لفقرات المقياس : إا الهدف من هذا التربيق ه لغرا التعرّف ع ى مدى و ح تع يما المقياس وفقراته ومالئمتها لمجتمع البحي بشكل أفضل، فضالً عن حسا الزمن الذ يتر به المختبرين لغرا إعمار إناباتهم ع ى المقياس وع يه طبق المقياس عن عينة تتألف من( 40 ) طالباً وطالبة من ط بة المرح ة الثان ية تّم اختيارهم برريقة عش ائية ومن عال التخصصين، وبناء ع ى نتائج التربيق فقد تبين أا فقرا المقياس وا حة ومفه مة وقد ب غ مت سط الزمن المستغرق في ا نابة ع ى المقياس( 15 )دقيقة . التحليل اإلحصائي لفقرات المقياس : 328 328 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 يعُدّ التح يل ا حصائي ل فقرا أعثر أهمية من التح يل المنرقي لهنا، إذ إا التح ينل المنرقني قند ال يكشنف عن صدق الفقرا ع ى نح دقيق رنه يعتمد ع ى الفحص الظاهر لهنا فقنط أ مث منا يبندو الاهريناً ل خبينر لذل فه أعثر عر ة ل تأثر بارحكام الذاتية ل فرد( ،فرج1980 : 331 - 332 ) يعُدّ التح يل ا حصائي ل فقرا أعثر أهمية من التح يل المنرقي لهنا، إذ إا التح ينل المنرقني قند ال يكشنف عن صدق الفقرا ع ى نح دقيق رنه يعتمد ع ى الفحص الظاهر لهنا فقنط أ مث منا يبندو الاهريناً ل خبينر لذل فه أعثر عر ة ل تأثر بارحكام الذاتية ل فرد( ،فرج1980 : 331 - 332 ) فتح يل الفقرا يمكننا من استقصاء الخصائص ا حصائية الستجابا المفح صين ع ى عل فقرة من فقرا أداة القياس(المقياس ) ومن بين هذه الخصائص ا حصائية التعرّف ع ى تمييز الفقرة . ( ،النبهاا2004 : 188 .) ولتحقيق ذل فقد تّم تربيق المقياس ع ى عينة عش ائية من ط بة المرح ة الثان ية ب غ ( 400 ) طالب وطالبة تم اختيارهم برريقة عش ائية من مجتمع البحي، وبعد تربيق المقياس ع ى العينة صحح إنابا ًالر بة ثّم استخرن الدرنة الك ية لكل فرد من أفراد عينة التح يل ا حصائي، ومن ثّم رتب الدرنا تنااليا ثم أخذ نسبة( 27 )%عمجم عة ع يا و ( 27 )% عمجم عة دنيا وقد أوصى عي ي(Kelly) عند تح يل مفردا االختبار االعتماد ع ى النسبة27 % من ارفراد في عل من المجم عتين الررفيتين واستبعد نسبة46 % ال سرى، ويشير إلى أا هذه النسبة تجعل المجم عتين في أفضل ما يك ا في الحجم والتباين(Kelly, 1955,p; 468) . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين 3.81 0.676 3.19 1.024 5.252 عالقة الفقرة بالدرجة الكلية للمقياس : يعتمد صدق المقياس اعتماداً مباشراً ع ى صدق مفرداته، وذل را أ ايادة في صدق المفردا ، تسد إلى ايادة صدق االختبار وتقاس صدق المفردا بحسا معامال ارتباطها بالميزاا وقد يك ا الميزاا داخ يّاً أو خارنيّاً، ونعني بالميزاا الداخ ي االختبار ه الذ يشمل ع ى ت المفردا وبالميزاا الخارني ه الذ نقيس به صدق المقياس نفسه، ويسمى الصدق الداخ ي أحياناً بالتجانس الداخ ي لالختبار رنه يقيس مدى تماس المفردا باختبارها وال يخت ف طريقة حسا الصدق الداخ ي عن طريق حسا الصدق ًوا حا اختالفاً منهما عل مفه م اخت ف واا الخارني . ( ،السيد1978 : 457 ) إذ إا االتساق بين درنة الفقرة والدرنة الك ية من خالر معامال االرتبناط الدالنة إحصنائياً تشنير إلنى أا فقرا االختبار متماسكة ومترابرة ومتسقة فيما بينها، وبالتالي فأا نميع الفقنرا تقنيس متغينر واحند، وهنذا مسشر مقب ر ع ى صدقها وصدق االختبار( ،معمرينة2009 : 152 - 153 ) ، بنل إا معامنل االتسناق النداخ ي مسشر من مسشرا صدق البناء، وقد تّم استعمار معامل ارتباط بيرس ا يجاد العالقة االرتباطية بين درننة عل فقرة مع الدرنة،الك ية ل مقياس وقد تبين من خنالر التح ينل أا أعثنر الفقنرا ممينزة عنند مسنت ى داللنة ( 0 05 )باستثناء الفقرة( 4 )وال جدور( 6 ) ي ح ذل مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 40 . 2.90 1.058 2.18 0.926 5.338 41 . 3.72 0.561 2.71 1.086 8.583 42 . 3.48 0.848 2.25 1.042 9.524 43 . 3.81 0.676 3.19 1.024 5.252 عالقة الفقرة بالدرجة الكلية للمقياس : يعتمد صدق المقياس اعتماداً مباشراً ع ى صدق مفرداته، وذل را أ ايادة في صدق المفردا ، تسد إلى ايادة صدق االختبار وتقاس صدق المفردا بحسا معامال ارتباطها بالميزاا وقد يك ا الميزاا داخ يّاً أو خارنيّاً، ونعني بالميزاا الداخ ي االختبار ه الذ يشمل ع ى ت المفردا وبالميزاا الخارني ه الذ نقيس به صدق المقياس نفسه، ويسمى الصدق الداخ ي أحياناً بالتجانس الداخ ي لالختبار رنه يقيس مدى تماس المفردا باختبارها وال يخت ف طريقة حسا الصدق الداخ ي عن طريق حسا الصدق ًوا حا اختالفاً منهما عل مفه م اخت ف واا الخارني . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين جدول( 5 ) ت المجموعة العليا المجموعة الدنيا القيمة التائية المستخرجة الوسط الحسابي االنحراف المعياري الوسط الحسابي االنحراف المعياري 1. 3.35 0.900 2.54 0.934 6.473 2. 3.58 0.787 2.80 1.039 6.275 3. 3.53 0.742 2.30 0.983 7.112 4. 2.52 1.009 2.30 1.087 1.557 5. 963 0.327 2.58 0.987 5.164 6. 2.59 1.103 2.02 1.004 4.000 7. 3.20 0.964 2.29 1.064 6.600 8. 3.52 0.881 2.84 1.025 5.200 9. 2.51 1.028 2.04 1.049 3.341 10 . 3.26 1.097 2.67 1.238 3,723 11 . 3.69 0.603 2.51 1.148 9.499 12 . 3.41 0,821 2,58 1.086 6.260 13 . 2.81 1.106 2.05 1.071 5.124 14 . 3.73 0.621 2.62 1.194 8.365 15 . 3.79 0.512 2.95 1.097 7.152 16 . 3.73 0.605 3.05 1.080 5.752 17 . 3.35 0.979 2.50 0.962 6.449 18 . 3.27 0.917 2.57 1.061 5.152 330 330 330 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 19 . 3.74 0.500 2.44 1.171 10.582 20 . 3.53 0.767 2.51 1.148 7.668 21 . 3.79 0.475 2.45 0.941 13.146 22 . 3.03 1.027 2.72 1.002 2.239 23 . 3.51 0.743 2.43 1.052 8.744 24 . 2.31 1.124 2.31 1.124 9.726 25 . 3.55 0.813 2.58 1.060 7.490 26 . 3.34 0.877 2.23 1.029 8.542 27 . 3.49 0,803 2.33 1.068 9.003 28 . 3.09 1.037 2.26 1.163 5.558 29 . 2.83 1.140 1.84 0.929 7.003 30 . 3.45 0.802 2.46 0.999 8.037 31 . 2.79 1.051 2.31 1.054 3.362 32 . 3.04 1.032 1.90 0.976 8.335 33 . 3.82 0.450 2.62 1.091 10.597 34 . 3.75 0.532 2.56 1.035 10.588 35 . 3.36 0.901 2.04 0.995 10.252 36 . 2.69 1.157 2.06 1.121 4.001 37 . 3.10 1.032 2.19 1.054 6.393 38 . 3.71 0.684 2.97 1.180 5.644 39 . 2.94 1.058 1.78 1.008 8.300 331 331 331 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 40 . 2.90 1.058 2.18 0.926 5.338 41 . 3.72 0.561 2.71 1.086 8.583 42 . 3.48 0.848 2.25 1.042 9.524 43 . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين ( ،السيد1978 : 457 ) إذ إا االتساق بين درنة الفقرة والدرنة الك ية من خالر معامال االرتبناط الدالنة إحصنائياً تشنير إلنى أا فقرا االختبار متماسكة ومترابرة ومتسقة فيما بينها، وبالتالي فأا نميع الفقنرا تقنيس متغينر واحند، وهنذا مسشر مقب ر ع ى صدقها وصدق االختبار( ،معمرينة2009 : 152 - 153 ) ، بنل إا معامنل االتسناق النداخ ي مسشر من مسشرا صدق البناء، وقد تّم استعمار معامل ارتباط بيرس ا يجاد العالقة االرتباطية بين درننة عل فقرة مع الدرنة،الك ية ل مقياس وقد تبين من خنالر التح ينل أا أعثنر الفقنرا ممينزة عنند مسنت ى داللنة ( 0.05 ) باستثناء الفقرة( 4 .)وال جدور( 6 ) ي ح ذل . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين ج دول( 6 ) م عامالت ارتباط درجة الفقرة بالدرجة الكلية مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 رقم الفقرة معامل ارتباط الفقرة رقم الفقرة معامل ارتباط الفقرة رقم الفقرة معامل ارتباط الفقرة رقم الفقرة معامل ارتباط الفقرة 1 0.386 12 0.366 23 0.408 34 0.529 2 0.376 13 0.309 24 0.471 35 0.486 3 0.410 14 0.474 25 0.452 36 0.360 4 0.045 15 0.478 26 0.436 37 0.354 5 0.263 16 0.436 27 0.481 38 0.329 6 0.270 17 0.412 28 0.351 39 0.352 7 0.318 18 0.327 29 0.361 40 0.241 8 0.386 19 0.529 30 0.426 41 0.448 9 0.331 20 0.422 31 0.391 42 0.390 10 0.245 21 0.571 32 0.368 43 0.295 11 0.542 22 0.233 33 0.492 عالقة درجة الفقرة بالدرجة الكلية لكل مجال : ر ر ر ر ب الفقرة ر ر ب الفقرة ر ر ب الفقرة 1 0.386 12 0.366 23 0.408 34 0.529 2 0.376 13 0.309 24 0.471 35 0.486 3 0.410 14 0.474 25 0.452 36 0.360 4 0.045 15 0.478 26 0.436 37 0.354 5 0.263 16 0.436 27 0.481 38 0.329 6 0.270 17 0.412 28 0.351 39 0.352 7 0.318 18 0.327 29 0.361 40 0.241 8 0.386 19 0.529 30 0.426 41 0.448 9 0.331 20 0.422 31 0.391 42 0.390 10 0.245 21 0.571 32 0.368 43 0.295 11 0.542 22 0.233 33 0.492 عالقة درجة الفقرة بالدرجة الكلية لكل مجال : يتفق المتخصص ا في مجار القياس النفسي ع ى أهمية الصدق في فقرا المقاييس النفسية، را صدق المقياس يعتمد بارساس ع ى صدق فقراته( ،عبدالرحمن1998 : 184 ) ، إذ أشار( انستاا ) إلى أا ارتباط الفقرة بمح داخ ي أو خارني مسشر لصدقها، وحينما ال يت فر مح خارني مناسب فإا الدرنة الك ية ل مجيب تمثل أفضل مح داخ ي في حسا هذه العالقة( Anastasi,1976,p.206 ) ، وع يه حُسب صدق الفقرا لمقياس الكفاءة الذاتية االنفعالية ولكل مجار ع ى حده عن طريق إيجاد العالقة بين درنة الفقرة والدرنة الك ية لكل مجار باستعمار معامل ارتباط بيرس ا . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين باالعتماد ع ى( 400 ) استمارة( وهي نفس االستمارا التي خضع ل تح يل في ء مجار المجم عتين المتررفتين ) ومن المعروف في بناء المقاييس انه ع ما ااد معامل ارتباط الفقرة بالمجم ع الك ي عاا تضمينها في المقياس يزيد من احتمار الحص ر ع ى يتفق المتخصص ا في مجار القياس النفسي ع ى أهمية الصدق في فقرا المقاييس النفسية، را صدق المقياس يعتمد بارساس ع ى صدق فقراته( ،عبدالرحمن1998 : 184 ) ، إذ أشار( انستاا ) إلى أا ارتباط الفقرة بمح داخ ي أو خارني مسشر لصدقها، وحينما ال يت فر مح خارني مناسب فإا الدرنة الك ية ل مجيب تمثل أفضل مح داخ ي في حسا هذه العالقة( Anastasi,1976,p.206 ) ، وع يه حُسب صدق الفقرا لمقياس الكفاءة الذاتية االنفعالية ولكل مجار ع ى حده عن طريق إيجاد العالقة بين درنة الفقرة والدرنة الك ية لكل مجار باستعمار معامل ارتباط بيرس ا . باالعتماد ع ى( 400 ) استمارة( وهي نفس االستمارا التي خضع ل تح يل في ء مجار المجم عتين المتررفتين ) ومن المعروف في بناء المقاييس انه ع ما ااد معامل ارتباط الفقرة بالمجم ع الك ي عاا تضمينها في المقياس يزيد من احتمار الحص ر ع ى 333 333 333 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 مقياس اعثر تجانسا( Allen &Yen, 1979, p.125 . ) وقد عان نميع معامال االرتباط دالة إحصائيا عند مقارنتها بالقيمة الجدولية لمعامل ارتباط بيرس اPerson) ) عند مست ى داللة( 0.05 ) ودرنة حرية( 399 ) ما عدا الفقرة( 4 ) والجدور( 7 ) ي ح ذل . مقياس اعثر تجانسا( Allen &Yen, 1979, p.125 . ) وقد عان نميع معامال االرتباط دالة إحصائيا عند مقارنتها بالقيمة الجدولية لمعامل ارتباط بيرس اPerson) ) عند مست ى داللة( 0.05 ) ودرنة حرية( 399 ) ما عدا الفقرة( 4 ) والجدور( 7 ) ي ح ذل . الفقرة( 4 ) والجدور( 7 ) ي ح ذل . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين جدول( 7 ) قيم معامالت االرتباط المحسوبة لفقرات مقياس الكفاءة الذاتية االنفعالية ت المجاالت رقم الفقرة قيمة معامل االرتباط الداللة 1 المعرفة االنفعالية 1 0.465 دالة 2 0.339 دالة 3 0.389 دالة 4 0.415 دالة 5 0.373 دالة 6 0.387 دالة 7 0.425 دالة 8 0.379 دالة 9 0.419 دالة 10 0.423 دالة 11 0.702 دالة 2 تنظيم االنفعاالت 12 0.535 دالة 13 0.350 دالة 14 0.578 دالة 15 0.605 دالة 16 0.615 دالة 17 0.531 دالة 18 0.412 دالة 19 0.631 دالة 20 0.534 دالة 21 0.591 دالة 3 إدارة االنفعاالت 22 0.242 دالة 23 0.457 دالة 24 0.548 دالة 25 0.530 دالة 26 0.523 دالة 27 0.579 دالة 28 0.473 دالة 29 0.517 دالة 30 0.531 دالة 31 0.265 دالة 32 0.424 دالة 33 0.469 دالة 4 المهارات االجتماعية 34 0.504 دالة 35 0.517 دالة 36 0.384 دالة 37 0.443 دالة 38 0.512 دالة 39 0.559 دالة 40 0.316 دالة 3 335 335 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 41 0.569 دالة 42 0.511 دالة لخصائص السيكومترية للمقياس : مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 41 0.569 دالة 42 0.511 دالة الخصائص السيكومترية للمقياس : خصائص السيكومترية للمقياس : -مؤشرات الصدق - مؤشرات الصدق - صدق األداة : يُعد الصدق من أكثر الصفات األساسية لالختبار أهمية، كذلك يعدّ أساس بناء االختبارات النفسية لكونه يساعد في تعرّف المكونات الداخلية لالختبار نفسه والتنبؤ فيما بعد بقدرات األفراد التعليمية والعملية ( ،النمر2008 : 69 .) أ يُعد الصدق من أكثر الصفات األساسية لالختبار أهمية، كذلك يعدّ أساس بناء االختبارات النفسية لكونه يساعد في تعرّف المكونات الداخلية لالختبار نفسه والتنبؤ فيما بعد بقدرات األفراد التعليمية والعملية ( ،النمر2008 : 69 .) والصدق مفهوم واسع له معان عدة تختلف وفقا الستعمال االختبار إال أن أولى معاني الصدق هو مدى نجاح االختبار في القياس وفي التشخيص والتنبؤ عن ميدان السلوك الذي وضع االختبار من أجله أي أن االختبار صادق ألنه يقيس ما وضع لقياسه( ،عوض1998 : 59 ) ول تحقق من صدق االختبار، تّم إيجاد ن عين من الصدق هما : ن عين من الصدق هما : 1 - الصدق الظاهري : - يعدّ هذا النوع من الخصائص المهمة في بناء االختبارات والمقاييس ويقصد به المظهر العام لالختبار من حيث المفردات وكيفية صياغتها ومدى وضوحها موضوعيتها ومدى مناسبة االختبار للغرض الذي وضع من أجله وان الوسيلة المفضلة للتأكد من الصدق الظاهري لالختبار هو أن يقوم عدد من الخبراء المختصين بتقرير كون الفقرات ممثلة للصفة المراد قياسها( ،العزاوي2008 : 94 .) وقد تحقق هذا الن ع من الصدق عند إعداد المقياس، إذ عر الباحثة المقياس ع ى مجم عة من المختصين في الع م الترب ية والنفسية(الم حق2 ) وقد حصل االختبار ع ى نسبة اتفاق80 .% 2 - صدق البناء : يطلق على صدق البناء أحيانا صدق المفهوم أو صدق التكوين الفرضي ويقصد به تحليل درجات المقياس استنادا للخاصية المراد قياسها، وتّم التحقق من هذا النوع من الصدق عن طريق إيجاد معامالت ارتباط الفقرات بالدرجة الكلية لالختبار، إذ تعدّ قوة االرتباط بين الفقرات المعدة لقياس السمة مؤشرا إحصائيا لصدق البناء( ،عودة1985 : 165 ) ً، فضالً عن حسا معامال التمييز التي تعدّ مسشرا آخر ع ى صدق البناء . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين جدول( 8 ) عينة الثبات موزعة على المدارس ت اسم المدرسة الصف الخامس العينة علمي أدبي ذكور إناث ذكور إناث 1 ثانوية البارودي للبنات - 5 - 5 10 2 ثانوية المستنصرية للبنات - 5 - 5 10 3 ثانوية عقبة بن نافع للبنين 5 - 5 - 10 4 ثانوية المرجان للبنات - 5 - 5 10 5 ثانوية عمرو بن جندب الغفاري للبنين 5 - 5 - 10 6 ثانوية خالد بن الوليد للبنين 5 - 5 - 10 مجموع الطلبة 30 30 60 - ا ستخراج الثبات بطريقتين : أ- طريقة إعادة االختبار( : Test-Retest ) قام الباحثة بتربيق المقياس ع ى عينة ب غ( 60 ) ، طالباً وطالبة من المدارس الثان ية في مدينة تكري ثم أعيد تربيق المقياس ذاته بعد مضي( 15 ) ي ماً ع ى العينة ذاتها، وبعد تصحيح ا نابا تّم إيجاد معامل االرتباط بين درنا الر بة في التربيق ارور ودرناتهم في التربيق الثاني وذل باستعمار معامل ارتباط بيرس ا فب غ( 0.81 )وع يه يعدّ معامل الثبا عارٍ مما يشير إلى أا المقياس له استقرار ثاب عبر الزمن . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين جدول( 8 ) عينة الثبات موزعة على المدارس ت اسم المدرسة الصف الخامس العينة علمي أدبي ذكور إناث ذكور إناث 1 ثانوية البارودي للبنات - 5 - 5 10 2 ثانوية المستنصرية للبنات - 5 - 5 10 3 ثانوية عقبة بن نافع للبنين 5 - 5 - 10 4 ثانوية المرجان للبنات - 5 - 5 10 5 ثانوية عمرو بن جندب الغفاري للبنين 5 - 5 - 10 6 ثانوية خالد بن الوليد للبنين 5 - 5 - 10 مجموع الطلبة 30 30 60 - ا ستخراج الثبات بطريقتين : أ- طريقة إعادة االختبار( : Test-Retest ) قام الباحثة بتربيق المقياس ع ى عينة ب غ( 60 ) ، طالباً وطالبة من المدارس الثان ية في مدينة تكري الثبات : - Reliability يُعد الثبات من المؤشرات الضرورية لالختبار لكونه يشير إلى االتساق في مجموعة درجات الفقرات التي تقيس فعال ما يجب قياسه( ،عودة والخليلي1993 : 345 ) ، فثبات درجات االختبار يقصد بها مدى خلوها من األخطاء غير المنتظمة التي تشوب القياس، أي مدى قياس االختبار للمقدار الحقيقي التي يهدف لقياسها فدرجات االختبار تكون ثابتة إذا كان االختبار يقيس سمة معينة قياسا في الظروف المختلفة التي قد تؤدي إلى أخطاء القياس فالثبات يعني االتساق أو الدقة في القياس( ،عالم 2000 : 131 ) .و يجاد الثبا قام الباحثة بتربيق المقياس ع ى عينة ب غ( 60 ) طالب وطالبة، وندور ( 8 ) ي ح ذل . يا ي ي ي ي ما 2000 : 131 ) .و يجاد الثبا قام الباحثة بتربيق المقياس ع ى عينة ب غ( 60 ) طالب وطالبة، وندور ( 8 ) ي ح ذل . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين يعدّ هذا النوع من الخصائص المهمة في بناء االختبارات والمقاييس ويقصد به المظهر العام لالختبار من حيث المفردات وكيفية صياغتها ومدى وضوحها موضوعيتها ومدى مناسبة االختبار للغرض الذي وضع من أجله وان الوسيلة المفضلة للتأكد من الصدق الظاهري لالختبار هو أن يقوم عدد من الخبراء المختصين بتقرير كون الفقرات ممثلة للصفة المراد قياسها( ،العزاوي2008 : 94 .) وقد تحقق هذا الن ع من الصدق عند إعداد المقياس، إذ عر الباحثة المقياس ع ى مجم عة من المختصين في الع م الترب ية والنفسية(الم حق2 )وقد حصل االختبار ع ى نسبة اتفاق80 .% 2 - صدق البناء : يطلق على صدق البناء أحيانا صدق المفهوم أو صدق التكوين الفرضي ويقصد به تحليل درجات المقياس استنادا للخاصية المراد قياسها، وتّم التحقق من هذا النوع من الصدق عن طريق إيجاد معامالت ارتباط الفقرات بالدرجة الكلية لالختبار، إذ تعدّ قوة االرتباط بين الفقرات المعدة لقياس السمة مؤشرا إحصائيا لصدق البناء( ،عودة1985 : 165 ) ً، فضالً عن حسا معامال التمييز التي تعدّ مسشرا آخر ع ى صدق البناء . 336 336 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 الثبات : - Reliability يُعد الثبات من المؤشرات الضرورية لالختبار لكونه يشير إلى االتساق في مجموعة درجات الفقرات التي تقيس فعال ما يجب قياسه( ،عودة والخليلي1993 : 345 ) ، فثبات درجات االختبار يقصد بها مدى خلوها من األخطاء غير المنتظمة التي تشوب القياس، أي مدى قياس االختبار للمقدار الحقيقي التي يهدف لقياسها فدرجات االختبار تكون ثابتة إذا كان االختبار يقيس سمة معينة قياسا في الظروف المختلفة التي قد تؤدي إلى أخطاء القياس فالثبات يعني االتساق أو الدقة في القياس( ،عالم 2000 : 131 ) .و يجاد الثبا قام الباحثة بتربيق المقياس ع ى عينة ب غ( 60 ) طالب وطالبة، وندور ( 8 ) ي ح ذل . 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين 337 337 337 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 ب- معامل االتساق الداخلي( ألفا كرونباخ( ) Cronbach Alpha Method ) تعد هذه الرريقة مفض ة لقياس الثبا فهي تقيس االتساق الداخ ي والتجانس بين فقرا المقياس( Anastasi & Urbina, 1988: 95 ) ، أ أا الفقرا نميعها تقيس فعالً الخاصية نفسها وهذا يتحقق عندما تك ا الفقرا مترابرة مع بعضها البعض داخل االختبار عذل ارتباط عل فقرة مع االختبار ع ه، وب غ معامل ثبا ألفا ( 0,78 )لم قياس الكفاءة الذاتية االنفعالية ا لفصل الرابع عرض النتائج ومناقشتها : يتضمن هذا الفصل عر ا ل نتائج التي تم الت صل اليها في هنذا البحني . وتمن مناقشتها في ء ا طار النظر والدراسا السابقة . وسيتم عرا النتائج تبعا رهداف البحي وع ى النحن االتي : - الهدف األول : التعرف على مستوى الكفاءة الذاتية االنفعالية لدى طلبة المرحلة ا لثانوية . تننم معالجننة البيانننا إحصننائياً باسننتعمار االختبننار التننائي لعينننة واحنندة فننأالهر النتننائج وننن د فننرق دار إحصننائياً بننين المت سننط الحسننابي لنندرنا أفننراد العينننة البننالغ( 123.51 ) درنننة بننانحراف معيننار قنندره ( 16.076 ) والمت سط الفر ي البالغ( 105 ) درنة وب غ القيمة التائية المحسن بة( 19.946 ) عنند مقارنتهنا بالقيمة الجدولية البالغة( 1.96 ) عند مست ى الداللة( 0.05 ) وبدرنة حرينة( 299 ) وتبنين إا مسنت ى الكفناءة الذاتية االنفعالية ه بدرنة ( مت سر ة) . والجدور( 19 ) ي ح ذل . جدول( 19 ) المتوسط الحسابي واالنحراف والقيمة التائية لمستوى الكفاءة الذاتية االنفعالية مقياس العدد المت سط الحسابي االنحننراف المعيار المت سط النظر القيمة التائية مست ى الداللة 0.05 الكفاءة الذاتية االنفعالية 300 123.51 16.076 105 المحس بة الجدولية 19.946 1.96 دالة لما عان النتيجة تشير إلى ونن د فنرق دار إحصنائياً أ أا مسنت ى الكفناءة الذاتينة االنفعالينة لندى أفنراد العينة ه بدرنة ( مت سر ة ) تشير هذه النتيجة إلى تمتنع الر بنة بمسنت ى منن الكفناءة الذاتينة االنفعالينة يمكنن 329 جدول( 5 ) القوة التمييزية لفقرات مقياس الكفاءة الذاتية االنفعالية بأسلوب المجموعتين المتطرفتين ( 8 )ح ي جدول( 8 ) عينة الثبات موزعة على المدارس ت اسم المدرسة الصف الخامس العينة علمي أدبي ذكور إناث ذكور إناث 1 ثانوية البارودي للبنات - 5 - 5 10 2 ثانوية المستنصرية للبنات - 5 - 5 10 3 ثانوية عقبة بن نافع للبنين 5 - 5 - 10 4 ثانوية المرجان للبنات - 5 - 5 10 5 ثانوية عمرو بن جندب الغفاري للبنين 5 - 5 - 10 6 ثانوية خالد بن الوليد للبنين 5 - 5 - 10 مجموع الطلبة 30 30 60 - ا ستخراج الثبات بطريقتين : ( )ح جدول( 8 ) عينة الثبات موزعة على المدارس ت اسم المدرسة الصف الخامس العينة علمي أدبي ذكور إناث ذكور إناث 1 ثانوية البارودي للبنات - 5 - 5 10 2 ثانوية المستنصرية للبنات - 5 - 5 10 3 ثانوية عقبة بن نافع للبنين 5 - 5 - 10 4 ثانوية المرجان للبنات - 5 - 5 10 5 ثانوية عمرو بن جندب الغفاري للبنين 5 - 5 - 10 6 ثانوية خالد بن الوليد للبنين 5 - 5 - 10 مجموع الطلبة 30 30 60 - ا ستخراج الثبات بطريقتين : أ- طريقة إعادة االختبار( : Test-Retest ) أ- طريقة إعادة االختبار( : Test-Retest ) قام الباحثة بتربيق المقياس ع ى عينة ب غ( 60 ) ، طالباً وطالبة من المدارس الثان ية في مدينة تكري ثم أعيد تربيق المقياس ذاته بعد مضي( 15 ) ي ماً ع ى العينة ذاتها، وبعد تصحيح ا نابا تّم إيجاد معامل االرتباط بين درنا الر بة في التربيق ارور ودرناتهم في التربيق الثاني وذل باستعمار معامل ارتباط بيرس ا فب غ( 0.81 )وع يه يعدّ معامل الثبا عارٍ مما يشير إلى أا المقياس له استقرار ثاب عبر الزمن . ا لفصل الرابع مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 ا لفصل الرابع 338 ا ( ) ( )ح جدول( 19 ) المتوسط الحسابي واالنحراف والقيمة التائية لمستوى الكفاءة الذاتية االنفعالية مقياس العدد المت سط الحسابي االنحننراف المعيار المت سط النظر القيمة التائية مست ى الداللة 0.05 الكفاءة الذاتية االنفعالية 300 123.51 16.076 105 المحس بة الجدولية 19.946 1.96 دالة لما عان النتيجة تشير إلى ونن د فنرق دار إحصنائياً أ أا مسنت ى الكفناءة الذاتينة االنفعالينة لندى أفنراد العينة ه بدرنة ( مت سر ة ) تشير هذه النتيجة إلى تمتنع الر بنة بمسنت ى منن الكفناءة الذاتينة االنفعالينة يمكنن جدول( 19 ) المتوسط الحسابي واالنحراف والقيمة التائية لمستوى الكفاءة الذاتية االنفعالية مقياس العدد المت سط الحسابي االنحننراف المعيار المت سط النظر القيمة التائية مست ى الداللة 0.05 الكفاءة الذاتية االنفعالية 300 123.51 16.076 105 المحس بة الجدولية 19.946 1.96 دالة لما عان النتيجة تشير إلى ونن د فنرق دار إحصنائياً أ أا مسنت ى الكفناءة الذاتينة االنفعالينة لندى أفنراد العينة ه بدرنة ( مت سر ة ) تشير هذه النتيجة إلى تمتنع الر بنة بمسنت ى منن الكفناءة الذاتينة االنفعالينة يمكنن 338 338 338 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 تفسير ذل في ء النظريا االنفعالية التي اعد ع ى ت فر امتالك الفرد قدراً عافيناً منن الن عي االنفعنالي والمهارا االنتماعية والتنظيم االنفعالي واستخدامها بالشكل االمثنل فني الم اقنف الحياتينة الي مينة فالرالنب يمت ال سي ة لتحقيق هذه المهارة من أنل النجاح والتف ق ع ى نميع المجناال االعاديمينة أو االنتماعينة او االنتماعيننة وتنمنني لننديهم القنندرة ع ننى م انهننة الصننع با وتسنناعدهم ع ننى تنظننيم مع منناتهم . تتفننق مننع, دراسة( آرامن, 2016 )ودراسة(عائشة, 2020 ) . الهدف الثاني : التعرف على طبيعة الفروق في مستوى الكفاءة الذاتية االنفعالية وفقا لمتغير الجـنس( ذكـور – إناث .) أشار نتائج المعالجة ا حصائية ل بيانا إلى ون د فرق دار إحصنائياً بنين النذع ر وا نناث فني الكفناءة الذاتية االنفعالية بين أفراد العينة إذ ب غ مت سط درنا الذع ر( 129.73 ) درننة وبنانحراف معينار قندره ( 10.329 ) في حين ب غ مت سط درنا ا نناث( 117.29 ) درننة بنانحراف معينار قندره( 18.279 ) وقند ب غ القيمة التائية المحس بة( 7.253 ) وعند مقارنتهنا بالقيمنة الجدولينة البالغنة( 1.96 ) عنند مسنت ى الداللنة ( 0.05 ) وبدرنة حرية( 298 )تبين ون د فرق دار إحصائياً وفقاً لمتغير الجننس ولصنالح النذع ر . والجندور ( 20 ) ي ح ذل . مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 تفسير ذل في ء النظريا االنفعالية التي اعد ع ى ت فر امتالك الفرد قدراً عافيناً منن الن عي االنفعنالي والمهارا االنتماعية والتنظيم االنفعالي واستخدامها بالشكل االمثنل فني الم اقنف الحياتينة الي مينة فالرالنب يمت ال سي ة لتحقيق هذه المهارة من أنل النجاح والتف ق ع ى نميع المجناال االعاديمينة أو االنتماعينة او االنتماعيننة وتنمنني لننديهم القنندرة ع ننى م انهننة الصننع با وتسنناعدهم ع ننى تنظننيم مع منناتهم . تتفننق مننع, دراسة( آرامن, 2016 )ودراسة(عائشة, 2020 ) . الهدف الثاني : التعرف على طبيعة الفروق في مستوى الكفاءة الذاتية االنفعالية وفقا لمتغير الجـنس( ذكـور – إناث .) أشار نتائج المعالجة ا حصائية ل بيانا إلى ون د فرق دار إحصنائياً بنين النذع ر وا نناث فني الكفناءة الذاتية االنفعالية بين أفراد العينة إذ ب غ مت سط درنا الذع ر( 129.73 ) درننة وبنانحراف معينار قندره ( 10.329 ) في حين ب غ مت سط درنا ا نناث( 117.29 ) درننة بنانحراف معينار قندره( 18.279 ) وقند ب غ القيمة التائية المحس بة( 7.253 ) وعند مقارنتهنا بالقيمنة الجدولينة البالغنة( 1.96 ) عنند مسنت ى الداللنة ( 0.05 ) وبدرنة حرية( 298 )تبين ون د فرق دار إحصائياً وفقاً لمتغير الجننس ولصنالح النذع ر . والجندور ( 20 ) ي ح ذل . 339 ب ي ي ب( 7.253 ) ج و ي ب ر ه ب ي و( 1.96 ) ا ى ( 0.05 ) وبدرنة حرية( 298 )تبين ون د فرق دار إحصائياً وفقاً لمتغير الجننس ولصنالح النذع ر . والجندور ( 20 ) ي ح ذل . ا لفصل الرابع ا لفصل الرابع ا لفصل الرابع عرض النتائج ومناقشتها : يتضمن هذا الفصل عر ا ل نتائج التي تم الت صل اليها في هنذا البحني . وتمن مناقشتها في ء ا طار النظر والدراسا السابقة . وسيتم عرا النتائج تبعا رهداف البحي وع ى النحن الهدف األول : التعرف على مستوى الكفاءة الذاتية االنفعالية لدى طلبة المرحلة ا لثانوية . تننم معالجننة البيانننا إحصننائياً باسننتعمار االختبننار التننائي لعينننة واحنندة فننأالهر النتننائج وننن د فننرق دار إحصننائياً بننين المت سننط الحسننابي لنندرنا أفننراد العينننة البننالغ( 123.51 ) درنننة بننانحراف معيننار قنندره ( 16.076 ) والمت سط الفر ي البالغ( 105 ) درنة وب غ القيمة التائية المحسن بة( 19.946 ) عنند مقارنتهنا بالقيمة الجدولية البالغة( 1.96 ) عند مست ى الداللة( 0.05 ) وبدرنة حرينة( 299 ) وتبنين إا مسنت ى الكفناءة الذاتية االنفعالية ه بدرنة ( مت سر ة) . والجدور( 19 ) ي ح ذل . مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 تظهر ب ح بينهم وهذا يرنع إلى حقائق ع مية وعن طريق البح ث الع مية والدراسنا التني اثبتن ذلن , يرنع ذل ع ا الرالبا في مرح ة عمرية يك نن ا بهنا أقنل قندرة ع نى فهنم انفعناالتهم وتنظيمهنا بالشنكل االيجنننابي نتيجنننة التغينننرا الفسننني ل نية, . ,عنننالوة ع نننى ذلننن طبيعنننة انتمائهنننا االنتمننناعي . اتفقننن ودراسة(حيدر, 2018 ) واخت ف مع دراسة(آيسن, 2017 ) ويعزى ذل إلى قاب ية الفنرد ع نى النتحكم بسن عه الخا أو غير المالئم واا الرالب يزداد لديه ال عي بعم ية اختيار الفعل في ن ء ذلن الن عي أا هنذا منن شأنه ا سهام في تنمية وتر ير امتالك الر بة ل مهارا الالامة ل تكيف مع هذه االو اع المخت فة . مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 جدول( 20 ) نتائج االختبار التائي لداللة الفروق في الكفاءة الذاتية االنفعالية وفقا لمتغير الجنس(ذكور-إناث) الجنس العدد مت سط الدرنا االنحراف المعيار القيمة التائية مست ى الداللة 0.05 المحس بة الجدولية ذع ر 150 129.73 10.329 7.253 1.96 دالة إناث 150 117.29 18.279 تشير هذه النتيجة وعما م ح في الجدور( 20 ) إلى ون د فروق في الكفاءة الذاتية االنفعالينة بنين متغينرا الجنس(ذع ر ,إناث )ولصالح الذع ر , ع ى الرغم منن أا عقن ر الجننس البشنر تكناد تكن ا متشنابهة إال أا هناك بالتأعيد بعض االختالفا بين الذع ر وا ناث فقد يكن ا الترعينب واحند بينمنا هنناك اختالفنا نفسنية تشير هذه النتيجة وعما م ح في الجدور( 20 ) إلى ون د فروق في الكفاءة الذاتية االنفعالينة بنين متغينرا الجنس(ذع ر ,إناث )ولصالح الذع ر , ع ى الرغم منن أا عقن ر الجننس البشنر تكناد تكن ا متشنابهة إال أا هناك بالتأعيد بعض االختالفا بين الذع ر وا ناث فقد يكن ا الترعينب واحند بينمنا هنناك اختالفنا نفسنية تشير هذه النتيجة وعما م ح في الجدور( 20 ) إلى ون د فروق في الكفاءة الذاتية االنفعالينة بنين متغينرا الجنس(ذع ر ,إناث )ولصالح الذع ر , ع ى الرغم منن أا عقن ر الجننس البشنر تكناد تكن ا متشنابهة إال أا هناك بالتأعيد بعض االختالفا بين الذع ر وا ناث فقد يكن ا الترعينب واحند بينمنا هنناك اختالفنا نفسنية مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 مجلة جامعة تكريت للعلوم االنسانية المجلد⦃ 30 ⦄ العدد⦃ 6 ⦄ ي لعام الجزء الثان 2023 1 Abdul Rahman, Saad, 1998: Psychometrics, Al Falah Library, Kuwait. 2- Odeh, Ahmed Suleiman (1998): Measurement and evaluation in 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https://openalex.org/W4394738140	https://hal.sorbonne-universite.fr/tel-04217074/document	French	null	Electrochemical etching and plating on silicon: application to junction delineation.	HAL (Le Centre pour la Communication Scientifique Directe)	1,991	cc-by	156	To cite this version: Corinne Dennig. Gravure et depots electrochimiques sur le silicium : application a la revelation de jonctions.. Chimie. Université Pierre et Marie Curie, 1991. Français. NNT : 1991PA066092. tel- 04217074 Gravure et depots electrochimiques sur le silicium : application a la revelation de jonctions. Corinne Dennig Distributed under a Creative Commons Attribution 4.0 International License HAL Id: tel-04217074 https://hal.sorbonne-universite.fr/tel-04217074v1 Submitted on 25 Sep 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License
https://openalex.org/W2962979297	https://www.aclweb.org/anthology/N18-2110.pdf	English	null	Detecting Linguistic Characteristics of Alzheimer’s Dementia by Interpreting Neural Models	null	2,018	cc-by	5,102	Detecting Linguistic Characteristics of Alzheimer’s Dementia by Interpreting Neural Models Sweta Karlekar Tong Niu Mohit Bansal UNC Chapel Hill {swetakar, tongn, mbansal}@cs.unc.edu guists and computer scientists to help quickly di- agnose people afﬂicted by this disease. Abstract Alzheimer’s disease (AD) is an irreversible and progressive brain disease that can be stopped or slowed down with medical treat- ment. Language changes serve as a sign that a patient’s cognitive functions have been im- pacted, potentially leading to early diagno- sis. In this work, we use NLP techniques to classify and analyze the linguistic characteris- tics of AD patients using the DementiaBank dataset. We apply three neural models based on CNNs, LSTM-RNNs, and their combina- tion, to distinguish between language samples from AD and control patients. We achieve a new independent benchmark accuracy for the AD classiﬁcation task. More importantly, we next interpret what these neural models have learned about the linguistic characteris- tics of AD patients, via analysis based on acti- vation clustering and ﬁrst-derivative saliency techniques. We then perform novel auto- matic pattern discovery inside activation clus- ters, and consolidate AD patients’ distinctive grammar patterns. Additionally, we show that ﬁrst derivative saliency can not only rediscover previous language patterns of AD patients, but also shed light on the limitations of neural models. Lastly, we also include analysis of gender-separated AD data. This task is challenging because it requires di- verse linguistic and world knowledge. For exam- ple, the sentence “Well...there’s a mother stand- ing there uh uh washing the dishes and the sink is overspilling...overﬂowing” is AD-positive. To distinguish this from a control sample, one needs to know that the word “overspill” is not com- mon in American English (Davies, 2009), and the speaker is correcting themselves by saying “overspilling...overﬂowing”, which hints on signs of confusion and memory loss (Duke et al., 2002). Moreover, different grammar patterns emerge based on the scenario at hand. In ad- dition, the characteristics of AD-affected speech vary between stages of disease progression (K¨onig et al., 2015), making it harder for feature-based ap- proaches to adapt. Motivated by the shortcomings of manual feature-engineering for such a diverse and com- plex task, we ﬁrst present three end-to-end neu- ral models to address it. The ﬁrst two are the widely adopted CNN and LSTM-RNN models, and the third is a stronger joint CNN-LSTM ar- chitecture. Our best-performing model requires only minimal feature engineering (namely auto- matic, commonly-used POS tags that are already present in the dataset) and establishes a new inde- pendent benchmark that outperforms previous AD classiﬁcation scores. Proceedings of NAACL-HLT 2018, pages 701–707 New Orleans, Louisiana, June 1 - 6, 2018. c⃝2018 Association for Computational Linguistics 1 Introduction Alzheimer’s dementia is the most common form of dementia, caused by Alzheimer’s disease (AD). AD cannot be cured or reversed (Glenner, 1990). However, medication can be used to slow or halt degeneration especially when detected at an early stage. Current diagnoses often involve lengthy medical evaluations. One of the early symp- toms of AD, cognitive impairment—which can be evidenced by issues with word-ﬁnding, im- paired reasoning or judgment, and changes in lan- guage (McKhann et al., 1984)—is motivating lin- More importantly, we next present interpreta- tion results to explain what AD-relevant linguis- tic features these neural models are learning, via two visualization techniques: Activation Cluster- ing (Girshick et al., 2014) and First Derivative Saliency (Simonyan et al., 2013), plus our novel approach of automatically discovering grammati- cal patterns common in different activation clus- ters. Furthermore, we split our dataset by gen- 701 Proceedings of NAACL-HLT 2018, pages 701–707 New Orleans, Louisiana, June 1 - 6, 2018. c⃝2018 Association for Computational Linguistics and uh mother is washing the dishes OUTPUT CONVOLUTION MAX POOLING RNN INPUT LAYER HIDDEN LAYERS CNN WORD VECTOR INPUT P (Alzheimer’s) P (Control) Figure 1: Our CNN-LSTM hybrid neural network. top of a CNN model. This model has been shown to perform better at sentiment classiﬁcation than either of its integral parts alone. CNN WORD VECTOR INPUT Visualization Techniques for Neural Models: There have been various visualization techniques proposed for neural networks in both Computer Vision (Krizhevsky et al., 2012; Simonyan et al., 2013; Zeiler and Fergus, 2014; Samek et al., 2017; Mahendran and Vedaldi, 2015) and NLP (Li et al., 2015; K´ad´ar et al., 2017). In this work, we adopt two visualization techniques: Activation Clus- tering (Girshick et al., 2014) following the po- liteness interpretation work of Aubakirova and Bansal (2016), which leads to insight on sentence- level patterns, and First Derivative Saliency (Si- monyan et al., 2013) following Li et al. (2015) and Aubakirova and Bansal (2016), which provides in- sight to the importance of each word in deciding the ﬁnal classiﬁcation label. Figure 1: Our CNN-LSTM hybrid neural network der and analyze the performance of our model on each subsample of the data to illustrate that the features we ﬁnd are not gender-speciﬁc. 1 Introduction These methods not only help rediscover AD linguistic features that have been found by previous works— including short answers, bursts of speech, re- peated requests for clariﬁcation, and starting with interjections—but also lead to new insights in AD characteristics via our automatic speech pattern extraction method. These ﬁndings could poten- tially help improve the accuracy and speed of med- ical diagnoses. 2 Related Works Our best performing model CNN-LSTM is closely related to C-LSTM by Zhou et al. (2015), where an LSTM is laid on 2 Related Works CNN: For each sentence, we apply an embed- ding and a convolutional layer, followed by a max- pooling layer (Collobert et al., 2011). The convo- lution features are obtained by applying ﬁlters of varying window sizes to each window of words. The result is then passed to a softmax layer that outputs probabilities over two classes. Language-based Alzheimer’s Detection: Previ- ous works using language to detect AD relied mainly on hand-crafted features from transcripts (Orimaye et al., 2017, 2015), occasionally us- ing acoustic data (K¨onig et al., 2015; Rudzicz et al., 2014). The challenge with feature-based approaches is that they rely heavily on the re- searchers’ linguistics and medical expertise, and are also hard to generalize to other progression stages and disease types, which may correspond to different linguistic features. Hand-picked fea- tures may also become outdated as language and culture evolves. Moreover, some features may be too nuanced for humans to detect, especially at early stages of AD. In order to address these is- sues, Orimaye et al. (2016) adopted a deep neu- ral network language model. However, a neural approach is usually a black-box and it is hard to interpret its reasoning for the ﬁnal classiﬁcation decisions. To make our approaches more inter- pretable while harvesting the beneﬁts of neural ap- proaches, we present three accurate neural models and include multiple visualization techniques to il- lustrate both their effectiveness and limitations. LSTM-RNN: CNNs are not specialized for cap- turing long-range sequential correlations (Pascanu et al., 2013a). We thus also experimented with an LSTM-RNN model, which consists of an embed- ding layer followed by an LSTM layer. The ﬁnal state, containing information from the entire sen- tence, is fed to a fully-connected layer followed by a softmax layer to obtain the output probabilities. CNN-LSTM: Observing that both models achieve results comparable to previous best performing ap- proach, and considering that they each have their own complementary strengths, we experimented with a combined architecture, laying an LSTM layer on top of CNN (See Figure 1). This CNN layer is identical to the vanilla CNN before the max-pooling layer, and the LSTM layer is identi- cal to the vanilla LSTM-RNN after the embedding layer. More details are provided in the appendix. CNN-LSTM on NL Classiﬁcation: CNN and LSTM (Hochreiter and Schmidhuber, 1997) have both been leveraged extensively for extracting fea- tures in natural language. 3We also tried using a bidirectional RNN, which gave 84.7%, 86.2% and 91.1% accuracies for our LSTM-RNN, CNN-RNN, and CNN-RNN-tagged models, respectively. 4 Experimental Setup Each transcript in DementiaBank comes with auto- matic morphosyntactic analysis, such as standard part-of-speech tagging, description of tense, and repetition markers.2 Note that these features are generic, automatically-extracted linguistic properties and are not AD-speciﬁc. We broke each transcript into individual utterances to use as data samples. Note that we also removed utterances that did not have accompanying POS tags. This balancing reduced the amount of data but ensured fair comparison between models with tagged and untagged setups. Based on error analysis of the POS-based CNN/RNN model’s classiﬁcation result, we found that almost all AD-positive results are classiﬁed correctly as AD-positive. However, there is more error in classifying non-AD samples, which could be due to the fact that DementiaBank includes pa- tients with probable and possible AD, each ex- hibiting various degrees of symptoms. Patients who are AD-positive may still have partially un- affected speech (similar to non-AD control pa- tients’ speech). However, because all utterances from AD-positive interviews are tagged as AD- positive, these seemingly unaffected utterances are still tagged AD-positive. To further understand the errors of our model, 10% of the wrongly classiﬁed non-AD examples were randomly selected and an- alyzed. Of this smaller sample, 36.3% were short utterances such as “okay”, “alright”, “oh my”, etc. These forms of speech are utterances that are present in both classes, but more commonly found in AD-positive cases. The remaining 63.7% were examples of speech that could be classiﬁed either way without surrounding context, such as “she’s Training Details Our CNN model was a 2-D convolutional neural network. Filter sizes of [3, 4, 5] were used. Our LSTM-RNN had 2 layers. The CNN-LSTM model had ﬁlter sizes [3, 4, 5, 6] and 1 LSTM hidden layer. For each model, all hyperparameters were tuned using the dev set. See appendix for dataset and training details. 4 Experimental Setup Dataset This study utilizes Dementia- Bank (Boller and Becker, 2005), the largest publicly available dataset of transcripts and 702 Model Details Accuracy 2D-CNN Non-Tagged Utterances 82.8 LSTM Non-Tagged Utterances 83.7 CNN-RNN Non-Tagged Utterances 84.9 CNN-RNN POS-tagged Utterances 91.1 Table 1: Accuracy results of models. Note that we downsampled the data to remove utterances that did not have accompanying POS tags, so as to allow fair com- parison between the tagged and untagged models. Figure 2: Boston cookie theft description task. Partic- ipants were asked to describe all events in the image. Table 1: Accuracy results of models. Note that we downsampled the data to remove utterances that did not have accompanying POS tags, so as to allow fair com- parison between the tagged and untagged models. Table 1: Accuracy results of models. Note that we downsampled the data to remove utterances that did not have accompanying POS tags, so as to allow fair com- parison between the tagged and untagged models. (see Table 1 for more details).3 Compared to other related works, Orimaye et al. (2015, 2017) used AUC instead of accuracy, and K¨onig et al. (2015) did not use DementiaBank data. Rudzicz et al. (2014) achieved an accuracy of 67.0% on the De- mentiaBank dataset using audio features as well as transcripts. Orimaye et al. (2016) achieved an accuracy of 87.5% but only used 36 transcripts. Their test set is thus different from ours, lead- ing to very little data and high variance. To the best of our knowledge, Orimaye et al. (2016) used the original transcripts, which include POS tags. Hence, most previous works on this topic are not directly comparable to our work, but we aim to establish a new independent, strong neural bench- mark and then more importantly focus on visual- ization and interpretability of neural models. Figure 2: Boston cookie theft description task. Partic- ipants were asked to describe all events in the image. audio recordings of AD (and control) patient interviews.1 Patients were asked to perform various tasks; for example, in the “Boston Cookie Theft” description task, patients were shown an image and asked to describe what they see (See Figure 2). Other tasks include the ‘Recall Test’ in which patients were asked to recall attributes of a story they had been told previously. 1http://dementia.talkbank.org 2http://talkbank.org/manuals/CHAT.docx 2http://talkbank.org/manuals/CHAT.docx 1http://dementia.talkbank.org 2 4Statistical insigniﬁcance calculated using the bootstrap test (Noreen, 1989; Efron and Tibshirani, 1994). We split our dataset based on gender and down-sampled the female data subset such that it had the same data-size and non-AD to AD data-ratio as the male data subset. 5POS tags used in this paper: v=verb, n=noun, pro=pronoun, adv=adverb, det=determiner, aux=auxiliary verb, prep=preposition, co=interjection, part=participle, presp=present participle. The frequencies of each POS tag are scaled based on the total number of tags in each cluster. 5 Results With untagged data, our CNN, LSTM and CNN- LSTM models achieved an accuracy of 82.8%, 83.7% and 84.9%, respectively. When fed with the given POS-tagged data, our best-performing CNN-LSTM model achieved 91.1% in accuracy, setting a new benchmark accuracy for this task 703 AD Non-AD POS Frequency POS Frequency n 0.20 n 0.15 det 0.14 det 0.13 adj 0.05 presp 0.07 adv 0.04 part 0.05 Non-AD Clusters - Cookie Task POS Freq POS Freq POS Freq n 0.15 n 0.13 n 0.15 det 0.13 det 0.13 det 0.13 presp 0.07 part 0.09 part 0.10 part 0.05 presp 0.09 presp 0.10 Table 2: Top POS tags and frequencies for three non- AD clusters for the Cookie task. Non-AD Clusters - Cookie Task POS Freq POS Freq POS Freq n 0.15 n 0.13 n 0.15 det 0.13 det 0.13 det 0.13 presp 0.07 part 0.09 part 0.10 part 0.05 presp 0.09 presp 0.10 Table 2: Top POS tags and frequencies for three non- AD clusters for the Cookie task. Table 2: Top POS tags and frequencies for three non- AD clusters for the Cookie task. Table 3: Top POS tags in AD cluster and non-AD cluster for Cookie task. 6.2.1 Rediscovering Existing Strategies drying dishes” and “stool’s tipping over”. Hence, future work could incorporate context from sur- rounding samples in each interview to help distin- guish between temporarily unaffected speech pat- terns in AD-positive patients and the continually unaffected speech of non-AD control patients. Our activation clusters corroborated previous stud- ies, forming clusters around known linguistic characteristics of Alzheimer’s disease (Watson, 1999; Rudzicz et al., 2014). Short Answers and Bursts of Speech Clusters formed around short answers, which have been split up by natural pauses in speech. {‘okay’, ‘and’, ‘yes’, ‘oh !’, ‘yes’, ‘ﬁne’} 6.2.2 Automatic Cluster Pattern Analysis Next, we extend activation clustering to perform novel automatic pattern discovery inside different clusters, as opposed to manually looking for pat- terns as in Aubakirova and Bansal (2016). Finding the most common POS tags in each cluster allows us to better understand which grammatical struc- tures are favored. No two clusters had exactly the same most-common POS tags, but many clusters shared similar top POS-tags on the same task. Control Clusters An example unaffected speech cluster for the Cookie task has the fol- lowing as the most common POS tags: [(‘n’, .15), (‘det’, .13), (‘presp’, .07), (‘part’, .05)].5 This pattern follows other clusters of unaffected speech, with nouns, determiners, and participles always found in the most-used POS tags. To illustrate this, two other control clusters found for the Cookie task have very similar top POS tags and frequencies: [(‘n’, .13), (‘det’, .13), 6 Analysis and Visualization We ﬁrst present gender analysis of our results and then present interpretation of the linguistic cues our CNN-LSTM model identiﬁed via two vi- sualization strategies: activation clustering (Gir- shick et al., 2014) and ﬁrst-derivative saliency heat maps (Simonyan et al., 2013). Repeated Requests for Clariﬁcation Another cluster formed around clariﬁcation questions and confusion about the task, speciﬁcally in the past tense. {‘Did I say facts ?’, ‘Did I get any ?’, ‘Did I say elephant ?’} Repeated Requests for Clariﬁcation Another cluster formed around clariﬁcation questions and confusion about the task, speciﬁcally in the past tense. {‘Did I say facts ?’, ‘Did I get any ?’, ‘Did I say elephant ?’} Repeated Requests for Clariﬁcation Another cluster formed around clariﬁcation questions and confusion about the task, speciﬁcally in the past tense. {‘Did I say facts ?’, ‘Did I get any ?’, ‘Did I say elephant ?’} Starting with Interjections Many clusters con- tain utterances that start with interjections such as “oh”, “well”, “so”, and “right”. {‘Well I gotta see it’, ‘Oh I just see a lot of uh...’, ‘So all the words that you can...’} 6.1 Gender Differences Many previous works have debated on the differ- ence in language for male versus female patients with Alzheimer’s (Bayles et al., 1999; McPher- son et al., 1999; Buckwalter et al., 1996; Ripich et al., 1995; Hebert et al., 2000; Heun and Kock- ler, 2002). In agreement with some of these pre- vious works, we found that the sets of the top ten most common POS-tags for both AD-positive men and women are the same, i.e., we did not detect a signiﬁcant difference in the language complexity or syntax of male and female patients with AD in our dataset. Moreover, our best performing model achieved 86.6% classiﬁcation accuracy on solely the male data and 86.2% accuracy on solely the female data, demonstrating that it found no sta- tistically signiﬁcant difference between the AD- positive language of men versus women.4 6.2 Activation Clusters Activation clustering (Girshick et al., 2014) treats the activation values of n neurons per input as coordinates in an n-dimensional space. K-means clustering is then performed to group together in- puts that maximally activate similar neurons. 704 0 50 100 150 200 250 and uh it seems that um True label: Alzheimer's, Predicted: Alzheimer's −0.4 −0.2 0 0.2 0.4 0 50 100 150 200 250 uh the boy is falling oﬀ that um stool True label: Control, Predicted: Control −0.4 −0.2 0 0.2 0.4 0.6 0 50 100 150 200 250 and the house with uh kitchen has space under sink True label: Alzheimer's, Predicted: Control −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 the the uh Figure 3: Left + Middle: ﬁrst derivative saliency heat maps for correctly classiﬁed Alzheimer’s and control examples. Right: ﬁrst derivative heat saliency maps for incorrectly classiﬁed Alzheimer’s example. 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Figure 3: Left + Middle: ﬁrst derivative saliency heat maps for correctly classiﬁed Alzheimer’s and control examples. Right: ﬁrst derivative heat saliency maps for incorrectly classiﬁed Alzheimer’s example. (‘part’, .09), (‘presp’, .09)] and [(‘n’, .15), (‘det’, .13), (‘part’, .10), (‘presp’, .10)] (see Table 2 for side-by-side comparison of these clusters). For the same task, non-AD clusters contain the same top four POS tags. (‘part’, .09), (‘presp’, .09)] and [(‘n’, .15), (‘det’, .13), (‘part’, .10), (‘presp’, .10)] (see Table 2 for side-by-side comparison of these clusters). For the same task, non-AD clusters contain the same top four POS tags. (‘part’, .09), (‘presp’, .09)] and [(‘n’, .15), (‘det’, .13), (‘part’, .10), (‘presp’, .10)] (see Table 2 for side-by-side comparison of these clusters). For the same task, non-AD clusters contain the same top four POS tags. words as heavily as Alzheimer’s patients. Instead, words that give structure to a sentence have the biggest impact on classiﬁcation, such as deﬁnite articles and determiners (e.g., “the” and “that”). Figure 3 (middle) shows that the most highlighted words are “the”, “that” and “is”. AD Clusters For the Recall task, one cluster of AD patients’ speech shows that the most common POS tags are [(‘n’, .15), (‘co’, .15), (‘v’, .06), (‘pro’, .06)]. Across samples from the Recall task, AD-marked clusters contained frequent interjec- tions and verbs. 6.3 First Derivative Saliency Heat Maps Saliency heat maps (Simonyan et al., 2013) illus- trate which words in an input had the biggest im- pact on the classiﬁcation of the whole sentence. This is done by taking the gradient of the ﬁnal scores w.r.t. the word embeddings of the inputs. Heat Map Analysis The ﬁller words “uh” and “um” are emphasized in Figure 3 (left), show- ing that they have a lot of inﬂuence on classiﬁ- cation. The initial “and” is highlighted as well, corroborating the results of the activation clusters in that starting with a coordinating conjunction is a trait of Alzheimer’s speech. However, in Figure 3 (middle), the “uh” ﬁller word is not highlighted, showing that most control patients do not use ﬁller 7 Conclusion We applied three models to the AD classiﬁcation task, and our CNN-LSTM model achieves a new benchmark accuracy in classifying AD using neu- ral models. We illustrate with two visualization techniques how these models capture unique lin- guistic features present in AD patients. We also discussed gender analysis. Potential future work includes using more conversational context and implementing multi-class classiﬁcation to differ- entiate among stages of AD. We also plan to apply this generalizable model to other similar neurolog- ical diseases, such as Diffuse Lewy Body disease and Huntington’s disease (Heindel et al., 1989). 6.2 Activation Clusters On the other hand, in regards to the Cookie task, the most common POS tags for the AD cluster found are [(‘n’, .20), (‘det’, .14), (‘adj’, .05), (‘adv’, .04)], i.e., more adjectives and adverbs. Hence, between different tasks such as Cookie and Recall, the most commonly used POS tags for AD clusters are distinct. Visualizing Limitations Furthermore, by visu- alizing an incorrectly classiﬁed example, we can learn about the limitations of our neural network. Figure 3 (right) illustrates a map of an incorrectly predicted sample. The model misclassiﬁed it due to the length of the utterance (activation clustering showed that AD patients tend to have short bursts of speech, see Section 6.2.1) and the heavy use of determiners. However, the repeated “um”s and starting with a coordinating conjunction strongly indicated AD, which confused our model. From Figure 3, the repeated use of ﬁller words (i.e. “uh”) had the second most inﬂuence on classiﬁ- cation. In future work, with more context data and advanced neural methods, our model’s next steps will be to learn how to better classify samples that strongly exhibit both AD and control features. 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For each model, the hyperparameters were tuned using the development set. Sylvester Olubolu Orimaye, Jojo Sze-Meng Wong, and Judyanne Sharmini Gilbert Fernandez. 2016. Deep- deep neural network language models for predicting mild cognitive impairment. In BAI@ IJCAI, pages 14–20. CNN We used a 2-D CNN. Filter sizes of [3, 4, 5] were used with 128 ﬁlters per ﬁlter size. Batch size was set to 128, and a dropout (Srivastava et al., 2014) of 0.80 was applied. Razvan Pascanu, Caglar Gulcehre, Kyunghyun Cho, and Yoshua Bengio. 2013a. How to construct deep recurrent neural networks. arXiv preprint arXiv:1312.6026. Razvan Pascanu, Tomas Mikolov, and Yoshua Bengio. 2013b. On the difﬁculty of training recurrent neural networks. In International Conference on Machine Learning, pages 1310–1318. LSTM Our LSTM had 2 layers with 128 hidden units. Batch size was 32, and a dropout of 0.70 was used. LSTM Our LSTM had 2 layers with 128 hidden units. Batch size was 32, and a dropout of 0.70 was used. CNN-LSTM Our CNN-LSTM model consisted of an LSTM on top of a CNN. The CNN had 100 ﬁlters per ﬁlter size of [3, 4, 5, 6]. Embedding dimensions of 300 were used. An LSTM with 300 hidden units was used. Both dropout on the CNN and recurrent dropout (Gal and Ghahramani, 2016) on the LSTM used a dropout rate of 0.65. Danielle N Ripich, Stephen A Petrill, Peter J White- house, and Elaine W Ziol. 1995. Gender differences in language of ad patients a longitudinal study. Neu- rology, 45(2):299–302. Frank Rudzicz, Leila Chan Currie, Andrew Danks, Te- jas Mehta, and Shunan Zhao. 2014. Automatically identifying trouble-indicating speech behaviors in alzheimer’s disease. In Proceedings of the 16th in- ternational ACM SIGACCESS conference on Com- puters & accessibility, pages 241–242. ACM. A Supplementary Materials Sylvester Olubolu Orimaye, Kah Yee Tai, Jojo Sze- Meng Wong, and Chee Piau Wong. 2015. 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BMC bioinformatics, 18(1):34. 706 A.2 Dataset Details Each transcript in DementiaBank comes with au- tomatic morphosyntactic analysis, such as stan- dard part-of-speech tagging, description of tense, and markers for repetitions. This automatic tag- ging is identical to that done on other datasets, such as the CHILDES TalkBank, and is thus not speciﬁc to DementiaBank. This dataset features transcripts of 104 different control patients, and 208 different diagnosed dementia patients. There is a total of 1017 Alzheimer’s transcripts and 243 control transcripts. Each of these transcripts were then broken down by sentences and inter- ruptions by the interviewer. We used each ut- terance by the patient as a data sample. Within the 14362 utterance samples, 11458 come from transcripts of Alzheimer’s-diagnosed interviewees and 2904 from those of control patients. There- fore, a majority-baseline classiﬁer that always guesses AD-positive will achieve an accuracy of 79.8% in our dataset. 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https://openalex.org/W4252652674	https://orca.cardiff.ac.uk/id/eprint/112674/1/c8cy90033g.pdf	English	null	Correction: <i>In situ</i> spectroscopic investigations of MoO<sub>x</sub>/Fe<sub>2</sub>O<sub>3</sub> catalysts for the selective oxidation of methanol	Catalysis science & technology	2,018	cc-by	753	a UK Catalysis Hub, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Oxon, OX11 0FA, UK. E-mail: catherine.brookes@rc-harwell.ac.uk, peter.wells@rc-harwell.ac.uk b Cardiff Catalysis Institute, School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT, UK c Department of Chemistry, University College London, 20 Gordon St, London, WC1H 0AJ, UK d Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxon, OX11 0DE, UK e Chemistry Department, Faculty of Science, Sohag University, SohagP.O.B 82524, Egypt a UK Catalysis Hub, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Oxon, OX11 0FA, UK. E-mail: catherine.brookes@rc-harwell.ac.uk, Catalysis Science & Technology CORRECTION Cite this: Catal. Sci. Technol., 2018, 8, 2998 DOI: 10.1039/c8cy90033g rsc.li/catalysis Correction: In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol Catherine Brookes,ab Michael Bowker,ab Emma K. Gibson,ac Diego Gianolio,d Khaled M. H. Mohammed,ace Stephen Parry,d Scott M. Rogers,ac Ian P. Silverwoodac and Peter P. Wellsac Correction for ‘In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol’ by Catherine Brookes et al., Catal. Sci. Technol., 2016, 6, 722–730. The authors wish to correct Fig. 10 of the manuscript. Fig. 10 should appear as follows: The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. Fig. 10 Magnitude component of the k2 weighted Fourier transform for the EXAFS data of the 1 and 6 ML MoOx/Fe2O3 catalyst reduced (procedure as in Fig. 9), and the associated simulated fit performed in Artemis. Open Access Article. Published on 18 May 2018. Downloaded on 6/21/2018 10:23:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Catalysis Science & Technology CORRECTION Cite this: Catal. Sci. Technol., 2018, 8, 2998 DOI: 10.1039/c8cy90033g rsc.li/catalysis Correction: In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol Catherine Brookes,ab Michael Bowker,ab Emma K. Gibson,ac Diego Gianolio,d Khaled M. H. Mohammed,ace Stephen Parry,d Scott M. Rogers,ac Ian P. Silverwoodac and Peter P. Wellsac Correction for ‘In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol’ by Catherine Brookes et al., Catal. Sci. Technol., 2016, 6, 722–730. The authors wish to correct Fig. 10 of the manuscript. Fig. 10 should appear as follows: The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. Fig. 10 Magnitude component of the k2 weighted Fourier transform for the EXAFS data of the 1 and 6 ML MoOx/Fe2O3 catalyst reduced (procedure as in Fig. 9), and the associated simulated fit performed in Artemis. Open Access Article. Published on 18 May 2018. Downloaded on 6/21/2018 10:23:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue CORRECTION Correction: In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol Catherine Brookes,ab Michael Bowker,ab Emma K. Gibson,ac Diego Gianolio,d Khaled M. H. Mohammed,ace Stephen Parry,d Scott M. Rogers,ac Ian P. Silverwoodac and Peter P. Wellsac Correction: In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol Catherine Brookes,ab Michael Bowker,ab Emma K. Gibson,ac Diego Gianolio,d Khaled M. H. Mohammed,ace Stephen Parry,d Scott M. Rogers,ac Ian P. Silverwoodac and Peter P. Wellsac Catherine Brookes,ab Michael Bowker,ab Emma K. Gibson,ac Diego Gianolio,d Khaled M. H. Mohammed,ace Stephen Parry,d Scott M. Rogers,ac Ian P. Silverwoodac and Peter P. Wellsac Correction for ‘In situ spectroscopic investigations of MoOx/Fe2O3 catalysts for the selective oxidation of methanol’ by Catherine Brookes et al., Catal. Sci. Technol., 2016, 6, 722–730. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. Fig. 10 Magnitude component of the k2 weighted Fourier transform for the EXAFS data of the 1 and 6 ML MoOx/Fe2O3 catalyst reduced (procedure as in Fig. 9), and the associated simulated fit performed in Artemis. Fig. 10 Magnitude component of the k2 weighted Fourier transform for the EXAFS data of the 1 and 6 ML MoOx/Fe2O3 catalyst reduced (procedure as in Fig 9) and the associated simulated fit performed in Artemis This article is licensed under a Fig. 10 Magnitude component of the k2 weighted Fourier transform for the EXAFS data of the 1 and 6 ML MoOx/Fe2O3 catalyst reduce (procedure as in Fig. 9), and the associated simulated fit performed in Artemis. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. 2998 \| Catal. Sci. Technol., 2018, 8, 2998 This journal is © The Royal Society of Chemistry 2018
https://openalex.org/W2233382046	https://www.nature.com/articles/srep19179.pdf	English	null	APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea	Scientific reports	2,016	cc-by	8,152	Jizhen Wei1,2, Min Zhang2, Gemei Liang1, Kongming Wu1, Yuyuan Guo1, Xinzhi Ni3 & Xianchun Li1,2 Lepidopteran midgut aminopeptidases N (APNs) are phylogenetically divided into eight clusters, designated as APN1–8. Although APN1 has been implicated as one of the receptors for Cry1Ac in several species, its potential role in the mode of action of Cry2Ab has not been functionally determined so far. To test whether APN1 also acts as one of the receptors for Cry1Ac in Helicoverpa zea and even for Cry2Ab in this species, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by RNAi (RNA interference) silencing of the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly increased the susceptibility of the three cell lines to Cry1Ac, but had no effects on their susceptibility to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines resistant to Cry1Ac, but didn’t change cell lines susceptibility to Cry2Ab. The findings from this study demonstrate that HzAPN1 is a functional receptor of Cry1Ac, but not Cry2Ab. Aminopeptidases N (APNs) are a class of broad-specificity metalloaminopeptidases that sequentially and pref- erentially remove neutral amino acids from the N-terminus of a range of peptides in microorganisms, plants and animals1. They share two conserved signature motifs: a catalytic zinc binding/gluzincin motif HEXXH-(X18)-E and an N-terminal amino acid substrate-binding exopeptidase motif GXMEN2,3. In the catalytic HEXXH- (X18)-E motif, the two histidine residues and the distant gluatamic acid residue constitute zinc ligands, whereas the gluatamic acid residue between the two histidine residues and zinc ion catalyze the sequential release of N-terminal neutral amino acids3. By virtue of the two conserved motifs, APNs efficiently retrieve amino acids from dietary proteins and endogenous proteins degraded during protein turnover and thus play important roles in nutrition, protein maturation, peptide hormone level regulation, stress response, cell signaling, cell cycle con- trol, and cellular processes involved in health and diseases1,4. p In recent years, lepidopteran midgut APNs are widely studied because of their potential role as one of the receptors for the Bacillus thuringiensis (Bt) crystal insecticidal toxins (Cry toxins)5,6, which are used extensively in sprays and transgenic crops to control lepidopteran insect pests7,8. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea OPEN received: 18 August 2015 accepted: 04 December 2015 Published: 12 January 2016 Jizhen Wei1,2, Min Zhang2, Gemei Liang1, Kongming Wu1, Yuyuan Guo1, Xinzhi Ni3 & Xianchun Li1,2 Results i di Binding profiles of Cry1Ac and Cry2Ab in H. zea larval midgut BBMV. As in H. zea midgut and fat body cell lines (Wei & Li, unpublished manuscript), ligand blot detected two binding bands of 210 and 130 kDa for Cry1Ac (Fig. 1A, left panel) and four binding proteins of 130 and 87, 57 and 42 kDa for Cry2Ab (Fig. 1A, right panel) in the brush border membrane vesicles (BBMV) isolated from H. zea larval midguts. Western blot analysis of H. zea larval midgut BBMV (Fig. 1B) with the antibody directed against APN1 from H. armigera30, a sister species of H. zea, revealed that the common binding band of 130 kDa by Cry1Ac and Cry2Ab was co-localized with H. zea APN1. Impact of heterologous expression of HzAPN1 on cytotoxicities of Cry1Ac and Cry2Ab. Western blot showed that Sf9 cells and H. zea midgut and fat body cells transfected with pAc-HzAPN1 produced signifi- cantly more APN1 proteins than the three cell lines transfected with the empty vector pAc (control cells) (Fig. 2; Pmidgut = 0.035, Pfat body = 0.004 and PSf9 = 0.048). More precisely, the protein level of APN1 in the pAc-HzAPN1 transfected midgut, fat body and Sf9 cells was 1.83, 1.55 and 1.97 times more than that in the corresponding pAc-transfected control cells. p When the above pAc- and pAc-HzAPN1-transfected cells were exposed to 15 μ g/ml of the activated Cry1Ac, mortality increased from none (− 2.64%) for pAc-transfected Sf9 cells to 48.05% for pAc-HzAPN1 transfected Sf9 cells (Table 1). Consistent with the higher APN1 increase in the pAc-HzAPN1 transfected midgut cells than in the pAc-HzAPN1 transfected fat body cells (Fig. 1), the mortality of the former increased by 55.95%, whereas it was 20.90% for the latter. When the concentration of activated Cry1Ac was doubled, mortality was increased significantly (for at least two to three times) from 24.19 (pAc control) to 76.17% (pAc-HzAPN1) for midgut cells, 23.77 to 51.57% for fat body cells and 10.61 to 44.35% for Sf9 cells, respectively (Table 1). When the pAc and pAc-HzAPN1 transfected cells were treated with 2.5 or 3.75 μ g/ml of the activated Cry2Ab, no significant mortality enhancement by heterologous expression of HzAPN1 was observed regardless of cell line and Cry2Ab concentration (Table 1). Effect of knocking down APN1 on cytotoxicities of Cry1Ac and Cry2Ab. Western blot detected 7.87-fold reduction in the protein level of APN1 in H. Jizhen Wei1,2, Min Zhang2, Gemei Liang1, Kongming Wu1, Yuyuan Guo1, Xinzhi Ni3 & Xianchun Li1,2 Lepidopteran midgut APNs are 100 to 180 kDa and mainly bound to microvillar membranes of midgut cells via a C-terminal glycosyl phosphatidyl inositol (GPI) anchor. Over 140 APN cDNAs have been cloned from more than 20 lepidopteran species (http://www.ncbi. nlm.nih.gov/). Phylogenetic analysis has resolved them into 8 clusters, designated as APN1–8 respectively, which are derived from multiple gene duplications9,10. Each lepidopteran species usually has no more than one APN for each cluster and their amino acid sequence identity ranges from 23 to 40% between different clusters of the same species and from 50 to 95% between members of the same cluster in different species9. pf p Although Cry1Ac and Cry2Ab are two of the most widely used Bt Cry toxins in transgenic crops8,11, studies on defining the role of insect APNs as a receptor for Cry toxins have been mostly focused on Cry1Ac. Among the 8 clusters of lepidopteran APNs, there is no evidence for involvement of APN4, APN7 and APN8 as a recep- tor for Cry1Ac, Cry2Ab, or other Cry toxins12–14. Conflicting evidence exists for APN2, because 1) it can bind to Cry1Ac in Helicoverpa armigera15, but not in Lymantria dispar16; and 2) it is not linked to Cry1Ac resistance in Plutella xylostella17. APN3 and APN5 can bind to Cry1Ac in H. armigera18 and P. xylostella17 respectively, but 1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China. 2Department of Entomology and BIO5 Institute, University of Arizona, Tucson, AZ 85721. 3USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA 31793, USA. Correspondence and requests for materials should be addressed to X.L. (email: lxc@email.arizona.edu) Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 1 1 www.nature.com/scientificreports/ Figure 1. Ligand blot detection of Cry1Ac and Cry2Ab binding proteins in H. zea larval midgut BBMV (A) and Western blot detection of HzAPN1 in H. zea larval midgut BBMV (B). Figure 1. Ligand blot detection of Cry1Ac and Cry2Ab binding proteins in H. zea larval midgut BBMV (A) and Western blot detection of HzAPN1 in H. zea larval midgut BBMV (B). their involvement in Cry1Ac resistance has not been documented. APN6 seems not to be a receptor for Cry1Ac and Cry2Ab because it can not bind to Cry1Ac in H. armigera19 and is not involved in Cry1Ac and Cry2Ab resistance in Trichoplusia ni14,20. Jizhen Wei1,2, Min Zhang2, Gemei Liang1, Kongming Wu1, Yuyuan Guo1, Xinzhi Ni3 & Xianchun Li1,2 In stark contrast, various studies with APN1 from Manduca sexta21, L. dispar22,23, P. xylostella24, H. armigera15,25, and T. ni20 consistently demonstrate that it is one of the midgut receptors for Cry1Ac. their involvement in Cry1Ac resistance has not been documented. APN6 seems not to be a receptor for Cry1Ac and Cry2Ab because it can not bind to Cry1Ac in H. armigera19 and is not involved in Cry1Ac and Cry2Ab resistance in Trichoplusia ni14,20. In stark contrast, various studies with APN1 from Manduca sexta21, L. dispar22,23, P. xylostella24, H. armigera15,25, and T. ni20 consistently demonstrate that it is one of the midgut receptors for Cry1Ac. y So far, the only study examined APN’s role in the mode of action of Cry2Ab shows that APN5 can bind to Cry2Ab in P. xylostella26. Whether APN1 also serves as one of the receptors for Cry2Ab still remains unclear. Presence of cross-resistance between Cry1Ac and Cry2Ab27–29 and detection of a Cry1Ac- and Cry2Ab-binding protein with a similar size (120 ~ 130 kDa) of APN1 in the midgut and fat body cell lines of H. zea by ligand blot suggest a possibility of APN1 as a shared receptor for Cry2Ab and Cry1Ac (Wei & Li, unpublished manuscript). To test this hypothesis, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by knocking down the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly enhanced the susceptibility of the three cell lines to Cry1Ac, but not to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines more tolerant to Cry1Ac, but didn’t change their susceptibility to Cry2Ab. The data demonstrate that APN1 is a receptor for Cry1Ac, but not for Cry2Ab. Results i di zea midgut cells transfected with 50 nM HzAPN1 dsRNA, compared with H. zea midgut cells transfected with 50 nM DsRed dsRNA (control cells; Pmidgut < 0.0001) (Fig. 3). Likewise, transfection of 50 nM HzAPN1 dsRNA also resulted in 2.47- (Pfat body = 0.0098) and 3.74-fold (PSf9 < 0.0001) decrease in the protein level of the endogenous APN1 in H. zea fat body cells and Sf9 cells, respec- tively (Fig. 3). Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 2 www.nature.com/scientificreports/ Figure 2. Expression level of APN1 in the cells transfected with pAc or pAc-HzAPN1 plasmids. For pAc (empty vector control) and pAc-HzAPN1 plasmids, three independent transfections were done for each cell line and the protein extracts from each of the three transfections were analyzed by Western blot. The lower panel picture of HzAPN1 and β -actin bands is a representative of the three Western blots. The average expression of HzAPN1 relative to that of β -actin calculated by Image J quantification of the three Western blots is showed in the upper panel. Each error bar represents the standard error of the mean from three transfection replicates. Significant differences in relative expression of HzAPN1 between pAc and pAc-HzAPN1 transfected cells of each cell line are indicated with an asterisk () (P < 0.05, Student’s t-test). Figure 2. Expression level of APN1 in the cells transfected with pAc or pAc-HzAPN1 plasmids. For pAc (empty vector control) and pAc-HzAPN1 plasmids, three independent transfections were done for each cell line and the protein extracts from each of the three transfections were analyzed by Western blot. The lower panel picture of HzAPN1 and β -actin bands is a representative of the three Western blots. The average expression of HzAPN1 relative to that of β -actin calculated by Image J quantification of the three Western blots is showed in the upper panel. Each error bar represents the standard error of the mean from three transfection replicates. Significant differences in relative expression of HzAPN1 between pAc and pAc-HzAPN1 transfected cells of each cell line are indicated with an asterisk () (P < 0.05, Student’s t-test). Results i di P values refer to the morality comparisons between pAc and pAc-HzAPN1 transfected cells of each cell line (Student’s t-test in JMP 8; SAS Institute, Inc.). Table 1. Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines transfected with pAc or pAc-HzAPN1 plasmids. Values in the table are average mortality ± standard errors of three replicates of three independent transfections of each plasmid per cell line. Mortality values of different treatments were arcsine transformed before analysis. P values refer to the morality comparisons between pAc and pAc-HzAPN1 transfected cells of each cell line (Student’s t-test in JMP 8; SAS Institute, Inc.). When the above HzAPN1 dsRNA-transfected H. zea midgut cells were exposed to 150 μ g/ml of the activated Cry1Ac, a 33.79% decrease in mortality was exhibited when compared with that of the DsRed dsRNA-transfected H. zea midgut cells (Table 2). Due to the lower knocking down activities of the endogenous APN1 in the other two cell lines (Fig. 3), HzAPN1 dsRNA only produced a 19.29% and a 13.12% reduction in mortality for H. zea fat body cells and Sf9 cells, respectively (Table 2). In contrast, no significant mortality reduction by knocking down of the endogenous APN1 was seen in the three insect cell lines when they were treated with 30 μ g/ml of the activated Cry2Ab (Table 2). Results i di Cell lines Treatments & P values Cry1Ac (30 μg/ml) Cry1Ac (15 μg/ml) Cry2Ab (3.75 μg/ml) Cry2Ab (2.5 μg/ml) Midgut cells pAc 24.19 ± 3.45 15.43 ± 1.93 56.25 ± 9.79 28.01 ± 3.98 pAc-HzAPN1 76.17 ± 3.57 71.38 ± 3.58 55.19 ± 1.99 31.91 ± 7.92 P 0.0005 0.0002 0.9214 0.8866 Fat body cells pAc 23.77 ± 3.32 14.46 ± 1.76 62.75 ± 3.44 34.81 ± 1.07 pAc-HzAPN1 51.57 ± 6.63 35.36 ± 6.81 55.80 ± 1.79 34.5 ± 3.86 P 0.02 0.041 0.1474 0.8087 Sf9 cells pAc 10.61 ± 0.77 − 2.64 ± 6.19 50.41 ± 4.45 20.22 ± 5.71 pAc-HzAPN1 44.35 ± 5.78 48.05 ± 6.57 57.86 ± 1.29 17.94 ± 7.18 P 0.0044 < 0.0001 0.1828 0.8165 Table 1. Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines transfected with pAc or pAc-HzAPN1 plasmids. Values in the table are average mortality ± standard errors of three replicates of three independent transfections of each plasmid per cell line. Mortality values of different treatments were arcsine transformed before analysis. P values refer to the morality comparisons between pAc and pAc-HzAPN1 transfected cells of each cell line (Student’s t-test in JMP 8; SAS Institute, Inc.). Cell lines Treatments & P values Cry1Ac (30 μg/ml) Cry1Ac (15 μg/ml) Cry2Ab (3.75 μg/ml) Cry2Ab (2.5 μg/ml) Midgut cells pAc 24.19 ± 3.45 15.43 ± 1.93 56.25 ± 9.79 28.01 ± 3.98 pAc-HzAPN1 76.17 ± 3.57 71.38 ± 3.58 55.19 ± 1.99 31.91 ± 7.92 P 0.0005 0.0002 0.9214 0.8866 Fat body cells pAc 23.77 ± 3.32 14.46 ± 1.76 62.75 ± 3.44 34.81 ± 1.07 pAc-HzAPN1 51.57 ± 6.63 35.36 ± 6.81 55.80 ± 1.79 34.5 ± 3.86 P 0.02 0.041 0.1474 0.8087 Sf9 cells pAc 10.61 ± 0.77 − 2.64 ± 6.19 50.41 ± 4.45 20.22 ± 5.71 pAc-HzAPN1 44.35 ± 5.78 48.05 ± 6.57 57.86 ± 1.29 17.94 ± 7.18 P 0.0044 < 0.0001 0.1828 0.8165 Table 1. Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines transfected with pAc or pAc-HzAPN1 plasmids. Values in the table are average mortality ± standard errors of three replicates of three independent transfections of each plasmid per cell line. Mortality values of different treatments were arcsine transformed before analysis. Table 1. Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines Discussion Cell lines Treatments & P values Cry1Ac (150 μg/ml) Cry2Ab (30 μg/ml) Midgut cells DsRed dsRNA 81.70 ± 1.85 86.19 ± 1.31 HzAPN1 dsRNA 47.91 ± 0.92 85.21 ± 4.11 P < 0.0001 0.831 Fat body cells DsRed dsRNA 87.38 ± 1.56 83.94 ± 2.50 HzAPN1 dsRNA 68.09 ± 2.89 80.71 ± 3.24 P 0.0042 0.4735 Sf9 cells DsRed dsRNA 61.20 ± 3.37 45.79 ± 2.90 HzAPN1 dsRNA 48.08 ± 3.24 48.86 ± 2.15 P 0.0486 0.4428 Table 2. Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines transfected with DsRed or HzAPN1 dsRNA. Values in the table are average mortality ± standard errors of three replicates of three independent transfections of each dsRNA per cell line. Mortality values of different treatments were arcsine transformed before analysis. P values refer to the morality comparisons between DsRed and HzAPN1 dsRNA transfected cells of each cell line (Student’s t-test in JMP 8; SAS Institute, Inc.). Cell lines Treatments & P values Cry1Ac (150 μg/ml) Cry2Ab (30 μg/ml) Midgut cells DsRed dsRNA 81.70 ± 1.85 86.19 ± 1.31 HzAPN1 dsRNA 47.91 ± 0.92 85.21 ± 4.11 P < 0.0001 0.831 Fat body cells DsRed dsRNA 87.38 ± 1.56 83.94 ± 2.50 HzAPN1 dsRNA 68.09 ± 2.89 80.71 ± 3.24 P 0.0042 0.4735 Sf9 cells DsRed dsRNA 61.20 ± 3.37 45.79 ± 2.90 HzAPN1 dsRNA 48.08 ± 3.24 48.86 ± 2.15 P 0.0486 0.4428 Table 2. Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines transfected with DsRed or HzAPN1 dsRNA. Values in the table are average mortality ± standard errors of three replicates of three independent transfections of each dsRNA per cell line. Mortality values of different treatments were arcsine transformed before analysis. P values refer to the morality comparisons between DsRed and HzAPN1 dsRNA transfected cells of each cell line (Student’s t-test in JMP 8; SAS Institute, Inc.). Cell lines Treatments & P values Cry1Ac (150 μg/ml) Cry2Ab (30 μg/ml) Midgut cells DsRed dsRNA 81.70 ± 1.85 86.19 ± 1.31 HzAPN1 dsRNA 47.91 ± 0.92 85.21 ± 4.11 P < 0.0001 0.831 Fat body cells DsRed dsRNA 87.38 ± 1.56 83.94 ± 2.50 HzAPN1 dsRNA 68.09 ± 2.89 80.71 ± 3.24 P 0.0042 0.4735 Sf9 cells DsRed dsRNA 61.20 ± 3.37 45.79 ± 2.90 HzAPN1 dsRNA 48.08 ± 3.24 48.86 ± 2.15 P 0.0486 0.4428 Table 2. Discussion Both Cry1Ac and Cry2Ab are expected to have two or more receptors in the larval midguts of Lepidopteran insects (Wei & Li, unpublished manuscript). Documentation of cross-resistance between the two toxins implies that they may share one common receptor27–29, although cross-resistance can occur through receptors-independent mechanisms. Recognition of one protein band with the same size of APN1 (120 ~ 130 kDa) by both Cry1Ac and Cry2Ab antibodies in the midgut and fat body cell lines of H. zea (Wei & Li, unpublished manuscript) and by Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 3 www.nature.com/scientificreports/ Figure 3. Expression level of APN1 in the cells transfected with DsRed or HzAPN1 dsRNA. For DsRed (dsRNA control) and HzAPN1 dsRNA, three independent transfections were conducted for each cell line and the protein extracts from each of the three transfections were analyzed by Western blot. The lower panel picture of APN1 and β -actin bands is a representative of the three Western blots. The average expression of HzAPN1 or SfAPN1 (Sf9 cells) relative to that of β -actin calculated by Image J quantification of the three Western blots is displayed in the upper panel. Each error bar represents the standard error of the mean from three transfection replicates. Significant differences in relative expression of HzAPN1 or SfAPN1 (Sf9 cells) between DsRed and HzAPN1 dsRNA transfected cells of each cell line are indicated with an asterisk () (P < 0.05, Student’s t-tests). Figure 3. Expression level of APN1 in the cells transfected with DsRed or HzAPN1 dsRNA. For DsRed Figure 3. Expression level of APN1 in the cells transfected with DsRed or HzAPN1 dsRNA. For DsRed (dsRNA control) and HzAPN1 dsRNA, three independent transfections were conducted for each cell line and the protein extracts from each of the three transfections were analyzed by Western blot. The lower panel picture of APN1 and β -actin bands is a representative of the three Western blots. The average expression of HzAPN1 or SfAPN1 (Sf9 cells) relative to that of β -actin calculated by Image J quantification of the three Western blots is displayed in the upper panel. Each error bar represents the standard error of the mean from three transfection replicates. Significant differences in relative expression of HzAPN1 or SfAPN1 (Sf9 cells) between DsRed and HzAPN1 dsRNA transfected cells of each cell line are indicated with an asterisk () (P < 0.05, Student’s t-tests). Methods I Insects and insect cell lines. Three insect cell lines and one susceptible strain of H. zea were used in this study. The H. zea susceptible strain was established with a few thousand eggs purchased from Benzon Research Inc. (Carlisle, PA) in October 2014 and maintained on wheat germ-containing diets at 27 ± 1 °C, 60 ± 10% RH and a photoperiod of 14 L:10 D40. The H. zea midgut cell line RP-HzGUT-AW1(MG) and H. zea fat body cell line BCIRL-HzFB33(FB), generously provided by Dr. Cynthia L.Goodman (BCIRL, USDA, ARS)41, were routinely maintained with Excell 420 insect serum-free medium (SAFC Bioscience, Lenexa, KS) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone-QB perbio, Logan, UT), 50 U/ml penicillin, 50 mg/ml strep- tomycin, and 12 mg/ml gentamycin (Invitrogen, CA) in an incubator at 28 °C. Sf9 cell line derived from pupal ovarian tissue of Spodoptera frugiperda was cultured in Sf-900 II SFM medium (GIBCO/BRL/Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and 12 mg/ml gentamycin in the same incubator. Toxins and antibody. Cry1Ac and Cry2Ab protoxin were supplied by Biotechnology Research Laboratory, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Activated Cry1Ac and Cry2Ab were prepared from the corresponding protoxin as described by Wei et al. (Wei & Li, unpublished manuscript). Anti-Cry1Ac antibody and anti-HaAPN1 antibody were obtained from Dr. Chenxi Liu of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences30. Preparation of anti-Cry2Ab antibody was described in Wei et al. (Wei & Li, unpublished manuscript). Ligand blot detection of CryAc and Cry2Ab binding proteins in H. zea larval midgut BBMV. Brush border membrane vesicles (BBMV) for ligand blot and Western blot (see below) were prepared from the 5th instar larvae midguts of the susceptible strain of H. zea using a differential centrifugation method42,43. When dissected out, midguts were open to remove peritrophic membranes and gut contents, cleaned in ice-cold 0.7% NaCl solution, placed on filter paper for a few seconds to remove excessive water, weighed and stored at − 80 °C. The frozen midguts taken out from the freezer were homogenized in nine-fold volume (w/v) of ice-cold buffer A (300 mM mannitol, 5 mM EGTA, 17 mM Tris-HCl, 1 mM PMSF), incubated with one volume of 24 mM MgCl2 on ice for 15 min, and centrifuged at 2500 × g and 4 °C for 15 min. Discussion Cytotoxcity of activated Cry1Ac and Cry2Ab to H. zea midgut, fat body and Sf9 cell lines transfected with DsRed or HzAPN1 dsRNA. *Values in the table are average mortality ± standard errors of three replicates of three independent transfections of each dsRNA per cell line. Mortality values of different treatments were arcsine transformed before analysis. P values refer to the morality comparisons between DsRed and HzAPN1 dsRNA transfected cells of each cell line (Student’s t-test in JMP 8; SAS Institute, Inc.). Cry2Aa antibody in Spodoptera littoralis BBMV31 suggest that the potential common receptor could be APN1 This present study was conducted to test whether this hypothesis is true or not.i h By ligand blot and Western blot, we confirmed that H. zea larval midgut BBMV, like H. zea midgut and fat body cell lines (Wei & Li, unpublished manuscript), also had a 130 kDa protein band that was recognized by Cry1Ac, Cry2Ab, and APN1 antibodies (Fig. 1). Heterlogous expression of HzAPN1 significantly increased the susceptibilities of Sf9, H. zea midgut and fat body cell lines to activated Cry1Ac (Fig. 2 and Table 1), whereas RNAi knocking down of the endogenous APN1 made the three cell lines significantly less susceptible to activated Cry1Ac (Fig. 3 and Table 2). In contrast, neither heterologous expression of HzAPN1 nor RNAi knocking down of the endogenous APN1 affected the susceptibilities of the three cell lines to activated Cry2Ab (Tables 1 and 2). The data suggest that the 130 kDa band recognized by Cry1Ac antibody is HzAPN1, but the 130 kDa band rec- ognized by Cry2Ab is an unknown protein. Moreover, the data directly demonstrate that HzAPN1 is one of the receptors for Cry1Ac, but not for Cry2Ab in H. zea. p y y While APN1 has been implicated as one of the receptors for Cry1Ac in a number of species15,21,23, this is the first report that functionally characterized APN1 as a receptor of Cry1Ac in H. zea. This is consistent with the Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 4 www.nature.com/scientificreports/ result of Sivakumar et al., who confirmed that APN1 from H. armigera (HaAPN1)32, the closest orthologue of HzAPN1, acted as a receptor of Cry1Ac in Sf21 cells. To our knowledge, the current study is also the first report that experimentally excluded APN1 as the basis of cross-resistance between the two toxins—the common recep- tor of Cry1Ac and Cry2Ab. Discussion However, this study cannot be extrapolated to rule out the possibility of other APNs as one of the receptors for Cry2Ab. First, the size of the protein band (120 ~ 130 kDa) recognized by Cry2Ab anti- body not only matches with the size of APN1, but also falls into the size range of other APNs. In addition, APN5 from P. xylostella (PxAPN5) was reported to bind to Cry2Ab26. Further functional analyses of HzAPN5 and other APNs from H. zea are needed to resolve this issue. The fact that RNAi knocking down of the endogenous APN1 protected not only midgut cells but also fat body and ovarian (Sf9) cells from the toxic effects of Cry1Ac (Fig. 3 and Table 2) suggests that APN1 may be expressed in both midgut and non-gut hemocoelic tissues at least in some species. Consistent with this notion, APN1 has been shown to be expressed in midgut, malpighian tubule, salivary gland and fat body in T. ni33, fat body, malpighian tubule and salivary gland in Achaea janata34, midgut, fat body, malpighian tubule, epidermis and hemolymph in S. exigua35, midgut, fat body, Malpighian tubule and carcass in Epiphyas postvittana36, and midgut and Malpighian tubule in B. mori37. The toxic effects of Cry1Ac and Cry2Ab on in vitro cultured fat body and ovarian (Sf9) cells observed in this study and Wei et al. (Wei & Li, unpublished manuscript) as well as the effects of intrahemocoelic injections of several Cry1 toxins on the growth and survival of Lymantria dispar and Neobellieria bullata larvae38 and of A. janata larvae34 further strengthen the notion of APNs expression in the above non-gut hemocoelic tissues. Apparently, these receptors-expressing non-gut hemocoelic tissues can serve as additional in vivo target tissues of Cry toxins, as long as these toxins can cross the insect midgut epithelium and reach the haemolymph when fed orally. Detection of small amounts of orally ingested Cry toxins in the haemolymph of Lygus Hesperus39 proves that Cry toxins can naturally cross the insect gut into the haemolymph at least in some species, but probably at a very low speed. Novel protein conjugation/fusion technology capable of speeding up the crossing of the haemocoelic-active Cry toxins across the insect gut into haemolymph is needed to further enhance the efficacy of Cry toxins by virtue of targeting both midgut and non-gut hemocoelic tissues. Methods I The supernatant was centrifuged at 30,000 × g and 4 °C for 30 min. The resulting pellet was suspended in buffer B (150 mM Mannitol, 2.5 mM EGTA, 8.5 mM Tris-HCl, 1 mM PMSF), left on ice up to 4 h, and then centrifuged at 30,000 g and 4 °C for 15 min. The final pellet was suspended in buffer C (150 mM NaCl, 5 mM EGTA, 1 mM PMSF, 20 mM Tris-HCl, 1% CHAPS) and used as the BBMV preparation. The protein concentrations of the BBMV preparations were determined by the Bradford protein assay, using bovine serum albumin (BSA) as a standard (Bradford, 1976). When con- ducted ligand blot, 8 μ g protein of the larval midgut BBMV preparation was separated on a 10% SDS-PAGE gel and electroblotted to a polyvinylidene difluoride (PVDF) membrane (Thermo scientific) in the transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The follow-up procedures for detection of Cry1Ac or Cry2Ab binding proteins using the anti-Cry1Ac antibody or anti-Cry2Ab antibody were the same as described in Wei et al. (Wei & Li, unpublished manuscript). Western blot detection of APN1 in larval midgut BBMV and cell line protein extracts. The lar- val midgut BBMV was prepared as above and the protein extracts of the three insect cell lines transfected with the control plasmid pAc, pAc-HzAPN1, DsRed double strand RNA (dsRNA), or HzAPN1 dsRNA (see prepa- ration and transfection of pAc-HzAPN1 plasmid and dsRNA below) were prepared as described by Wei et al. Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 5 www.nature.com/scientificreports/ (Wei & Li, unpublished manuscript). Six μ g protein of the larval midgut BBMV and/or the protein extract of each cell line transfected with pAc, pAc-HzAPN1, DsRed dsRNA, or HzAPN1 dsRNA were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and electroblotted to a polyvinylidene diflu- oride (PVDF) membrane (Thermo scientific) in the transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membrane was blocked with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) containing 0.1% Tween 20 (Bio-Rad) (PBST buffer) and 5% milk (Amresco) at 4 °C overnight or room temperature for at least 2 h. Methods I The PCR reaction (20 μ L) was composed of 1 μ L of HzAPN1-PGEMT, 1 μ L of LongAmp® Taq DNA polymerase (New England Biolabs, Ipswich, MA), 1.6 μ L of dNTP (5 mM), 2 μ L of primer mix (25 mM each), 4 μ L of 5× longAmp Taq reaction buffer and 10.4 μ L ddH2O. The PCR conditions were 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s and 65 °C for 1 min, and a final extension of 5 min at 65 °C. The resultant PCR product was TA-cloned into the pGEM-T Easy-vector to generate the template plas- mid HzAPN1-pGEMT for production of HzAPN1 dsRNA. Likewise, a 480-bp cDNA sequence of DsRed was PCR-amplified from pBac [3 × P3-DesRedaf] with the primers DsRED-F (TGCAGGTGACCAAGGGC) and DsRED-R (CGTTGTGGGAGGTGATGT) and TA-cloned into the pGEM-T Easy-vector to generate the template plasmid DsRed-pGEMT for production of the DsRed dsRNA (i.e. control dsRNA). p p p ( ) We used two pairs of universal primers complementary to the flanking sequence of the multiple clon- ing sites of the pGEM-T Easy-vector to PCR-amplify two in vitro transcription templates (one for sense RNA, one for antisense RNA) from the template plasmids DsRed-pGEMT and HzAPN1-pGEMT, respectively. The primer pair T7 pGEMTeasy Fwd (5′ -GGTGTAATACGACTCACTATAGGG-3′ ) and pGEMTeasy Rev (5′ -CAAGCTATGCATCCAACGCGTTGGGAG-3′ ) were used to PCR-amplify the sense template that had a T7 RNA polymerase promoter sequence at the 5′ end of its sense strand. Another primer pair T7 pGEMTeasy Rev (5′ -GGTGTAATACGACTCACTATAGGGCAAGCTATGCATCCAACGCGTTGGGAG-3′ ) and pGEMTeasy Fwd (5′ -CGAATTGGGCCCGGACGTCGCA-3′ ) were used to PCR-amplify the antisense template that had a T7 RNA polymerase promoter sequence at the 5′ end of its antisense strand. The resulting sense and antisense tem- plate PCR products were cleaned, quantified, and combined at 1:1 ratio. Then the combined sense and antisense templates were used to make dsRNA by simultaneous in vitro transcription of both the sense and antisense RNA using the RiboMax Large Scale RNA Production System (Promega) according to the product manual. Following in vitro transcription, the DsRed and HzAPN1 dsRNA products were cleaned with MEGAclear Kit (Ambion Inc.), quantified and stored at −80 °C for subsequent transfection. Cell transfection and toxicity bioassay. H. zea midgut, fat body and Sf9 cells were seeded onto a 12-well plate (9 × 105 cells/well) and incubated at 28 °C, respectively. Methods I The membrane was then hybridized at 4 °C with the primary antibody anti-HaAPN1 antiserum (adding to the blocking buffer at 1:15,000 dilution) for about 14 h, washed three times of 15 min each with PBST buffer, probed at 4 °C with the horseradish peroxidase (HRP)-conjugated secondary antibody (ZSGB-BIO, China) (1:15,000) for 2 h, washed three times of 15 min each with PBST buffer, and visualized with immobilon western chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA, USA)30. ) A duplicate SDS-PAGE gel of the larval midgut BBMV and/or cell line protein extracts was electrophoresed simultaneously to detect the reference protein β -actin. The procedure for detection of β -actin was identical to that for APN1 except that the membrane blot was hybridized with anti-β -actin antibody (sc-69879, Santa Cruz Biotechnology, USA) (1:3000) for 2 h at room temperature with constant shaking and then probed with a goat anti-mouse IgM-HRP (Santa Cruz Biotechnology, USA) (1:3000) for 1 h at room temperature with constant shaking. Construction of pAC-HzAPN1 construct. The open reading frame (ORF) of HzAPN1 was PCR-amplified from the plasmid HzAPN1-pGEMT (Zhang & Li, unpublished manuscript) with the gene-specific primers APN-5′ ASCI (5′ -GGCGCGCCGACCAGCGTGGAGTCACAA-3′ ) and APN-3′ NOTI (5′ -GCGGCCGCTTAAGCCATATTAACAACGAGAGTCA-3′ ). PCR reaction (20 μ L) contained 1 μ L of HzAPN1-pGEMT (Zhang & Li, unpublished manuscript), 1 μ L of LongAmp® Taq DNA polymerase (New England Biolabs, Ipswich, MA), 1.6 μ L of dNTP (5 mM), 2 μ L of primer mix (25 mM each), 4 μ L of 5× longAmp Taq reaction buffer and 10.4 μ L ddH2O. The PCR reaction was initiated at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 65 °C for 1 min, and a final extension of 5 min at 65 °C. The PCR products were TA-cloned into pGEM® -T vector (Promega, Madison, WI), released by double digestion with ASCI and NOTI (New England Biolabs, Ipswich, MA), and subcloned into the expression vector pAc/V5-His A (Invitrogen) via the ASCI and NOTI enzyme sites. Production of HzAPN1 and DsRed dsRNA. A 658 bp cDNA sequence of HzAPN1 was PCR-amplified from HzAPN1-pGEMT (Zhang & Li, unpublished manuscript) using the primers HZAPN1 dsRNA-F (5′ -GGATCCTGT CTGACTCCCTTGACTCTGCTC-3′ ) and HzAPN1 dsRNA-R (5′ -CGGTTGTATTC GTGGATTGATAGCCT-3′ ). Methods I For heterologous expression of HzAPN1 in the three cell lines, cells were transfected with 2 μ g/well of pAc (control, empty vector) or pAC-HzAPN1 plasmids using Cellfectin (Invitrogen; 8 μ l per well) for 5 h. For RNA interference (RNAi) silencing of endogenous APN1 in the three cell lines, cells were transfected with 50 nM of DsRed (control) or HzAPN1 dsRNA for 5 h. We then replaced the transfection mixture of each well with 1.5 ml of supplemented Excell 420 (midgut and fat body cells) or Sf-900 II SFM medium (Sf9 cells) and incubated the cells at 28 °C for 64 hours. The cells in each independent transfection replicate were reseeded onto three wells in a 96-well micro-plate at 10000 cells in 100 μ L per well. After 2 h of attachment, the cells were treated with activated Cry1Ac or activated Cxry2Ab. The concentrations for bioassays of the cells transfected with pAc (control) or pAc-HzAPN1 plasmids were 15 and 30 μ g/ml for acti- vated Cry1Ac and 2.5 and 3.75 and μ g/ml for activated Cry2Ab), respectively. The concentrations for bioassays Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 6 www.nature.com/scientificreports/ of the cells transfected with DsRed dsRNA (control) and HzAPN1 dsRNA were 150 μ g/ml for activated Cry1Ac and 30 μ g/ml for activated Cry2Ab, respectively. Cell mortality was determined by replacing 35 μ L medium from each well with 35 μ L of 0.4% trypan blue (AMRESCO®, BioExpress, Ohio, USA) and counting the numbers of stained (dead cells) and unstained (live cells) in two 400x fields of view from the central parts of each well under an inverted microscope (VistaVision, VWR) after 4.5 h for Cry1Ac or 5.5 hours for Cry2Ab(Wei & Li, unpub- lished manuscript). Each plasmid or dsRNA was independently transfected into each cell three times and each independent transfection was bioassayed three times with control or a given concentration of Cry1Ac or Cry2Ab (3 × 3 = 9 replicates). 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Statistical Analysis Cell mortality was calculated by dividing the number of dead cells (the number of cells before toxin treatment – the number of unstained cells after treatment) by the number of cells before treatment and corrected with the Abbott formula44. Significant differences in mortality between the control plasmid pAc (or the DsRed control dsRNA) and pAc-HzAPN1 (or HzAPN1 dsRNA) were compared using Student t-test with α = 0.05 (JMP 8; SAS Institue, Inc.). Mortality values of different treatments were arcsine transformed before analysis.h yf y The band intensities of the target protein APN1 and the reference protein β -actin on all the Western blot pictures of the protein extracts from the three cell lines transfected with pAc, pAc-HzAPN1, DsRed dsRNA, or HzAPN1 dsRNA were quantified by densitometry using Image J software (NIH, v1.46). 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Chem. 282, 7312–7319 (2007). g y insecticidal protein Cry1Ac. J. Biol. Chem. 282, 7312–7319 (2007 p y 33. Nan, X. Gene cloning﹅localization and expression of midgut aminopeptidase N isozymes from the Cabbage Looper, Trichoplus Northwest University of Science and Technology, PhD thesis (2012). y gy ( ) 34. Ningshen, T. J., Aparoy, P., Ventaku, V. R. & Dutta-Gupta, A. Functional interpretation of a non-gut hemocoelic aminopeptidase N (APN) in a Lepidopteran insect pest Achaea janata. Author Contributions X.L. and J.W. conceived and designed the experiments. J.W. and M.Z. performed the experiments. X.L. and J.W. analyzed the data. X.L., J.W., G.L., K.W., Y.G. and X.N. wrote the manuscript. J.W. and X.L. shared in the scoping and writing responsibilities. All authors have read and approved the manuscript for publication. X.L. and J.W. conceived and designed the experiments. J.W. and M.Z. performed the experiments. X.L. and J.W. analyzed the data. X.L., J.W., G.L., K.W., Y.G. and X.N. wrote the manuscript. J.W. and X.L. shared in the scoping and writing responsibilities. All authors have read and approved the manuscript for publication. Additional Informationihi Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 Additional Information Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Wei, J. et al. APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea. Sci. Rep. 6, 19179; doi: 10.1038/srep19179 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:19179 \| DOI: 10.1038/srep19179 8
https://openalex.org/W2972739731	http://addi.ehu.es/bitstream/10810/37408/1/agronomy-09-00542-v2.pdf	English	null	Potential Benefits and Risks for Soil Health Derived From the Use of Organic Amendments in Agriculture	Agronomy	2,019	cc-by	16,054	Received: 15 July 2019; Accepted: 10 September 2019; Published: 12 September 2019 Received: 15 July 2019; Accepted: 10 September 2019; Published: 12 September 2019 Abstract: The use of organic amendments in agriculture is a common practice due to their potential to increase crop productivity and enhance soil health. Indeed, organic amendments of diﬀerent origin and composition (e.g., animal slurry, manure, compost, sewage sludge, etc.) can supply valuable nutrients to the soil, as well as increase its organic matter content, with concomitant beneﬁts for soil health. However, the application of organic amendments to agricultural soil entails a variety of risks for environmental and human health. Organic amendments often contain a range of pollutants, including heavy metals, persistent organic pollutants, potential human pathogens, and emerging pollutants. Regarding emerging pollutants, the presence of antibiotic residues, antibiotic-resistant bacteria, and antibiotic-resistance genes in agricultural amendments is currently a matter of much concern, due to the concomitant risks for human health. Similarly, currently, the introduction of microplastics to agricultural soil, via the application of organic amendments (mainly, sewage sludge), is a topic of much relevance, owing to its magnitude and potential adverse eﬀects for environmental health. There is, currently, much interest in the development of eﬃcient strategies to mitigate the risks associated to the application of organic amendments to agricultural soil, while beneﬁting from their numerous advantages. Keywords: agricultural systems; food security; sustainable intensiﬁcation; soil health; antibiotic resistance; soil conservation Agronomy 2019, 9, 542; doi:10.3390/agronomy9090542 agronomy agronomy agronomy Potential Beneﬁts and Risks for Soil Health Derived From the Use of Organic Amendments in Agriculture Julen Urra 1 , Itziar Alkorta 2 and Carlos Garbisu 1,* 1 NEIKER-Tecnalia, Department of Conservation of Natural Resources, Soil Microbial Ecology Group, c/ Berreaga 1, E-48160 Derio, Spain; jurra@neiker.eus 2 Instituto BIOFISIKA (CSIC, UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain; itzi.alkorta@ehu.es * Correspondence: cgarbisu@neiker.eus; Tel.: +34-94-403-43-00 1 NEIKER-Tecnalia, Department of Conservation of Natural Resources, Soil Microbial Ecology Group, c/ Berreaga 1 E 48160 Derio Spain; jurra@neiker eus 1 NEIKER-Tecnalia, Department of Conservation of Natural Resources, Soil Microbial Ecology Group, c/ Berreaga 1, E-48160 Derio, Spain; jurra@neiker.eus 2 Instituto BIOFISIKA (CSIC, UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain; itzi.alkorta@ehu.es * Correspondence: cgarbisu@neiker.eus; Tel.: +34-94-403-43-00 agronomy agronomy www.mdpi.com/journal/agronomy 1. Ecological Intensiﬁcation In order to feed the constantly growing human population, it was estimated that food production will have to be doubled within the next few decades [1]. The Green Revolution, which introduced new crop varieties and livestock breeds along with the extensive use of irrigation, machinery, and synthetic agrochemicals (fertilizers, pesticides), led to sharp increases in food production from agricultural systems since the beginning of the 1960s. This global increase in food production was underpinned by intensiﬁcation rather than spread of agricultural land [2]. Seeking for enhanced crop productivity, agricultural intensiﬁcation was sustained by indiscriminate inputs of synthetic agrochemicals, an overuse of water, and the alteration of the soil ecosystem, at great expense to the environment. Indeed, replacing soil internal processes with external inputs resulted in the progressive deterioration of the fundamental properties of those soils, including the potential for self-regulation [3]. Soil is a multi-functional, extremely complex, and highly dynamic three-dimensional system in which solid, liquid, and gaseous components interact in multiple physical, chemical, and biological processes. On the other hand, soil is a non-renewable resource at the human scale [4]. Healthy soils support a multitude of functions [5] and the delivery of key ecosystem services. Soil health/soil quality Agronomy 2019, 9, 542; doi:10.3390/agronomy9090542 www.mdpi.com/journal/agronomy 2 of 23 Agronomy 2019, 9, 542 is recurrently deﬁned as the capacity of a given soil to perform its functions. Although both terms are often used interchangeably, soil quality is normally associated with a soil’s ﬁtness for a speciﬁc use, whereas soil health is frequently used in a broader sense to indicate “the capacity of soil to function as a vital living system to sustain biological productivity, promote environmental quality, and maintain plant and animal health” [6]. The recovery and conservation of soil health is, thus, of utmost importance for the preservation of life on earth, justifying the concerns of the European Commission in developing a soil legislation framework [7], which was unfortunately withdrawn in 2014. g y Developing strategies and tools to promote agricultural sustainability whilst maximizing crop yields will be a major challenge for the next decades, in an attempt to meet the abovementioned goal of food production while protecting the integrity of our environment. 1. Ecological Intensiﬁcation In this context, ecological intensiﬁcation was advocated as a suitable approach to integrate ecological processes into agricultural practices, in order to simultaneously enhance the delivery of ecosystem services and reduce, or even replace, the external anthropogenic inputs [8]. This innovative approach does not display a consolidated set of guidelines, but rather a suite of alternative models for sustainable intensiﬁcation, based on a greater reliance on ecological processes and ecosystem services, so as to minimize external anthropogenic inputs without adversely aﬀecting crop productivity. Promising ecological intensiﬁcation models combine technological advances in agricultural science, such as precision agriculture [9], the use of sensors [10], and state-of-the-art technologies for agricultural waste reduction and reutilization, with sustainable practices and methodologies aimed at protecting the integrity of the soil ecosystem and, speciﬁcally, its valuable biodiversity. Relying on minimum soil disturbance, a permanent soil organic cover, and crop diversiﬁcation, conservation agriculture was shown to deliver a variety of essential ecosystem services, such as soil carbon (C) storage and sequestration, water regulation, soil erosion control, etc. [11]. However, in certain cases and situations, conservation agriculture was shown to result in a reduction in crop productivity, as compared to conventional agriculture [12]. Organic farming relies on natural ecological processes to maintain the integrity of the soil ecosystem and, concomitantly, the provision of ecosystem services, and it is particularly focused on long-term agricultural productivity [13]. Furthermore, organic farming aims to exclude the use of synthetic fertilizers and claims for their systematic substitution by organic amendments, thus contributing to the valorization of organic waste [14]. Nevertheless, in many cases, organic amendments may harbor traditional and emerging pollutants and, therefore, cause toxicity problems [15–17], thus entailing a potential risk to human and ecosystem health. The aim of this review article (focusing, but not exclusively, on research papers published in the last 10 years) is to highlight the potential beneﬁts and drawbacks associated to the use of organic amendments as agricultural fertilizers, while addressing the existing strategies and technologies to mitigate the potential downsides. 2. Organic Amendments Our current production systems and transformation processes, designed to create useful goods and services, usually entail the continuous generation and disposal of massive amounts of waste. The required transition toward more ecological and sustainable production systems demands changing the current linear production model, where resources are converted into products and waste, to a circular model which, in a way, attempts to mimic the principles and functioning of natural ecological processes and cycles [18]. The paradigm of circular economy is based on closed-loop models, in which waste and by-products are eﬀectively integrated into the system as valuable assets, thereby reducing natural resource utilization and waste production [19]. This transition was advocated by the European Commission in several documents, such as the Roadmap to a Resource-Eﬃcient Europe, which appeals for sustainable production and an eﬃcient use of resources [20], and the European Union (EU) Action Plan for the Circular Economy, which establishes actions covering the whole lifecycle of products and encourages to “close the loop” through greater recycling and re-use [21]. 3 of 23 Agronomy 2019, 9, 542 Agronomy 2019, 9, 542 Within this circular economy paradigm, the reutilization of organic waste and by-products as soil amendments is gaining much interest, since it poses a realistic, cost-eﬀective, and environmentally sound alternative to landﬁll disposal (the least preferred option for waste management) [22]. Organic amendments, such as composts, animal manures, slurries, crop residues, digestates from the anaerobic treatment of waste, biosolids, etc., are extensively applied to agricultural soil as fertilizers [23,24] or, alternatively, as amendments in soil remediation and reclamation initiatives [25–28]. Biofertilizers, deﬁned as “mixtures of selected beneﬁcial microorganisms and/or other organic substances (plant growth hormones, vitamins, etc.) for sustainable soil management and plant productivity” [29], are broadly applied worldwide given their promising potential [30]. Recently, “biofertilization techniques” were included within the group of “organic residues most commonly used as soil amendments” [31]. Nonetheless, other authors [23,32] diﬀerentiated “microbial inoculants” from organic waste-derived “organic amendments” or “organic fertilizers”. Undeniably, microbial inoculants are of an organic nature and may potentially exert beneﬁcial eﬀects on plant growth and health. However, it is not the purpose of this review article to debate whether or not microbial inoculants should be categorized as “organic amendments”, nor to discuss their potential beneﬁcial or adverse eﬀects. 2.1. Crop Residues and Green Manures Crop residues are deﬁned as the “non-edible part of the plant that is left in the ﬁeld after harvest” [33], while the term green manure refers to “speciﬁc forage or crop varieties that are incorporated into the soil while green or soon after maturing” [34]. These plant-based amendments are a valuable source of organic matter (OM) and are considered “the greatest source of soil organic matter (SOM)” for agricultural soils [35]. Moreover, they can provide protection against soil erosion, suppress weeds [36], improve soil physicochemical and biological properties, and enhance soil fertility [37]. 2.2. Animal manures Composed of feces, urine, and animal bedding, animal manure was long used as soil organic amendment since it can enhance soil fertility through the supply of essential macro- and micronutrients, as well as OM [34,38]. The application of animal manure can improve soil structure by reducing bulk density and increasing soil porosity, water inﬁltration/percolation rate, and aggregate stability [38,39]. Furthermore, manure-based amendments can stimulate soil microbial activity and biomass, as well as alter the composition and diversity of soil microbial communities [40,41]. 2. Organic Amendments Given that organic amendments may be (i) originated from diﬀerent sources (agriculture, urban, industry), (ii) subjected or not to treatments (composting, anaerobic digestion, etc.), and (iii) presented in diﬀerent stages of matter (solid, liquid), it is not surprising that they can have a wide variety of diﬀerent properties and agronomic potentialities. The most common organic amendments belong to the categories below. 2.3. Biosolids Biosolids (also referred to as sewage sludge) are solid organic residues originated in wastewater treatment plants [42]. Given the load of macro- and micronutrients that these organic amendments contain, their application to agricultural soil can be highly beneﬁcial for soil fertility [43]. Indeed, the application of biosolids to soil was shown to enhance its physicochemical and biological properties [44,45], and was proposed as a suitable practice for C sequestration in agricultural soil [46]. 2.5. Anaerobic Digestion Anaerobic digestion is a biological process via which organic waste is stabilized in the absence of oxygen, resulting in the formation of biogas and an organic by-product known as digestate [52,53]. A broad range of organic waste can be subjected to the process of anaerobic digestion, which entails the degradation and mineralization of the labile organic constituents, thereby increasing the stability of the resulting by-product [53,54]. Given their nutrient-rich composition, digestates, which may then be separated into a liquid and solid fraction, can be used as organic amendments and agricultural fertilizers [55]. Moreover, the anaerobic digestion of organic waste was reported to eﬀectively reduce the load of potential human pathogens and pollutants within the digested organic material [56,57]. Regardless of the speciﬁc category of amendment, the beneﬁcial or adverse eﬀects that any given organic amendment exert on the (agricultural) soil ecosystem depend on many diﬀerent factors, ranging from intrinsic characteristics of the amendment (composition, stability, maturity, etc.) to application times and rates, soil type and properties (both physicochemical and biological), cropping system, climatic conditions, etc. [25,37]. Hence, in order to properly assess the suitability of a given organic amendment for speciﬁc agricultural purposes, an exhaustive characterization of the amendment itself and the agricultural soil and crop needs should be carried out prior to its application. In any event, it is important to emphasize that, in many rural areas of the world (e.g., regions in southeast Asia, sub-Saharan Africa, etc.), there is a dearth of adequate sources of OM for application to agricultural soil [58]. As an example, agricultural soils in Bangladesh and Nepal are in great need of larger amounts of OM to maintain and improve their fertility, which was for decades driven by chemical fertilizers; however, regrettably, the organic fertilizer subsector in these countries is still at a very early stage of development [58]. Similarly, in sub-Saharan Africa, where agricultural soils often present a high level of degradation and poor fertility, organic inputs are customarily in short supply in smallholder farming systems due to limited aﬀordability and/or accessibility [59]. One of the principles of integrated soil fertility management (ISFM)—the combined application of fertilizer and organic resources—can contribute to minimize this limitation and, in general, to the sustainable intensiﬁcation needed in sub-Saharan Africa to address rural poverty and natural resource degradation [59]. 2.4. Compost The decomposition of OM under controlled aerobic conditions can lead to a stable, humus-like end product known as compost [47]. Compost can be produced from a wide array of organic materials, including agrarian (crop residues, animal manures) and municipal solid waste and sewage sludge. In fact, compost constitutes the most commonly used organic amendment for agricultural 4 of 23 Agronomy 2019, 9, 542 fertilization [48]. Composted amendments incorporate OM into agricultural soil, thereby improving soil porosity, aeration, water holding capacity, aggregate stability, and nutrient availability [39], as well as stimulating soil microbial activity and biomass [49,50]. Composted amendments contain more recalcitrant organic fractions than the raw components themselves, leading to longer-term positive eﬀects on soil health [51]. 3. Beneﬁcial Eﬀects of Organic Amendments Aiming to increase crop productivity while protecting agroecosystem health, organic farming was shown, through the proper application of organic amendments, to enhance soil health, as compared to conventional farming which relies on the extensive use of synthetic fertilizers and pesticides [13,38,63–65]. The beneﬁcial or adverse eﬀects of any given agricultural practice on soil health are usually evaluated and monitored through a wide array of indicators, which include physical (e.g., structure, bulk density, porosity, aggregate stability, water holding capacity), chemical (e.g., contents of plant macro- and micronutrients, OM, pH, cation exchange capacity), and biological (e.g., enzyme activities, respiration, potentially mineralizable nitrogen, microbial biomass C and nitrogen, microbial functional and structural diversity, diversity of macro- and mesofauna) soil properties. The latter group (biological properties) and, in particular, soil microbial indicators gained much attention lately, owing to their sensitivity, fast response, integrative characteristic, and ecological relevance [66–69]. Indeed, soil microorganisms, which comprise a major fraction of the soil total living biomass, play a key role in soil functioning and the delivery of crucial ecosystem services [70–72]. In this sense, belowground soil biodiversity was recognized as the main driver of many critical soil processes [3,73–75], as well as being responsible, to a great extent, for the stability (resistance and resilience) of the soil ecosystem [76]. The soil ecosystem is known to harbor an overwhelmingly high biodiversity and is then characterized by a high level of functional redundancy. Accordingly, it was suggested [77] that shifts in microbial community composition might not entail relevant changes in soil ecosystem functioning. This assumption can certainly be true for some natural soils, but may nonetheless fall short at low levels of soil biodiversity, as is the case for many agricultural soils [78], where increasing species diversity and a higher number of functional groups were reported to improve soil ecosystem functioning [79]. Assessing belowground soil biodiversity is, thus, imperative when measuring soil ecosystem functionality and, concomitantly, soil health. Unfortunately, the assessment of structural and functional biodiversity in such a complex ecosystem can be an extremely diﬃcult and daunting task [80,81]. Aiming to facilitate this somewhat overwhelming task, the use of molecular and, particularly, “omics” methods and techniques is currently being increasingly promoted. 2.5. Anaerobic Digestion ISFM is deﬁned as “a set of soil fertility management practices that necessarily include the use of fertilizer, organic inputs, and improved germplasm combined with the knowledge on how to adapt these practices to local conditions, aiming at maximizing agronomic use eﬃciency of the applied nutrients and improving crop productivity” [60]. Within the ISFM framework, emphasis is placed on the suitable combination of agronomic practices with mineral and organic inputs and other amendments that are tailored for speciﬁc cropping systems and socioeconomic proﬁles [59]. Finally, in impoverished rural areas of developing countries, it is imperative to urgently emphasize the critical importance of paying much more attention to SOM, agroecological practices, and the value chains that can provide organic fertilizer in large enough quantities. Finally, when applying organic amendments to increase the content of SOM, it must always be remembered that SOM is a complex, dynamic, and highly variable soil constituent. In any case, the persistence of OM in soil clearly indicates the existence of protective mechanisms that slow or prevent their decomposition by soil microorganisms. Thus, some OM inputs are accessed easily by soil microorganisms, and mineralized within minutes, hours, or days. In contrast, OM that becomes protected from microbial activity can remain in soils for years, decades, centuries, or even millennia. There are three main mechanisms that stabilize soil organic C (SOC): (i) physical protection via aggregation, which decreases the accessibility of organics to microorganisms and enzymes; (ii) 5 of 23 Agronomy 2019, 9, 542 chemical protection, through the formation of organo-mineral complexes; and (iii) biochemical protection, through the chemical recalcitrance of organic molecules [61]. From these mechanisms, four soil C pools are often considered during the quantiﬁcation of the soil C sequestration capacity: unprotected (free particulate OM), physically protected (microaggregate-associated C), chemically protected (silt- and clay-associated C), and biochemically protected (nonhydrolyzable C) [61,62]. This latter pool, the biochemically protected SOC pool, is also known as the passive or recalcitrant pool in SOM models. These SOM stabilization mechanisms are of great relevance in studies on soil C-saturation [62]. 3. Beneﬁcial Eﬀects of Organic Amendments Nevertheless, many of these molecular biology tools still have many technical limitations and constraints, which points out the need to be very cautious when drawing conclusions about the responses of soil microbial communities to the application of agricultural practices, including, of course, the application of organic amendments [81,82]. One of the main beneﬁts of the application of exogenous OM to agricultural soil is the restoration and maintenance of the SOM content, which greatly contributes to long-term soil fertility and functioning [83,84]. SOM is, possibly, the most important soil property, as it sustains the physical, chemical, and biological dimensions of soil fertility and health [85]. Moreover, given that SOM simultaneously contributes to both soil fertility and soil C sequestration (and, hence, climate change mitigation), its enhancement was strongly promoted in international food security and climate forums [86]. Regarding the potential beneﬁts of organic amendments for the biological properties of agricultural soils, it is a well-known fact that organic amendments may directly stimulate microbial growth by providing energy and essential nutrients, or indirectly by promoting plant growth and, consequently, 6 of 23 Agronomy 2019, 9, 542 the amount of root exudates in the rhizosphere [23,87]. Apart from increasing microbial growth and biomass, the presence of diverse substrates susceptible to enzymatic hydrolysis within the amendments themselves leads to the stimulation of soil microbial activities [88]. A higher availability of nutrients and growth substrates may also aﬀect soil microbial diversity and composition, by increasing the number of ecological niches and promoting a variety of ecological interactions such as competition and/or antagonism between organisms [26,89]. Biodiversity shifts may then lead to functional changes related, for instance, to plant growth promotion and disease suppression [49,90]. Moreover, increasing structural and functional soil diversity may strengthen the stability of the soil ecosystem, promoting its resistance and resilience against natural and anthropogenic stresses and disturbances [91,92]. These beneﬁcial eﬀects of organic amendments on the biomass, activity, and diversity of soil organisms, in turn, exert a long-term beneﬁcial impact on soil health [93] and also contribute to the provision of key ecosystem services (C and nutrient cycling, disease suppression, etc.). Yet, it is important to highlight that microbial responses to the application of organic amendments vary greatly depending on the nature and lability of the OM present in the amendments themselves [94]. Positive eﬀects on soil biological properties following the application of organic amendments were substantially evidenced in many studies. 3. Beneﬁcial Eﬀects of Organic Amendments A great deal of investigations on the use of organic amendments [41,49,50,82,95–105] reported increases in soil microbial activity and biomass, as well as changes in microbial community composition (with potential concomitant eﬀects on soil functioning) and, to a lesser extent, in microbial diversity. In this sense, a 10-year ﬁeld experiment was conducted [106] to study the eﬀects of replacing a mineral nitrogen fertilizer by an organic amendment (fermented pig manure), at diﬀerent substitution ratios (0, 25, 50, 75, and 100%), on agricultural soil properties. Interestingly, the authors [106] reported an increase in soil bacterial diversity at increasing ratios of chemical fertilizer substitution. In another study [107], the long-term eﬀects of organic versus conventional fertilization on soil microbial communities were investigated, ﬁnding out that the former modulated microbial community composition while increasing microbial richness and diversity. Similarly, other authors [108] reported that organic farming promotes soil microbial diversity and the abundance of beneﬁcial soil microorganisms, with concomitant beneﬁcial eﬀects on the stability of the soil ecosystem. An improvement in soil microbial structural and functional diversity, as well as an increase in bacterial richness and evenness, was reported [109] after the application of organic amendments to agricultural soil. These authors [109] concluded that organic manures can “engineer” the soil ecosystem by selectively modifying the environment, thus enhancing ecosystem sustainability. The beneﬁcial impact of increasing microbial diversity and activity, through the continuous application of organic amendments, on the restoration of saline soils was recently highlighted [110]. The eﬀect of 10 and 20 years of continuous organic farming versus conventional farming practices on agricultural soil health was studied [111], reporting a stimulation of soil ecosystem functioning under organic management which was driven by the alteration of the soil microbial composition rather than by changes in species richness. A lack of long-term impact of organic amendments on soil microbial alpha-diversity, in the presence of signiﬁcant shifts in soil microbial community structure, was observed by other authors [82,112]. Notably, changes in soil microbial structural and functional diversity were reported after the incorporation of a wide variety of organic amendments, including crop residues [113,114], manures [110,115], biosolids [103,116], composted waste [112,117], and digestates [106,118]. These changes were identiﬁed through the utilization of diﬀerent techniques: community-level physiological proﬁling [64,115,119], phospholipid fatty-acid analysis [89,100,115], Sanger sequencing [109], and next-generation sequencing [64,111,117,120]. In addition to improving soil biological properties, organic amendments are also known to positively inﬂuence soil chemical properties. 3. Beneﬁcial Eﬀects of Organic Amendments In fact, the abovementioned positive eﬀects of organic amendments on soil microbial communities are often linked to changes in soil chemical characteristics driven by the application of amendments [45,108,120]. Several authors [121,122] evidenced the key role of soil pH for both microbial community structure and function. Indeed, as described above, 7 of 23 Agronomy 2019, 9, 542 organic amendments can have a direct eﬀect on soil fertility by supplying a wide variety of macro- and micronutrients, which support plant and microbial growth [38]. In addition, organic amendments may aﬀect soil pH and alter cation exchange capacity, thus indirectly inﬂuencing nutrient availability, microbial activity, and, hence, soil fertility [23]. Variations in the composition and maturity of the organic amendments may alter their impact on soil pH. Some amendments contain high quantities of calcium and/or magnesium, which may then cause a kind of “liming eﬀect” on acidic soils, increasing their pH [123]. Liming was shown to signiﬁcantly increase soil microbial activity (as reﬂected by the values of soil dehydrogenase activity) in acid soils [124]. On the other hand, the application of organic amendments can also result in a decrease in soil pH, owing to the release of humic acids derived from the degradation of the organic C pool provided by the amendment [88], and/or due to the nitriﬁcation of the ammonium present in the amendment [96]. The addition of organic amendments may enhance the soil cation exchange capacity, mainly through the increase of the soil C pool, leading to enhanced nutrient availability and reduced nutrient leaching [125]. Nutrient availability can be aﬀected by the biochemical composition of the amendment. In particular, its carbon-to-nitrogen (C/N) ratio can limit soil microbial growth and activity and, thus, inﬂuence the rate of OM decomposition and the patterns of nutrient release [126]. Another beneﬁcial aspect of organic amendments is their ability to immobilize heavy metals through the formation of chemically stable metallo-humic complexes and aggregates [127], or by increasing soil pH (metal bioavailability in soil is commonly reduced at higher pH values) [128]. This beneﬁcial eﬀect was evidenced in many studies [42,96,129]. Also, organic amendments were shown to stimulate the degradation and/or mineralization of organic pollutants by providing nutrients and energy to soil degrading microbial populations [130]. The soil physical characteristics can also be positively inﬂuenced by the application of organic amendments. 3. Beneﬁcial Eﬀects of Organic Amendments In this sense, the incorporation of exogenous OM to soil was shown to improve soil structure (better porosity and aggregate stability) [131,132] and water retention capacity, with concomitant positive eﬀects for soil functioning and crop productivity [133]. Likewise, the stimulation of soil microbial communities through the application of organic amendments may indirectly improve soil structure, since microbial activity (through, for instance, the secretion of exopolysaccharides) and, particularly, hyphal growth can markedly inﬂuence soil aggregation and aggregate stability [134,135]. On the other hand, an increase in soil porosity often reduces soil crusting and bulk density, which could restrict the movement of water and air through the soil matrix [136,137]. In turn, this facilitates the development of the rooting matrix and improves the quality of the habitable space for soil biological communities. Furthermore, organic amendments can aﬀect particle size distribution and the total surface area within the soil, increasing the number and types of available niches for biological colonization. 4.1. Traditional Risks In spite of all the aforementioned beneﬁts, the application of organic amendments to agricultural soil may also exert some detrimental eﬀects on soil ecosystem health. For instance, organic amendments can harbor potentially harmful constituents such as human pathogens, heavy metal(loid)s, organic pollutants, emerging contaminants (antibiotic-resistance genes, endocrine disruptors, microplastics), etc. [103,129,138,139]. Moreover, the inappropriate application and/or overuse of organic amendments may result in other undesired environmental risks, including an excess of nutrients (eutrophication), immobilization of essential nutrients, contamination of underground water, emission of greenhouse gases, and soil acidiﬁcation or salinization [25,39,140]. Altogether, these adverse side-eﬀects threaten the safe usage of organic amendments for agricultural purposes and pose a potential risk to environmental and human health [34]. 8 of 23 Agronomy 2019, 9, 542 Agronomy 2019, 9, 542 Aiming to prevent these potential drawbacks, several legislative tools arose in Europe, including, among others, the Waste Directive (EU) 2018/851, the Directive on the Landﬁll of Waste (1999/31/EC), the Animal Waste Directive (90/667/EEC), and the Sewage Sludge Directive (86/278/EEC) [26]. These regulations provide guidelines on waste disposal and, interestingly, set threshold values for the contaminants present in organic waste. Nevertheless, there still exist concerns about the quality of these legislations, speciﬁcally regarding the lack of data and regulation for most emerging contaminants. On the other hand, it is widely accepted that bioavailable contaminant concentrations are more signiﬁcant for environmental risk assessment than total contaminant concentrations. The potential negative eﬀects exerted by, for instance, toxic heavy metals on soil health are known to depend upon their bioavailable concentrations [141], which, in many cases, are not correlated with total concentration values [72]. In spite of this well-known fact, in most countries, the existing legislation on soil contamination still relies on the values of total contaminant concentration. Owing to their lack of biodegradability, heavy metals have an extremely long persistence in the soil environment [142]. Therefore, the regular application of organic amendments may lead to metal accumulation in soil, with concomitant risks of metal bioaccumulation and biomagniﬁcation along the trophic chain [143]. As previously addressed, the application of organic amendments can enhance the formation of soil aggregates and metallo-humic complexes, which can then reduce the bioavailability of heavy metals. In contrast, the decomposition and mineralization of OM may increase metal bioavailability due to the disintegration of soil aggregates and the formation of soluble organic metal carriers [144,145]. 4.1. Traditional Risks In this regard, a disruption of nutrient cycling processes derived from the metal toxicity caused by the repeated application of biosolids was reported [88]. Reductions in soil microbial biomass were also observed by several authors [146,147], following the application of organic amendments. In addition to inorganic contaminants, organic amendments can incorporate organic pollutants into the soil ecosystem which, in some cases, may also show a high level of persistence and recalcitrance [129,148,149]. Moreover, the breakdown products and secondary metabolites produced during the degradation of these organic pollutants may happen to be even more toxic and persistent than the parent compounds themselves [148]. Furthermore, little is known regarding the breakdown rates of many of these organic pollutants and their transformation products in the soil ecosystem, as well as concerning their potential toxic eﬀects on the soil biota. Therefore, there is an urgent need to determine the potential ecotoxicity of those organic pollutants present in organic amendments, in order to ensure the long-term sustainability and safety of this agronomic practice [150]. Some organic amendments, particularly those derived from raw, unstable animal by-products or biosolids, can contain potentially pathogenic organisms [151,152], including enteric bacteria, parasites, viruses, and fungi [149]. In this regard, it was suggested that Bacillus anthracis and Bordetella pertussis may be dominant human pathogens in animal manure [153], and Escherichia coli and Klebsiella pneumoniae in biosolids [154]. The possibility of pathogen incorporation to agricultural soil through the application of organic amendments is a risk that must be thoroughly prevented given its serious implications for human health. An exhaustive biological characterization of the organic amendments is, thus, imperative in order to minimize, or better avoid, this potential biohazard. As an example, Bibby and Peccia [155] investigated the viral pathogen load of diﬀerent biosolids, identifying >40 diﬀerent human viruses. An excessive and inappropriate application of organic amendments may also result in an excess of nutrients (e.g., phosphorus, nitrogen), which can eventually cause negative environmental consequences such as contamination of watercourses and eutrophication [140,156,157]. On the other hand, organic amendments with a high C/N ratio can entail the immobilization of mineral nitrogen within the soil microbial biomass, since microorganisms are generally more eﬀective than plants at competing for nutrients [158]. In addition, the application of organic amendments to soil may trigger the release of gases to the atmosphere, including ammonia and greenhouse gases, most relevantly methane and nitrous oxides [39,140,159,160]. 4.2. Emerging Contaminants Microplastics (<5 mm in size) arise from the weathering and fragmentation of plastics into smaller particles [162]. Microplastics are extremely or completely resistant to biodegradation, and may cause potential detrimental eﬀects on soil ecosystem functioning and, in particular, on soil organisms via their ingestion and accumulation [163]. Furthermore, microplastics can interact with soil contaminants, altering their ecotoxicity and mobility/bioavailability (many contaminants can become adsorbed onto microplastics) [164,165]. Domestic and industrial wastewaters can carry substantial loads of potentially harmful microplastics [163], which eventually end up in the corresponding wastewater treatment plant. Wastewater treatment plants are very eﬀective at removing microplastics from the treated water [166], resulting in the accumulation of microplastics in the biosolids themselves [167]. The application of diﬀerent rates of biosolids, as drivers of microplastic contamination, into agricultural soil was studied [168], ﬁnding detectable levels of these potentially harmful emerging contaminants in the amended soils. Nevertheless, existing data on the impact of microplastics on the soil ecosystem are still very scarce [169]. On the other hand, in the last few decades, the amount of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in the environment increased substantially due to anthropogenic activities, resulting in their current identiﬁcation as emerging environmental contaminants [170]. Indeed, the overuse and misuse of antibiotics for human and veterinary applications resulted in a proliferation of clinically relevant ARB and ARGs in the environment. Actually, antibiotic resistance is increasingly being recognized as one of the greatest threats for global health, as evidenced by the high-level policy initiatives that recently arose, e.g., the Transatlantic Taskforce on Antimicrobial Resistance, the Global Antibiotic Resistance Partnership, the Joint Programming Initiative on Antimicrobial Resistance [171], endorsed by the World Health Organization [172], and the Political Declaration on AMR of the United Nations [173]. In Europe, the European Commission published the Action Plan Against the Rising Threats from Antimicrobial Resistance [174], which contains 12 actions seeking to palliate the detrimental eﬀects of antimicrobial resistance. This Action Plan was later updated by the publication of the EU One Health Action Plan against Antimicrobial Resistance [175]. Guidelines, actions, restrictions, and objectives are urgently needed, since it was estimated that antibiotic-resistant infections could cause 10 million deaths per year by 2050 [176]. Antibiotics are known to be poorly metabolized in the human and animal body. 4.1. Traditional Risks The emission of these gases depends upon (i) the type of 9 of 23 Agronomy 2019, 9, 542 organic waste, (ii) the applied treatments (composting, anaerobic digestion), (iii) the timing, dose, and method of application, etc. Finally, soil acidiﬁcation and salinization may occur following the application of organic amendments to agricultural soil which can, in turn, aﬀect soil structure, as well as nutrient availability, and, importantly, the mobility and bioavailability of pollutants, thus threatening agricultural productivity and ecosystem health. Some organic amendments can indeed increase the soil’s electrical conductivity (higher salinity and sodicity), with concomitant detrimental eﬀects for crop yield and soil biological activity [161]. Conversely, the use of acid organic amendments or the generation of humic acids (or the activity of some biological processes such as nitriﬁcation) may result in soil acidiﬁcation, often resulting in increased solubility, mobility and bioavailability of soil contaminants [88,96]. 4.2. Emerging Contaminants These authors [181] highlighted the importance of pretreating the raw organic waste and/or establishing oﬀset times between amendment incorporation and crop harvest for safe consumption. Dolliver et al. [198] found that corn, lettuce, and potato crops were able to accumulate sulfamethazine from manured soils, pointing out the concerns about the consumption of low levels of antibiotics from crops grown in manured soils. The dissemination of ARGs among bacteria is mainly driven by horizontal gene transfer (HGT). Indeed, HGT is the main mechanism for genetic variation in prokaryotic organisms, allowing their adaptation to changing environmental conditions and disturbances. HGT facilitates the colonization of ecological niches [188,189] through the acquisition of genes via mobile genetic elements (MGEs), such as plasmids, integrons, and transposons. Although there are three main mechanisms of intercellular DNA movement (transformation, conjugation, transduction) [190], conjugative plasmid-mediated HGT is considered the most relevant mechanism for the dissemination of ARGs among bacteria [191]. MGEs often carry integrons, which act as natural cloning systems and expression vectors of gene cassettes encoding functions of potential adaptive signiﬁcance, e.g., antibiotic resistance [192]. Relevantly, integrons have a key role in the dissemination of ARGs in manure- and biosolid-amended soils [193,194]. In this regard, the rhizosphere was addressed as a major hotspot for HGT [195]. Interestingly, the phyllosphere was also shown to be conducive to conjugative plasmid transfer [196]. Consequently, crops harvested from manure- or biosolid-amended soils can potentially carry ARGs, representing a potential route of exposure to ARB for animals and humans [170,183]. The abundance and diversity of ARGs in organically versus conventionally produced lettuce was investigated by high-throughput quantitative PCR [197], detecting 134 ARGs in the phyllosphere and leaf endophytes of lettuce samples, which were signiﬁcantly enriched in the organically produced lettuces. The same research group conducted an analogous study [183] with lettuce and endive crops in manure-amended soils, obtaining similar results. Other authors [181] detected ARGs and MGEs in vegetables grown in both manured and inorganically fertilized soils. Some antibiotic determinants were exclusively detected in the manured soils. These authors [181] highlighted the importance of pretreating the raw organic waste and/or establishing oﬀset times between amendment incorporation and crop harvest for safe consumption. Dolliver et al. [198] found that corn, lettuce, and potato crops were able to accumulate sulfamethazine from manured soils, pointing out the concerns about the consumption of low levels of antibiotics from crops grown in manured soils. 4.2. Emerging Contaminants Interestingly, antibiotic resistance is frequently associated with metal resistance [199], as the molecular mechanisms underpinning resistance to both antibiotics and heavy metals are often similar [199]. This phenomenon is due to the evolutionary mechanism of co-selection, which drives the simultaneous resistance to diﬀerent pollutants (e.g., metals, antibiotics, biocides) through co-resistance (when diﬀerent genes encoding for metal and antibiotic resistance are allocated in the same genetic determinant) or cross-resistance (when the same gene provides resistance to both antibiotics and metals) mechanisms [200]. Co-selection is a most relevant mechanism for the abovementioned risk of the appearance and dissemination of ARGs associated with the application of organic amendments to agricultural soil, since the presence of heavy metals in the amendments may enhance antibiotic resistance or select for ARB [201]. 4.2. Emerging Contaminants Hence, a considerable amount of these emerging contaminants are excreted unchanged or as active metabolites of the parent species [177], resulting in the presence of a high amount of antibiotics in many wastewaters [178]. Not surprisingly, both livestock manure and wastewater treatment plants are acknowledged as important reservoirs for ARB and ARGs [179,180]. In this sense, the long-term application of animal manure and biosolids to agricultural soil may lead to the introduction, proliferation, and dissemination of these emerging contaminants in the environment [181–185]. It was reported that the repeated exposure of the soil environment to amendment-borne ARGs correlates with the emergence and proliferation of ARGs in indigenous soil bacteria [186,187]. 10 of 23 Agronomy 2019, 9, 542 Agronomy 2019, 9, 542 The dissemination of ARGs among bacteria is mainly driven by horizontal gene transfer (HGT). Indeed, HGT is the main mechanism for genetic variation in prokaryotic organisms, allowing their adaptation to changing environmental conditions and disturbances. HGT facilitates the colonization of ecological niches [188,189] through the acquisition of genes via mobile genetic elements (MGEs), such as plasmids, integrons, and transposons. Although there are three main mechanisms of intercellular DNA movement (transformation, conjugation, transduction) [190], conjugative plasmid-mediated HGT is considered the most relevant mechanism for the dissemination of ARGs among bacteria [191]. MGEs often carry integrons, which act as natural cloning systems and expression vectors of gene cassettes encoding functions of potential adaptive signiﬁcance, e.g., antibiotic resistance [192]. Relevantly, integrons have a key role in the dissemination of ARGs in manure- and biosolid-amended soils [193,194]. In this regard, the rhizosphere was addressed as a major hotspot for HGT [195]. Interestingly, the phyllosphere was also shown to be conducive to conjugative plasmid transfer [196]. Consequently, crops harvested from manure- or biosolid-amended soils can potentially carry ARGs, representing a potential route of exposure to ARB for animals and humans [170,183]. The abundance and diversity of ARGs in organically versus conventionally produced lettuce was investigated by high-throughput quantitative PCR [197], detecting 134 ARGs in the phyllosphere and leaf endophytes of lettuce samples, which were signiﬁcantly enriched in the organically produced lettuces. The same research group conducted an analogous study [183] with lettuce and endive crops in manure-amended soils, obtaining similar results. Other authors [181] detected ARGs and MGEs in vegetables grown in both manured and inorganically fertilized soils. Some antibiotic determinants were exclusively detected in the manured soils. 5. Overcoming the Drawbacks In addition to stabilizing nutrients and OM, which results in a longer-term availability of essential nutrients and a positive eﬀect on soil microbial activity and biomass [51], composting is a well-known mechanism for minimizing or eliminating many unwanted eﬀects of the application of raw organic waste to agricultural soil [25]. Composting, through the hygienization of organic waste, can signiﬁcantly mitigate the risk of incorporation of potential human pathogens into the soil ecosystem, although it may not entirely prevent the regrowth of pathogenic strains [152,202,203]. Moreover, composting is acknowledged to be an eﬀective measure to alleviate antimicrobial resistance during the application of organic amendments to agricultural soil [204,205]. In this sense, total or partial degradation of antibiotic residues through composting processes was widely reported [206–208]. Moreover, as composting processes entail changes in the physicochemical characteristics of the organic waste, the bioavailability of the antimicrobial compounds may be reduced [209]. A reduction in the amount of antibiotics or their bioavailability may eventually lead to a decrease in the load of ARGs. The relatively high temperatures reached during many composting processes may also decrease the load of ARB and ARGs [205,207]. 11 of 23 Agronomy 2019, 9, 542 Agronomy 2019, 9, 542 The anaerobic digestion of organic waste was also proposed as an eﬀective mechanism to reduce the negative consequences of the application of organic waste to agricultural soil. Indeed, the anaerobic digestion of organic waste was often reported to eﬀectively reduce the levels of organic pollutants and potential human pathogens present in organic amendments [57,210]. Furthermore, many authors [211–213] reported the potential of the anaerobic digestion for the removal of antibiotic residues and antibiotic determinants in organic waste. As with composting, this process entails the stabilization of the OM and may then inﬂuence the bioavailability of organic pollutants by promoting sorption processes [56]. Relevantly, physical adsorption was identiﬁed as a key mechanism for the removal of antibiotic residues from organic materials [214]. The anaerobic digestion of organic waste may be carried out under mesophilic or thermophilic conditions, the former being the most widely applied process [215]. However, under thermophilic conditions, better results are obtained regarding the removal of antibiotic-resistance determinants [216,217]. However, the anaerobic digestion of organic waste was repeatedly reported to ineﬃciently remove ARGs [218]. 5. Overcoming the Drawbacks A previous treatment, consisting of applying a thermal hydrolysis prior to the process of anaerobic digestion, was proposed to reduce more eﬃciently the load of ARB and ARGs [215], since the high pressure and thermal conditions yielded by this process promote cell lysis and, thus, the release of degradable components [219]. In any event, both composting and anaerobic digestion recurrently showed their potential for the removal of antibiotic-resistance determinants. Masse et al. [220] concluded that composting was more eﬀective than anaerobic digestion for reducing antibiotic residues from organic waste. Other authors [221] also found that composted manure contained up to seven orders of magnitude less antibiotic-resistance determinants than the one treated with other aerobic and anaerobic treatments. According to these results, composting appears the best option for reducing the reservoir of antibiotic resistance present in raw organic waste. Nonetheless, some authors showed inconsistent results regarding the positive eﬀect of composting on the reduction of ARGs. For example, Peng et al. [222] compared the abundance and diversity of tetracycline (tet) resistance genes in agricultural soils after six years of continuous fresh versus composted manure application. They found nine classes of tet genes, and two of them were signiﬁcantly more abundant in soils amended with composted manure (no reduction in the total abundance of tet genes after manure composting was detected). As described above, owing to the energy and nutrient content of organic waste, many scientists traditionally investigated possible treatment options for such waste, mainly through anaerobic digestion or composting. Currently, within the ﬁelds of waste treatment and waste valorization, the utilization of organic waste as substrates for producing insects, mainly as a protein source for the livestock sector or as a source of fats for biodiesel production, appears a most promising alternative [223]. Processing of (bio)waste with larvae, such as for instance ﬂy larvae, is becoming a promising waste treatment technology. Nonetheless, compared to more conventional waste treatment technologies such as composting or anaerobic digestion, the process performance is variable and the mechanisms driving the decomposition of the organic waste are still poorly understood [224]. The larvae grown on the (bio)waste can then be used for animal feed production, thus providing a protein source to help alleviate the rising global demand for animal feed [225] and, interestingly, revenues for ﬁnancially viable waste management systems [224]. 5. Overcoming the Drawbacks In particular, black soldier ﬂy (Hermetia illucens L.; Diptera: Stratiomyidae) biowaste processing is a treatment technology that received much attention over the last few decades [226–228]. Interestingly, a recent study [225] concluded that black soldier ﬂy biowaste treatment oﬀers an environmentally relevant alternative, with very low direct emissions of greenhouse gases and potentially high reduction in global warming potential. Finally, the possibility of using CRISPR/Cas (clustered regularly interspaced short palindromic repeats), a prokaryotic immune system which protects bacteria and archaea against phage attack and undesired plasmid replication [229], is being investigated to selectively remove ARGs from bacterial populations. Some studies [230,231] indeed conﬁrmed the potential of this methodology to remove ARGs and/or the plasmids that encode those genes. Nevertheless, this technology still exhibits several important drawbacks [232]: (i) ﬁnding an appropriate delivery vector, since phages or conjugative 12 of 23 Agronomy 2019, 9, 542 plasmids normally show narrow host ranges; (ii) unpredictability of the response of the microbial community following the introduction of a delivery vector, due to the inherent complexity of microbial communities; (iii) evolution of resistance to CRISPR/Cas through mutation of the target hosts and/or by exhibiting anti-CRISPR activity (selection for arc genes); and (iv) legislative and social barriers regarding the release of gene-editing systems to the environment, as well as a lack of unanimous acceptance by the scientiﬁc community. 6. Conclusions Acknowledgments: The authors thank the Basque Government for ﬁnancial support through the URAGAN pro Acknowledgments: The authors thank the Basque Government for ﬁnancial support through the URAGAN project. Acknowledgments: The authors thank the Basque Government for ﬁnancial support through the URAGAN project. Conﬂicts of Interest: The authors declare no conﬂicts of interest Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. 4. Hakeem, K.; Sabir, M.; Ozturk, M.; Mermut, A. Soil Remediation and Plants-Prospects and Challenges, 1st ed.; Academic Press & Elsevier: New York, NY, USA, 2014; p. 724. 6. Conclusions In the search for suitable strategies to optimize agricultural environmental sustainability while maximizing crop productivity and production, the paradigm of ecological intensiﬁcation recently gained much interest, in an attempt to enhance the provision of ecosystem services through the consideration of natural ecological processes during the design and implementation of agricultural practices and management systems. In this regard, the application of organic waste and by-products as agricultural soil amendments is a common practice, given their potential to increase crop productivity while enhancing the health of the soil ecosystem. Moreover, the integration of organic waste into the value chain as valuable assets meets the current circular economy paradigm. Organic amendments can be obtained from a wide range of organic materials and origins. Their potential positive eﬀects on soil ecosystem functioning depend upon many factors including their composition, stability, maturity, frequency and rate of utilization, soil type, cropping system, climatic conditions, etc. Therefore, an exhaustive characterization of both the organic amendment and the agroecosystem itself must be performed prior to its application, in order to identify the potentialities and limitations of any given organic amendment for soil and crop health. Composting and anaerobic digestion are acknowledged to be eﬃcient for overcoming some of the potential adverse impacts that organic amendments may exert on the soil ecosystem and, in general, the environment. Pertaining to the potential adverse eﬀects that organic amendments can exert on the soil ecosystem, some emerging contaminants, such as ARB, ARGs, and MGEs, are currently causing much concern as they pose a serious risk to environmental and human health. Given that biological emerging contaminants such as these (ARGs, MGEs, ARB) can persist in the environment and, worse, make copies of themselves and be transferred by HGT to other biological receptors, there is an urgent need to develop more eﬀective treatments of organic waste, which must go beyond the typical hygienization and bacterial disinfection and eﬀectively destroy DNA. 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https://openalex.org/W3124751011	https://europepmc.org/articles/pmc4404741?pdf=render	English	null	A Theory of Social Thermoregulation in Human Primates	Social Science Research Network	2,014	cc-by	19,896	A theory of social thermoregulation in human primates 1 Department of Clinical Psychology, VU University, Amsterdam, Netherlands, 2 Department of Psychology, University of Virginia, Charlottesville, VA, USA, 3 Department of Social Psychology, Tilburg University, Tilburg, Netherlands, 4 Department of Developmental Psychology, Tilburg University, Tilburg, Netherlands, 5 Department of Sociology, University of Groningen, Groningen, Netherlands Beyond breathing, the regulation of body temperature—thermoregulation—is one of the most pressing concerns for many animals. A dysregulated body temperature has dire consequences for survival and development. Despite the high frequency of social thermoregulation occurring across many species, little is known about the role of social thermoregulation in human (social) psychological functioning. We outline a theory of social thermoregulation and reconsider earlier research on people’s expectations of their social world (i.e., attachment) and their prediction of the social world. We provide support and outline a research agenda that includes consequences for indi- vidual variation in self-regulatory strategies and capabilities. In our paper, we discuss physiological, neural, and social processes surrounding thermoregulation. Emphasizing social thermoregulation in particular, we appeal to the economy of action principle and the hierarchical organization of human thermoregulatory systems. We close with future directions of a crucial aspect of human functioning: the social regulation of body temperature. Edited by: Andreas B. Eder, University of Würzburg, Germany Reviewed by: Sascha Topolinski, University of Cologne, Germany Adam Fay, State University of New York at Oswego, USA *Correspondence: Hans IJzerman, Department of Clinical Psychology, VU University, Van der Boechorststraat 1, 1081 BT Amsterdam, Netherlands h.ijzerman@gmail.com Keywords: social thermoregulation, embodiment, social cognition, attachment theory, development, neural reuse, economy of action Introduction Beyond breathing, thermoregulation—the regulation of body temperature—is the most immediate problem for many animals. Food can be stored and budgeted. Mating depends on survival. But survival depends first on oxygen and an adequately heated (or cooled) body. Indeed, a dysregulated body temperature has dire consequences. Normal development in infants requires an optimal internal body temperature across a wide variety of mammalian species—especially in infants born prematurely. Hibernating animals, which maintain a very low body temperature during hibernation, can die during abnormally long winters (Boyles and Brack, 2009). And elderly humans—whose ability to regulate body temperature is often compromised (Van Someren et al., 2002; Shibasaki et al., 2013)—sometimes die during abnormally hot or cold seasonal variations (Mallet, 2002). Finally, in early development, infants require body heat from the caregiver to stay alive, for some animals even to the detriment of the caregiver, who risks death in exchange (Adels and Leon, 1986; Gilbert et al., 2007). Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Received: 02 January 2015 Accepted: 31 March 2015 Published: 21 April 2015 Received: 02 January 2015 Accepted: 31 March 2015 Published: 21 April 2015 IJzerman H, Coan JA, Wagemans FMA, Missler MA, van Beest I, Lindenberg S and Tops M (2015) A theory of social thermoregulation in human primates. Front. Psychol. 6:464. doi: 10.3389/fpsyg.2015.00464 HYPOTHESIS AND THEORY published: 21 April 2015 doi: 10.3389/fpsyg.2015.00464 Citation: The most basic function of social thermoregulation is to help warm another agent, either through touch, or through a warming of the skin in the supporter, where the skin-warming could be a mediating effect while touching the agent. These effects occur in many animals, including humans. We think that higher order social cognitions and behaviors—like attachment, social emotional functioning, and affect regulation—are scaffolded on top of these more basic functions. And some empirical support indeed reveals that social thermoregulation has broader consequences for social cognition, such as attachment (mental models of self and others), emotional functioning, and the degree to which people possess the capacity to self-regulate (consciously or not; cf. Holt-Lunstad et al., 2008). We will discuss these higher order social cognitions in more detail at the end of this review.3 The ultimate purpose of our review is to explain basic social thermoregulation and how early social thermoregulation serves the development of higher-level cognition. In order to do so, we discuss what kind of innate expectations humans are likely to have in regards to being social thermoregulated in the early stages of their life, and how these innate mechanisms have come to rely on core features required for the effective regulation of resources. The most prominent innate mechanism (though certainly not the only one) we think is social thermoregulation.4 We will also suggest that thermoregulatory development can be altered through care- giving style, in a way that allows a new look at the classical idea of attachment theory. In our review we use heuristics to understand how parts of the brain may have matured, and how some more novel functions After discussing the integration of social thermoregulation dur- ing development and what this means for people’s predictive mod- els of others, we discuss how self-regulatory processes in humans may be influenced to a considerable degree by thermoregulation and the processes around it. To date, we are not aware of any other thermoregulation-based model of social cognition, and we hope that our comprehensive review contributes to much needed theory formation related to temperature cues. Finally, we think that our theory provides a first explication of how (and why) social thermoregulation contributes to attachment, emotional function- ing, and the capacity to self-regulate. Citation: IJzerman H, Coan JA, Wagemans FMA, Missler MA, van Beest I, Lindenberg S and Tops M (2015) A theory of social thermoregulation in human primates. Front. Psychol. 6:464. doi: 10.3389/fpsyg.2015.00464 Across many animal species, animals to seek and maintain thermoneutrality—a strategy that can be characterized as seeking temperature-specific homeostasis—by maintaining a gradient between the core temperature and the body’s peripheral temperature (Cannon, 1929; Mallet, April 2015 \| Volume 6 \| Article 464 1 Frontiers in Psychology \| www.frontiersin.org Human social thermoregulation IJzerman et al. of social cognition and behavior—like providing social support to another—have built up from more plesiomorphic social ther- moregulatory functions (Mandler, 1992). That is, we think that social behavior is partially embedded within a thermoregulatory system that (a) includes both reactive and predictive cues (from adjusting to environmental temperatures to giving a sad friend a warm hug), and is (b) hierarchically organized in terms of bioenergetic cost (Satinoff, 1978, 1982; Proffitt, 2006; Beckes and Coan, 2011; Morrison and Nakamura, 2011). More broadly, our contribution is based on four propositions, and we propose a number of different conjectures that we hope will guide research in this area. The first proposition is that much of animal life is not possible without proper thermoregulation. Second, ther- moregulation is bioenergetically costly. Third, energy expendi- tures are diminished by social thermoregulation, when the costs of thermoregulating are shared within social groups or dyads. Fourth, efficient energy conservation through social thermoreg- ulation happens both throughout development and in later life, and both have consequences for the development of physiological mechanisms supporting attachment, affect, perception, and social cognition (Coan et al., 2006; Coan, 2008; IJzerman and Koole, 2011). 2002).1 A striking feature of thermoregulation is its social nature:across many species, fellow animals help to warm in times of danger, disease, and distress, because the net energy cost of thermoregulation decreases when it is done through bodily contact with others. Typically, social thermoregulation occurs within obvious ingroups or among close kin (like mother–child relationships, or between romantic partners), what others have called a Communal Sharing relationship (cf. Fiske, 1991, 1992).2 In our paper, we summarize how social thermoregulation functions across many animal species, discuss how social thermoregulation functions in the human body and brain (in particular its role in the generalized regulation of bioenergetic resources; q.v. Beckes and Coan, 2011), and consider broader implications for psychological functioning. What do we mean by social thermoregulation? 4Despite its centrality in the early life of the infant, we clearly do not intend to say that social thermoregulation is the only feature that is crucial to early attachment, as other “perceptual building bricks” are clearly also important to the life of the infant (like smell, moving in synchrony, and responsive interactions by the caregiver). 1Thermoneutrality, or the thermal neutral zone, is the temperature at which the organism does not need to regulate its body temperature (in near naked male human subjects this is between 33°C and 35°C; Savage and Brengelmann, 1996). 2 Frontiers in Psychology \| www.frontiersin.org Behavioral Thermoregulation and its Physiological and Neural Mechanisms These perceptual building bricks should first determine children’s expectations, and then afford people to let close oth- ers share the risks of living in dangerous (e.g., cold) environ- ments and help carry energetic burdens (e.g., Beckes and Coan, 2011). Both basic predictive and reactive homeostatic control mecha- nisms are foundational to what Tops et al. (2014b) have dubbed predictive and reactive control systems (PARCS). When a chal- lenge or task is perceived as predictable and controllable, predic- tive homeostasis is maintained. By contrast, situational novelty and unpredictability require effortful feedback processing and can trigger reactive physiological responses that are subjectively effortful and potentially incur health costs (Romero et al., 2009). In terms of thermoregulation, reactive control (e.g., the experi- ence of psychological stress) increases core body temperature and decreases skin temperature by restricting blood flow to extrem- ities (e.g., Rimm-Kaufman and Kagan, 1996: Porges, 2001). In this way, reactive control serves to avert physical harm. But its implementation comes at an energetic cost. Besides harm avoidance, energy acquisition and storage are important prerequisites for reproductive success (Bronson, 1989). Thus, in most species, behavioral sequences are organized so that a period of eating and fattening often proceeds mating and caring for offspring. This is particularly important in habitats where food availability fluctuates or is unpredictable (Schneider et al., 2013). In species representing every vertebrate taxa and even in some invertebrates, many putative “satiety” or “hunger” mod- ulators including oxytocin and serotonin, function to schedule ingestive behavior in order to optimize reproductive success in environments where energy availability fluctuates (de Matos Feijó et al., 2011; Schneider et al., 2013). The same modulators such as oxytocin and serotonin are also implicated in thermoregulation. For example, the secretion of oxytocin contributes to vasodilation, a state in which the blood flows to the periphery and leads to an increased skin temperature (Lowry et al., 2009). These sub- stances are also reliably related to a wide variety of social behav- iors, with its main function to be motivated to return to social predictability. But despite Bowlby’s (1969) stress on building bricks (activated through for example touching or clinging), thus far we only have rudimentary understandings of the exact mechanisms that allow almost reflexive responses in Communal Sharing relationships, a feature that has been regarded central to the attachment litera- ture. Behavioral Thermoregulation and its Physiological and Neural Mechanisms The relationship literature is replete with studies that approximate a fuller understanding of how people relate to one another. The diversity of these approaches is enormous: They have included cognitive representations of non-close vs. close others through metaphorical “inclusions” of “others into the self” (Aron et al., 1992) or mechanisms that are central to the maintenance of com- munal sharing relationships through verbal capitalizations (Reis et al., 2010), and they have extended to co-regulatory physiological processes (Sbarra and Hazan, 2008; Waters et al., 2014), and so forth. Our focus predominantly explicates a level lower than most of these approaches, and we focus on the closest of people’s relationships: Communal Sharing relationships. Central to the notion of hierarchical organization—and crucial for the principle of economy of action—are generalized imper- atives to predict and control one’s environment that should be visible in all regulation mechanisms described here. Challenges to prediction and control trigger a host of neural and physiological responses (Sapolsky, 2005; Clark, 2013). Physiological responses to such challenges take one of two forms: reactive homeostatic responses arise to feedback signaling changes in physiological variables that have already occurred or were not predicted. Predic- tive homeostatic responses emerge in anticipation of predictably timed challenges (Moore-Ede, 1986; Romero et al., 2009; cf. Landys et al., 2006). Many related approaches have thus already focused on lower level processes in service of formation and maintenance of the communal sharing relationships, but we believe we offer a very basic—and novel—regulatory mechanism. We already know a considerable amount regarding the secretion of neuromodulators such as oxytocin and vasopressin in relation to the maintenance of close relations (e.g., Insel and Young, 2001). Case in point is a variety of research on breast-feeding (e.g., Uvnäs-Moberg and Eriksson, 2008) and intercourse (e.g., Carter, 1992). Our approach focuses on a related process that we see—as a per- ceptual mechanism—crucial to understanding social relations. Bowlby (1969) thought attachment to first rely on “perceptual building bricks,” that are “activated by stimuli falling within one or more broad ranges, [are] terminated by stimuli falling within other broad ranges, and [are] strengthened or weakened by stim- uli of yet other kinds” (p. 625; see also IJzerman and Koole, 2011). A Framework of Reactive and Predictive Temperature Homeostasis cognition still remain informed conjecture, but we set an agenda to investigate these important links. cognition still remain informed conjecture, but we set an agenda to investigate these important links. As a general rule, we presume that thermoregulatory mechanisms in many animals are (physiologically and cognitively) hierarchi- cally organized by bioenergetic costs, which follows from the more general principle of biological and cognitive systems referred to as the “economy of action,” as well as the tendency to re-use more ancient brain mechanisms for more novel purposes (e.g., Anderson, 2010). The economy of action principle manifests as a general tendency toward energy conservation where possible, due to the uncertainty of resources and the need to bring in more resources than are expended (Davies et al., 2012). Citation: In order to track progress regarding our theoretical presumptions, we have created a project page on the Open Science Framework and we encourage col- leagues to post studies related to our theory (https://osf.io/7d94r/). We propose social thermoregulation to be a key feature of social life, and many existing findings can be interpreted in light of our framework. We do realize that many links between physiology and 2It is not impossible that social thermoregulation is implicated in other rela- tional models, like Authority Ranking. In fact, whether marmots take greater risks in providing warmth to others is partly related to their status in the group, with subordinate marmots needing to provide greater warmth by being on the outside of the group (Armitage, 1999). Similarly so, thermoregulation may even take place in Equality Matching relationships (for example when people meet a potential romantic interest for the first time, which then may move into Communal Sharing; cf. Clark and Mills, 1993). However, we think that, evolutionarily speaking, social thermoregulation primarily developed in the communal sharing context. Social thermoregulation allows for direct survival, and it would have thus been very beneficial to share metabolic resources “to be relatively kind and altruistic to people of [one’s] own kind” (Fiske, 1992, p. 691). 3When we allude to predictive models in this paper, we refer to a predictive control system (as opposed to a reactive control system) that guides behaviors through internal models of the environment, which are formed in long-term memory and kept stable by slow learning of the environment’s predictability. The idea of the internal model originated with Craik (1943) and was later applied by Bowlby (1969), and the ability to apply the internal model to predict future events was noted by Craik (1943) to be an adaptive property of human thought. This idea of the internal model has recently been applied to so-called “situation models” (Zwaan and Radvansky, 1998), and its application in pre- dictive control systems relies on brain networks like the default mode network (that includes the medial prefrontal cortex, posterior cingulate cortex and precuneus) and the dorsal executive network (that includes the dorsolateral prefrontal cortex; see Tops et al., 2014b). April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 2 Human social thermoregulation IJzerman et al. 5There is an additional kind of thermoregulation, called kleptothermy. Klep- totherms are organisms that regulate their own body temperature by stealing heat from other organisms. Kleptothermy mostly happens in poikilotherms and is unilateral, but also happens to some degree in homeotherms. In homeotherms, kleptothermy is mostly reciprocal (Brischoux et al., 2009). We do think that kleptothermy is an interesting class of thermoregulation, poten- tially in relation to communal sharing relationships. However, the complexity of this topic means that it deserves an entirely separate discussion, which is why we do not further elaborate on it here. Behavioral Thermoregulation and its Physiological and Neural Mechanisms Our perception-oriented focus on social thermoregulation further allows comprehension of how people communicate their intentions to bond, how these intentions affect (early) memo- ries of their social relations, and how people engage in bonding experiences—even when they do not have control over language. Furthermore, these building bricks should play an important role in the formation of the internal model of relationships (cf. Craik, 1943). In all of these, we suggest, social thermoregulation plays an important role. But to understand whether thermoregula- tion plays a role at all in the higher order social behaviors and cognitions, we need to know how neurological and physiologi- cal mechanisms behave in the context of behavioral and social thermoregulation. April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org 3 IJzerman et al. Human social thermoregulation shivering through the contraction of skeletal muscles, or sweat- ing through the catabolization of brown adipose tissue (BAT); Grigg et al., 2004; Ivanov, 2006]. This way of regulating body temperature allows the skin temperature to drop, so that core body temperature can be conserved (Akin, 2011). Another way of regulating body temperature for nocturnal animals, like skunks, is to enter torpor—a state that is characterized by decreased body temperature and minimizing energy loss—from midnight until dawn (Hwang et al., 2006).5 In predictable environments, models can be formed that opti- mize predictive control. Predictive control, in turn, allows ener- getic expenditure to be scheduled, precluding the need for costly reactive control. The activation of reactive or predictive control mechanisms depends on perceptions of threats to physiological (e.g., food) and/or social resources, as well as generalized pre- dictability (cf. Stearns, 1992). We reason that early social expe- riences not only facilitate the formation of internal predictive models of attachment, but because of this formation it may induce general inclinations toward either reactive or predictive control. g p A shift from reactive toward predictive temperature control is particularly important because reactive thermoregulation can be so costly. This shift may be seen as one from reactive ther- moregulation for survival toward predictive thermoregulation for care and attachment for sharing the thermoregulatory load (cf. Coan and Sbarra, 2014). In evolutionary terms, the development of more predictable forms of social thermoregulation may have been based on the importance of thermoregulation in the care for offspring. It is likely that parental thermoregulation of offspring evolved initially to reactively control unpredictable and dangerous environments. Behavioral Thermoregulation and its Physiological and Neural Mechanisms In line with prevailing theories that mechanisms of adult attachment and affiliation evolved from mechanisms of caregiving or mother-offspring attachment (Van IJzendoorn, 1995), the development of predictive thermoregulatory control via social channels may have involved an expansion and generaliza- tion from care for offspring to adult attachment. Increased social thermoregulatory control may in turn have increased the pre- dictability of prevailing environmental circumstances, and with that, possibilities for extended investment of time and resources toward offspring, facilitating their predictive model development. Before expanding on this last possibility, we now turn to how temperature control works at different hierarchically nested levels. Neural Mechanisms Underlying Behavioral Thermoregulation One of the most important tasks of the body outside of main- taining an oxygen supply is the regulation of body temperature. In fact, the range of tolerable temperatures within the brain and bodily core is small (Kurz, 2008). Although the brain in particular is capable of surviving for short periods at relatively low temper- atures—a condition followed by generalized torpor and disorien- tation—even small increases in temperature will cause neuronal dysfunction and death (Burger and Fuhrman, 1964; Cabanac and Caputa, 1979). In order for the organism to survive, it thus needs to be able to cool its own body by itself. Interestingly, unlike the maintenance of a continuous supply of oxygen via the lungs, there is no “temperature” organ in the body, despite the fraught nature of temperature in the struggle to survive. Thus, the brain and body contain multiple, often redundant capacities for predicting and adapting to changes in ambient and core temperature, in order to keep those temperatures within optimal limits. These capacities range from prefrontal systems tasked with predicting changes in ambient temperature (frequently related to predictive models) to cutaneous effectors capable of communi- cating ambient temperature information to the central nervous system and thermosensitive cells within the central nervous sys- tem capable of detecting even small changes in the temperature of circulating blood (Nakayama et al., 1961; Hori, 1991). Planning for future potential temperature shifts, mediated by cortical pre- frontal and midline structures, are part of an all-purpose method of maintaining optimal conditions for health and well-being. These processes are capable of contingency planning, prediction and vigilance, and offer the advantage of precluding the need for other systems dedicated to reactive thermoregulatory effort (like while watching the weather report and bring an extra coat for use during a chilly day to warm up). Frontiers in Psychology \| www.frontiersin.org Behavioral Thermoregulation for the Solitary Animal The most basic component in our hierarchy of thermoregulation is behavioral thermoregulation, a way in which many animals exert reactive control to adapt their own temperature, and several types of animals achieve this in different ways (simply in their attempts to stay alive by conserving energy). Related to the notion of predictive and reactive control—and its relation to energy expenditure—an organism’s environmental temperature plays a crucial role in shaping metabolic activity, while it also influences sexual reproductive behaviors across species (Bronson, 1989). But what are some ways in which animals thermoregulate themselves? The most basic form of thermoregulation can be engaged in by poikilotherms (environmentally dependent, cold-blooded animals), who regulate their temperature using their external environment. For example, wood turtles move from one area to the other (from forest clearings into streams) to use the envi- ronmental temperature to regulate their own body temperature (Dubois et al., 2009; Akin, 2011). Homeotherms (animals with constant, environment independent and typically high body tem- peratures), like poikilotherms, can use their environment to regula their temperature. In addition, they rely on internal physiolog- ical mechanisms to regulate body temperature when external temperatures are within non-threatening limits [such as through The most basic component in our hierarchy of thermoregulation is behavioral thermoregulation, a way in which many animals exert reactive control to adapt their own temperature, and several types of animals achieve this in different ways (simply in their attempts to stay alive by conserving energy). Related to the notion of predictive and reactive control—and its relation to energy expenditure—an organism’s environmental temperature plays a crucial role in shaping metabolic activity, while it also influences sexual reproductive behaviors across species (Bronson, 1989). But what are some ways in which animals thermoregulate themselves? Those on-line methods for detecting internal and external fluc- tuations in temperature are themselves complex. The mechanisms through which the body responds to challenges to core body temperature are more complex still, and, as we cannot emphasize enough, they are organized hierarchically in terms of their own The most basic form of thermoregulation can be engaged in by poikilotherms (environmentally dependent, cold-blooded animals), who regulate their temperature using their external environment. For example, wood turtles move from one area to the other (from forest clearings into streams) to use the envi- ronmental temperature to regulate their own body temperature (Dubois et al., 2009; Akin, 2011). Behavioral Thermoregulation for the Solitary Animal Homeotherms (animals with constant, environment independent and typically high body tem- peratures), like poikilotherms, can use their environment to regula their temperature. In addition, they rely on internal physiolog- ical mechanisms to regulate body temperature when external temperatures are within non-threatening limits [such as through April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org 4 Human social thermoregulation IJzerman et al. energy demands. What follows here is a brief overview of these systems and processes. In subsequent sections we review how these systems and processes interact with others known to be involved in social contact and social regulation, as well as some conjectures about how (and why) the brain may be designed to minimize its own bioenergetic expenditures in maintaining desirable core body temperatures through proximity to reliable social resources. Throughout, the primary focus is on mechanisms of increasing body temperature, as that is the most frequently used form of thermoregulation to involve the use of social mech- anisms—after all, an increase in body temperature is immediately dangerous, and a response by others would be too slow [curiously, cold (vs. warm) receptors are also found 3–10 times more likely in most areas of the body; Guyton, 1991]. changes in the temperature of circulating blood directly through a small proportion (approximately 30% for warm, less than 5% for cold) of thermosensitive neurons. These neurons are capable of altering their activity in response to even quite small changes in blood temperature and play a role in both heat retention and heat loss relevant to the brain and core (Nakayama et al., 1961; Guyton, 1991). Temperature Regulation The brain and body are capable of mounting many defenses against undesirable challenges to core body temperature. Given the diverse reactive mechanisms that are involved across the body, these strategies are often very expensive in terms of energy expen- diture. Once temperature changes are detected by any or all of the means described above, regulatory mechanisms are active almost immediately. Again, central to these regulatory mechanisms is that they are hierarchically organized in terms of their relative bioenergetic costs, following from this general principle referred to as the “economy of action” (Proffitt, 2006; Davies et al., 2012). Temperature Detection Body temperature is set as a function of core bodily needs—such as optimal enzyme activity, the maintenance of metabolic needs, and defense against infection. Because a small window of core body temperature is necessary for optimal functioning, the brain and periphery have multiple means of predicting and detecting change both in and outside of the skin. The first and probably least expensive strategy to regulate tem- perature for humans involves the strategies we have alluded to above: Prior predictions and contingency planning—in short, pre- dictive ambient temperature control with technology (heating and cooling systems) or by using thermoregulatory clothing (coats, shorts, etc.). Like other ways of outsourcing mental functions to the environment, abilities to prepare for predicted changes in ambient temperature derive from a mechanism altogether differ- ent from the mechanisms related to more proximal thermoregula- tory adjustments, including prefrontal and cingulate mechanisms related to vigilance, working memory, and executive control. By being able to predict temperature in advance, it precludes more downstream thermoregulatory efforts. Being able to predict temperature in advance allows the organism to schedule energy expenditure in service of thermoregulation more efficiently. It is also exactly in the management of this costly ability that we will later locate social thermoregulatory strategies. Humans can detect and predict temperature in many ways. The most complex of these—likely a uniquely human trait—is the ability to predict changes in ambient temperature long before such changes could potentially impact temperature in the body’s core. Detection of temperature can thus go via predictive systems outside of one’s own physical body. Culture affords people to use technology, and do this many days in advance—an advantage that is hard to overstate, since such advanced prediction capa- bilities enable people to alter behaviors and environments such that more proximal (and bioenergetically expensive) detection and adjustment measures are unnecessary. A way related to reactive temperature control runs via com- plex physiological systems of temperature detection and response systems. A critical central nervous system mechanism for this appears to be the preoptic area (POA) of the anterior hypotha- lamus. The POA serves many integrative functions, including the utilization of multiple streams of temperature-related information sources for the purpose of both up and down regulating core body temperature as needed, and maintaining core body temperature “settings” through a variety of mechanisms. Frontiers in Psychology \| www.frontiersin.org Social Thermoregulation Saves Energy: Thermoregulation across Relationships and Development Thermoregulation to warm the body is a costly business for many solitary animals, in part because it is tied to reactive control, but also because of the many regulatory mechanisms involved across the body. This may be best exemplified through understanding energy regulation in the case of temperature, glucose availabil- ity, and torpor (which manifests in decreased activity and lower temperature). For example, the onset of torpor is tied to glucose availability in deer mice (Nestler, 1991; Stamper and Dark, 1997). Furthermore, daily torpor has been suggested to be common in sunny regions for the marsupial Sminthopsis macroura, sugges- tively because the environmental temperature helps the animal to conserve energy (Geiser and Drury, 2003). Comparably, Sockeye salmon have been suggested to initiate torpor more frequently when food resources are limited (Brett, 1971; Lillywhite et al., 1973). Keeping all this in mind, the most efficient way to con- serve energy is by seeking an optimal environment in terms of energy savings before changes in temperature occur, in which the organism can attain “thermoneutrality,” which all depends on an idiosyncratic level of comfort for the animal. In other words, the level of comfort relies on metabolic activity (Terrien et al., 2011), which differs across organisms, across the lifespan, and across social contexts.8 One of the key principles throughout our hierar- chy of thermoregulation is that the regulation of body temperature is closely tied to limiting energy expenditure, and thus, solitary thermoregulatory activity is best minimized if possible, or, at the least, made as efficient as possible. Thermogenesis by BAT activity is fairly well characterized, especially in rodents, as a regulation mechanism for cold-induced thermogenesis (Saito, 2013). In humans, most of what is known about the vital thermoregulatory role of BAT concerns early infancy, and only recently we know that BAT plays an important role in adult humans (Nedergaard et al., 2007; Cypess et al., 2009; Virtanen et al., 2009). Remarkably, BAT recruitability in humans has even been linked to incidental cold exposures (Lee et al., 2014)6. Unsurprisingly, thermoregulatory activity within BAT is mediated in part through the POA, but also through circuits within the medulla and spinal column. Metabolic activity within BAT is higher than in many bodily tissues, and one consequence of metabolism is the generation of heat as chemical bonds are broken down. 6Interestingly enough, Lee et al. (2014) suggest that the change in BAT radio density may well be due to a process they refer to as “fat-browning” or the transition of white adipose tissue into brown adipose tissue. Based on this, we can ask the question to what degree social factors, such as the predictability of one’s social environment, play a role in fat-browning, and to what degree BAT is similarly organized in terms of predictive and reactive temperature homeostasis. Temperature Detection For example, during threat responding piloerection may serve as part of a warning signal to conspecifics (a warning that is obviously vestigial in humans), and vasoconstriction may buffer the impact of injuries by limiting potential blood loss during conflict. core body temperature, at least at the level of thermosensitive neurons in the POA and elsewhere. Although effective, shivering is a very costly strategy, and thus best avoided if possible. Impor- tantly, the precise mechanisms by which shivering is initiated as a defensive thermoregulatory response remain largely unknown, but are likely to include the POA, the parabrachial nucleus, and circuits of the medulla—all systems and circuits implicated in other thermoregulatory efforts.7 By the time changes in temperature are detectible in the core (and indeed in the POA directly via thermosensitive neurons), the thermoregulatory strategies outlined above can be considered insufficient, and the body is thus forced to take reactive mea- sures that are more bioenergetically expensive. Among the options at this stage are thermogenesis via increased metabolic activity within BAT, sympathetically mediated increases in heart rate, and shivering—the rapid, small muscle movements in and around the visceral core that increase local body temperature (Golozoubova et al., 2006). 7What we are thus not trying to say is that individual differences in ther- moregulatory efforts/abilities do not occur across individuals and even across cultures. It is likely that the body has adapted to its temperature environment and to the direct social exigencies. There are clear indications that there are differences in thermoregulatory abilities across cultural/ethnic groups (Daa- nen, 2003; Maley et al., 2014), gender (Davies, 1979), individuals (Havenith, 2001), and lifespan (Davies, 1979; McDonald et al., 1989; Scarpace et al., 1994). To what degree these thermoregulatory differences rely on specific cultural, social, or physiological factors, remains to be seen. 8Within the social psychological literature, it is common to revert to explana- tions based on positive or negative affectivity, also in relation to temperature cues (Zajonc, 1980). Beyond that this alternative process has been empirically negated (see, e.g., Szymkow et al., 2013, for one example), from the idea that we propose here it also follows that skin and ambient temperature are co- dependent and the valence of a temperature cue depends on the demand of the organism and the environment, and it is likely that preferences rely on thermoregulatory inferences. Temperature Detection The skin is an impor- tant initial defense against changes in core body temperature resulting from changes in ambient temperatures. As with many homeostatic systems, changes in skin temperature are detectable before—sometimes long before—threats to core body tempera- ture are detected. Although we still have limited knowledge of the precise mechanisms by which effectors within the skin relay information to the POA regarding ambient temperature, the effect is so rapid and pervasive that brain and core temperatures rarely change in response to ambient cooling of the skin (Guyton, 1991). At the proximal thermoregulatory level, one of the first reactive mechanisms recruited to maintain optimal levels of core body temperature is also the one most associated with predictive tem- perature outcomes—the modulation of blood to the periphery of the body. On the one hand, when ambient temperatures are very high, vasodilation can allow more blood out to the periphery, on the assumption that there is little danger that circulating blood will adjust internal temperature. On the other hand, vasoconstriction is an early defensive against ambient chill, such that smaller amounts of blood are delivered to the periphery, where cooling might threaten core temperatures (though, in extreme cases of cold, vasodilation has been observed; Daanen, 2003, 2009). Impor- tantly, this is thought to be implemented in a predictive fashion, such that cutaneous effectors within the skin detecting changes in ambient temperature cause vasoconstriction before—often long before—any such changes are detectable in the body’s core. What is known is that cutaneous sensory information about ambient temperature is mediated through the dorsal horn of the spinal column and the lateral parabrachial nucleus of the mid- brain—mechanisms that also deliver temperature information from the viscera, though precisely how this is accomplished is not as well known. Interestingly, the POA is capable of detecting Often consonant with vasoconstriction strategies is piloerec- tion, otherwise commonly known as “goosebumps.” Piloerection refers to the constriction of cutaneous muscles that cause hairs in the skin to stand erect. Importantly, it also causes the skin to “bunch” such that it is less porous with prevailing ambient April 2015 \| Volume 6 \| Article 464 5 Human social thermoregulation IJzerman et al. conditions, thus conserving body heat. Importantly, both vaso- constriction and piloerection are sensitive to psychological as well as ambient temperature conditions. mmunal Ways to Thermoregulate Socially Communal Ways to Thermoregulate Socially The strategy of huddling has been detected across different rodent families. The overview by Ebensperger (2001) shows that hud- dling strategies in the service of energy conservation has been detected across many different rodent species.11 In humans, there may be comparable strategies to reduce the energetic costs of thermoregulation. Most of the research efforts in humans on huddling (which in humans focuses on touch) to date however have been dedicated to other (related) areas of research, such as social judgment (Erceau and Guéguen, 2007), prosocial behaviors (Crusco and Wetzel, 1984; Hornik and Ellis, 1988; Field, 2010), valence of affect (Vallbo and Johansson, 1984), and emotional communication (Hertenstein et al., 2006, 2009; for reviews, see Gallace and Spence, 2010; Tjew-A-Sin and Koole, 2013). , ) Beyond these direct empirical examples, a review considering eight hypotheses on the evolutionary causes for group living amongst rodents found that social thermoregulation was con- sidered as one of only two with considerable empirical support (reducing risk of predation was the other one; Ebensperger, 2001). Ebensperger (2001) cites two types of research findings in support of the social thermoregulation hypothesis. First, sociality and communal nesting amongst species prevails for those in relatively colder areas or seasons (e.g., grouping and burrow sharing). Sec- ond, energy expenditure amongst rodents decreases if they are allowed to huddle in experiments with their conspecifics. A final compelling experimental example has been provided amongst the Octodon degus (a Chilean rodent), whose basic metabolic rate at 15°C is 40% lower when housed with three or five vs. alone, while the surface temperature of the huddled animal significantly increased (Nuñez-Villegas et al., 2014). Together this supports the idea that huddling causes energetic demands to be reduced, providing a co-regulation of body temperature (see also, Akin, 2011), in part through the reduction of the area of the animals that is exposed to the environment. We will first address this strategy in the following section. Later we also discuss thermoregulation throughout development and caregiving, and apply reactive and predictive control to forming predictive models of social ther- moregulation from early infancy to later life. p j ) Yet, surprisingly little research has been devoted to the role of touch, emotion regulation, and the basic metabolic rate amongst humans. 9We note that they “upregulate” as the cooling of the body does not nearly pose as much as a threat to the body as an increase in temperature. Downregulating body temperature through others is thus not likely to be quick enough to save the organism from sustaining brain damage. 10One reason why net energy expenditure is lower is because being ther- moregulated indicates an increased predictability of the environment and is related to activity of the vagus nerve. Specifically, a decreased predictability of the environment is associated with an increased activity of people’s reactive control systems, which is typically more costly than the activation of predictive systems. Supporting this idea, vagal tone is associated with social wellbeing and social support seeking (Geisler et al., 2013). 11Evidence on huddling strategies was cited by Ebensperger (2001) on Bathy- ergidae (Heterocephalus glaber—Withers and Jarvis, 1980), Muridae (Abrothrix andinus, Abrothrix lanosus—Canals et al., 1997; Apodemus agrarius—Tertil, 1972; Apodemus flavicollis—Fedyk, 1971; Clethrionomys glareolus—Górecki, 1968; Gebczyński, 1969; Gebczyńska and Gebczyński, 1971; Eligmodontia typus—Canals et al., 1997; Meriones unguiculatus—Contreras, 1984; Mus musculus—Prychodko, 1958; Martin et al., 1980; Contreras, 1984; Microtus townsendii—Andrews et al., 1987; Ochrotomys nuttalli—Springer et al., 1981; Ondatra zibethicus—Bazin and MacArthur, 1992; Peromyscus leucopus— Glaser and Lustick, 1975; Vogt and Lynch, 1982; Peromyscus maniculatus— Andrews and Belknap, 1986; Phyllotis darwini—Canals et al., 1997; and Rei- throdontomys megalotis—Pearson, 1960), Myocastoridae (Myocastor coypus— Moinard et al., 1992), Octodontidae (Octodon degus—Canals et al., 1989), and Sciuridae (Ammospermophilus leucurus—Karasov, 1983; Glaucomys volans— Stapp et al., 1991), but also for round-tailed ground squirrels Spermophilus tereticaudus (Dunford, 1977), southern flying squirrels Glaucomys volans (Stapp et al., 1991; Layne and Raymond, 1994), gray squirrels (Koprowski, 1996), house mice (Morton, 1978), meadow voles Microtus pennsylvanicus (Madison et al., 1984), red-backed voles Clethrionomys rutilus (West, 1977), white-footed mice (Nicholson, 1941), and wood mice Apodemus sylvaticus (Wolton, 1985) during cold parts of the year (while converging support has also been detected in deer mice, white-footed mice, and free-ranging alpine marmots Marmota marmota; Sealander, 1952; Andrews and Belknap, 1986; Arnold, 1988, 1990a,b). Social Thermoregulation Saves Energy: Thermoregulation across Relationships and Development The POA plays a similar role in activating sympathetically mediated cardiac thermogenesis, particularly through increases in heart rate—increases that also impact overall metabolism and, hence, body heat. Indeed, increases in heart rate are mediated through many mechanisms similar or identical to those mediating BAT thermogenesis (Astrup et al., 1985), and can probably be considered alongside BAT in terms of bioenergetic cost. g g By contrast—and in line with our earlier thoughts on how energetically expensive solitary thermoregulation actually is—shivering can in many ways be seen as the body’s last resort for reactive temperature homeostasis. Experimental support sug- gests the thermal threshold needed for shivering to commence is lower than that of any other known thermoregulatory mechanism (Badjatia et al., 2008), as heat production can be increased two- to threefold, and from an energy perspective, its efficiency is only about 10% (Yousef, 1987; Höppe, 1993). In shivering, motor neu- rons are rapidly fired in small rhythmic bursts that actually leave the body shaking and that generate a great deal of heat through increases in local metabolism. Indeed, by the time the body has begun shivering, there likely have been noticeable changes in Thus, solitary thermoregulation is very costly and it would be cost efficient if the body had cheaper mechanisms in order to stay alive. Evolution has resolved this predicament through April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org 6 Human social thermoregulation IJzerman et al. another level in our hierarchy of thermoregulation, that is, the co-regulation of body temperature through conspecifics, which allows formation of models for predictive control of the temper- ature environment. Interestingly enough, in human brain mecha- nisms for social interaction (like sex) are largely overlapping with those of behavioral thermoregulation (Satinoff, 1982). Behav- iorally, conspecifics help upregulate temperature to thermoneu- trality or homeostasis through huddling, and this strategy has been detected across a variety of animals.9 This principle is best illustrated by the social behavioral strategies that, across species, have evolved to reduce energetic costs to warm one’s body (all resulting in a reduction of the net costs of total thermoregulation for groups of animals). As one example, research reveals that African Four-Striped Grass mice utilize less energy per individual in large groups as compared to smaller groups when they are below their thermoneutral zones in the laboratory (Scantlebury et al., 2006).10 for infants. Social Thermoregulation Saves Energy: Thermoregulation across Relationships and Development Further, many animals also do so reciprocally, as adult birds and mammals shield each other from harsh climatic environments through huddling (Gilbert et al., 2007; Terrien et al., 2011). We now turn to examples from the animal literature where huddling as a means for social thermoregulation has been detected. mmunal Ways to Thermoregulate Socially One study has linked face and body temperature specif- ically to the role of touch in sexual arousal, where skin tem- perature of the face increased after highly intimate (face and chest) contact (Hahn et al., 2012). There are some other studies that were documented on the role of skin-to-skin contact and its relation to skin temperature which may show the connection between touch and skin temperature, to which we return when we discuss developmental thermoregulation. More indirect links have been provided between close relationships and the regula- tion of metabolic resources: Henriksen et al. (2014) found that being socially integrated is related to a lesser consumption of drinks containing sugar during pregnancy, while thinking about a romantic partner (who is likely to be a source of warmth) That thermoregulation is done socially can be found across homeotherms and poikilotherms, and research observations have revealed that close kin do so to support the weakest in the group, both throughout development as well as incidentally: Homeotherms for example engage in longer lasting social ther- moregulation like nest-sharing to increase the chance of survival April 2015 \| Volume 6 \| Article 464 7 Frontiers in Psychology \| www.frontiersin.org IJzerman et al. Human social thermoregulation leads to a slight increase in glucose levels (Stanton et al., 2014). The first part of a research agenda on social thermoregulation should thus focus on uncovering the relationship between social integration, thermoregulation, and metabolism regulation in humans. In general, the huddling strategies that we have discussed in the previous section also prove their use in the care of human neonates. Infants across many animal species do not yet have the surface to body area to be able to thermoregulate their own body. Young infants have a stress hyporesponsive period (SHRP), and maternal thermoregulation maintains this SHRP, protecting the developing brain of the infant (at least in rats), further allowing it to mature for predictive capacity (Suchecki et al., 1993). On the flipside, maternal separation (and the inevitable coldness and unpredictability of the environment) leads to elevated stress levels. For a human infant these elevated stress levels immediately pan out in a greater deviation between core and skin temperature, and this thermoregulatory risky state can even lead to the infant’s death (Mori et al., 2010). Thermoregulation Throughout Development Thermoregulation Throughout Development The first research example on social thermoregulation that is well known to psychologists is that of Harry Harlow’s rhesus monkeys. In his most famous contribution on this topic, The Nature of Love, Harlow (1958) discussed rhesus monkeys that he raised either with a surrogate mother made out of wire, or one made out of terrycloth (the surrogate, non-soft wire mother caused greater psychological problems in the rhesus infants’ later lives). In a lesser-known contribution, Harlow and Suomi (1970) discussed their observation of a rhesus monkey raised with a physically warm surrogate mother. After switching to a physically cold surro- gate mother, the researchers noted that the infant rhesus monkey displayed remarkably less affiliative behaviors with this cold sur- rogate mother, and was also much less explorative. Once Harlow and Suomi (1970) switched back the warm mother, the rhesus monkey quickly returned to the original level of affiliative and explorative behavior and “forgave” the surrogate for her coldness. Conversely, for the infant rhesus monkey that was originally raised with a cold surrogate mother the level of affiliative behavior never reached that level of the rhesus monkey originally raised with a warm mother displayed. Harlow’s studies on rhesus monkeys showed how the infants had a preference for warm surrogate mothers, or cotton cloth mothers that provided physical comfort. The explanation for these studies—in line with our reasoning—is that the warm, comfort- able mother provided metabolism regulation. Comparably, rat dams choose an environment (a warm one) in which it is less costly in terms of thermoregulation to provide their caregiving. Reminiscent of Harlow’s studies, this even occurs when their nest is in a colder environment (i.e., they prefer warmer environ- ments over their own nests). Interestingly enough, these rats were observed to optimize their expenditure of resources: When they could choose their thermal environment, the rat dams chose envi- ronments that were somewhat below their thermoneutral zone. They did so because typically require less feeding in somewhat colder environments. The author argued that this helped the rat dam to conserve energy (see, McFarland, 1977). Together, this thus further supports the idea that that thermoregulation is closely tied to being as energy efficient as possible. mmunal Ways to Thermoregulate Socially Thermoregulation by the caregiver is required for survival and we thus suspect that the same process of brain maturation due to maternal thermoregulation also occurs in humans. To close this section, the supposed link between social ther- moregulation and energy conservation is not without exception: The support for energy conservation as dependent on seasonal changes in ambient temperature through group formation was not detected in prairie voles Microtus ochrogaster (Getz et al., 1993; Getz and McGuire, 1997; but see Getz and Hofmann, 1986; Getz et al., 1987), or seems unrelated to seasonal changes in ambient temperature (Blumstein and Arnold, 1998). We think of these latter (rare) findings as outliers in the role of social ther- moregulation in the service of energy conservation, and poten- tially helpful in understanding the boundary conditions of social thermoregulation. Additionally, for altricial mammals, episodes of hypothermia have been suggested to reduce growth through a reduction of biochemical activities. Again, the reasons for instability in ther- moregulation and a following insufficient growth are likely to be found in the problem of energy expenditure. Mothers have typically “solved” this problem by having a body that is evolved to deal with this. For example, the neuromodulator oxytocin is typically involved in nursing behaviors, such as touching and breastfeeding. Additionally, energy intake (at least in mother rats) can be increased through increased hunger that is related to the secretion of oxytocin. Further support for this idea is that in caregiving rats, if the neural pathways for milk ejection are lesioned, energy intake is decreased back to their normal levels (Uvnäs-Moberg and Eriksson, 2008). Thermoregulating the Young The anecdote by Harlow and Suomi (1970) provides fascinating directions to explore how thermoregulation functions in early development. One of the ways to explain their observation may well be that one of the most stressful and risky periods that animals have in their lives is just after being born and the period thereafter. And, in fact, the security that a warm feeling offers against cold stress should help regulating the infant, mostly to be protected from hyperthermia. The length that an infant needs to be ther- moregulated by a caregiver in its quest to survive varies species- by-species. As one example, golden hamsters are able to regulate their own body temperatures by the age of 14 days (Leonard, 1982). Human infants are even more fragile and need to rely on older individuals for a considerable time for the regulation of body temperature to prevent falling prey to reactive thermoregulation (Winberg, 2005). Just like incidental forms of thermoregulation (e.g., to shield from harsh environments), developmental thermoregulation is typically achieved through huddling. For example, Norway rats follow economy of action principles in caregiving through form- ing a huddle, which decreases the exposed surface area of the developing pups relative to their internal heat producing system (Woodside et al., 1981). Shielding young animals from harsh environments is very important in the context of reproduction, April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org 8 Human social thermoregulation IJzerman et al. smallest temperature decrease was found for the newborns that were held skin-to-skin with their mother (Bystrova et al., 2003). which is why many species use this strategy to protect their young. In marmots, juveniles profit most from huddling as they typically have the lowest fat reserves (Armitage et al., 1976; Arnold, 1988). Also for infants of emperor penguins, a species that assumes collective breeding tasks during extremely cold Antarctic winters, huddling increases the chances of survival (Gilbert et al., 2007). We can reconsider earlier research conducted by one of our own labs (IJzerman et al., 2013) and other labs (Fay and Maner, 2012) in light of our theory. In both studies, people’s perceptions of the reliability of their social world (their attachment styles) moderated the effects of warm (vs. cold) cues. Evidence in Humans of Development Thermoregulation There are some indications that skin-to-skin contact closely relates to the maintenance of body temperature. One research example revealing the importance of skin-to-skin contact finds that feet of babies that were held skin-to-skin, as compared to those that were removed from the skin of the mother (and swad- dled in six cotton cloths and a cotton blanket as an outer layer) revealed a greater increase in skin temperature. Beyond that this provides first support for the co-regulation of body temperature throughout development, the researchers proceeded to conclude that young infants are equipped with some kind of innate program to seek warm comfort from their caregiver (Bystrova et al., 2007; see also Caporael, 1997; Jonas et al., 2007; IJzerman et al., 2012). Consequences for Psychological Mechanisms Most of what we have provided until here can be explained through people’s direct interaction with the environment, and much of it can be understood through a direct coupling of action and agents that can mostly rely on biosocial models (e.g., Chemero, 2009; Beckes et al., 2014). But we would like to extend our model further, and provide a first consideration into what our ideas may mean for more complex cognitive systems. We think that the consequences are twofold: First, and most obvious to psychologists, there should be considerable consequences of including our model of social thermoregulation into a predictive model of self and others (i.e., attachment style). We will reinter- pret existing research and discuss the formation of self, and the formation of the predictive model for social thermoregulation. Furthermore, we discuss earlier research in light of our theory, and indicate how people use temperature estimates for prospection. Finally, our theory should also more closely tie such predictive models to basic physiological mechanisms, and these should in turn be heavily influenced by their social context. Risk for Caregivers in Provision of Social Thermoregulation This kind of thermoregulation is rightfully communal, as it is not without risk for the caregiver: Taking care of the infant’s body temperature increases the chances for disturbances in the caregiver’s thermoregulation processes. Lactating Norway rats for example are vulnerable to acute hyperthermia when in contact with their pups (Woodside et al., 1981; Adels and Leon, 1986). Furthermore, loss of mass is greater and survivorship is lower for marmots that have to care for their own infants (without the help of subordinate marmots), while also the loss of mass for both the caregivers and subordinate marmots is greater with the presence of infants (Armitage, 1999). Although in humans these severe risks seem less likely because of the way our cultures have evolved, these findings for animals suggest that social thermoregulation first evolved in the context of Communal Sharing relationships (= in the context of close kin, providing for the other, even at the risk of death for the self). Still, while there may be no immediate risk of death for humans, such communal thermoregulation for humans is still heavy in terms of energy demands, and should mostly occur with predictable relationship partners (a finding for which we have some preliminary support from our own lab; Wagemans et al., 2014). Thermoregulating the Young This is important, because the review by Winberg (2005) further suggested—but provided little empirical evidence for—the idea that skin-to-skin contact in the infants’ early life stages also contributed to a better relationship with the caregiver (and potentially to later social relationships). The research examples we cite provide first such evidence for the hypothesis Winberg (2005) proposed. While early research support thus exists, we add as an agenda point the importance of testing the increased predictability of energy provision through social thermoregulation. Frontiers in Psychology \| www.frontiersin.org Social Thermoregulation and the Development of Higher Order Cognition The development of higher order cognition makes it possible for human beings to live the way they do. Higher order cognitive func- tions, like predictive analysis, inhibitory control, self-reflective consciousness, abstract thinking, willed action, and a theory of mind are on top of the hierarchical structure and typically are thought to correlate with activity in the prefrontal cortex (e.g., Dietrich, 2003). These types of activities allow people to partic- ipate in complex cultural structures (Baumeister, 2005), partake in complex social normative systems, and choose environments that support these abilities (Lindenberg, 2013). After reviewing such experimental studies on skin-to-skin con- tact, Winberg (2005) suggested that keeping the infant against the skin has major effects on the feelings of security of the infant, with infants crying less and having a smaller reduction in skin tem- peratures. For example, children that were just born showed dif- ferences in skin temperatures depending on treatment condition. Children were swaddled in six layers of cotton cloth, placed in the mothers’ arms, or held skin-to-skin with the mother. The decrease in skin temperature, which accompanied an increase in crying and interpreted by the authors as being a result of the “stress of being born,” was greatest in the swaddling condition, then the condition in which newborns were placed in their mothers’ arms, and the We believe that effective social thermoregulation is one cru- cial facet that makes this all possible, by allowing growth of the prefrontal cortex combined with the development of internal models of one’s social environments. The growth of these higher order structures—metabolically speaking—is expensive. We think that the infant brain makes predictions about the availability of April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org 9 Human social thermoregulation IJzerman et al. notions that we consider central to predictive models have been discussed, viz., conceptual metaphor theory (Lakoff and John- son, 1999), neural reuse theory (Anderson, 2010), and perceptual symbol systems (Barsalou, 1999, 2008). We build on perceptual symbol systems and neural reuse theory here (for explanations for our preference, see IJzerman and Koole, 2011; Beckes et al., 2014). Building on these theories and Tops et al.’s (2010) theory of Predictive and Reactive Controls Systems, we explicate a general reliance on predictive control to understand how we form models of our social world. Evolved Simulators: Innate Expectations p Others have argued that people are born with systems that have evolved throughout different generations through repeated assemblies (Caporael, 1997). Because of the importance of procre- ating and giving warm care, infants should have “evolved simula- tors” to seek warm contact in early life stages that provides an envi- ronment that is predictable for their regulation of their metabolic resources (see also Barsalou, 1999, 2008; IJzerman et al., 2012).13 ( , , ; J , ) There is some support for the idea that if infants do not meet “warm” experiences, they will face negative consequences. In a relatively controversial experimental study, Bystrova et al. (2009) found that early separation of the infant from the skin of the mother led to poorer self-regulation in the infant 1 year thereafter, very suggestively a sign that the separation of the warm skin of the caregiver altered their predictive models of the world. And, indeed, some early support exists that the infant has connected their predictive model (i.e., attachment styles) with temperature cues (Fay and Maner, 2012; IJzerman et al., 2013). Attachment has been directly linked to social thermoregulation in relation to attachment and predictability as well: Infants ranging in age from eight to 16 weeks showed drops in skin temperature in response to their attachment figure (the mother) leaving the room (the typical set up in the famous Strange Situation paradigm; e.g., Ainsworth and Bell, 1970), and the presence of a non-attachment figure (a stranger) without the attachment figure caused the same effect. Importantly, these effects were solely detectable in skin temperature, but not in differential responses in smiling or crying (Mizukami et al., 1990; see also Mizukami et al., 1987). There is thus some early support for linking attachment and affiliation to thermoregulation. An important point on the research agenda for social thermoregulation is thus to find out whether infants are predisposed to seek for specific thermo-related cues, and whether The hypothesis that emerges then that greater skin-to-skin con- tact (like the examples we cited earlier) should stimulate people’s capacities to self-regulate. This has been found: regular skin-to- skin contact in early life improves executive functioning for a child (Feldman et al., 2014). 13Different scholars have identified these systems with different names. Fiske (2000, p. 82) identifies these as mods. He defines mods as “an innate proclivity to coordinate social interaction in a universally struc- tured yet culturally organized way. It is an evolved but incomplete atten- tional–cognitive–learning–motivational system for organizing social interac- tion that cannot function without a congruent cultural complement.” We are in complete agreement with his statement. However, Fiske (2000) also identifies mods to be functionally neurologically modular (which disagrees with our position on neural reuse; Anderson, 2010). Thomsen et al. (2011) and IJzerman and Cohen (2011) identify these innate proclivities as “innate input analyzers” and “pre-wired embodiments.” We use “evolved simulator” as this most closely aligns with the broader literature on the formation of predictive models. Evolved Simulators: Innate Expectations While such early research support exists, our ideas are still quite tentative, and the research agenda should now focus on establishing (1) more direct links between social thermoregulation and the development of higher order cognitive functions, (2) whether responsive thermoregulation to the infant’s stress improves self-control/executive functioning in later life (mediated by improved BAT functioning and greater growth in predictive systems), and comparatively whether (3) animals that engage in “more effective” thermoregulation, also have greater brain mass related to predictive functioning (see, e.g., Dunbar, 1998). Social Thermoregulation and the Development of Higher Order Cognition The discussion of this support may also help understand why we think that thermoregulation is causally involved in the formation of the internal model. energy in early life. When the caregiver is available for reliable thermoregulation, it allows for dedication of the excess energy for the maturation of the infant brain, which should considerably impact the growth of structures related to higher order cognitive functioning (Carter, 2014). Indeed, as we noted before, maternal thermoregulation protects the developing brain of the infant (at least in rats), allowing it to mature for greater predictive capacity. Because harsh environ- ments may stimulate BAT growth, the bioenergetic resources typ- ically devoted to thermoregulation could potentially be invested into growth of other areas of the brain that allow for higher order control.12 In turn, we predict that adverse early social experiences will affect these sociophysiological mechanisms. And there is indeed some evidence that the degree to which people are thermoregulated in their early days shapes higher order cognitive functions, like levels of self and/or executive con- trol. Attachment security—which is typically related to predictive control—correlates with enhanced self-regulation (e.g., Mikulin- cer et al., 1993; Mikulincer and Florian, 1995), and also with a more coherent self and with greater self-complexity (Mikulincer, 1995). We interpret this evidence tentatively as meaning that the “self” and its regulation emerges from the interaction with and being regulated by close others (see for comparable reasonings Häfner and IJzerman, 2011; and also Righetti et al., 2013), and that one of the earliest mechanisms of these is through social thermoregulation. Formation of the Predictive Model In further elucidating this developmental link, we need to under- stand the organization of the predictive model, which is likely to rely on (at least) two factors, one that is reliant on basic physi- ological development and another that is part of the organism’s cognitive development (a point we already discussed above). We think that early social development contributes to the way the organism’s body develops later on, and one important facet of this development should entail the earlier discussed BAT (see Cannon and Nedergaard, 2004). The link between oxytocin secretion and touch is well known (e.g., Uvnäs-Moberg et al., 2015). Curiously, deficiencies in the oxytocin receptor gene have been associated with problems in the development of BAT in mice (Takayanagi et al., 2008). We thus think that BAT, seen as important for thermogenesis, could even play a role in determining meaning- ful psychological individual difference patterns, and that a place to examine this is in individual difference patterns of people’s thermoneutral zones (Kingma et al., 2012, 2014). There is thus support that social thermoregulation is at least involved in the internal model of the close relationship. But that does not mean that social thermoregulation is the only factor in forming predictive models of relationships. Going back to Damasio’s (1989) idea, convergence zones are “uninformed as to the content of the representations they assist in attempting to construct” (p. 46), and thus rely on the multiple—and bi- directional—inputs across body and brain that are crucial for predictive models of relationships. But what is the content of internal models that are formed on the basis of this process? And how should they update? We think they are likely slow in updating, and based on what we have reasoned there should be an important role for thermal cues (the content of our knowledge about rela- tionships) through reactive control systems into predictive control systems—including the predictive model that provides us with a report of the social “weather,” which can both be longer term (predictive) or help us respond in the here and now (reactive). In making our model even more detailed, we further explain how Tops et al.’s (2010, 2014a,b) theory of PARCS can explain some of the earlier evidence that has been collected. ( g ) We believe that BAT may develop in different ways in case of early aversive postnatal experiences—through ways that are still to be explored. 14There is also growing support for gender differences in brown adipose tissue thermogenesis. This suggest that this process is enhanced in females leading to differential experiences in warmth between huddling male and female mice of 8 days old and thus possible gender differences through early interaction experiences (Harshaw et al., 2014). 15Convergence zones were first discussed both by Barsalou (1999, 2008) and Damasio (1989) as their role being “to enact formulas for the reconstitution of fragment-based momentary representations of entities or events in sensory or motor cortices” (p. 46). Forming Predictive Models of Relationships—Attachments One of the hallmark features of attachment theory has been the concept of the predictive model of others (e.g., Craik, 1943; Bowlby, 1969; Hazan and Shaver, 1987; Mikulincer and Shaver, 2003; but see also Beckes et al., 2014). In the recent past, several 12Curiously enough, the development of the infant’s body to be able to thermoregulate solitarily seems to go hand in hand with the development of this specialized organ, which has been implicated in internal thermoreg- ulation (Cannon and Nedergaard, 1985; Grigg et al., 2004; Ivanov, 2006). Indeed, in oxytocin receptor deficient mice brown adipose tissue was impaired (Takayanagi et al., 2008), tentatively suggesting that brown adipose tissue develops differently in accordance with one’s (innate) social abilities, and possibly one’s social experiences. More potently, mice that interact in larger groups (and are thus better thermoregulated) have less BAT, which suggests greater “fat-browning” in harsher environments (Heldmaier, 1975). April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 10 Human social thermoregulation IJzerman et al. differences in contingent thermoregulation from the mother to the child leads to differences in attachment styles. idea that people utilize cognitions that are reliant on the activation of recent other stimuli (Neely, 1977; Tulving and Schacter, 1990; Bargh and Chartrand, 1999; Bargh et al., 2012). And indeed, a warm cup makes people judge others as more sociable, and makes themselves more generous (Williams and Bargh, 2008), while warmth (vs. cold) also makes people perceive more relation- ships in their environment and construe an experimenter as more overlapping with the self, and makes people more likely to use more relational language, be more cooperative, and trust others more (IJzerman and Semin, 2009; Kang et al., 2011; Storey, 2013; Schilder et al., 2014). 16Criticism has arisen in regards to dual process models (see, e.g., Keren and Schul, 2009). We do not see PARCS as a comparable dual process model. Indeed, Tops et al. (2014a,b) use “the terms reactive and predictive as labels to refer to functional systems that combine a set of features that together are optimal in controlling behavior and physiology in unpredictable or pre- dictable circumstances. For example, some features of the reactive system do not fit at all to the label reactive (e.g., sustained attentional control or active maintenance of task goals by the reactive system). Moreover, each system contains certain features that are evolutionarily older and seem more primitive (e.g., related to reactivity, impulsivity, stimulus controlled) while other features of the same system are evolutionarily more recent and appear at a higher (e.g., cognitive control, reflective) level. Predictive control by internal models—and the chances of focusing one’s mental state inward—may have evolved later and at first glance appear more “sophisticated” than reactive control. However, the point we want to make is that both types of systems are continuing to evolve and have both developed higher-level control.” Moreover, Formation of the Predictive Model Understanding the development of BAT will be important for also further understanding attachment, as the absence of reliable and physically warm caregiving may even lead to changes in BAT development, affecting social thermoregu- lation abilities and even the range within which the predictive model can operate.14 In other words, some effects (and specif- ically—though not only—of temperature cues) that previously have been attributed to predictive models (i.e., attachment styles) alone may be due to differential physiological factors—and this should be part of a research agenda for social thermoregulation. And for interpersonal relationships a predictive model is more complex still. A predictive model for relationships should rely on what has been previously coined as convergence zones to form coherent representations of the social world.15 Convergence zones for close relationships are likely formed through repeated caregiv- ing behaviors (like breastfeeding), which are likely to contribute to predictive models through the activation of opioid and oxy- tocin systems (Pedersen and Prange, 1979; Uvnäs-Moberg, 1996; Winberg, 2005), and we think that these convergence zones allow people to run simulations on what their social world will be like, by merely thinking about caregiving, having sex, or about being excluded. The Further Formation of the Predictive Model Predictive and reactive control systems specifies two levels of control—one predictive and one reactive—and the theory allows for a way to integrate thermal cues into broader representations, which are formed through the slow updating of information regarding one’s social world. From this perspective, motivational control can be shifted between predictive systems and reactive systems. Broadly speaking, this suggests that reactive control systems evolved early in evolutionary history for the purpose of behavioral control in unpredictable environments, like harsh climates16. The reactive control system is thought to specialize in 16Criticism has arisen in regards to dual process models (see, e.g., Keren and Schul, 2009). We do not see PARCS as a comparable dual process model. Indeed, Tops et al. (2014a,b) use “the terms reactive and predictive as labels to refer to functional systems that combine a set of features that together are optimal in controlling behavior and physiology in unpredictable or pre- dictable circumstances. For example, some features of the reactive system do not fit at all to the label reactive (e.g., sustained attentional control or active maintenance of task goals by the reactive system). Temperature Estimates and the Regulation of Metabolic Resources the processing of novelty (cf. Whalen, 2007), biological salience (cf. Adolphs, 2010), and urgent environmental stimuli in order to react to exigencies, such as is the case for newborn infants and their (thermoregulatory) expectations of their social world. The reactive system functions in a feedback-guided manner to the immediate situation and focuses attention narrowly on the local situation. In this manner it can take new information—whether the parent will regulate their temperature or not—and is able to communicate with the predictive systems to update predictive models promoting greater predictive control in the future (Hasher and Zacks, 1979; Tops et al., 2014a,b). the processing of novelty (cf. Whalen, 2007), biological salience (cf. Adolphs, 2010), and urgent environmental stimuli in order to react to exigencies, such as is the case for newborn infants and their (thermoregulatory) expectations of their social world. The reactive system functions in a feedback-guided manner to the immediate situation and focuses attention narrowly on the local situation. In this manner it can take new information—whether the parent will regulate their temperature or not—and is able to communicate with the predictive systems to update predictive models promoting greater predictive control in the future (Hasher and Zacks, 1979; Tops et al., 2014a,b). To what degree then is PARCS supported with regard to social thermoregulation? If indeed the reactive system helps guide the agent through gaging momentary resources, then one should see differences in estimation of ambient temperature in relation to social resources, and second, there should be a link between ther- moregulation and the regulation of social behavior. The ability to gage resources could be something that we have referred earlier to as the “weather report.” And indeed, there are a number of reports that show the link between the “weather report” (i.e., the gage of resources) and close relations: Primes of social/physical similarity (vs. social/physical distance and exclusion) lead people to estimate temperature as higher, whether this is about the relationship (Zhong and Leonardelli, 2008; IJzerman and Semin, 2010), the self/other (Szymkow et al., 2013), or consumer products (IJzer- man et al., 2014). IJzerman et al. (2014) even find that temperature cues are causally implicated in the willingness to purchase the consumer products that make them feel warmer (for comparable findings, see Van Acker et al., 2015). Temperature Estimates and the Regulation of Metabolic Resources The reactive system helps guide people to gage and act on their momentary resources [that are specified by people’s gage for (social) warmth vs. coldness], due to an integration of early perceptual cues into the predictive models. These should—at least situationally—pan out in “working models of relationships” (Craik, 1943; Bowlby, 1969). Repeated patterns should lead to a slow updating of such a model. Earlier we have referred to the formation of higher order cognitive functions as a result of maternal thermoregulation. The brain areas associated with these higher order cognitions (of which the predictive system is a part) are thus believed to largely be an outgrowth of evolu- tionary pressures that emerged in highly predictable and stable environments (Tops et al., 2014a,b). The predictive system sup- ports a variety of cognitive functions that are representational and semantic in nature, one of the most important of which is related to thermoregulation, because it is so essential to many animals’ survival. These findings should tell us that the participants perceive the social world to be filled (or not) with resources, and that higher (lower) estimates of temperature may suggest to them that their world is socially more (less) predictable. And people can act on momentary resources, and self-regulate their own feelings that relate to a lack of being close to others: Holding a warm object has been foundtoalleviate thedetrimental effects associated with brief social exclusion (Bargh and Shalev, 2012; IJzerman et al., 2012).18 The direct experience of physical coldness has also been found to lead to a desire to be with others (vs. being alone) and an increased preference for romance movies, and—crucially—the latter only occurred for people associating these movies with psychological warmth (Hong and Sun, 2012; Lee et al., 2013). In addition, among PTSD patients, loneliness has been found to positively relate to a preference for warm foods (Li and Liao, 2013). Finally, in colder conditions people self-regulate through socially warm experiences: Nostalgia is triggered by coldness (Zhou et al., 2012; Sedikides et al., 2014), people seek socially warm experiences when they are cold (Zhang and Risen, 2014), and they judge houses to be more homely when they are colder (Van Acker et al., 2015; for a review, see Raison et al., 2015). 18For skepticism regarding the shower studies, see Donnellan et al. (2014) and Ferrell et al. (2014), and for skepticism of this skepticism, see Shalev and Bargh (2014). Formation of the Predictive Model Moreover, each system contains certain features that are evolutionarily older and seem more primitive (e.g., related to reactivity, impulsivity, stimulus controlled) while other features of the same system are evolutionarily more recent and appear at a higher (e.g., cognitive control, reflective) level. Predictive control by internal models—and the chances of focusing one’s mental state inward—may have evolved later and at first glance appear more “sophisticated” than reactive control. However, the point we want to make is that both types of systems are continuing to evolve and have both developed higher-level control.” Moreover, But how can we know that thermoregulation is causally involved with the internal model? In social psychology, such ques- tions are typically resolved through priming methods, which is the April 2015 \| Volume 6 \| Article 464 11 Frontiers in Psychology \| www.frontiersin.org Human social thermoregulation IJzerman et al. PARCS does not describe just two systems (one being reactive and the other being predictive), nor does it only include systems that are either purely reactive or purely predictive (Tops et al., 2010, 2014a,b; Tops and Boksem, 2011). Temperature Estimates and the Regulation of Metabolic Resources There is even some preliminary support that skin temperature is responsive to someone else’s stress (Vuorenkoski et al., 1969; Wagemans et al., 2014), which we think serves in social emotion regulation. It is still crucial however to show that skin temperature of a supporter is causally involved with the regulation of another’s emotion. These findings should tell us that the participants perceive the social world to be filled (or not) with resources, and that higher (lower) estimates of temperature may suggest to them that their world is socially more (less) predictable. And people can act on momentary resources, and self-regulate their own feelings that relate to a lack of being close to others: Holding a warm object has been foundtoalleviate thedetrimental effects associated with brief social exclusion (Bargh and Shalev, 2012; IJzerman et al., 2012).18 The predictive system is further involved in cognitive tasks with internally focused attention such as imagining a different time or space (Buckner and Carroll, 2007), or another person’s per- spective (Waytz and Mitchell, 2011). Craik (1943) suggested that using predictive models allows for testing alternative possibilities, and making better predictions regarding situational outcomes. In the same sense, PARCS suggests that the predictive system’s function is to run simulations to predict future events. Thus, the predictive system engages in creating internal models that predict future outcomes through simulations, and updates those models slowly, in line with the idea that it responds to environ- mental predictability. Together, this means that the predictive system helps people in better scheduling metabolism, and thus better using the “weather report” they have obtained of their social environment. The way that perceptual simulations thus are likely to function is by calling on the relevant information from named convergence zones, which would include relevant features of the entire thermoregulatory state of the processes we have discussed above (for how simulations work, see Barsalou, 1999, 2008).17 We regard this wide array of findings as supportive of our model. Nevertheless, whether these mechanisms lead to the devel- opment of stable individual differences remains an informed con- jecture at this stage and we also add this to the social thermoreg- ulation research agenda. First, of course, there is the potential for differences in growth of the infant brain, and having greater control over reactive cues (vs. predictive cues) may be adap- tive for the individual organism in unpredictive environments. PARCS does not describe just two systems (one being reactive and the other being predictive), nor does it only include systems that are either purely reactive or purely predictive (Tops et al., 2010, 2014a,b; Tops and Boksem, 2011). 17As Barsalou (1999, 2008) stated, these simulations are not likely to function like a complete simulation of the original perceptual state, but are likely to be incomplete (and function much like a Gestalt), while specific goals are likely to call on activating different parts of the convergence zones we have alluded to. References Bargh, J. A., Schwader, K. L., Hailey, S. E., Dyer, R. L., and Boothby, E. J. (2012). Automaticity in social-cognitive processes. Trends Cogn. Sci. 16, 593–605. doi: 10.1016/j.tics.2012.10.002 Adels, L. E., and Leon, M. (1986). Thermal control of mother-young con- tact in Norway rats: factors mediating the chronic elevation of mater- nal temperature. Physiol. Behav. 36, 183–196. doi: 10.1016/0031-9384(86) 90094-6 j Bargh, J. A., and Shalev, I. (2012). The substitutability of physical and social warmth in daily life. Emotion 12, 154. doi: 10.1037/a0023527 in daily life. Emotion 12, 154. doi: 10.1037/a0023527 Barsalou, L. W. (1999). Perceptions of perceptual symbols. Behav. Brain Sci. 22, 637–660. doi: 10.1017/S0140525X99532147 Adolphs, R. (2010). Emotion. Curr. Biol. 20, R549–R552. doi: 10.1016/j.cub. 2010.05.046 Barsalou, L. W. (2008). Grounded cognition. Annu. Rev. Psychol. 59, 617–645. doi: 10.1146/annurev.psych.59.103006.093639 Ainsworth, M. D. S., and Bell, S. M. (1970). Attachment, exploration, and separa- tion: illustrated by the behavior of one-year-olds in a strange situation. Child Dev. 41, 49–67. doi: 10.2307/1127388 Baumeister, R. F. (2005). The Cultural Animal: Human Nature, Meaning, and Social Life. Oxford: Oxford University Press. Akin, J. A. (2011). Homeostatic processes for thermoregulation. Nat. Educ. Knowl. 3, 7. Bazin, R. C., and MacArthur, R. A. (1992). Thermal benefits of huddling in muskrat (Ondatra zibethicus). J. Mammal. 73, 559–564. doi: 10.2307/1382023 Anderson, M. L. (2010). Neural reuse: a fundamental organizational principle of the brain. Behav. Brain Sci. 33, 245–313. doi: 10.1017/S0140525X10000853 Beckes, L., and Coan, J. A. (2011). Social baseline theory: the role of social proximity in emotion and economy of action. Soc. Personal. Psychol. Compass 5, 976–988. doi: 10.1111/j.1751-9004.2011.00400.x Andrews, R. V., and Belknap, R. W. (1986). Bioenergetic benefits of huddling by deer mice (Peromyscus maniculatus). Comp. Biochem. Physiol. 85A, 775–778. doi: 10.1016/0300-9629(86)90294-X Beckes, L., IJzerman, H., and Tops, M. (2014). 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In so doing, we have described a kind of temperature homeostasis that relies on predictive and reactive control systems, from behavioral thermoregulation to the formation of predictive models of self and others. We have also noted quite a number of empirical findings that are supportive of our model, but realize Temperature Estimates and the Regulation of Metabolic Resources Specifically, deficiencies in the oxytocin gene receptor have been implicated in poor development of BAT in mice (Takayanagi et al., 2008), and if mice are poorly thermoregulated, they develop more BAT (Heldmaier, 1975). Basically, it could be that unreliable warmth- related caregiving in the infant’s early life contributes to impaired development of BAT, and therefore also to a predictive system that relies on a different physiology. Temperature Estimates and the Regulation of Metabolic Resources 17As Barsalou (1999, 2008) stated, these simulations are not likely to function like a complete simulation of the original perceptual state, but are likely to be incomplete (and function much like a Gestalt), while specific goals are likely to call on activating different parts of the convergence zones we have alluded to. 18For skepticism regarding the shower studies, see Donnellan et al. (2014) and Ferrell et al. (2014), and for skepticism of this skepticism, see Shalev and Bargh (2014). April 2015 \| Volume 6 \| Article 464 Frontiers in Psychology \| www.frontiersin.org 12 Human social thermoregulation IJzerman et al. that much remains to be explored. Nevertheless, we think that our theory of social thermoregulation will achieve broad applicability across domains in psychology. For example, to what degree are findings that we detect in attachment studies due to predictive models, and to what degree do they rely on the development of BAT? Do the predictive models for social thermoregulation also play a role in other types of relational models than communal sharing (see, e.g., Fiske, 1992)? How does social thermoregula- tion relate to findings that have been typically implicated in the domain of self-regulation, like obesity? And how does sexual behavior in humans relate to social thermoregulation, and how can it aid vs. impede the formation of communal bonds? Finally, if social interactions are at least in part predicated upon social thermoregulation, then do novel technological developments that help humans resolve energy regulation decrease a direct need for (face-to-face?) interpersonal relationships? At any rate, we hope that our theory will become truly generative, and that it will help the research community to explore many of these questions. 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https://openalex.org/W2921229466	https://europepmc.org/articles/pmc6478207?pdf=render	English	null	Zika Virus in Rectal Swab Samples	Emerging infectious diseases	2,019	cc-by	2,891	Zika Virus in Rectal Swab Samples Camila Helena Aguiar Bôtto-Menezes, Armando Menezes Neto, Guilherme Amaral Calvet, Edna Oliveira Kara, Marcus Vinícius Guimarães Lacerda, Marcia da Costa Castilho, Ute Ströher, Carlos Alexandre Antunes de Brito, Kayvon Modjarrad, Nathalie Broutet, Patrícia Brasil, Ana Maria Bispo de Filippis, Rafael Freitas Oliveira Franca, ZIKABRA Study Team1 detectable viremia with subsequent testicular damage and congenital defects in the offspring of pregnant mice (5). These findings indicate that the anorectal mucosa may rep resent an infection route for Zika virus. However, whether Zika virus can be detected in the anorectal mucosa of natu rally infected human patients remains largely unknown. To clarify the kinetics of Zika virus infection across biologi cal compartments and to devise rational measures for pre venting transmission of the virus, in July 2017 we began a cohort study of men and women >18 years of age with Zika virus infection in Brazil; the study will continue until mid-2020. Written informed consent was obtained from all enrolled participants. We detected Zika virus RNA in rectal swab samples from 10 patients by using real-time reverse transcription PCR, and we isolated the virus from 1 patient. The longest inter val from symptom onset to detection was 14 days. These findings are applicable to diagnosis and infection prevention recommendations. 1Team members are listed at the end of the article. Author affiliations: Amazonas State University, Manaus, Brazil (C.H.A. Bôtto-Menezes); Tropical Medicine Foundation Doctor Heitor Vieira Dourado, Manaus (C.H.A. Bôtto-Menezes, M.V.G. Lacerda, M. da Costa Castilho); Institute Aggeu Magalhães, Recife, Brazil (A.M. Neto, C.A. Antunes de Brito, R.F.O. Franca); Evandro Chagas National Institute of Infectious Diseases, Rio de Janeiro, Brazil (G.A. Calvet, P. Brasil); World Health Organization, Geneva, Switzerland (E.O. Kara, U. Ströher, N. Broutet); Oswaldo Cruz Foundation, Manaus (M.V.G. Lacerda); Walter Reed Army Institute of Research, Silver Spring, Maryland, USA (K. Modjarrad); Oswaldo Cruz Institute, Rio de Janeiro (A.M. Bispo de Filippis) The Study To assess the persistence of Zika virus in different body fluids of persons with confirmed infection, we conducted a multicenter prospective cohort study (the ZIKABRA Study). Laboratory confirmation of infection was based on real-time reverse transcription PCR (rRT-PCR) per formed on samples (urine, blood, or both) from persons in whom a rash developed <48 hours after initial symptom onset. We also invited household contacts or sex partners to participate in the study and enrolled those with posi tive rRT-PCR results for Zika virus. Full details about the study protocol, including ethics approval, are described by Calvet et al. (7). I n early 2015, Zika virus was identified in Brazil and spread across nearly the whole continent, affecting thou sands of persons (1). This outbreak was associated with microcephaly and other congenital abnormalities resulting from infection of the mother during pregnancy (2). For dif ferent periods after infection, Zika virus RNA can be found in diverse body fluids such as saliva, amniotic fluid, urine, cerebrospinal fluid, blood, semen, and tears (3); the longest period of viral RNA shedding has been identified in semen (>1 year after symptom onset) (4). f I We collected samples (saliva, blood, urine, vagi nal, and rectal swabs) at specific intervals from patients identified as Zika virus positive after the screening visit (Figure). Vaginal and rectal swab samples were diluted in 1 mL of sterile Hank’s Balanced Salt Solution (Ther moFisher Scientific, https://www.thermofisher.com) and stored at −80°C until processing. After collection, speci mens were kept refrigerated and transported within 2 hours to the laboratory, where they were maintained at −80°C. Zika virus detection was performed by rRT-PCR by processing 200 µL of each specimen for RNA extrac tion through an automated nucleic acid purification plat form by using the Maxwell 16 Viral Total Nucleic Acid Purification Kit (Promega Corporation, https://www. promega.com) in a final volume of 70 µL. For rRT-PCR, we used the commercially available ZDC Kit from Insti tuto de Tecnologia em Imunobiológicos Biomanguinhos, approved by Agência Nacional de Vigilância Sanitária/ Although Zika virus RNA has been detected in differ ent body fluids, we found only 1 report of Zika virus elimi nation through feces from 1 naturally infected person (5). Experimentally, Zika virus is able to infect mice and adult macaques (6) through the anorectal mucosa, leading to Author affiliations: Amazonas State University, Manaus, Brazil (C.H.A. The Study Bôtto-Menezes); Tropical Medicine Foundation Doctor Heitor Vieira Dourado, Manaus (C.H.A. Bôtto-Menezes, M.V.G. Lacerda, M. da Costa Castilho); Institute Aggeu Magalhães, Recife, Brazil (A.M. Neto, C.A. Antunes de Brito, R.F.O. Franca); Evandro Chagas National Institute of Infectious Diseases, Rio de Janeiro, Brazil (G.A. Calvet, P. Brasil); World Health Organization, Geneva, Switzerland (E.O. Kara, U. Ströher, N. Broutet); Oswaldo Cruz Foundation, Manaus (M.V.G. Lacerda); Walter Reed Army Institute of Research, Silver Spring, Maryland, USA (K. Modjarrad); Oswaldo Cruz Institute, Rio de Janeiro (A.M. Bispo de Filippis) 951 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 DISPATCHES Figure. Detection of Zika virus RNA in human biological specimens from 8 patients, according to Ct and days after disease onset. Patient identification numbers above charts correspond to numbers in the Table. Horizontal dashed lines indicate real-time reverse transcription PCR cutoff Ct of 38. Disease onset is day 0 (screening visit), defined after interviewing patients about symptoms. Ct, cycle threshold. Figure. Detection of Zika virus RNA in human biological specimens from 8 patients, according to Ct and days after disease onset. Patient identification numbers above charts correspond to numbers in the Table. Horizontal dashed lines indicate real-time reverse transcription PCR cutoff Ct of 38. Disease onset is day 0 (screening visit), defined after interviewing patients about symptoms. Ct, cycle threshold. ANVISA (registry no. 80142170032; https://www.bio. fiocruz.br). We considered positive those samples that displayed positive amplification in the ZDC Kit internal control reaction (which consists of an RNA virus–like particle individually added to each specimen before RNA extraction) and those samples in which the target ampli fication was detected within 38 amplification cycles, as previously described (8). ANVISA (registry no. 80142170032; https://www.bio. fiocruz.br). We considered positive those samples that displayed positive amplification in the ZDC Kit internal control reaction (which consists of an RNA virus–like particle individually added to each specimen before RNA extraction) and those samples in which the target ampli fication was detected within 38 amplification cycles, as previously described (8). value (Ct) was 34.92 in plasma (interquartile range [IQR] 33.57–36.78) and 31.57 in urine (IQR 30.34–34.92). The longest duration of persistence in saliva was 21 days. Zika virus RNA was detected in rectal swab samples from all patients (median Ct 34.36 [IQR 30.08–36.07]); positive results were obtained at >1 time for 4 patients. Vaginal swab sample results were positive for 2 patients (Figure). The Study We attempted virus isolation in Vero E6 cells from all rectal swab specimens positive by rRT-PCR. After fil tering samples through a 0.22-µm syringe filter, a rectal swab sample from patient 191002 (at 7 days after symp tom onset) was positive in Vero E6 cells, inducing cyto pathic effect and returning a Ct of 28.77 after 6 days of incubation at 37°C. We report 10 Zika virus–infected patients from 2 locations in Brazil. Of these, 3 patients were identified in Recife, northeastern Brazil, and 7 were from Manaus, northern Brazil. The targeted Zika virus amplicons were found in the plasma of 9 patients and in the urine of 7 patients, all tested at screening visits. We also tested all plasma and urine samples for dengue and chikungunya virus by rRT-PCR; results were negative. Of the 10 Zika virus–infected patients, 9 were symptomatic and 1 (a Zika virus–positive household contact) was asymptomatic at enrollment but subsequently reported muscular weakness and irritability on day 5 and arthralgia on day 7 after Zika virus identification. Median patient age was 31.5 years; 7 patients were nonpregnant women, and 3 were men (Ta ble). All patients were negative for HIV-1, hepatitis B and C, and syphilis. Conclusions Clinical signs and symptoms reported at enrollment visit in study of persistence of Zika virus in different body fluids of persons with confirmed infection Brazil* Recently, 2 studies explored the mucosa as an ex perimental entry site for Zika virus. Macaques exposed to high doses of the virus by direct inoculation on palatine tonsils, nasal mucosa, and conjunctival mucosa became infected, whereas virus-naive animals exposed to saliva from Zika virus–infected macaques, containing a 20-fold lower virus concentration, remained uninfected (6). In mice, intra-anal inoculation resulted in viremia, replica tion in multiple organs, and Zika virus RNA detection in feces (5). However, it seems that high viral loads are required for successful experimental infection through mucosal tissues. In humans, a report of a man infected through anal intercourse implies that the anorectal mu cosa is an entry site for Zika virus (10). Also, a rapidly progressive fatal case, with secondary nonsexual trans mission to a close contact who reported having had no contact with blood or other body fluids except tears from the original patient, raised the hypothesis of Zika virus transmission through mucosa (11). Although mucosal tis sues have been extensively explored as a potential site of Zika virus infection, the amount of infectious particles in these tissues remains unclear. ZIKABRA Study Team members (in alphabetical order): André Luiz de Abreu, Adele Schwartz Benzaken, Ximena Pamela Diaz Bermudez, Camila Helena Aguiar Bôtto-Menezes, Patrícia Brasil, Carlos Alexandre Antunes Brito, Nathalie Jeanne Nicole Broutet, Guilherme Ama ral Calvet, Marcia da Costa Castilho, Ana Maria Bispo de Filippis, Rafael Freitas Oliveira Franca, Silvana Pereira Giozza, Ndema Habib, Edna Oliveira Kara, Marcus Vini cius Guimarães Lacerda, Sihem Landoulsi, Morganna Cos ta Lima, Noemia Lima, Maeve Brito de Mello, Ana Izabel Costa de Menezes, Robyn Meurant, Kayvon Modjarrad, Armando Menezes Neto, Cristina Pimenta, Casey Storme, Ute Ströher, Anna Thorson, Lydie Trautman. Conclusions Our detection of Zika virus RNA in human rectal swab samples demonstrates the presence of virus RNA in the anorectal mucosa of naturally infected patients. Because the anorectal mucosa is a major entry site for HIV-1 and other sexually transmitted disease organisms (9), this finding may have implications for Zika virus transmis sion. Direct contact with infected mucosa could present a risk for virus transmission. Moreover, recovery of in fectious Zika virus from anorectal mucosa samples may be associated with active virus replication in this body compartment, implicating its permissiveness to Zika vi rus infection. For all patients, Zika virus RNA was detected >1 time in different body fluids. The longest interval for Zika virus–positive results by rRT-PCR in plasma and urine was 14 days after symptom onset; median cycle threshold 952 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 Zika Virus in Rectal Swab Samples Table. Clinical signs and symptoms reported at enrollment visit in study of persistence of Zika virus in different body fluids of persons 8 with confirmed infection, Brazil* Patient ID Age, y/sex Symptoms at enrollment 191001 21/M None (asymptomatic household contact) 191002 25/M Erythema and vesicular rash, nasal congestion, sweating, upper limb muscle spasms 291001 22/F Itchy macular rash, conjunctival hyperemia, prostration, chills, taste alteration, lower back pain, arthralgia, periarticular edema, nausea, irritability 151001 38/M Itchy macular rash, headache, photophobia, retro-orbital pain, burning eyes, arthralgia (wrists, metacarpals, and phalanges), tingling hands 251001 31/F Itchy macular rash, conjunctival hyperemia, anorexia 251002 36/F Itchy macular rash, headache, conjunctival hyperemia, arthralgia (shoulders, elbows, wrists, knees, ankles), abdominal pain, periarticular edema (ankles) 251003 43/F Itchy macular rash, photophobia, retro-orbital pain, oropharyngeal pain, arthralgia (elbows and ankles) 251005 19/F Itchy macular rash, fever, headache, photophobia, conjunctival hyperemia, retro-orbital pain, muscle weakness, numbness, irritability, appetite loss, nausea 251006 65/F Itchy macular rash, fever, headache, photophobia, arthralgia (wrists, phalanges, heel, cervical spine), muscle weakness, prostration, numbness, tingling, drowsiness, abdominal pain, nausea 251007 32/F Itchy macular rash, fever, headache, retro-orbital pain, photophobia *Reported signs and symptoms were registered after an initial rash episode following laboratory confirmation of Zika virus infection. All signs and symptoms were registered <2 d after the initial rash (except for 1 asymptomatic contact patient). Fever (>38°C) was any febrile episode reported within 30 d before the medical examination. ID, identification. Table. Acknowledgments We thank all patients for their willingness to participate in the study and all collaborating staff for their invaluable help in designing the protocol. The research leading to these results received funding from Wellcome Trust grant no. 206522/Z/17/Z, World Health Organization (UNDP-UNFPA-UNICEF-WHO-World Bank Special Programme of Research, Development and Research Training in Human Reproduction), World Health Emergency Programme Organization, Brazilian Ministry of Health (Convênio no. 837059/2016, Processo 25000162039201616), US National Institutes of Health National Institute of Allergy and Infectious Diseases (award no. R21AI139777). This work was supported by a cooperative agreement (W81XWH-18-2-0040) between the Henry M. Jackson Foundation for the Advancement of Military Medicine and the US Department of the Army. The work was funded by the US Defense Health Agency (0130602D16). The funders had no role in study design, data collection, analysis decision to publish or preparation of the manuscript In patients with yellow fever, mucocutaneous bleed ing with virus presence has been reported (12). However, no mucosal bleeding was observed in the patients we re port, despite extensive medical examination. In addition, in 5 of 9 patients, the virus had already cleared from the blood at the time of anorectal detection, which supports the concept of local virus replication. Our finding of Zika virus in the anorectal mucosa of naturally infected per sons may influence the recommendations for prevention of Zika virus transmission. We suggest the use of rectal swabbing, a noninvasive method, for diagnosing infection with Zika virus, among other emerging viruses (13). analysis, decision to publish, or preparation of the manuscript. 953 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 DISPATCHES References 1. Faria NR, Azevedo RDSDS, Kraemer MUG, Souza R, Cunha MS, Hill SC, et al. Zika virus in the Americas: early epidemiological and genetic findings. Science. 2016;352:345–9. 1. Faria NR, Azevedo RDSDS, Kraemer MUG, Souza R, Cunha MS, Hill SC, et al. Zika virus in the Americas: early epidemiological and genetic findings. Science. 2016;352:345–9. 10. Deckard DT, Chung WM, Brooks JT, Smith JC, Woldai S, Hennessey M, et al. Male-to-male sexual transmission of Zika virus—Texas, January 2016. MMWR Morb Mortal Wkly Rep. 2016;65:372–4. http://dx.doi.org/10.15585/mmwr.mm6514a3 2. Miranda-Filho DB, Martelli CMT, Ximenes RA, Araújo TV, Rocha MA, Ramos RC, et al. Initial description of the presumed congenital Zika syndrome. Am J Public Health. 2016;106:598–600. http://dx.doi.org/10.2105/AJPH.2016.303115 2. Miranda-Filho DB, Martelli CMT, Ximenes RA, Araújo TV, Rocha MA, Ramos RC, et al. Initial description of the presumed congenital Zika syndrome. Am J Public Health. 2016;106:598–600. http://dx.doi.org/10.2105/AJPH.2016.303115 11. Swaminathan S, Schlaberg R, Lewis J, Hanson KE, Couturier MR. Fatal Zika virus infection with secondary nonsexual transmission. N Engl J Med. 2016;375:1907–9. http://dx.doi.org/ 10.1056/NEJMc1610613 3. Paz-Bailey G, Rosenberg ES, Doyle K, Munoz-Jordan J, Santiago GA, Klein L, et al. Persistence of Zika virus in body fluids–preliminary report. N Engl J Med. 2017;379:1234–43. 12. Monath TP. Yellow fever: an update. Lancet Infect Dis. 2001; 1:11–20. http://dx.doi.org/10.1016/S1473-3099(01)00016-0 4. Barzon L, Percivalle E, Pacenti M, Rovida F, Zavattoni M, Del Bravo P, et al. Virus and antibody dynamics in travelers with acute Zika virus infection. Clin Infect Dis. 2018;66:1173–80. http://dx.doi.org/10.1093/cid/cix967 13. Uyeki TM, Mehta AK, Davey RT Jr, Liddell AM, Wolf T, Vetter P, et al.; Working Group of the U.S.–European Clinical Network on Clinical Management of Ebola Virus Disease Patients in the U.S. and Europe. Clinical management of Ebola virus disease in the United States and Europe. N Engl J Med. 2016;374:636–46. http://dx.doi.org/10.1056/NEJMoa1504874 5. Li C, Deng Y-Q, Zu S, Quanquin N, Shang J, Tian M, et al. Zika virus shedding in the stool and infection through the anorectal mucosa in mice. Emerg Microbes Infect. 2018;7:169. http://dx.doi.org/10.1038/s41426-018-0170-6 Address for correspondence: Rafael F.O. Franca, Oswaldo Cruz Foundation–FIOCRUZ, Department of Virology and Experimental Therapy, Av. Professor Moraes Rego, s/n–Campus da UFPE, Cidade Universitária Recife, Pernambuco 50740-465, Brazil; email: rafael.franca@cpqam.fiocruz.br Address for correspondence: Rafael F.O. Franca, Oswaldo Cruz Foundation–FIOCRUZ, Department of Virology and Experimental Therapy, Av. Professor Moraes Rego, s/n–Campus da UFPE, Cidade Universitária Recife, Pernambuco 50740-465, Brazil; email: rafael.franca@cpqam.fiocruz.br 6. About the Author on the persistence of Zika virus (ZIKV) in body fluids of patients with ZIKV infection in Brazil. BMC Infect Dis. 2018;18:49. http://dx.doi.org/10.1186/s12879-018-2965-4 on the persistence of Zika virus (ZIKV) in body fluids of patients with ZIKV infection in Brazil. BMC Infect Dis. 2018;18:49. http://dx.doi.org/10.1186/s12879-018-2965-4 Dr. Bôtto-Menezes is a research associate at Fundacao de Medicina Tropical Doutor Heitor Vieira Dourado and professor at Amazonas State University. Her research interests are tropical medicine, infectious diseases, and epidemiology. 8. 8. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008;14:1232–9. http://dx.doi.org/10.3201/eid1408.080287 9. Workowski KA, Bolan GA; Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep. 2015 Jun 5;64(RR-03):1–137. References Haddow AD, Nalca A, Rossi FD, Miller LJ, Wiley MR, Perez-Sautu U, et al. High infection rates for adult macaques after intravaginal or intrarectal inoculation with Zika virus. Emerg Infect Dis. 2017;23:1274–81. http://dx.doi.org/10.3201/eid2308.170036 p g 7. Calvet GA, Kara EO, Giozza SP, Bôtto-Menezes CHA, Gaillard P, de Oliveira Franca RF, et al.; ZIKABRA Study Team. Study p g 7. Calvet GA, Kara EO, Giozza SP, Bôtto-Menezes CHA, Gaillard P, de Oliveira Franca RF, et al.; ZIKABRA Study Team. Study Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 954
https://openalex.org/W4386803459	https://bmcchem.biomedcentral.com/counter/pdf/10.1186/s13065-023-01003-3	English	null	Chalcone/1,3,4-Oxadiazole/Benzimidazole hybrids as novel anti-proliferative agents inducing apoptosis and inhibiting EGFR & BRAFV600E	BMC chemistry	2,023	cc-by	16,667	RESEARCH Open Access BMC Chemistry BMC Chemistry BMC Chemistry Hagar et al. BMC Chemistry (2023) 17:116 https://doi.org/10.1186/s13065-023-01003-3 Abstract Introduction One of the most robust global challenges and difficulties in the 21st century is cancer. Treating cancer is a goal which continues to motivate researchers to innovate in design and development of new treatments to help battle the disease. Objectives Our objective was developing new antiapoptotic hybrids based on biologically active heterocyclic motifs "benzimidazole–oxadiazole-chalcone hybrids'' that had shown promising ability to inhibit EGFR and induce apoptosis. We expected these scaffolds to display anticancer activity via inhibition of BRAF, EGFR, and Bcl-2 and induction of apoptosis through activation of caspases. Methods The new hybrids 7a-x were evaluated for their anti-proliferative, EGFR & BRAFV600E inhibitory, and apoptosis induction activities were detected. Docking study & dynamic stimulation into EGFR and BRAFV600E were studied. Results All hybrids exhibited remarkable cell growth inhibition on the four tested cell lines with IC50 ranging from 0.95 μM to 12.50 μM. which was comparable to Doxorubicin. Compounds 7k-m had the most potent EGFR inhibitory activity. While, compounds 7e, 7g, 7k and 7l showed good inhibitory activities against BRAFV600E. Furthermore, Compounds 7k, 7l, and 7m increased Caspases 3,8 & 9, Cytochrome C and Bax levels and decreased Bcl-2 protein levels. Compounds 7k-m received the best binding scores and showed binding modes that were almost identical to each other and comparable with that of the co-crystalized Erlotinib in EGFR and BRAF active sites. Conclusion Compounds 7k-m could be used as potential apoptotic anti-proliferative agents upon further optimization. Keywords Benzimidazole, Oxadiazole, Chalcone, Apoptosis, Anticancer Agents, EGFR, BRAFV600E 2Pharmacology Department, College of Pharmacy, Jouf University, Sakaka 72314, Saudi Arabia 3Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt 4Pharmacognosy Department, Faculty of Pharmacy, Nahda University, Beni-Suef 62513, Egypt Correspondence: Dalia Abdelhamid dalia_abdelhameed@mu.edu.eg 1Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt Correspondence: Dalia Abdelhamid dalia_abdelhameed@mu.edu.eg 1Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt Chalcone/1,3,4-Oxadiazole/Benzimidazole hybrids as novel anti-proliferative agents inducing apoptosis and inhibiting EGFR & BRAFV600E Fatma Fouad Hagar1, Samar H. Abbas1, Hesham A. M. Gomaa2, Bahaa G. M. Youssif3, Ahmed M. Sayed4, Dalia Abdelhamid1* and Mohamed Abdel-Aziz1 Introduction development and progression of different carcinomas including lung, colorectal, breast, brain, and pancreas [15]. Therefore these genetic alterations showed high probability to respond to EGFR small molecule tyrosine kinase inhibitors (TKI). Additionally, a recent study has shown that suppression of EGFR signaling was correlated to induction of intrinsic apoptosis in sensitive non-small cell lung cancer (NSCLC) EGFR-mutant cell lines [16]. However, EGFR inhibitors have exhibited limited effica cies and have been challenged by innate and acquired resistance in the clinic [17]. Treating cancer is a goal which continues to motivate researchers to innovate in design and development of new treatments to help battle the disease. Despite these efforts to combat cancer it is spreading quickly to all age groups and it is the second leading cause of death [1]. The global burden of cancer is estimated to climb to 28.4 mil lion in 2040, representing a 47% increase over 2020 [2]. Lung, liver, stomach, breast, and colon cancer were the top five causes of cancer-related mortality worldwide [3]. Lung and breast cancers alone represent nearly one- fourth of all newly diagnosed cancer patients in 2020 [4 with lung cancer attributing to 18% of cancer mortality and breast cancer as the main cause of cancer mortality among women [5]. Erlotinib, is one of the most popular EGFR inhibitors which is currently marketed to treat many types of cancer with EGFR gene mutations (including non-small lung and pancreatic cancer) [18]. Its anticancer activity drives from suppressing intracellular phosphorylation of TK at the ATP binding site of the receptor, inhibiting JAK2V617F; a mutant version of JAK2 and inducing apoptotic cell death pathways [19]. Despite its high potency, selectivity, and acceptable safety profile, patients rapidly develop resis tance within 8–12 months from the start of treatment via mutation in the ATP binding pocket of the EGFR kinase domain [20]. Apoptosis is an intriguing target for development of innovative cancer therapeutics. Normally, the apoptotic pathway becomes activated by DNA damage or uncon trolled proliferation [6]. There are two pathways that activate apoptosis: the intrinsic and extrinsic pathways which correlate with intracellular and extracellular sig nals [7]. The intrinsic pathway is activated in response to DNA damage and cytokine deprivation, whereas the extrinsic pathway is triggered by the immune system. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party materil in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Introduction The two pathways converge at the executioner caspases [3, 6, 7] which are a class of cysteine proteases that cleave tar get proteins required for normal cell function [8]. Acti vation of caspases results in plasma membrane changes and shrinking of apoptotic cells that eventually lead to cell death [9]. The intrinsic pathway is regulated by B-cell lymphoma-2 (Bcl-2) protein family which include pro apoptotic effector proteins, proapoptotic BH3-only pro teins, and antiapoptotic Bcl-2 proteins. Bcl-2 proteins inhibit apoptosis through inhibition of the proapoptotic Bcl-2 proteins, Bcl-2-associated X protein (BAX) and Bcl-2 homologous antagonist killer (BAK) [10]. BH3-only proteins inhibit the antiapoptotic Bcl-2 proteins [11]. EGFR signaling is a part of a complex network that has been the target of effective cancer therapies. However, emergence of resistance is a major hurdle to developing an effective anticancer regimen [21]. The combination of an EGFR inhibitor and a BRAF inhibitor synergizes and improves anticancer activity through induction of apoptosis, as demonstrated by the combination of dab rafenib with cetuximab in colorectal cells [22]. Therefore, combination therapy that combines an anti-EGFR, anti- BRAF, and apoptotic agent may exhibit a multi-pronged approach that can be developed into a highly attractive and specific molecular oriented remedy.f i In our continued research efforts to develop antiapop totic hybrids based on biologically active heterocyclic motifs we have discovered that benzimidazole–oxadi azole-chalcone hybrids have shown promising ability to inhibit EGFR and induce apoptosis. [23–25]. Individu ally each motif has been reported as an effective antican cer scaffold with mechanisms of action as either EGFR inhibitor, BRAF inhibitor, or inducer of apoptosis [24, 26, 27]. In this study we explored new chemical architectures attaching the benzimidazole directly to oxadiazole and chalcone. In these compounds variably substituted aro matic rings were attached to the 2-position of benzimid azole. We envisioned this structural modification would afford improved compounds capable of interacting with EGFR and stimulating caspase-3 to induce apoptosis. Apoptosis evasion is essential for survival of can cer cells and leads to increasing invasiveness, stimulat ing angiogenesis, deregulation of cell proliferation, and interference with differentiation. Usually, the predomi nant methods of apoptosis evasion include inhibition of intrinsic pathway and caspase function, overexpression of antiapoptotic Bcl-2 proteins, and loss of BAX and/or BAK [12]. This results in resistance to any intrinsic apop totic stimuli which includes some anticancer drugs [13]. Introduction Epidermal growth factor receptor (EGFR) and B-RAF play a role in regulating the mitogen-activated protein (MAP) kinase pathway which affects cell division, differ entiation, and could lead to abnormal cell proliferation [14]. Signaling pathways of EGFR control angiogen esis, activation, and regulation of cellular proliferation. Overexpression of the EGFR gene and mutations of the EGFR tyrosine kinase domain were reported in the Apoptosis evasion is essential for survival of can cer cells and leads to increasing invasiveness, stimulat ing angiogenesis, deregulation of cell proliferation, and interference with differentiation. Usually, the predomi nant methods of apoptosis evasion include inhibition of intrinsic pathway and caspase function, overexpression of antiapoptotic Bcl-2 proteins, and loss of BAX and/or BAK [12]. This results in resistance to any intrinsic apop totic stimuli which includes some anticancer drugs [13]. Epidermal growth factor receptor (EGFR) and B-RAF play a role in regulating the mitogen-activated protein (MAP) kinase pathway which affects cell division, differ entiation, and could lead to abnormal cell proliferation [14]. Signaling pathways of EGFR control angiogen esis, activation, and regulation of cellular proliferation. Overexpression of the EGFR gene and mutations of the EGFR tyrosine kinase domain were reported in the Based on the previous silico molecular docking simu lations using MOE software for the most active com pounds and Erlotinib into ATP binding sites it was found that Erlotinib forms 2 H-bond interactions with Met769 Hagar et al. BMC Chemistry (2023) 17:116 Page 3 of 22 Page 3 of 22 Hagar et al. BMC Chemistry compounds 7a-x was accomplished by coupling the oxa diazole and acetylated chalcone in the presence triethyl amine and acetonitrile as solvent in good yields ranging from 73–89% [30]. The structure of the new compounds was confirmed via NMR and mass spectral analyses. 1 H NMR spectra of compounds showed singlet signal or two singlets due to rotamers at δ: 4.14–4.46 ppm related to (S-CH2-CO). On other hand, in all compounds, the two protons of chalcone appear in the aromatic region as two doublet signals at δ: 7.56–7.74 ppm and δ: 7.80–8.16 ppm with coupling constant range J = 15.2–15.6. Additionally, the amide proton of the linker NH appears as singlet sig nal at δ: 10.83–11.16 ppm and benzimidazole proton NH appears as singlet signal at δ: 13.10-13.48 ppm. Introduction 13 C NMR spectra of compounds showed two carbonyl groups related to C = O of chalcone and N-C = O appeared at δ: 186.96-189.12 ppm and 160.22–169.50 ppm, further more, SCH2 appears at δ: 43.63–47.58 ppm. and Gln767 and one cation…π interaction with Leu 820. None of the earlier benzimidazole- chalcone hybrids showed identical binding interactions as Erlotinib with the same amino acid residues. Moreover, the most potent EGFR inhibitors had one H‐bonding interaction with Met769 as Erlotinib which meant this interaction was essential for activity. The new design moved the aromatic ring to a distal substitution on benzimidazole to intro duce more flexibility in the structure. This modification allows the benzimidazole ring to be in closer proximity to more amino acids which are important for interaction with Erlotinib. Herein, we report the design, synthesis and vari ous biological evaluations of hybrid molecules formed from benzimidazole, oxadiazole, and chalcone deriva tives aimed to improve anticancer activity by synergetic effect, decrease side effects, and minimize emergence of drug resistance. We expected these scaffolds to display anticancer activity via inhibition of BRAF, EGFR, and Bcl-2 and induction of apoptosis through activation of caspases. We present the synthetic strategy for prepar ing twenty-four new compounds, the results of detailed anticancer screening, and molecular docking. Lastly, mechanistic investigations of the most active compounds 7k, 7L, and 7m regarding EGFR and BRAF inhibition and induction of caspases are described. Biological evaluation Anti-proliferative assays d In vitro one-dose anti-proliferative screening in NCI The twenty-four synthesized compounds were submitted to the National Cancer Institute (NCI), USA. Eighteen hybrids 7a-h, 7k-r, 7u, and 7v were selected by NCI for one-dose assessment of their anti-proliferative activities at 10 µM dose by Sulforhodamine B colorimet ric assay. The assessment was performed in 60 cell lines derived from nine tumor subpanels, comprising lung, leu kemia, melanoma, colon, renal, CNS, prostate, breast, and ovarian cancer cell lines. Results and discussion Chemistryh The series of chemical reactions that were employed to prepare targeted benzimidazole-chalcone hybrids 7a-x is illustrated in Fig. 1. First, substituted 2-phe nyl-1 H-benzo[d]imidazole-5-carboxylic acids 1a-e were synthesized through condensation of 3,4-diaminobenzoic acid with the appropriate aldehyde adduct in the pres ence of DMF for 3–6 h [28–29]. Fisher esterification of the resulting benzimidazole carboxylic acids 1a-e with absolute ethanol using catalytic amount of concentrated H2SO4 as a dehydrating agent with heating for 20 h at reflux afforded the corresponding ethyl ester compounds 2a-e [30]. Carbohydrazide compounds 3a-e were pre pared by heating the esters 2a-e with hydrazine mono hydrate for 6–7 h at reflux [30]. Consequently, the carbo hydrazide compounds 3a-e were converted to 1,3,4 oxa diazole-benzimidazole derivatives 4a-e by heating with carbon disulfide and KOH in absolute ethanol overnight [23, 29]. The one-dose NCI results revealed that the tested hybrids displayed remarkable anti-proliferative activities especially hybrids 7e, 7k, and 7m-o (Figs. 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 and 88 in supporting information). The most potent compound was hybrid 7k which displayed moderate to high anti- proliferative activities against eighteen cell lines includ ing lung, leukemia, melanoma, colon, renal, breast, and CNS cancer cell lines with growth percentages ranging from 13 to 67% (Fig. 2). Moreover, eight compounds 7g, 7h, 7k-m, 7p, 7q, and 7v exhibited strong anti-prolifer ative activities in LOX IMVI with growth percentages ranging from − 32 to 18%. While 7e and 7n showed mod erate activities against LOX IMVI with growth percent ages 56% and 44%, respectively. In addition, 7e and 7k demonstrated moderate anti-proliferative activity against the CCRF-CEM cell line, with growth percent rang ing from 38 to 52%. Compounds 7e, 7g, 7k, and 7n also displayed moderate anti-proliferative activities against the SR cell line with growth percentages ranging from 26 to 50%. Moreover, compounds 7c, 7e, 7g, 7k, 7m, 7p, and 7v exhibited notable anti-proliferative activity against the RPMI-8226 cell line with growth percentage Chalcone intermediates 5a-e were prepared by Claisen Schmidt condensation in the presence of KOH. A series of 2-bromo-N-(4-cinnamoylphenyl)acetamide deriva tives [30].6a-e were synthesized by reaction of the appro priate chalcone with bromoacetyl bromide at 0o C in a biphasic medium (DCM/water) using K2CO3 as a base [30]. Finally, the alkylation reaction that affords target (2023) 17:116 Hagar et al. Results and discussion Chemistryh BMC Chemistry (2023) 17:116 Page 4 of 22 Hagar et al. BMC Chemistry range 4.95–56%. Furthermore, compound 7e, 7k, and 7n showed moderate anti-proliferative activities in K-562 cell line with growth percent range 58%, 55% and 47% respectively. On the other hand, compounds 7e, 7g, 7k, 7n, 7o, and 7u-v displayed moderate activities against MCF-7 cell line with growth percentages ranging from Fig. 1 Scheme for synthesis of benzimidazole/chalcone hybrids 7a-x. Reagent and reaction conditions: (a) The appropriate sodium hydroxy(phenyl) methanesulfonate adduct, DMF, reflux 3-6 h; (b) EtOH, Conc. H2SO4, reflux 20 h; (c) NH2NH2, EtOH, reflux 3-7 h; (d) 1- CS2, KOH, EtOH, reflux 12 h.; 2- Conc HCl, (60.0-87.0%).; (e) KOH (60%), EtOH, stirring at 0 C for 3 h, then stirring at rt overnight; (f) BrCH2COBr, CH2Cl2, K2CO3, H2O, stirring overnight; (g) TEA, Acetonitrile, rt, 24-48 h Fig. 1 Scheme for synthesis of benzimidazole/chalcone hybrids 7a-x. Reagent and reaction conditions: (a) The appropriate sodium hydroxy(phenyl) methanesulfonate adduct, DMF, reflux 3-6 h; (b) EtOH, Conc. H2SO4, reflux 20 h; (c) NH2NH2, EtOH, reflux 3-7 h; (d) 1- CS2, KOH, EtOH, reflux 12 h.; 2- Conc HCl, (60.0-87.0%).; (e) KOH (60%), EtOH, stirring at 0 C for 3 h, then stirring at rt overnight; (f) BrCH2COBr, CH2Cl2, K2CO3, H2O, stirring overnight; (g) TEA, Acetonitrile, rt, 24-48 h Fig. 1 Scheme for synthesis of benzimidazole/chalcone hybrids 7a-x. Reagent and reaction conditions: (a) The appropriate sodium hydroxy(phenyl) methanesulfonate adduct, DMF, reflux 3-6 h; (b) EtOH, Conc. H2SO4, reflux 20 h; (c) NH2NH2, EtOH, reflux 3-7 h; (d) 1- CS2, KOH, EtOH, reflux 12 h.; 2- Conc HCl, (60.0-87.0%).; (e) KOH (60%), EtOH, stirring at 0 C for 3 h, then stirring at rt overnight; (f) BrCH2COBr, CH2Cl2, K2CO3, H2O, stirring overnight; (g) TEA, Acetonitrile, rt, 24-48 h Fig. 1 Scheme for synthesis of benzimidazole/chalcone hybrids 7a-x. Reagent and reaction conditions: (a) The appropriate sodium hydroxy(phenyl) methanesulfonate adduct, DMF, reflux 3-6 h; (b) EtOH, Conc. H2SO4, reflux 20 h; (c) NH2NH2, EtOH, reflux 3-7 h; (d) 1- CS2, KOH, EtOH, reflux 12 h.; 2- Conc HCl, (60.0-87.0%).; (e) KOH (60%), EtOH, stirring at 0 C for 3 h, then stirring at rt overnight; (f) BrCH2COBr, CH2Cl2, K2CO3, H2O, stirring overnight; (g) TEA, Acetonitrile, rt, 24-48 h range 4.95–56%. Furthermore, compound 7e, 7k, and 7n showed moderate anti-proliferative activities in K-562 cell line with growth percent range 58%, 55% and 47% respectively. Results and discussion Chemistryh Also, compounds 7e and 7k, displayed mod erate anti-proliferative activities against MOLT-4 cell line (growth percent 30% and 47%, respectively). Concerning HCT-116 cell line, four compounds; 7e, 7k, 7n, and 7o exhibited moderate activity against it with growth per centages equal to 40.76%, 58.69%, 57.96%, and 50.04%, range 4.95–56%. Furthermore, compound 7e, 7k, and 7n showed moderate anti-proliferative activities in K-562 cell line with growth percent range 58%, 55% and 47% respectively. Also, compounds 7e and 7k, displayed mod erate anti-proliferative activities against MOLT-4 cell line (growth percent 30% and 47%, respectively). Concerning HCT-116 cell line, four compounds; 7e, 7k, 7n, and 7o exhibited moderate activity against it with growth per centages equal to 40.76%, 58.69%, 57.96%, and 50.04%, respectively. On the other hand, compounds 7e, 7g, 7k, 7n, 7o, and 7u-v displayed moderate activities against MCF-7 cell line with growth percentages ranging from 42.88 to 59.08%. Additionally, compound 7e displayed higher potency against proliferation of RXF 393 cell line with growth percent equal to -6.89%. Moreover, com pounds 7c and 7k showed significant anti-proliferative activities against T-47D cell line with growth percentages equal to 52.35% and 42.05%, respectively. (2023) 17:116 Page 5 of 22 Hagar et al. BMC Chemistry Fig. 2 One dose mean graph for compound 7k on nine different cancer cell line panels at Conc 10 µM Fig. 2 One dose mean graph for compound 7k on nine different cancer cell line panels at Conc 10 µM Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. Results and discussion Chemistryh BMC Chemistry Page 6 of 22 Table 1 IC50 of 7g, 7L, 7p, 7q, 7v, and Staurosporine in melanoma LOX-IMVI cell line upon 48 h incubation Compound 7g 7l 7p 7q 7v Staurosporine IC50 ± SEM (µM) 1.05 ± 0.10 0.80 ± 0.10 1.20 ± 0.10 2.50 ± 0.20 0.90 ± 0.10 7.10 ± 0.05 Table 2 Cell viability % of MCF-10 A cells upon treatment with 50 µM of compounds 7a-x for 96 h and IC50 of compounds 7a-x & Doxorubicin in A-549, MCF-7, Panc-1 and HT-29 after 48 h incubation Compound Cell viability % in MCF-10 A (50 µM) IC50 ± SEM (µM) A-549 MCF-7 Panc-1 HT-29 7a 91 9.90 ± 1.10 9.20 ± 1.10 10.40 ± 1.30 11.15 ± 1.40 7b 88 8.20 ± 0.90 7.90 ± 0.80 8.40 ± 1.00 9.10 ± 1.10 7c 87 4.70 ± 0.50 4.15 ± 0.50 5.20 ± 0.40 5.50 ± 0.50 7d 89 9.70 ± 1.10 8.90 ± 1.10 10.30 ± 1.30 11.10 ± 1.40 7e 87 1.60 ± 0.30 1.25 ± 0.08 1.70 ± 0.30 1.95 ± 0.30 7f 87 2.15 ± 0.50 2.05 ± 0.50 2.40 ± 0.60 2.70 ± 0.50 7g 91 1.80 ± 0.60 1.45 ± 0.30 1.90 ± 0.50 2.10 ± 0.60 h 87 1.95 ± 0.60 1.75 ± 0.40 2.05 ± 0.50 2.25 ± 0.60 7i 84 9.70 ± 1.10 9.20 ± 1.00 10.15 ± 1.20 10.20 ± 1.30 7j 89 9.95 ± 1.10 9.40 ± 1.20 10.30 ± 1.20 10.70 ± 1.10 7k 91 1.20 ± 0.20 0.95 ± 0.08 1.30 ± 0.20 1.40 ± 0.20 7l 89 1.45 ± 0.30 1.10 ± 0.10 1.50 ± 0.30 1.65 ± 0.30 7m 94 1.40 ± 0.50 1.20 ± 0.08 1.50 ± 0.20 1.85 ± 0.40 7n 92 1.90 ± 0.60 1.65 ± 0.30 2.05 ± 0.50 2.20 ± 0.60 7o 89 7.60 ± 0.30 7.10 ± 0.60 8.20 ± 1.20 8.60 ± 0.40 7p 87 2.50 ± 0.50 2.10 ± 0.20 3.10 ± 0.40 3.30 ± 0.40 7q 94 3.40 ± 0.40 3.02 ± 0.30 3.80 ± 0.50 3.70 ± 0.50 7r 91 3.60 ± 0.50 3.10 ± 0.50 3.90 ± 0.50 4.20 ± 0.50 7s 87 3.80 ± 0.60 3.15 ± 0.50 4.30 ± 0.60 4.55 ± 0.60 7t 88 10.60 ± 1.10 10.10 ± 1.00 10.90 ± 1.20 10.80 ± 1.20 7u 90 2.25 ± 0.50 2.10 ± 0.08 2.40 ± 0.20 2.70 ± 0.50 7v 87 2.45 ± 0.50 2.20 ± 0.08 2.60 ± 0.20 2.90 ± 0.50 7w 91 10.90 ± 1.10 10.20 ± 1.20 11.15 ± 1.20 11.20 ± 1.30 7x 85 11.50 ± 1.20 10.800 ± 1.10 11.80 ± 1.20 12.50 ± 1.50 Doxorubicin - 1.21 ± 0.10 0.90 ± 0.10 1.41 ± 0.10 1.01 ± 0.10 of 7g, 7L, 7p, 7q, 7v, and Staurosporine in melanoma LOX-IMVI cell line upon 48 h incubation as a positive control against four cancer cell lines; pan creatic cancer cell line (Panc-1), epithelial line cancer cell (A-549), breast cancer cell line (MCF-7), and colon cancer cell line (HT-29). Results and discussion Chemistryh Further, it was noted that for optimal activ ity with p-chlorine on phenyl ring the substitution order on the chalcone phenyl ring was p- chlorine > p-methoxy >>> H. benzimidazole ring with an effective substitution order of p-methoxy group > p-chlorine atom >>> 3,4-dime thoxy groups. Further, it was noted that for optimal activ ity with p-chlorine on phenyl ring the substitution order on the chalcone phenyl ring was p- chlorine > p-methoxy >>> H. Results and discussion Chemistryh In vitro anti-proliferative assay in LOX-IMVI NCI in vitro anti-proliferative screening of compounds 7a-x showed preferable activity against melanoma LOX-IMVI cell line over other cell lines. Specifically, five compounds; 7g, 7l, 7p, 7q, and 7v showed remarkable anti-prolifera tive activities against it with growth percentages equal to -24.15%, -29.94%, -11.79%, 3.17%, and − 31.58%, respec tively. Consequently, the half-maximal inhibitory con centration (IC50) of these compounds was determined in LOX-IMVI cell line using propidium iodide (PI)31, 32 with Staurosporine (broad spectrum protein kinase inhibitor) as a reference. Interestingly, the five compounds displayed an IC50 lower than that of Staurosporine (as shown in Table 1). It is noteworthy that four of these compounds contain the para-chloro chalcone motif (except 7p). As presented in Table 2, all tested hybrids exhibited remarkable cell growth inhibition on the four tested cell lines with IC50 ranging from 0.95 µM to 12.50 µM which was comparable to Doxorubicin (IC50 ranging from 0.90 µM to 1.41 µM). The most effective compound was 7k with IC50 ranging from 0.95 µM to 1.40 µM and the sec ond most active one was 7L with IC50 ranging from 1.10 µM to 1.65 µM. It was observed that the highest activity was displayed by compounds; 7k, 7L, and 7m in which R1 = 4-OCH3. Additionally, these hybrids showed similar effects on the Panc-1, A-549, and HT-29 cell lines with slight variation in IC50 values. Our structure-activity relationship (SAR) analysis revealed that the anticancer activity is reliant on pattern of substitution in both phe nyl rings (Fig. 3). Anticancer activity was correlated to substitution on phenyl ring attached to position two of In vitro anti-proliferative assays in Panc-1, A-549, MCF-7, and HT-29 cells The propidium iodide (PI) tests31, 32 were further used for assessment of the IC50 of all synthesized compounds and compared to Doxorubicin (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Page 7 of 22 Hagar et al. BMC Chemistry Fig. 3 The effect of compounds derivatization on anti-proliferative activity showed cell viability of more than 80% (Table 2.). The cytotoxic activity of 7k against MCF-7 was 96-fold higher than its toxicity against MCF-10 A. Similarly compounds 7L and 7v were 111 and 97-fold more selective towards LOX-IMVI cell line respectively. benzimidazole ring with an effective substitution order of p-methoxy group > p-chlorine atom >>> 3,4-dime thoxy groups. Apoptosis induction activityhf Consequently, compounds 7k, 7L, and 7m were con sidered promising agents which could be used as poten tial anti-proliferative agents targeting EGFR-TK after optimization. Cytochrome C assay The amount of cytochrome C in the cell is critical for caspase activation and the beginning of the intrinsic apoptosis process [31]. The results of test ing compounds 7k, 7l, and 7m as Cytochrome C activa tors were shown in Table 4. Compounds 7k, 7l, and 7m increased Cytochrome C levels by 14, 13, and 11 times, respectively, when compared to untreated control cells. These results support the assumption that Cytochrome C overexpression and the activation of intrinsic apoptotic pathway were the causes of apoptosis. EGFR inhibitory activity assay compounds is mediated through inhibition of both EGFR and BRAFV600E. Apoptosis induction activityhf Caspases assays The effects of the most active com pounds; 7k, 7l, and 7m on caspases (3, 8, and 9) were evaluated and compared to Doxorubicin treated and untreated cells [38]. The results showed that the tested compounds increased the level of active caspase-3 by 7–8 folds when compared to control cells; and that com pounds 7k, 7l, and 7m induced remarkable overexpres sion of caspase-3 protein level (501.60 ± 4.00, 492.50 ± 4.00 and 473.60 ± 3.50 pg/mL, respectively) and were compa rable to Doxorubicin (503.50 ± 4.50 pg/mL). Compared to the untreated cells, the most active anti-proliferative derivative 7k showed an 8-fold increase in caspase-3 level. The effect of compounds 7k, 7l, and 7m on caspases 8 and 9 were also investigated to underline the role of intrinsic and extrinsic apoptotic pathways in these com pounds’ anti-proliferative actions. As shown in Table 4, compound 7k increased caspase 8 and 9 levels in com parison to untreated cells by 10 and 17 folds, respectively. Compound 7l increased caspase 8 and 9 levels by 9 and 16 folds, respectively. These results indicated that the new hybrids were capable of activating both the intrinsic and extrinsic pathways, with a stronger impact on the intrinsic pathway because caspase 9 levels were higher. effective compound 7k (R1 = 4-OCH3, R2 = H) demon strated the greatest inhibitory efficacy against EGFR with an IC50 of 0.55 µM, being approximately 7-fold less potent than Erlotinib (IC50 = 0.08 µM). Compounds 7L and 7m rank second and third in activity, with IC50 val ues of 0.80 µM and 0.90 µM, respectively. The remaining compounds displayed weak inhibitory activity against EGFR, with IC50 values ranging from 1.15 µM to 3.90 µM. Consequently, compounds 7k, 7L, and 7m were con sidered promising agents which could be used as poten tial anti-proliferative agents targeting EGFR-TK after optimization. effective compound 7k (R1 = 4-OCH3, R2 = H) demon strated the greatest inhibitory efficacy against EGFR with an IC50 of 0.55 µM, being approximately 7-fold less potent than Erlotinib (IC50 = 0.08 µM). Compounds 7L and 7m rank second and third in activity, with IC50 val ues of 0.80 µM and 0.90 µM, respectively. The remaining compounds displayed weak inhibitory activity against EGFR, with IC50 values ranging from 1.15 µM to 3.90 µM. EGFR inhibitory activity assay To explore the molecular mechanism of the synthesized hybrids, an assessment of the EGFR inhibitory ability of the most potent ten compounds was performed using the EGFR-TK assay [35, 36]. The most active compounds; 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v were selected to determine their enzymatic inhibitory activity and compared to Erlo tinib and the findings are included in Table 3. The results showed that the tested compounds inhibited EGFR with IC50 values ranging from 0.55 µM to 3.90 µM. The most In vitro toxicity assay in normal cells (cell viability assay) To check the safety of prepared compounds and evaluate their selectivity towards tumor cells over nor mal ones, the toxicity of compounds 7a-x were evaluated by 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazol (MTT) assay on the normal human mammary epithelial cell line (MCF-10 A) [33, 34]. The human mammary epi thelial cell lines were incubated for four days with a con centration of 50 µM of compounds 7a-x. All compounds Page 8 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry effective compound 7k (R1 = 4-OCH3, R2 = H) demon strated the greatest inhibitory efficacy against EGFR with an IC50 of 0.55 µM, being approximately 7-fold less potent than Erlotinib (IC50 = 0.08 µM). Compounds 7L and 7m rank second and third in activity, with IC50 val ues of 0.80 µM and 0.90 µM, respectively. The remaining compounds displayed weak inhibitory activity against EGFR, with IC50 values ranging from 1.15 µM to 3.90 µM. Consequently, compounds 7k, 7L, and 7m were con sidered promising agents which could be used as poten tial anti-proliferative agents targeting EGFR-TK after optimization. Table 3 Inhibitory activities of selected compounds against EGFR and BRAFV600E Compound EGFR Inhibition IC50 ± SEM (µM) BRAF V600E Inhibition IC50 ± SEM (µM) 7e 1.15 ± 0.20 0.90 ± 0.10 7g 1.70 ± 0.20 1.00 ± 0.20 7h 2.30 ± 0.20 2.70 ± 0.30 7k 0.55 ± 0.10 1.70 ± 0.20 7L 0.80 ± 0.10 1.90 ± 0.20 7m 0.90 ± 0.10 2.20 ± 0.20 7n 1.40 ± 0.20 3.70 ± 0.20 7p 3.50 ± 0.30 4.20 ± 0.30 7q 3.90 ± 0.30 4.70 ± 0.30 7v 2.80 ± 0.30 3.10 ± 0.20 Erlotinib 0.08 ± 0.01 0.06 ± 0.01 Table 3 Inhibitory activities of selected compounds against EGFR and BRAFV600E compounds is mediated through inhibition of both EGFR and BRAFV600E. BRAFV600E inhibitory activity An in vitro study was carried out to assess the anti-BRAFV600E activity of ten compounds namely, 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v and compared to Erlotinib [24, 37]. The enzyme assay revealed that all ten compounds inhibited BRAFV600E with IC50 values ranging from 0.90 µM to 4.70 µM (as shown in Table 3). Compounds 7e, 7g, 7k and 7l showed good inhibitory activities against BRAFV600E (IC50 = 0.90 µM, 1.00, µM, 1.70 µM, and 1.90 µM, respectively) and they were revealed to be potent inhibitors of cancer cell proliferation as well as promising EGFR inhibitors (IC50 = 1.15 µM, 1.70 µM, 0.55 µM and 0.80 µM respectively). These encouraging results sug gest that the favourable anti-proliferative activity of these Expression levels of BAX and Bcl-2 proteins The effects of compounds 7k, 7l, and 7m on BAX and Bcl-2 protein levels in the MCF-7 breast cancer cell lines were studied further using Doxorubicin as a reference for comparison [39]. The results in Table 5 showed that as compared to Table 4 Effects of compound 7k, 7l, and 7m on caspases activity and amount of Cytochrome C in MCF-7 breast cancer cells Compound Caspase-3 Caspase-8 Caspase-9 Cytochrome C Conc (pg/mL) Fold change Conc (ng/mL) Fold change Conc (ng/mL) Fold change Conc (ng/mL) Fold change 7k 501.600 ± 4.00 7.60 1.680 9.90 15.900 17.00 0.660 14.00 7l 492.500 ± 4.00 7.50 1.570 9.30 15.100 16.00 0.595 13.00 7m 473.600 ± 3.50 7.20 1.350 7.90 13.900 15.00 0.515 11.00 Doxorubicin 503.200 ± 4.20 7.65 1.750 10.00 16.200 17.40 0.604 13.10 Control 65.600 1.00 0.170 1.00 0.930 1.00 0.046 1.00 Page 9 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry Fig. 5 Apoptosis and necrosis percentage in MCF-7 cells after incubation with DMSO (control) and with IC50 of compound 7k (0.95 µM) for 24 h Fig. 5 Apoptosis and necrosis percentage in MCF-7 cells after incubation with DMSO (control) and with IC50 of compound 7k (0.95 µM) for 24 h Table 5 Effects of compound 7k-m on the protein expression levels of BAX and Bcl-2 Compound BAX Bcl-2 Conc (pg/mL) Fold change Conc (ng/ mL) Fold reduction 7k 279.500 33.0 0.940 5.4 7l 271.600 32.0 0.975 5.2 7m 268.200 31.5 1.050 4.9 Doxorubicin 276.500 32.5 0.985 5.1 Control 8.500 1.0 5.080 1.0 Fig. BRAFV600E inhibitory activity 4 Results of cell cycle analysis in untreated MCF-7 cells (Control) and MCF-7 cells treated with IC50 (0.95 µM) of hybrid 7k for 24 h Table 5 Effects of compound 7k-m on the protein expression levels of BAX and Bcl-2 Fig. 5 Apoptosis and necrosis percentage in MCF-7 cells after incubation with DMSO (control) and with IC50 of compound 7k (0.95 µM) for 24 h method40, 41 was used to investigate its capability to induce apoptosis. In this assay MCF-7 cell line was treated with IC50 concentration (0.95 µM) of 7k and stained with Annexin V/PI then incubated for 24 h. Results showed that compound 7k increased the level of both early and late apoptosis to 28.71% and 12.63% respectively whereas necrosis level was only 3.05% (Figs. 5 and 6). These cumu lative results further indicate that compound 7k showed potent anti-proliferative activity by induction of apoptosis. Fig. 4 Results of cell cycle analysis in untreated MCF-7 cells (Control) and MCF-7 cells treated with IC50 (0.95 µM) of hybrid 7k for 24 h Docking study into EGFR and BRAFV600Eh Doxorubicin, the compounds 7k, 7l, and 7m caused a significant increase in BAX levels. Compound 7k induc tion of BAX level (279.50 pg/mL) was similar to Doxo rubicin (276 pg/mL) with a 33-fold increase over control untreated cancer cell, followed by compound 7l (271 pg/ mL and 32-fold rise). Finally, compound 7k reduced anti apoptotic Bcl-2 protein levels in MCF-7 cells to 0.94 ng/ mL, followed by compound 7l (0.97 ng/mL) in compari son to Doxorubicin (0.98 ng/mL). Doxorubicin, the compounds 7k, 7l, and 7m caused a significant increase in BAX levels. Compound 7k induc tion of BAX level (279.50 pg/mL) was similar to Doxo rubicin (276 pg/mL) with a 33-fold increase over control untreated cancer cell, followed by compound 7l (271 pg/ mL and 32-fold rise). Finally, compound 7k reduced anti apoptotic Bcl-2 protein levels in MCF-7 cells to 0.94 ng/ mL, followed by compound 7l (0.97 ng/mL) in compari son to Doxorubicin (0.98 ng/mL). The structures of the most bioactive derivatives (i.e., 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v) were docked inside the active sites of EGFR and BRAF to investigate their bind ing interactions with these proteins (Figs. 7 and 8). The resulting docking scores of these derivatives were conver gent (i.e., docking scores ranged from − 8.4 to -10.7 kcal/ mol, Table 6) and comparable with that of the co-crystal ized ligands Erlotinib and vemurafenib, respectively. With regards to EGFR, compounds 7k, 7l, and 7m received the best binding scores (-10.6, -10.7, and − 10.4, respectively). Additionally, they showed binding modes that were almost identical to each other (RMSD = 0.79 Å) and comparable with that of the co-crystalized Erlotinib (docking score = -9.7 kcal/mol). Cell cycle analysis and apoptosis detection C ll l l i C d k Cell cycle analysis and apoptosis detection Cell cycle analysis Compound 7k was applied to MCF-7 cancer cell line with IC50 concentration (0.95 µM) to assess its effect on cell cycle progression and apoptosis induction. Data obtained from cell cycle analysis (Fig. 4) revealed that 7k significantly increased the percentage of accumulation of MCF-7 cells in G0-G1 and S phase 61.95% and 34.86% respectively as compared to 58.27% and 29.51% in untreated cells. On other hand, incubation with 7k decreased percentage accumulation in the G2/M phase to 3.19% versus 12.22% in control. From the pre vious results, it was concluded that compound 7k might arrest cells at G1/S and prevent it from entering G2/M phase as indicated by decreased percentage of accumula tion of cells in G2/M phase compared to control. As shown in Fig. 7, each structure was able to form 2 H-bonds with MET-769 and ASP-831 amino acid resi dues, while the co-crystalized Erlotinib formed a single H-bond with MET-769. Additionally, the three structures (i.e., 7k, 7l, & 7m) showed the same hydrophobic inter actions with PHE-699, VAL-702, LYS-721, LEU-820, and LEU-834 which also interact with Erlotinib. Similarly, 7k, 7l, and 7m were also the best-scoring compounds upon docking inside BRAF’s active site (docking scores = -9.8, -9.9, and − 9.9 kcal/mol, respec tively). Moreover, they showed identical binding modes (RMSD = 0.87 Å) comparable with that of the co-crys talized inhibitors Vemurafenib and Erlotinib (docking scores = -9.6 and − 9.5 kcal/mol, respectively) (Fig. 8). Apoptosis detection To further study the root for cyto toxic activity of compound 7k, Annexin V-FITC/PI assay Hagar et al. BMC Chemistry (2023) 17:116 Page 10 of 22 Hagar et al. BMC Chemistry Fig. 6 Cell cycle and apoptosis induction analysis of MCF-7 cells after incubation with IC50 of compound 7k (0.95 µM) for 24 h using Annexin V/PI com pared to control untreated MCF-7 Fig. 6 Cell cycle and apoptosis induction analysis of MCF-7 cells after incubation with IC50 of compound 7k (0.95 µM) for 24 h using Annexin V/PI com pared to control untreated MCF-7 The structures of 7k, 7l, and 7m were able to establish the same hydrophilic interactions (i.e. H-bonds) with THR-81, CYS-84, HIS-91, except for compound 7m which formed additional H-bond through its additional methoxy group with SER-87 (Fig. 8A). Furthermore, the three structures also shared hydrophobic interactions with ILE-15, VAL-23, and LEU-66. Cell cycle analysis and apoptosis detection C ll l l i C d k Vemurafenib (the co- crystalized inhibitor) established a slightly different bind ing mode, where it formed 2 H-bonds with GLN-82 and CYS-84, and four hydrophobic interactions with ILE-15, VAL-23 (similarly to compounds 7k-m), LYS-35, and PHE-48 (Fig. 8B). In silico prediction of physicochemical and pharmacokinetic properties In this study, we used two web servers Swiss ADME (http://www.swissadme.ch/index.php) and PKCSM (http://biosig.unimelb.edu.au/pkcsm/) to investigate the physicochemical and pharmacokinetic features of compounds 7k, 7l and 7m. Swiss ADME affords infor mation about SILICOS-IT, MLOGP, iLOGP, XLOGP3, and WLOGP, the distinct models that predict lipophi licity. Also, Log of Consensus Po/w is calculated by tak ing their arithmetic mean [43]. BOILED Egg is a map of polarity expressed in TPSA, another model for predict ing lipophilicity. The yolk reflects the potential for BBB permeability in the BOILED Egg plot (Fig. 10), whereas the white represents the possibility for GI absorption. Finally, a bioavailability radar map is a plot of six differ ent physicochemical parameters: size, polarity, flexibility, solubility, saturation, and lipophilicity. The pink hexagon in the center of the figure represents the optimal range for excellent oral bioavailability (Fig. 11) [44, 45]. To further validate the docking results, the best dock ing poses for 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF were subjected to 100 ns-long MD simulations. As shown in Fig. 9, the structures of the three compounds (i.e., 7k, 7L, and 7m) exhibited signifi cant stability inside the active sites of both enzymes with low deviations from their initial state (i.e., docked poses) (Average RMSDs ranged from 1.9 Å to 2.2 Å). Accord ingly, the calculated absolute binding free energy (ΔGBind) for each compound using the MM-PBSA method were convergent and comparable with the co-crystalized inhibitors (Erlotinib and Vemurafenib, respectively) (Table 7) [42]. Judging from the previous in silico struc tural analysis, it can be concluded that the derivatives 7k, 7L, and 7m are considered promising structure motifs acting as EGFR and BRAF dual inhibitors. PKCSM affords important information about pharma cokinetic parameters of the drug such as: Caco-2 per meability, volume of distribution at steady state (VDss), Pgp I and II inhibitors, total clearance, central nervous system (CNS) permeability, AMES toxicity, renal organic cation transporter 2 (OCT2) substrate, maximum recom mended tolerated dose (MRTD) human, oral rat acute toxicity (LD50) and chronic toxicity-lowest observed Page 11 of 22 Page 11 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. In silico prediction of physicochemical and pharmacokinetic properties BMC Chemistry Table 6 Results of the docking compounds 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v into the active sites of EGFR (PDB ID: 1M17) and BRAFV600E (PDB ID: 3OG7) in comparison with their co-crystallized ligands Compound Docking score EGFR Docking score BRAFV600E Interactions H-bonding Hydrophobic EGFR BRAF EGFR BRAF 7e -8.4 -8.5 - SER-87, HIS-91 PHE-699, VAL-702 VAL-23, LEU-66 7g -9.2 -9.4 - SER-87, HIS-91 PHE-699, LEU-834, LEU-820, ILE-15, VAL-23 7h -9.2 -9.9 - CYS-84, SER-87, HIS-91 VAL-702, LEU-834 ILE-15, VAL-23, LEU-66 7k -10.6 -9.8 MET-769, ASP-831 THR-81, CYS-84, SER-87, HIS-91 PHE-699, VAL-702, LEU-820, LEU-834 ILE-15, VAL-23, LEU-66 7l -10.7 -9.9 MET-769, ASP-831 THR-81, CYS-84, HIS-91 PHE-699, VAL-702, LEU-820, LEU-834 ILE-15, VAL-23, LEU-66 7m -10.4 -9.9 MET-769, ASP-831 THR-81, CYS-84, PHE-699, VAL-702, LEU-820, LEU-834 ILE-15, VAL-23, LEU-66 7n -10.1 -9.5 MET-769 THR-81, CYS-84 PHE-699, LEU-820, LEU-834 ILE-15, VAL-23, LEU-66 7p -9.1 -9.0 - THR-81 PHE-699, VAL-702, LEU-820 ILE-15, VAL-23 7q -8.8 -9.1 MET-769 CYS-84 PHE-699, VAL-702, LEU-820 VAL-23, LEU-66 7v -9.3 -9.1 ASP-831 THR-81, CYS-84 PHE-699, LEU-820, LEU-834 VAL-23, LEU-66 Erlotinib -9.7 -9.5 MET-769 - LEU-694, VAL-702 LEU-820 - Vemurafenib - -9.6 - GLN-82, CYS-84 - ILE-15, VAL-23, LYS-35, PHE-48 Table 6 Results of the docking compounds 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v into the active sites of EGFR (PDB ID: 1M17) and BRAFV600E (PDB ID: 3OG7) in comparison with their co-crystallized ligands PKCSM results were slightly different from Swis sADME results. The difference in prediction outcomes between web servers are most likely due to changes in the modules and algorithms utilized on each web server. As a result, it is critical to receive ADMET characteristics from many web servers rather than just one [47]. adverse effect (LOAEL), skin sensitization, hepatotoxic ity, Tetrahymena pyriformis toxicity, and LC50 by fathead minnow toxicity [46]. SwissADME results of compounds 7k, 7l, 7m, and Erlotinib are presented in Table 8. These results indi cated that compounds 7k and 7m obeyed Lipinski rule with one violation (M.wt > 500) while 7l did not; with two violations: M,wt > 500 and MLOGP of 4.17 (> 4.15). Com pound 7k calculated octanol/water partition coefficient (log P) is 3.28 which is equal to Erlotinib while the log P values for 7l and 7m were 5.87 and 5.41, respectively. The three compounds showed low molecular flexibility (rotat able bond (RB) > 10) which may indicate poor absorption of the compounds. In silico prediction of physicochemical and pharmacokinetic properties The results shown in Table 9, revealed that the com pounds 7k, 7l, and 7m displayed good values for oral absorption. Erlotinib was predicted to have a cellular per meability of 97.99% while compounds 7k, 7l, and 7m val ues ranged from 93.26 to 98.11%. The three compounds showed higher water solubility equal to -2.89 log mol/L than Erlotinib − 5.11 log mol/L. and displayed good skin permeability but low permeability in Caco2 (human colon adenocarcinoma). It was also predicted that 7k, 7l, and 7m may be a substrate of the ATP-binding cassette transporter P-glycoprotein, which was found to have an inhibitory effect in epithelial cells. All three compounds showed promising cell permea bility through diverse biological membrane as topological polar surface area (TPSA) results ranged from 148.30 to 157.53 while Erlotinib was 74.73. Page 12 of 22 (2023) 17:116 Hagar et al. BMC Chemistry Fig. 7 A: Binding modes of compounds 7k, 7l, 7m (green, cyan, and brick red structures, respectively) inside the binding site of kinase domain of human EGFR (PDB ID: 1M17) B: Kinase domain of EGFR with the co-crystalized Erlotinib (orange-colored structure) Fig. 7 A: Binding modes of compounds 7k, 7l, 7m (green, cyan, and brick red structures, respectively) inside the binding site of kinase domain of human EGFR (PDB ID: 1M17) B: Kinase domain of EGFR with the co-crystalized Erlotinib (orange-colored structure) Fig. 8 A: Binding modes of compounds 7k, 7l, 7m (green, cyan, and brick red structures, respectively) inside the binding site of kinase domain of human BRAF (PDB ID: 3OG7) B: Kinase domain of EGFR with the co-crystalized Erlotinib/Vemurafenib (orange-colored structure) Fig. 8 A: Binding modes of compounds 7k, 7l, 7m (green, cyan, and brick red structures, respectively) inside the binding site of kinase domain of human BRAF (PDB ID: 3OG7) B: Kinase domain of EGFR with the co-crystalized Erlotinib/Vemurafenib (orange-colored structure) Furthermore, the software predicted toxicity profiles including mutagenicity and cardiotoxicity. The three compounds and Erlotinib showed similar safety profile and acceptable level profile hERG I (human ether-a-go- go-related gene I) but displayed hepatotoxicity. Based on the in-silico ADME/Tox prediction, we may infer that compounds 7k, 7l, and 7m may possess an acceptable pharmacokinetic and safety profile, validating its promise as an oral therapy for cancer. Complete ADMET results of testing with PKCSM are presented in Table 9. Fig. 9 RMSDs of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF (A and B, respectively) in comparison with the previously reported co- crystalized inhibitors (i.e., Erlotinib and Vemurafenib, respectively) over the course of 100 ns-long MD simulations Table 7 The calculated ΔGBind (in kcal/mol) of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF in comparison with the previously reported co-crystalized inhibitors (i.e., Erlotinib and Vemurafenib, respective Table 7 The calculated ΔGBind (in kcal/mol) of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF (A and B, respectively) in comparison with the previously reported co-crystalized inhibitors (i.e., Erlotinib and Vemurafenib, respectively) Table 7 The calculated ΔGBind (in kcal/mol) of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF (A and B, respectively) in comparison with the previously reported co-crystalized inhibitors (i.e., Erlotinib and Vemurafenib, respectively) p p y p y p y Energy Component 7k 7l 7m Erlotinib Vemurafenib EGFR BRAF EGFR BRAF EGFR BRAF EGFR BRAF ΔG gas -49.3409 -53.6574 -54.9876 -53.6723 -41.8203 -49.5564 -48.1047 -48.4837 ΔG solv 14.8213 16.4637 16.7483 17.3443 11.2943 14.4463 13.6452 16.3827 ΔG TOTAL -34.5196 -38.2393 -38.2393 -36.328 -30.526 -35.1101 -34.4595 -32.101 Conclusion Swiss ADME and PKCSM. The results revealed that the compounds had an ADMET profile that is comparatively similar to Erlotinib. In this study we explored the apoptotic activity of new chemical architectures comprised of substituted benz imidazole moiety directly linked to an oxadiazole ring which is tethered to a chalcone derivative. The new hybrids’ NCI assay results revealed that they had remark able anti-proliferative activities, principally hybrids 7e, 7k, and 7m-o. Furthermore, in vitro anti-proliferative assays revealed promising inhibitory activity against a panel of four cancer cell lines, with IC50 values compara ble to Doxorubicin. Compounds 7k, 7l, and 7m displayed promising activity in activation of caspases, inhibiting EGFR and/or BRAFV600E, induction of BAX and inhibi tion of Bcl-2 proteins expression. Further docking, MD simulations, and ΔGBind experiments were conducted to study the binding modes and stability of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF. These results indicated that these compounds could be used as potential apoptotic anti-proliferative agents upon further optimization. ADMET properties of compounds 7k, 7l, and 7m were predicted successfully by two web servers General procedure for synthesis of 2-{5-[2-aryl-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3- arylacryloyl]phenyl}acetamide 7a-x [25, 30] TEA (0.18 mL/mol) was added as a base to an equimolar mixture of the proper 2-bromo-N-{4-[(E)-3-arylacryloyl] phenyl}acetamides 6a-e (0.09 mmol) and the proper oxa diazoles 4a-e (0.09 mmol) in acetonitrile. The reaction mixture was left to stir from 48 to 72 h at room tem perature till the formation of the precipitate. The target hybrids were obtained in a good yield by filtration, dry ing, and recrystallization of the formed precipitate from acetonitrile. In silico prediction of physicochemical and pharmacokinetic properties Concerning distribution, the compounds showed low volume of distribution at a steady state at a value of -0.011, -0.009 and-0.038 log L/kg which was comparable to Erlotinib value of 0.167 log L/kg. Distribution parame ter revealed that the compounds were poorly distributed to blood brain barrier and displayed safety in CNS. It was predicted that the three compounds have no or little drug-drug interactions as they were shown to be CYP3A4 but not CYP2D6 substrates, and CYP2C19, CYP2C9, and CYP3A4 but not CYP2D6 inhibitors. According to excretion parameters all compounds showed better results for total clearance from liver and kidney with values of 0.678, 0.669 and 0.703 log ml/min/ kg compared to Erlotinib with 0.572 log ml/min/kg value. (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Page 13 of 22 Hagar et al. BMC Chemistry Chemistry General Details: See Appendix A. General Details: See Appendix A. General procedure for synthesis of 2-{5-[2-aryl-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3- arylacryloyl]phenyl}acetamide 7a-x [25, 30] TEA (0.18 mL/mol) was added as a base to an equimolar mixture of the proper 2-bromo-N-{4-[(E)-3-arylacryloyl] phenyl}acetamides 6a-e (0.09 mmol) and the proper oxa diazoles 4a-e (0.09 mmol) in acetonitrile. The reaction mixture was left to stir from 48 to 72 h at room tem perature till the formation of the precipitate. The target hybrids were obtained in a good yield by filtration, dry ing, and recrystallization of the formed precipitate from acetonitrile. (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Page 14 of 22 Hagar et al. BMC Chemistry 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4- oxadiazol-2-ylthio}-N-{4-[(E)-3-phenylacryloyl] phenyl}acetamide (7a) Off-white crystal (0.21 g, 80% yield); m.p: 273–275 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 4.44 (2 H, s, SCH2), 7.46 (4 H, d, J = 5.4 Hz, Ar-H), 7.52–7.60 (3 H, m, Ar-H), 7.74 (1 H, d, J = 15.6 Hz, CH = CH), 7.82 (3 H, d, J = 8.4 Hz, Ar-H), 7.86–7.90 (2 H, m, Ar-H), 7.95 (1 H, d, J = 15.6 Hz, CH = CH), 8.19–8.25 (5 H, m, Ar-H), 10.86 (1 H, s, NH), 13.31 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.16, 112.88, 117.44, 117.66, 118.79, 120.29, 121.43, 122.44, 126.95, 127.35, 129.51, 129.73, 129.98, 130.48, 130.86, 133.25, 134.99, 143.44, 144.07, 153.85, 153.97, 154.32, 163.15, 165.57, 166.45, 188.32; HRMS (ESI) m/z [M + H]+ calcd for C32H23N5O3S: 558.1594, found: 558.1536. 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4- oxadiazol-2-ylthio}-N-{4-[(E)-3-(4-chlorophenyl) acryloyl]phenyl}acetamide (7b) Off-white crystal (0.19 g, 80% yield); m.p: 263–264 °C; 1 H NMR (500 MHz, DMSO d ) δ ( ) 4 43 (2 H SCH2) 7 52 (3 H d J = 8.2 Hz, Ar-H), 7.53–7.60 (3 H, m, Ar-H), 7.71 (1 H, d, J = 15.4 Hz, CH = CH), 7.81 (2 H, d, J = 8.5 Hz, Ar-H), 7.92 (2 H, d, J = 8.5 Hz, Ar-H), 7.97 (1 H, d, J = 15.4 Hz, CH = CH), 8.16–8.24 (6 H, m, Ar-H), 10.86 (1 H, s, NH), 13.32 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 47.58, 113.59, 117.84, 119.55, 121.26, 123.20, 127.52, 128.96, 129.47, 129.76, 130.38, 130.89, 131.48, 133.20, 134.04, 134.31, 135.14, 137.74, 138.06, 142.82, 144.02, 151.09, 153.12, 165.87, 166.46, 188.31; HRMS (ESI) m/z [M + H]+ calcd for C32H22ClN5O3S: 592.1205, found: 592.1160. Chemistry 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4-oxa diazol-2-ylthio}-N-{4-[(E)-3-(4-methoxyphenyl)acry loyl]phenyl}acetamide (7c) Beige crystal (0.19 g, 79% yield); m.p: 175–177 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.83 (3 H, s, OCH3), 4.26 (2 H, s, SCH2), 6.92–7.14 (3 H, m, Ar-H), 7.54–7.65 (5 H, m, 4 Ar-H + CH = CH), 7.73 (1 H, s, Ar-H), 7.80–7.90 (5 H, m, Ar-H), 8.16 (1 H, d, J = 15.4 Hz, CH = CH), 8.23–8.39 (3 H, m, Ar-H), 10.91 (1 H NH) 13 34 (1 H NH) 13 C NMR (125 MH Fig. 10 Forecast Boiled-Egg plotting from Swiss ADME online website for scaffolds 7k, 7l, 7m, and Erlotinib Fig. 10 Forecast Boiled-Egg plotting from Swiss ADME online website for scaffolds 7k, 7l, 7m, and Erlotinib Fig. 10 Forecast Boiled-Egg plotting from Swiss ADME online website for scaffolds 7k, 7l, 7m, and Erlotinib 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4- oxadiazol-2-ylthio}-N-{4-[(E)-3-phenylacryloyl] phenyl}acetamide (7a) Off-white crystal (0.21 g, 80% yield); m.p: 273–275 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 4.44 (2 H, s, SCH2), 7.46 (4 H, d, J = 5.4 Hz, Ar-H), 7.52–7.60 (3 H, m, Ar-H), 7.74 (1 H, d, J = 15.6 Hz, CH = CH), 7.82 (3 H, d, J = 8.4 Hz, Ar-H), 7.86–7.90 (2 H, m, Ar-H), 7.95 (1 H, d, J = 15.6 Hz, CH = CH), 8.19–8.25 (5 H, m, Ar-H), 10.86 (1 H, s, NH), 13.31 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.16, 112.88, 117.44, 117.66, 118.79, 120.29, 121.43, 122.44, 126.95, 127.35, 129.51, 129.73, 129.98, 130.48, 130.86, 133.25, 134.99, 143.44, 144.07, 153.85, 153.97, 154.32, 163.15, 165.57, 166.45, 188.32; HRMS (ESI) m/z [M + H]+ calcd for C32H23N5O3S: 558.1594, found: 558.1536. H-benzo[d]imidazol-5-yl]-1,3,4- J = 8.2 Hz, Ar-H), 7.53–7.60 (3 H, m, Ar-H), 7.71 (1 H, d, J = 15.4 Hz, CH = CH), 7.81 (2 H, d, J = 8.5 Hz, Ar-H), 7.92 (2 H, d, J = 8.5 Hz, Ar-H), 7.97 (1 H, d, J = 15.4 Hz, CH = CH), 8.16–8.24 (6 H, m, Ar-H), 10.86 (1 H, s, NH), 13.32 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 47.58, 113.59, 117.84, 119.55, 121.26, 123.20, 127.52, 128.96, 129.47, 129.76, 130.38, 130.89, 131.48, 133.20, 134.04, 134.31, 135.14, 137.74, 138.06, 142.82, 144.02, 151.09, 153.12, 165.87, 166.46, 188.31; HRMS (ESI) m/z [M + H]+ calcd for C32H22ClN5O3S: 592.1205, found: 592.1160. Chemistry 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4-oxa diazol-2-ylthio}-N-{4-[(E)-3-(4-methoxyphenyl)acry loyl]phenyl}acetamide (7c) Beige crystal (0.19 g, 79% yield); m.p: 175–177 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.83 (3 H, s, OCH3), 4.26 (2 H, s, SCH2), 6.92–7.14 (3 H, m, Ar-H), 7.54–7.65 (5 H, m, 4 Ar-H + CH = CH), 7.73 (1 H, s, Ar-H), 7.80–7.90 (5 H, m, Ar-H), 8.16 (1 H, d, J = 15.4 Hz, CH = CH), 8.23–8.39 (3 H, m, Ar-H), 10.91 (1 H, s, NH), 13.34 (1 H, s, NH); 13 C NMR (125 MHz, 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4-oxa diazol-2-ylthio}-N-{4-[(E)-3-(4-methoxyphenyl)acry loyl]phenyl}acetamide (7c) Beige crystal (0.19 g, 79% yield); m.p: 175–177 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.83 (3 H, s, OCH3), 4.26 (2 H, s, SCH2), 6.92–7.14 (3 H, m, Ar-H), 7.54–7.65 (5 H, m, 4 Ar-H + CH = CH), 7.73 (1 H, s, Ar-H), 7.80–7.90 (5 H, m, Ar-H), 8.16 (1 H, d, J = 15.4 Hz, CH = CH), 8.23–8.39 (3 H, m, Ar-H), 10.91 (1 H, s, NH), 13.34 (1 H, s, NH); 13 C NMR (125 MHz, 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4- oxadiazol-2-ylthio}-N-{4-[(E)-3-(4-chlorophenyl) acryloyl]phenyl}acetamide (7b) Off-white crystal (0.19 g, 80% yield); m.p: 263–264 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 4.43 (2 H, s, SCH2), 7.52 (3 H, d, Page 15 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Fig. 11 Radar bioavailability diagram from Swiss ADME website for scaffolds 7k, 7l, 7m, and Erlotinib. The pink area characterizes the optimum property values range for the oral bioavailability, and the red lines represent the forecasted characters Page 15 of 22 Page 15 of 22 Hagar et al. BMC Chemistry Fig. 11 Radar bioavailability diagram from Swiss ADME website for scaffolds 7k, 7l, 7m, and Erlotinib. The pink area characterizes the optimum property values range for the oral bioavailability, and the red lines represent the forecasted characters Fig. 11 Radar bioavailability diagram from Swiss ADME website for scaffolds 7k, 7l, 7m, and Erlotinib. The pink area characterizes the optimum property values range for the oral bioavailability, and the red lines represent the forecasted characters (1 H, s, NH), 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.89, 56.06, 56.22, 111.12, 111.95, 118.87, 119.13, 119.93, 124.28, 125.29, 127.20, 127.97, 129.29, 129.49, 129.88, 129.95, 130.22, 130.77, 133.42, 134.03, 143.20, 143.45, 144.37, 149.52, 151.70, 154.21, 163.06, 166.18, 166.55, 187.79; HRMS (ESI) m/z [M + K]+ calcd for C34H27N5O5S: 656.1364, found 656.1407. Chemistry 2-{5-[2-(4-Chlorophenyl)-1 H-benzo[d]imidazol- 5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-phenyl acryloyl]phenyl}acetamide (7f) Beige crystal (0.21 g, 84.5% yield); m.p: 161–163 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 4.45 (2 H, s, SCH2), 7.40–7.50 (4 H, m, Ar-H), 7.63 (2 H, d, J = 8.3 Hz, Ar-H), 7.73 (1 H, d, J = 15.6 Hz, CH = CH), 7.84 (3 H, d, J = 8.3 Hz, Ar-H), 7.88 (3 H, d, J = 8.3 Hz, Ar-H), 7.94 (1 H, d, J = 15.6 Hz, CH=CH) 8 19 (2 H d J=8 6 Hz Ar H) 8 24 (2 H d Table 9 ADMET Results of the test with PKCSM for the target compounds 7k, 7l, 7m and Erlotinib Properties 7k 7l 7m Erlotinib LOGP 6.9154 7.5688 6.924 3.4051 Surface area 250.582 260.885 262.060 169.532 Water Solubility -2.894 -2.894 -2.894 -5.114 Caco2 Permeability 0.133 0.026 0.114 0.877 Intestinal absorption 96.941 98.107 93.255 97.995 Skin permeability -2.735 -2.735 -2.735 -2.802 P-glycoprotein (Pgp) substrate Yes Yes Yes No Pgp1 inhibitor Yes Yes Yes Yes Pgp2 inhibitor Yes Yes Yes Yes VDss -0.011 -0.009 -0.038 0.167 BBB permeability -1.353 -1.534 -1.563 -0.604 CNS Permeability -2.881 -2.824 -3.043 -3.531 CYP2D6 substrate No No No No CYP3A4 substrate Yes Yes Yes Yes CYP1A2 inhibitor Yes Yes Yes No CYP2C19 inhibitor Yes Yes Yes Yes CYP2C9 inhibitor Yes Yes Yes Yes CYP2D6 inhibitor No No No No CYP3A4 inhibitor Yes Yes Yes Yes Total clearance 0.678 0.669 0.703 0.572 Renal OCT2 substrate Yes Yes No No AMES toxicity Yes Yes Yes No Max tolerated dose MRTDs log 0.339 0.324 0.315 0.375 hERG I inhibitor No No No No hERG II inhibitor Yes Yes Yes Yes Oral Rat Acute Toxicity LD50 2.479 2.479 2.479 3.155 LOAEL 2.165 2.095 1.895 1.477 Hepatotoxicity Yes Yes Yes Yes T. Pyriformistoxicity 0.285 0.285 0.285 0.356 Minnow toxicity -1.122 -1.579 -1.875 -2.252 Table 9 ADMET Results of the test with PKCSM for the target compounds 7k, 7l, 7m and Erlotinib 8.32 (1 H, d, J = 8.5 Hz, Ar-H), 10.89 (1 H, s, NH), 13.30 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.99, 56.62, 60.62, 106.92, 106.95, 107.12, 118.8, 121.49, 126.56, 127.12, 127.24, 127.26, 129.01, 129.03, 129.48, 129.72, 130.17, 130.47, 130.75, 139.20, 139.63, 140.36, 144.45, 145.40, 153.56, 162.28, 168.69, 186.96; HRMS (ESI) m/z [M + H]+ calcd for C35H29N5O6S: 648.1911, found: 648.1963. Chemistry BMC Chemistry (2023) 17:116 Page 16 of 22 Page 16 of 22 Hagar et al. BMC Chemistry Table 8 ADMET Results of the test with SwissADME for the target compounds 7k, 7l, 7m, and Erlotinib Compound M.Wt Fraction Csp3 RB HBA HBD MR TPSA Log P GI absorption BBB perm. Lip. V. Bio. Sc. 7k 587.65 0.06 11 7 2 166.67 148.30 3.28 Low No Yes 0.55 7l 622.09 0.06 11 7 2 171.68 148.30 5.87 Low No No 0.17 7m 617.67 0.09 12 8 2 173.17 157.53 5.41 Low No Yes 0.55 Erlotinib 393.44 0.27 10 6 1 111.40 74.73 3.28 High Yes Yes 0.55 e 8 ADMET Results of the test with SwissADME for the target compounds 7k, 7l, 7m, and Erlotinib pound M.Wt Fraction Csp3 RB HBA HBD MR TPSA Log P GI absorption BBB perm. Lip. V. Bio. Sc. 587.65 0.06 11 7 2 166.67 148.30 3.28 Low No Yes 0.55 622.09 0.06 11 7 2 171.68 148.30 5.87 Low No No 0.17 617.67 0.09 12 8 2 173.17 157.53 5.41 Low No Yes 0.55 inib 393.44 0.27 10 6 1 111.40 74.73 3.28 High Yes Yes 0.55 8.32 (1 H, d, J = 8.5 Hz, Ar-H), 10.89 (1 H, s, NH), 13.30 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.99, 56.62, 60.62, 106.92, 106.95, 107.12, 118.8, 121.49, 126.56, 127.12, 127.24, 127.26, 129.01, 129.03, 129.48, 129.72, 130.17, 130.47, 130.75, 139.20, 139.63, 140.36, 144.45, 145.40, 153.56, 162.28, 168.69, 186.96; HRMS (ESI) m/z [M + H]+ calcd for C35H29N5O6S: 648.1911, found: 648.1963. Chemistry DMSO-d6) δ (ppm): 43.63, 55.84, 114.86, 114.89, 114.91, 119.87, 119.88, 119.94, 127.06, 127.12, 127.24, 129.01, 129.03, 129.49, 129.51, 129.53, 129.62, 131.16, 131.38, 138.12, 139.15, 144.01, 145.35, 153.41, 161.97, 163.32, 187.81; HRMS (ESI) m/z [M + H]+ calcd for C33H25N5O4S: 588.1700, found: 588.1620. 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4-oxa diazol-2-ylthio}-N-{4-[(E)-3-(3,4-dimethoxyphenyl) acryloyl]phenyl}acetamide (7d) Dark brown (0.20 g, 83% yield); m.p: 156–158 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.82 (3 H, s, OCH3), 3.87 (3 H, s, OCH3), 4.45 (2 H, s, SCH2), 7.02 (1 H, d, J = 8.4 Hz, Ar-H), 7.38 (1 H, d, J = 8.4 Hz, Ar-H), 7.54 (1 H, s, Ar-H), 7.56–7.60 (3 H, m, 2 Ar-H + CH = CH), 7.69 (1 H, d, J = 15.4 Hz, CH = CH), 7.76 (1 H, d, J = 8.4 Hz, Ar-H), 7.84 (3 H, d, J = 8.4 Hz, Ar-H), 7.86 (1 H, s, Ar-H), 7.89–7.96 (1 H, m, Ar-H), 8.20 (2 H, d, J = 8.0 Hz, Ar-H), 8.24 (2 H, d, J = 8.0 Hz, Ar-H), 11.01 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4-oxa diazol-2-ylthio}-N-{4-[(E)-3-(3,4,5-trimethoxyphenyl) acryloyl]phenyl}acetamide (7e) Dark brown (0.21 g, 85% yield); m.p: 143–144 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.73 (3 H, s, OCH3), 3.88 (6 H, s, 2OCH3), 4.26 (1.20 H, s, SCH2), 4.45 (0.80 H, s, SCH2), 7.24 (2 H, s, Ar-H), 7.54 (1 H, d, J = 7.1 Hz, Ar-H), 7.57–7.60 (4 H, m, Ar-H), 7.70–7.74 (2 H, m, 1 Ar-H + CH = CH), 7.84 (1 H, d, J = 8.5 Hz, Ar-H), 7.94 (1 H, d, J = 15.3 Hz, CH = CH), 8.18 (1 H, d, J = 8.5 Hz, Ar-H), 8.23 (3 H, d, J = 8.5 Hz, Ar-H), 2-{5-[2-Phenyl-1 H-benzo[d]imidazol-5-yl]-1,3,4-oxa diazol-2-ylthio}-N-{4-[(E)-3-(3,4,5-trimethoxyphenyl) acryloyl]phenyl}acetamide (7e) Dark brown (0.21 g, 85% yield); m.p: 143–144 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.73 (3 H, s, OCH3), 3.88 (6 H, s, 2OCH3), 4.26 (1.20 H, s, SCH2), 4.45 (0.80 H, s, SCH2), 7.24 (2 H, s, Ar-H), 7.54 (1 H, d, J = 7.1 Hz, Ar-H), 7.57–7.60 (4 H, m, Ar-H), 7.70–7.74 (2 H, m, 1 Ar-H + CH = CH), 7.84 (1 H, d, J = 8.5 Hz, Ar-H), 7.94 (1 H, d, J = 15.3 Hz, CH = CH), 8.18 (1 H, d, J = 8.5 Hz, Ar-H), 8.23 (3 H, d, J = 8.5 Hz, Ar-H), Hagar et al. Chemistry 2-{5-[2-(4-Chlorophenyl)-1 H-benzo[d]imidazol- 5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-phenyl acryloyl]phenyl}acetamide (7f) Beige crystal (0.21 g, 84.5% yield); m.p: 161–163 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 4.45 (2 H, s, SCH2), 7.40–7.50 (4 H, m, Ar-H), 7.63 (2 H, d, J = 8.3 Hz, Ar-H), 7.73 (1 H, d, J = 15.6 Hz, CH = CH), 7.84 (3 H, d, J = 8.3 Hz, Ar-H), 7.88 (3 H, d, J = 8.3 Hz, Ar-H), 7.94 (1 H, d, J = 15.6 Hz, CH = CH), 8.19 (2 H, d, J = 8.6 Hz, Ar-H), 8.24 (2 H, d, Page 17 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry J = 8.6 Hz, Ar-H), 11.03 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.88, 117.50, 119.03, 122.39, 128.81, 128.98, 129.10, 129.26, 129.35, 129.59, 130.36, 130.45, 130.96, 133.11, 135.20, 135.67, 136.25, 143.42, 143.65, 143.92, 149.29, 155.80, 162.98, 166.37, 166.46, 188.02; HRMS (ESI) m/z [M + H]+ calcd for C32H22ClN5O3S: 592.1205, found: 592.1152. J = 8.6 Hz, Ar-H), 11.03 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.88, 117.50, 119.03, 122.39, 128.81, 128.98, 129.10, 129.26, 129.35, 129.59, 130.36, 130.45, 130.96, 133.11, 135.20, 135.67, 136.25, 143.42, 143.65, 143.92, 149.29, 155.80, 162.98, 166.37, 166.46, 188.02; HRMS (ESI) m/z [M + H]+ calcd for C32H22ClN5O3S: 592.1205, found: 592.1152. Ar-H), 8.36 (2 H, d, J = 8.3 Hz, Ar-H), 10.96 (1 H, s, NH), 13.48 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.02, 56.08, 56.25, 111.25, 112.06, 113.58, 119.08, 119.99, 124.31, 124.36, 124.43, 124.47, 127.47, 127.78, 127.85, 128.08, 130.27, 130.33, 133.08, 133.12, 133.50, 143.31, 144.42, 144.47, 149.52, 151.71, 164.14, 165.30, 166.07, 187.95; Anal. calcd. for C34H26ClN5O5S: C 62.62, H 4.02, N 10.74, S 4.92. Found: C 62.84, H 4.19, N 11.03, S 5.01. Chemistry 2-{5-[2-(4-Chlorophenyl)-1 H-benzo[d]imidazol- 5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(4- chlorophenyl)acryloyl]phenyl}acetamide (7g) Pale brown powder (0.19 g, 79.5% yield); m.p: 215–217 °C; 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 4.44 (2 H, s, SCH2), 7.19–7.24 (1 H, m, Ar-H), 7.50 (2 H, d, J = 8.1 Hz, Ar-H), 7.57–7.61 (1 H, m, Ar-H), 7.69 (1 H, d, J = 15.5 Hz, CH = CH), 7.79 (2 H, d, J = 8.3 Hz, Ar-H), 7.88–7.95 (3 H, m, 2 Ar-H + CH = CH), 7.97 (1 H, s, Ar-H), 8.11 (2 H, d, J = 8.0 Hz, Ar-H), 8.18 (2 H, d, J = 8.3 Hz, Ar-H), 8.33 (2 H, d, J = 8.0 Hz, Ar-H), 10.89 (1 H, s, NH), 13.35 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.10, 111.76, 113.41, 118.96, 119.08, 120.91, 123.15, 124.44, 127.47, 127.83, 128.75, 129.40, 130.54, 130.98, 131.37, 133.08, 134.20, 135.45, 142.47, 143.56, 145.13, 151.96, 164.14, 165.29, 166.11, 187.92; Anal. calcd. for C32H21Cl2N5O3S: C 61.35, H 3.38, N 11.18, S 5.12. Found: C 61.62, H 3.52, N 11.40, S 5.29. 2-{5-[2-(4-Chlorophenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (7j) Beige powder (0.20 g, 82% yield); m.p: 223–225 °C; 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 3.71 (3 H, s, OCH3), 3.86 (6 H, s, OCH3), 4.32 (0.40 H, s, SCH2), 4.45 (1.60 H, s, SCH2), 7.16–7.28 (4 H, m, Ar-H), 7.56–7.62 (1 H, m, Ar-H), 7.67 (1 H, d, J = 15.5 Hz, CH = CH), 7.81 (2 H, d, J = 8.8 Hz, Ar-H), 7.89 (1 H, d, J = 15.5 Hz, CH = CH), 8.14 (2 H, d, J = 8.4 Hz, Ar-H), 8.19 (2 H, d, J = 8.8 Hz, Ar-H), 8.35 (2 H, d, J = 8.4 Hz, Ar-H), 10.89 (1 H, s, NH), 13.39 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.18, 56.63, 60.62, 107.00, 113.44, 119.09, 121.60, 122.96, 124.46, 126.91, 127.49, 127.78, 127.85, 130.42, 130.46, 130.77, 133.11, 133.33, 140.22, 143.41, 143.47, 144.48, 153.59, 164.16, 165.31, 166.08, 166.32, 188.02; Anal. calcd. for C35H28ClN5O6S: C 61.63, H 4.14, N 10.27, S 4.70. Found: C 61.83, H 4.32, N 10.53, S 4.83. Chemistry 2-{5-[2-(4-Chlorophenyl)-1 H-benzo[d]imidazol-5-yl]- 1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(4-methoxyphe nyl)acryloyl]phenyl}acetamide (7h) Beige crystal (0.19 g, 82% yield); m.p: 228–230 °C; 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 3.81 (3 H, s, OCH3), 4.26 (0.60 H, s, SCH2), 4.46 (1.40 H, s, SCH2), 7.01 (2 H, d, J = 8.8 Hz, Ar-H), 7.22–7.24 (2 H, m, 1 Ar-H + CH = CH), 7.60–7.66 (2 H, m, Ar-H), 7.69 (1 H, d, J = 15.5 Hz, CH = CH), 7.78– 7.84 (4 H, m, Ar-H), 8.12–8.17 (4 H, m, Ar-H), 8.37–8.41 (2 H, m, Ar-H), 11.00 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.51, 55.42, 113.69, 114.44, 118.66, 119.46, 122.53, 122.59, 123.68, 127.03, 127.12, 127.27, 127.42, 129.89, 130.76, 133.02, 133.15, 142.90, 143.54, 149.98, 161.33, 163.66, 164.84, 164.92, 165.68, 168.97, 187.53; Anal. calcd. for C33H24ClN5O4S: C 63.71, H 3.89, N 11.26, S 5.15. Found: C 63.98, H 4.05, N 11.51, S 5.23. 2-{5-[2-(4-Methoxyphenyl)-1 H-benzo[d]imidazol- 5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-phenylac ryloyl]phenyl}acetamide (7k) Orange crystal (0.11 g, 74% yield); m.p: 178–180 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.86 (3 H, s, OCH3), 4.34 (2 H, s, SCH2), 7.14 (2 H, d, J = 8.3 Hz, Ar-H), 7.37–7.46 (5 H, m, Ar-H), 7.73 (1 H, d, J = 15.4 Hz, CH = CH), 7.78 (2 H, d, J = 8.3 Hz, Ar-H), 7.83–7.89 (3 H, m, Ar-H), 7.95 (1 H, d, J = 15.4 Hz, CH = CH), 8.15–8.21 (4 H, m, Ar-H), 10.79 (1 H, s, NH), 13.10 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.12, 55.85, 114.93, 114.95, 119.05, 119.09, 121.71, 122.42, 128.73, 128.76, 128.84, 129.27, 129.33, 129.36, 129.76, 130.44, 130.48, 130.97, 131.00, 133.08, 133.10, 135.22, 143.53, 143.94, 165.32, 166.32, 188.03; HRMS (ESI) m/z [M + H]+ calcd. for C33H25N5O4S: 588.1700, found: 588.1643. Chemistry Ar-H + CH = CH), 7.83 (1 H, d, J = 8.2 Hz, Ar-H), 7.92 (2 H, d, J = 8.4 Hz, Ar-H), 7.98 (1 H, d, J = 8.4 Hz, Ar-H), 8.15– 8.19 (3 H, m, Ar-H), 8.28 (2 H, m, Ar-H), 8.34 (1 H, d, J = 8.4 Hz, Ar-H), 10.98 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.86, 55.86, 114.37, 115.00, 115.52, 119.04, 119.26, 123.14, 125.22, 125.76, 126.34, 129.37, 129.81, 130.17, 130.44, 130.48, 130.59, 130.94, 132.98, 134.19, 135.41, 143.12, 143.64, 166.17, 166.46, 167.29, 187.81; Anal. calcd. for C33H24ClN5O4S: C 63.71, H 3.89, N 11.26, S 5.15. Found: C 63.52, H 4.03, N 11.52, S 5.23. 2-{5-[2-(4-Methoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-(4-methoxyphenyl)acryloyl]phenyl}acetamide (7m) Beige Crystal (0.13 g, 81% yield); m.p: 247–249 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.82 (3 H, s, OCH3), 3.85 (H, s, OCH3), 4.25 (1.25 H, s, SCH2), 4.43 (0.75 H, s, SCH2), 6.95–7.03 (2 H, m, Ar-H), 7.12–7.15 (3 H, m, Ar-H), 7.71 (1 H, d, J = 15.3 Hz, CH = CH), 7.80–7.85 (5 H, m, Ar-H + CH = CH), 8.09–8.20 (6 H, m, Ar-H), 10.90 (1 H, s, NH), 13.32 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.05, 55.84, 55.85, 114.84, 114.94, 116.83, 116.86, 119.06, 119.86, 120.78, 122.38, 127.85, 128.83, 128.90, 130.30, 131.15, 133.27, 133.43, 143.20, 144.01, 154.06, 154.36, 161.55, 162.94, 166.00, 166.51, 169.50, 187.80; HRMS (ESI) m/z [M + H]+ calcd. for C34H27N5O5S: 618.1806, found: 618.1737. 2-{5-[2-(3,4-Dimethoxyphenyl)-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-phenylacryloyl]phenyl}acetamide (7p) Orange pow der (0.18 g, 89.32% yield); m.p: 185–186 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.85 (3 H, s, OCH3), 3.90 (3 H, s, OCH3), 4.26 (0.60 H, s, SCH2), 4.43 (1.40 H, s, SCH2), 7.16 (1 H, s, Ar-H), 7.40–7.55 (3 H, m, Ar-H), 7.65 (1 H, s, Ar-H), 7.74 (1 H, d, J = 15.4 Hz, CH = CH), 7.77– 7.83 (5 H, m, Ar-H), 7.88–7.97 (3 H, m, 2 Ar-H + CH = CH), 8.08–8.20 (2 H, m, Ar-H), 8.26–8.28 (1 H, m, Ar-H), 10.88 (1 H, s, NH), 13.31(1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.01, 56.10, 56.23, 110.41, 112.31, 112.32, 116.86, 117.16, 119.09, 120.16, 122.22, 122.36, 129.27, 129.32, 129.36, 129.40, 129.77, 130.48, 131.10, 131.40, 133.27, 135.13, 137.82, 143.49, 143.95, 149.41, 151.29, 163.10, 166.29, 188.03; HRMS (ESI) m/z [M + H]+ calcd. Chemistry for C34H27N5O5S: 618.1806, found: 618.1865. 2-{5-[2-(4-Methoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3- (3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (7n) Pale brown crystal (0.19 g, 78% yield); m.p: 222– 224 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.81 (3 H, s, OCH3), 3.87 (6 H, s, 2OCH3), 4.35 (0.87 H, s, SCH2), 4.45 (1.13 H, s, SCH2), 7.00 (1 H, d, J = 8.6 Hz, Ar-H), 7.18 (1 H, d, J = 8.4 Hz, Ar-H), 7.23 (1 H, d, J = 8.4 Hz, Ar-H), 7.36 (1 H, d, J = 8.6 Hz, Ar-H), 7.53 (1 H, s, Ar-H), 7.67 (1 H, d, J = 15.6 Hz, CH = CH), 7.69–7.85 (4 H, m, Ar-H + CH = CH), 7.91 (1 H, d, J = 8.6 Hz, Ar-H), 8.00 (1 H, d, J = 8.6 Hz, Ar-H), 8.15–8.19 (2 H, m, Ar-H), 8.25 (1 H, s, Ar-H), 8.30 (1 H, d, J = 8.8 Hz, Ar-H), 11.00 (1 H, s, NH), 13.30 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 44.37, 56.05, 56.16, 56.21, 110.97, 112.10, 114.18, 115.29, 115.51, 119.02, 119.32, 119.86, 124.40, 125.73, 127.90, 129.35, 129.81, 130.17, 130.29, 130.32, 133.56, 142.97, 144.31, 149.37, 151.24, 151.76, 152.90, 162.28, 163.10, 167.65, 187.50; Anal. calcd. for C35H29N5O6S: C 64.90, H 4.51, N 10.81, S 4.95. Found: C 65.07, H 4.69, N 11.05, S 5.01. 2-{5-[2-(3,4-Dimethoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(4- chlorophenyl)acryloyl]phenyl}acetamide (7q) Brown powder (0.12 g, 84.62% yield); m.p: 194–195 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.85 (3 H, s, OCH3), 3.91 (3 H, s, OCH3), 4.14 (1.20 H, s, SCH2), 4.36 (0.80 H, s, SCH2), 7.15 (1 H, d, J = 6.6 Hz, Ar-H), 7.52 (2 H, d, J = 8.8 Hz, Ar-H), 7.71 (1 H, d, J = 15.2 Hz, CH = CH), 7.75 (1 H, s, Ar-H), 7.80 (2 H, d, J = 8.0 Hz, Ar-H), 7.84 (3 H, d, J = 8.8 Hz, Ar-H), 7.92 (2 H, d, J = 8.0 Hz, Ar-H), 7.97 (1 H, d, J = 15.2 Hz, CH = CH)), 8.12 (1 H, s, Ar-H), 8.15–8.22 (2 H, d, J = 8.8 Hz, Ar-H), 10.98 (1 H, s, NH), 13.28 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.80, 56.10, 56.15, 110.49, 112.29, 116.98, 119.01, 119.10, 120.34, 120.36, 122.49, 123.16, 129.31, 129.38, 130.45, 130.49, 130.96, 132.95, 133.01, 134.20, 134.59, 135.42, 142.42, 143.63, 143.66, 149.39, 151.24, 162.00, 166.53, 187.79; HRMS (ESI) m/z [M + H]+ calcd. Chemistry 2-{5-[2-(4-Chlorophenyl)-1 H-benzo[d]imidazol- 5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(3,4- dimethoxyphenyl)acryloyl]phenyl}acetamide (7i) Pale brown powder (0.23 g, 83% yield); m.p: 208–210 °C; 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 3.81 (3 H, s, OCH3), 3.86 (3 H, s, OCH3), 4.45 (2 H, s, SCH2), 7.01 (1 H, d, J = 8.3 Hz, Ar-H), 7.25 (1 H, d, J = 8.3 Hz, Ar-H), 7.36 (1 H, d, J = 8.3 Hz, Ar-H), 7.52 (1 H, s, Ar-H), 7.72–7.78 (2 H, m, 1 Ar-H + CH = CH), 7.80–7.84 (4 H, m, 3 Ar-H + CH = CH), 8.13 (2 H, d, J = 8.5 Hz, Ar-H), 8.18 (2 H, d, J = 8.5 Hz, 2-{5-[2-(4-Methoxyphenyl)-1 H-benzo[d]imidazol- 5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(4-chlo rophenyl)acryloyl]phenyl}acetamide (7l) Pale brown crystal (0.23 g, 80% yield); m.p: 245–247 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.89 (3 H, s, OCH3), 4.26 (1 H, s, SCH2), 4.35 (1 H, s, SCH2), 7.17–7.24 (2 H, m, Ar-H), 7.51 (2 H, d, J = 8.2 Hz, Ar-H), 7.70 (1 H, d, J = 15.6 Hz, CH = CH), 7.76–7.81 (2 H, m, 1 Page 18 of 22 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry 156 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.71 (3 H, s, OCH3), 3.84 (3 H, s, OCH3), 3.86 (6 H, s, 2OCH3), 4.39 (0.30 H, s, SCH2), 4.46 (1.70 H, s, SCH2), 7.15 (2 H, d, J = 8.8 Hz, Ar-H), 7.23 (2 H, s, Ar-H), 7.67 (1 H, d, J = 8.0 Hz, Ar-H), 7.71 (1 H, d, J = 15.5 Hz, CH = CH), 7.86 (2 H, d, J = 8.8 Hz, Ar-H), 7.91 (1 H, d, J = 15.5 Hz, CH = CH), 8.20 (3 H, d, J = 8.5 Hz, Ar-H), 8.25 (3 H, d, J = 8.5 Hz, Ar-H), 11.16 (1 H, s, NH), 13.38 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 44.32, 55.94, 56.63, 57.81, 106.96, 114.81, 114.96, 114.99, 115.02, 116.87, 119.03, 119.06, 121.72, 129.15, 129.22, 129.25, 130.43, 130.74, 132.98, 140.00, 140.15, 143.48, 144.31, 153.52, 153.55, 161.76, 166.02, 168.18, 188.00; HRMS (ESI) m/z [M + H]+ calcd. for C36H31N5O7S: 678.2017, found: 678.2090. Chemistry for C34H26ClN5O5S: 652.1416, found: 652.1367. 2-{5-[2-(4-Methoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (7o) Pale brown Crystal (0.21 g, 86% yield); m.p: 154– Hagar et al. BMC Chemistry (2023) 17:116 Page 19 of 22 Hagar et al. BMC Chemistry (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.05, 56.10, 56.59, 56.65, 60.60, 107.24, 110.15, 112.31, 116.65, 118.80, 121.20, 121.50, 127.89, 128.43, 129.77, 130.59, 131.12, 133.25, 139.46, 139.98, 140.29, 143.19, 143.49, 145.35, 145.65, 146.69, 149.08, 153.72, 159.07, 164.44, 166.54, 189.12; HRMS (ESI) m/z [M + H]+ calcd. for C37H33N5O8S: 708.2123, found 708.2062. (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.05, 56.10, 56.59, 56.65, 60.60, 107.24, 110.15, 112.31, 116.65, 118.80, 121.20, 121.50, 127.89, 128.43, 129.77, 130.59, 131.12, 133.25, 139.46, 139.98, 140.29, 143.19, 143.49, 145.35, 145.65, 146.69, 149.08, 153.72, 159.07, 164.44, 166.54, 189.12; HRMS (ESI) m/z [M + H]+ calcd. for C37H33N5O8S: 708.2123, found 708.2062. 2-{5-[2-(3,4-Dimethoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(4- methoxyphenyl)acryloyl]phenyl}acetamide (7r) Beige powder (0.15 g, 78.62% yield); m.p: 170–171 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.83 (3 H, s, OCH3), 3.86 (3 H, s, OCH3), 3.90 (3 H, s, OCH3), 4.34 (1.60 H, s, SCH2), 4.43 (0.40 H, s, SCH2), 7.02 (2 H, d, J = 8.5 Hz, Ar-H), 7.16 (1 H, d, J = 8.5 Hz, Ar-H), 7.64 (1 H, s, Ar-H), 7.70 (1 H, d, J = 15.6 Hz, CH = CH), 7.76–7.84 (7 H, m, Ar-H + CH = CH), 7.86 (1 H, s, Ar-H), 8.13–8.20 (3 H, m, Ar-H), 10.83 (1 H, s, NH), 13.29 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 45.98, 55.84, 56.11, 56.12, 110.43, 110.44, 112.31, 114.86, 115.52, 116.88, 118.89, 119.02, 119.03, 119.86, 119.89, 120.18, 120.21, 122.59, 124.69, 124.72, 124.89, 125.04, 127.86, 130.27, 131.16, 143.70, 149.39, 151.02, 161.75, 168.17, 187.79; Anal. calcd. for C35H29N5O6S: C 64.90, H 4.51, N 10.81, S 4.95. Found: C 64.79, H 4.63, N 11.02, S 4.92. Chemistry 2-{5-[2-(3,4,5-Trimethoxyphenyl)-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-phenylacryloyl]phenyl}acetamide (7u) Brown crys tal (0.13 g, 73.35% yield); m.p: 188–190 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.92 (6 H, s, 2OCH3), 3.94 (3 H, s, OCH3), 4.36 (1.25 H, s, SCH2), 4.46 (0.75 H, s, SCH2), 7.42–7.46 (4 H, m, Ar-H), 7.71 (2 H, d, J = 8.6 Hz, Ar-H), 7.75 (1 H, d, J = 15.6 Hz, CH = CH), 7.80–7.84 (2 H, m, 1 Ar-H + CH = CH), 7.88 (2 H, s, Ar-H), 7.93 (1 H, d, J = 6.6 Hz, Ar-H), 7.95–8.01 (1 H, m, Ar-H), 8.16 (3 H, d, J = 8.6 Hz, Ar-H), 10.90 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 44.46, 56.68, 57.58, 110.16, 112.69, 114.10, 119.25, 120.67, 122.08, 122.98, 124.11, 124.40, 124.62, 129.77, 130.59, 130.88, 131.18, 132.08, 135.43, 141.41, 143.71, 148.56, 151.11, 153.64, 154.23, 162.72, 165.35, 187.79; Anal. calcd. for C35H29N5O6S: C 64.90, H 4.51, N 10.81, S 4.95. Found: C 65.12, H 4.68, N 10.97, S 5.03. 2-{5-[2-(3,4-Dimethoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3- (3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (7s) Brown powder (0.20 g, 86.30% yield); m.p: 190– 193 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.82 (3 H, s, OCH3), 3.85 (3 H, s, OCH3), 3.87 (3 H, s, OCH3), 3.90 (3 H, s, OCH3), 4.26 (0.50 H, s, SCH2), 4.43 (1.50 H, s, SCH2), 6.98–7.07 (1 H, m, Ar-H), 7.15 (1 H, d, J = 8.2 Hz, Ar-H), 7.37–7.44 (2 H, m, Ar-H), 7.56 (1 H, d, J = 15.6 Hz, CH = CH), 7.62–7.69 (1 H, m, Ar-H), 7.71 (1 H, s, Ar-H), 7.82 (4 H, d, J = 8.8 Hz, Ar-H), 7.87 (1 H, d, J = 15.6 Hz, CH = CH), 8.13 (1 H, s, Ar-H), 8.20 (1 H, d, J = 8.8 Hz, Ar-H), 8.31 (1 H, d, J = 8.8 Hz, Ar-H), 10.88 (1 H, s, NH), 13.32 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.02, 56.05, 56.10, 56.21, 110.35, 110.41, 111.01, 112.00, 112.05, 112.30, 112.35, 116.87, 118.59, 118.95, 119.04, 119.88, 119.92, 122.39, 124.34, 128.06, 130.32, 130.35, 144.52, 149.41, 149.47, 151.13, 151.30, 151.65, 151.90, 165.74, 166.18, 166.62, 188.01; HRMS (ESI) m/z [M + H]+ calcd. for C36H31N5O7S: 678.2017, found 678.1952. Chemistry BMC Chemistry Compounds 7a-x were incubated with cancer cells for 2 days at different concentrations. See Appendix A. CH = CH), 7.83 (2 H, d, J = 6.6 Hz, Ar-H), 7.84–7.87 (3 H, m, Ar-H + CH = CH), 8.15–8.18 (3 H, m, Ar-H), 11.06 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 44.23, 55.86, 56.99, 60.41, 105.01, 114.71, 118.65, 119.85, 122.26, 122.67, 127.23, 128.35, 129.47, 130.89, 130.94, 135.90, 142.61, 142.82, 144.03, 148.83, 149.09, 152.50, 153.11, 153.13, 161.62, 162.43, 162.73, 166.16, 187.79; HRMS (ESI) m/z [M + H]+ calcd. for C36H31N5O7S: 678.2017, found: 678.1996. CH = CH), 7.83 (2 H, d, J = 6.6 Hz, Ar-H), 7.84–7.87 (3 H, m, Ar-H + CH = CH), 8.15–8.18 (3 H, m, Ar-H), 11.06 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 44.23, 55.86, 56.99, 60.41, 105.01, 114.71, 118.65, 119.85, 122.26, 122.67, 127.23, 128.35, 129.47, 130.89, 130.94, 135.90, 142.61, 142.82, 144.03, 148.83, 149.09, 152.50, 153.11, 153.13, 161.62, 162.43, 162.73, 166.16, 187.79; HRMS (ESI) m/z [M + H]+ calcd. for C36H31N5O7S: 678.2017, found: 678.1996. Cell viability determination MTT assay was done to determine the effect of the synthesized compounds on the viability of mammary epithelial cells (MCF-10 A) [33, 34]. Compounds 7a-x were incubated with MCF-10 A cells for 4 days at 50 µM concentration, and the viability of cells was determined. See Appendix A. 2-{5-[2-(3,4,5-Trimethoxyphenyl)-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-(3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (7x) Brown crystal (0.21 g, 86.50% yield); m.p: 212–215°C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.82 (3 H, s, OCH3), 3.87 (3 H, s, OCH3), 3.92 (6 H, s, 2OCH3), 3.93 (3 H, s, OCH3), 4.36 (1.25 H, s, SCH2), 4.45 (0.75 H, s, SCH2), 7.02 (1 H, d, J = 8.8 Hz, Ar-H), 7.36–7.39 (1 H, m, Ar-H), 7.66–7.72 (3 H, m, 2 Ar-H + CH = CH), 7.76–7.79 (2 H, m, 1 Ar-H + CH = CH), 7.81 (2 H, s, Ar-H), 8.83 (2 H, d, J = 7.5 Hz, Ar-H), 8.15–8.19 (3 H, m, Ar-H), 11.03 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm); 44.46, 54.42, 56.06, 56.22, 56.97, 104.78, 110.45, 111.88, 117.22, 119.16, 119.34, 120.02, 122.57, 124.10, 124.47, 124.63, 128.08, 130.26, 132.90, 133.40, 139.78, 139.97, 144.97, 145.30, 148.64, 149.41, 151.39, 151.59, 160.22, 164.69, 167.57, 187.78; Anal. calcd. for C37H33N5O8S: C 62.79, H 4.70, N 9.90, S 4.53. Found: C 62.95, H 4.82, N 10.16, S 4.60. Biology Screening of anti-proliferative activity by NCIh BAX activation assay The most potent caspase activa tors 7k, 7l, and 7m were investigated for their influence on BAX level in a breast cancer cell line (MCF-7) using doxorubicin as a control according to reported assay [38]. See Appendix A. The methodology of the NCI anti-proliferative screening has been described in details elsewhere (http://www.dtp. nci.nih.gov). Cytotoxic activity using MTT assay and determination of IC50 Assay for anti-proliferative effect in melanoma LOX- IMVI cell lines Using the propidium iodide fluorescence assay with Staurosporine as the reference drug, the anti proliferative activities of compounds 7g, 7l, 7p, 7q, and 7v, was carried out according to a previously reported procedure31, 32 to detect IC50 against LOX-IMVI cell lines. compounds 7g, 7l, 7p, 7q, and 7v were incubated with LOX-IMVI cells for 2 days. See Appendix A. Bcl-2 Inhibition assay The most potent caspase acti vators 7k, 7l, and 7m were examined for their influence on Bacl-2 level in a breast cancer cell line (MCF-7) using doxorubicin as a control according to reported assay [38]. See Appendix A. Cell apoptosis assay Apoptosis was determined by flow cytometry based on the Annexin-V-fluoresce in isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, USA) [40, 41] See Appendix A. Assay for anti-proliferative effect Using the propidium iodide fluorescence assay with doxorubicin as the refer ence drug, IC50 of the 24 compounds 7a-x, was carried out according to a previously reported procedure31, 32 against a panel of four human cancer cell lines; epithelial cancer cell line (A-549), breast cancer cell line (MCF-7), colon cancer cell line (HT-29) and pancreas cancer cell line (Panc-1). Chemistry 2-{5-[2-(3,4,5-Trimethoxyphenyl)-1 H-benzo[d]imid azol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)-3-(4- chlorophenyl)acryloyl]phenyl}acetamide (7v) Brown crystal (0.16 g, 77.54% yield); m.p: 196–198 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.75 (6 H, s, OCH3), 3.76 (3 H, s, 2OCH3), 4.25 (0.75 H, s, SCH2), 4.43 (1.25 H, s, SCH2), 7.51–7.52 (5 H, m, 4 Ar-H + CH = CH), 7.72 (2 H, d, J = 6.6 Hz, Ar-H), 7.77–7.82 (2 H, m, 1 Ar-H + CH = CH), 7.94 (2 H, s, Ar-H), 7.97 (2 H, d, J = 6.6 Hz, Ar-H), 8.15 (1 H, d, J = 8.5 Hz, Ar-H), 8.20 (1 H, d, J = 6.6 Hz, Ar-H), 10.92 (1 H, s, NH); 13 C NMR (125 MHz, DMSO-d6) δ (ppm): 46.02, 56.47, 60.78, 117.17, 119.03, 121.50, 123.13, 123.16, 123.19, 128.46, 129.38, 130.06, 130.29, 130.95, 130.97, 131.01, 134.32, 135.41, 135.43, 135.52, 136.48, 142.15, 142.45, 153.72, 153.74, 163.32, 164.66, 188.00; HRMS (ESI) m/z [M + H]+ calcd. for C35H28ClN5O6S: 682.1522, found: 682.1471. 2-{5-[2-(3,4-Dimethoxyphenyl)-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4-[(E)- 3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acet amide (7t) Brown crystal (0.165 g, 79.35% yield); m.p: 190–193 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.72 (3 H, s, OCH3), 3.85 (3 H, s, OCH3), 3.87 (6 H, s, 2OCH3), 3.90 (3 H, s, OCH3), 4.24 (0.75 H, s, SCH2), 4.43 (1.25 H, s, SCH2), 7.14–7.17 (1 H, m, Ar-H), 7.23 (2 H, s, Ar-H), 7.26 (1 H, d, J = 8.6 Hz, Ar-H)), 7.65–7.70 (2 H, m, 1 Ar-H + CH = CH), 7.78 (1 H, s, Ar-H), 7.81 (3 H, d, J = 8.6 Hz, Ar-H), 7.84 (1 H, s, Ar-H), 7.90 (1 H, d, J = 15.4 Hz, CH = CH), 8.21 (1 H, d, J = 8.4 Hz, Ar-H), 8.31 (1 H, d, J = 8.4 Hz, Ar-H), 10.91 (1 H, s, NH), 13.24 2-{5-[2-(3,4,5-Trimethoxyphenyl)-1 H-benzo[d] imidazol-5-yl]-1,3,4-oxadiazol-2-ylthio}-N-{4- [(E)-3-(4-methoxyphenyl)acryloyl]phenyl}acet amide (7w) Brown crystal (0.163 g, 76.65% yield); m.p: 200–204 °C; 1 H NMR (500 MHz, DMSO-d6) δ (ppm): 3.76 (3 H, s, OCH3), 3.82 (3 H, s, OCH3), 3.93 (6 H, s, 2OCH3), 4.26 (0.70 H, s, SCH2), 4.45 (1.30 H, s, SCH2), 7.00-7.02 (2 H, m, Ar-H), 7.69 (2 H, d, J = 6.6 Hz, Ar-H), 7.77 (2 H, d, J = 6.6 Hz, Ar-H), 7.80 (1 H, d, J = 15.4 Hz, Hagar et al. BMC Chemistry (2023) 17:116 Hagar et al. BMC Chemistry (2023) 17:116 Page 20 of 22 Hagar et al. EGFR inhibitory assayhfi The inhibitory efficacy of of the most active compounds 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v against EGFR was evalu ated using the EGFR-TK assay [35, 36]. See Appendix A. Apoptosis-induction activity detectionhf Caspases assays The effect of compounds; 7k, 7l, and 7m on caspases-3,8,9 was determined and compared to doxorubicin as a control according to reported assays [38]. See Appendix A. Cytochrome C assay Compounds 7k, 7l, and 7m were evaluated as Cytochrome C activators in the MCF-7 human breast cancer cell line according to previously reported assays [39]. See Appendix A. BRAF kinase assay Compounds 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v were further examined for their ability to inhibit the V600E mutant BRAF using kinase assay against BRAFV600E. 24, 37 See Appendix A. Data Availability D t d t i l 16. Gao F, Li M, Liu W, Li W. 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Involved in writing the details of biological and biochemical tests and data in the manuscript.4- Bahaa Youssif Principal investigator, a committee member involved with the design and execution of biological investigations and contributed in revising the manuscript.5- Ahmed Sayed Carried out molecular modelling studies. Involved in writing the details of docking studies in the manuscript.6- Dalia Abdelhamid* Principal investigator, a committee member involved with the design and execution of organic synthetic procedures and schemes and principal contributor in writing and revising the manuscript. Responsible for all journal communications.7- Mohamed Abdel-Aziz Principal investigator, a committee member involved with the design and execution of organic synthetic procedures and schemes and principal coordinator of the whole research project. Involved in revising the manuscript. 7. Carneiro BA, El-Deiry WS. Targeting apoptosis in Cancer Therapy. 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https://openalex.org/W3117684333	https://link.springer.com/content/pdf/10.1007/JHEP04(2021)186.pdf	English	null	Geometrizing non-relativistic bilinear deformations	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	19,452	Published for SISSA by Springer Published for SISSA by Springer Received: January 30, 2021 Revised: March 9, 2021 Accepted: March 21, 2021 Published: April 20, 2021 Received: January 30, 2021 Revised: March 9, 2021 Accepted: March 21, 2021 Published: April 20, 2021 Open Access, c⃝The Authors. Article funded by SCOAP3. Keywords: 2D Gravity, Field Theories in Lower Dimensions, Integrable Field Theories Geometrizing non-relativistic bilinear deformations JHEP04(2021)186 Dennis Hansen,a Yunfeng Jiangb,c and Jiuci Xud,e Dennis Hansen,a Yunfeng Jiangb,c and Jiuci Xud,e aInstitut für Theoretische Physik, Eidgenössische Technische Hochschule Zürich, Wolfgang-Pauli-Strasse 27, 8093 Zürich, Switzerland bDepartment of Theoretical Physics, CERN, 1 Esplanade des Particules, Geneva 23, CH-1211, Switzerland cShing-Tung Yau Center and School of physics, Southeast University, Nanjing 210096, China dDepartment of Physics, University of California, Santa Barbara, CA 93106, U.S.A. eUniversity of Science and Technology of China, 96 Jinzhai Road 230026, Hefei, Anhui, China E-mail: dehansen@phys.ethz.ch, jinagyf2008@gmail.com, Jiuci_Xu@umail.ucsb.edu Dennis Hansen,a Yunfeng Jiangb,c and Jiuci Xud,e aInstitut für Theoretische Physik, Eidgenössische Technische Hochschule Zürich, Wolfgang-Pauli-Strasse 27, 8093 Zürich, Switzerland bDepartment of Theoretical Physics, CERN, 1 Esplanade des Particules, Geneva 23, CH-1211, Switzerland cShing-Tung Yau Center and School of physics, Southeast University, Nanjing 210096, China dDepartment of Physics, University of California, Santa Barbara, CA 93106, U.S.A. eUniversity of Science and Technology of China, 96 Jinzhai Road 230026, Hefei, Anhui, China E-mail: dehansen@phys.ethz.ch, jinagyf2008@gmail.com, Jiuci_Xu@umail.ucsb.edu Abstract: We deﬁne three fundamental solvable bilinear deformations for any massive non-relativistic 2d quantum ﬁeld theory (QFT). They include the TT deformation and the recently introduced hard rod deformation. We show that all three deformations can be interpreted as coupling the non-relativistic QFT to a speciﬁc Newton-Cartan geometry, similar to the Jackiw-Teitelboim-like gravity in the relativistic case. Using the gravity for- mulations, we derive closed-form deformed classical Lagrangians of the Schrödinger model with a generic potential. We also extend the dynamical change of coordinate interpretation to the non-relativistic case for all three deformations. The dynamical coordinates are then used to derive the deformed classical Lagrangians and deformed quantum S-matrices. Keywords: 2D Gravity, Field Theories in Lower Dimensions, Integrable Field Theories ArXiv ePrint: 2012.12290 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP04(2021)186 1 3 3 5 5 6 7 8 9 10 10 11 14 16 16 17 19 1 Introduction 1 2 Newton-Cartan gravity in two dimensions 3 2.1 Geometric content 3 2.2 Flat Newton-Cartan spacetime 5 2.3 Covariant derivatives and matter actions 5 2.4 Matter currents 6 3 Bilinear deformations and Newton-Cartan gravity 7 3.1 Simple examples 8 3.2 The bilinear deformations 9 4 Deformed classical Lagrangians 10 4.1 Schrödinger model and conserved currents 10 4.2 Deformed Lagrangian I. The direct approach 11 4.3 Deformed Lagrangian II. 1 Introduction Recent studies of solvable irrelevant deformations of relativistic quantum ﬁeld theories have extended our understanding of them. The most studied example of such deformations is the TT deformation [1, 2], which can be deﬁned for any Lorentz invariant 2d QFT with a local stress energy tensor. For theories with additional symmetries, similar deformations such as the J ¯T [3], JTa deformations [4] and the ones constructed by higher conserved currents [5, 6] have been studied. All these deformations share similar features. They all modify the UV behavior of the QFTs and lead to non-local theories, yet they are under good analytical control due to the deformations’ solvability. JHEP04(2021)186 Surprisingly, such deformations can be deﬁned not only for 2d relativistic QFTs but also for a much broader class of theories. These include non-Lorentz invariant QFTs [7, 8], non-relativistic quantum many body systems such as the Bose gas [9] and lattice models like quantum spin chains [10–12]. Furthermore, it was recently shown [8, 9] that the de- formed 1d Bose gas share many qualitative features with TT deformed relativistic QFTs, such as the break down of UV physics for the spectrum and the Hagedorn behavior for thermodynamics. These ﬁndings hint that what we have seen so far from solvable deforma- tions of relativistic QFTs is only the tip of the iceberg — the structure and main features of such deformations can be extended to a much wider setting. One of the important lessons we learned from relativistic QFT is that TT deformation can be reformulated as coupling the QFT to certain special 2d topological gravity theory, both in ﬂat [13, 14] and curved space [15, 18], at least classically. The topological gravity one encounters in the relativistic case is similar to Jackiw-Teitelboim gravity [16, 17] on ﬂat spacetime, but it is not exactly the same gravity theory. Similar interpretations also holds for JTa deformation [4] where the gravity theory also involves an additional U(1) gauge ﬁeld. The 2d gravity formulation is tightly related to the random geometry interpre- tation of TT deformation [19] and the dynamical change of coordinates [18, 20–22]. These formulations oﬀer us a more geometrical understanding of the TT deformation. It is natural to ask whether similar geometrical interpretations exist for other settings. This paper is the ﬁrst step towards such an interesting goal by giving an aﬃrmative answer in the context of non-relativistic QFTs. Geometrizing non-relativistic bilinear deformations The gravity approach 14 5 Dynamical coordinates and gauge ﬁelds 16 5.1 The dynamical coordinates 16 5.2 Deformed Lagrangian III. Dynamical coordinates 17 5.3 Deformed quantum S-matrices 19 6 Conclusions and discussions 23 A Review of D-dimensional Newton-Cartan geometry 24 A.1 Newton-Cartan geometries 25 A.1.1 Type I 26 A.1.2 Type II 26 A.2 Connections and curvatures 27 A.3 Matter currents of ﬁeld theories on Newton-Cartan backgrounds 29 B Non-relativistic groups 30 C Null reductions 31 C.1 Lorentzian spacetimes with a null Killing vector 31 C.2 Klein-Gordon theory 32 JHEP04(2021)186 – i – 1 Introduction It is probably not too surprising that geometrical interpretations exist for non-Lorentz invariant QFTs, as the random geometry interpreta- tion was already pointed out in [7]. Nevertheless, notice that the random geometry picture follows directly from a Hubbard-Stratonovich transformation of the TT operator. It is not at all obvious from this what the gravity theory the QFT couples to is. Besides, given that the seed and deformed theories are not Lorentz invariant, the gravity theory cannot be a usual Einstein-Hilbert type gravity. A more natural candidate, in this case, is Newton- Cartan geometry, which is manifestly covariant under non-relativistic symmetry [23–27]. We will show that for non-relativistic QFTs, an elegant gravity interpretation for T¯T- and two other solvable deformations to be deﬁned shortly indeed exists in the framework of Type I Newton-Cartan geometry. For the TT deformation, the gravity theory written in the ﬁrst-order formalism takes the same form as its relativistic counterpart [14, 15]. The gravity theory for the other two deformations is similar to the ones for JTa deformed – 1 – relativistic QFTs [4]. From the gravity formulation, the dynamical change of coordinates interpretation naturally follows [15, 18]. The dynamical coordinates provide a powerful tool to compute several important quantities, such as the deformed classical Lagrangian and quantum S-matrices. Apart from extending what we have learned from relativistic QFTs to the non-relativistic settings, the gravity formulation for the non-relativistic QFTs is also in- teresting in its own right. To start with, most ﬁeld theories one encounters in condensed matter physics are not Lorentz invariant. TT deformations for Galilean or Bargmann (the central extension of the Galilei group [28]) invariant QFTs are deﬁnitely interesting to study. In particular, such QFTs are closely related to condensed matter systems that can be realized in experiments, one may gain more physical intuitions about TT deformation by studying such systems. Notice that Newton-Cartan gravity has already played a role in condensed matter systems such as quantum Hall eﬀect [29] and unitary Fermi gas [30]. It would be fascinating to make connections to these ﬁelds. JHEP04(2021)186 From the gravity point of view, Newton-Cartan geometry is diﬀerent from Einstein- Hilbert gravity and exhibits new features. One important diﬀerence is that there is a built- in U(1) symmetry in massive non-relativistic QFTs, which corresponds to the conservation of mass or particle number. 1To be more precise, this is true for one sign of the deformation parameter. For the other sign, the hard rod deformation increases the distance between particles. 1 Introduction Correspondingly, an U(1) gauge ﬁeld is an essential ingredient in so-called Type I Newton-Cartan geometry, which displays local Bargmann symmetry. Therefore, any Bargmann invariant 2d QFTs have at least three fundamental local sym- metry currents, which correspond to mass, momentum, and energy conservation. This implies, apart from the TT deformation, we can always deﬁne two other solvable bilinear deformations constructed from the mass current with the momentum or energy currents. We shall call these two deformations the hard rod deformation and the JE deformation, re- spectively. As we alluded before, these two deformations also allow a gravity interpretation in Newton-Cartan geometry. We study these three fundamental deformations in parallel. The hard rod and JE deformations are new in non-relativistic QFTs. They are similar to the JTa deformation of relativistic QFTs. The diﬀerence is that in the non-relativistic setting such U(1) symmetry is built-in and does not require further assumptions. Interestingly, the hard rod deformation was constructed very recently in [8, 9]. It is found that this deformation has the eﬀect of deforming the point particles to ﬁnite size hard rods.1 We will conﬁrm this intuition and show that it can be formulated equivalently as coupling the undeformed theory to a speciﬁc Newton-Cartan geometry. Intuitively, this relation can be understood as follows. Traditionally, the hard rod model can be solved by performing a change of coordinates which eliminates the sizes of the rods and leaves only the free space (see for example [31]). In the new coordinate, the rods become point particles and the model can be solved readily. Therefore, the crucial point here is the change of coordinates. Since this change amounts to a redeﬁnition of the length, it can be formulated as putting the theory on a diﬀerent geometry where the metric is deﬁned diﬀerently. A covariant way to formulate this intuition is precisely coupling the original – 2 – theory to the proper Newton-Cartan geometry. We will see that the change of coordinate we mentioned above appear naturally from the 2d gravity formulation in section 5. The TT deformation can be understood in the same way. Under TT deformation, point particles also become hard rods.2 The only diﬀerence from the hard rod deformation is that now the size of each rod is no longer a ﬁxed number, but depends on the energy of the particle (or the rod), which needs to be determined self-consistently. 1 Introduction Therefore we obtain certain ‘dynamical’ hard rod model. This new kind of hard rod model can also be solved by a change of coordinate, but now the new coordinates depend on the stress-energy tensor of the theory and thus become ﬁeld-dependent, or dynamical. This gives a clear physical understanding of the dynamical change of coordinates, both in the non-relativistic case and the Lorentz invariant case. This also explains why the TT deformed theory is non-local. It is simply because we are describing ﬁnite size objects such as strings or hard rods in the deformed theory. JHEP04(2021)186 The paper is structured as follows: In section 2, we give a pedagogical review of Type I Newton-Cartan gravity in 2d. In section 3, we derive the gravity formulations for the three bilinear deformations. This is achieved by viewing these deformations’ deﬁnitions as equations for the classical action and solving them formally by a heat-kernel-like approach. In section 4, we apply and test our gravity formulation by deriving closed-form classical Lagrangians. We compare the results with the ones obtained from a direct approach and ﬁnd a perfect match. In section 5, we derive the dynamical change of coordinates from the gravity formulation. The dynamical coordinates provide yet another approach to derive the deformed Lagrangian. In addition, we derive the deformed quantum S-matrices using the dynamical coordinates. We conclude in section 6 and discuss future directions. Appendix A is dedicated to a more detailed introduction to Newton-Cartan geometry. 2Again for the sign where the eﬀective length of the system becomes smaller. For the other sign, the space between the particles is increased. 2 Newton-Cartan gravity in two dimensions We give a pedagogical and minimal review on Newton-Cartan geometry suitable for our aims in the subsequent sections. Only the formalism required for that is introduced. A general review can be found in appendix A. 2.1 Geometric content Newton-Cartan (NC) geometry is the natural framework to study non-relativistic ﬁeld the- ories and their coupling to gravity. It is a covariant formulation where the Galilean group is a subgroup of the local symmetry group. It was ﬁrst obtained as a geometrization of Newtonian spacetime by Cartan [23, 24], but has seen a revival in recent years. There is extensive literature on the subject and its applications to a wide selection of areas such as generalized holography [26, 32–42], condensed matter theory [29, 43–47], hydrodynam- ics [48–54], string theory [55–63] and more [64–68]. We shall consider the 2-dimensional gravity relevant to our aims. – 3 – In 2d NC geometry, there are two fundamental tensors: • the clock-form τµ, giving the local ﬂow of time and • the clock-form τµ, giving the local ﬂow of time and • the spatial vector eµ, giving the local space direction. • the spatial vector eµ, giving the local space direction. Newton-Cartan geometry is deﬁned by requiring that they satisfy the fundamental orthog- onality relation (2.1) τµeµ = 0. (2.1) This implies that there is indeed a preferred direction of time in NC geometry, and the structure is implemented covariantly. However, we may only deﬁne projective inverses −vµ, eµ that preserves (2.1). The ambiguity in selecting the inverses is exactly the Galilean boost freedom, which is also referred to as Milne boosts in the literature [43]. JHEP04(2021)186 JHEP04(2021)186 When grouped together, the ﬁelds naturally form Galilean zweibeine deﬁned as EA µ = (τµ, eµ) , Eµ A = (−vµ, eµ) , (2.2) (2.2) and satisfying the completeness relations EA µ Eµ B = δA B, EA µ Eν A = δν µ. (2.3) (2.3) Written out in components these are Written out in components these are τµeµ = 0, τµvµ = −1, eµeµ = 1, eµvµ = 0, −τµvν + eµeν = δν µ. (2.4) The zweibeine transform under diﬀeomorphisms and local Galilean transformations as δτµ = Lξτµ, δeµ = Lξeµ + η τµ, (2.5) δvµ = Lξvµ + η eµ, δeµ = Lξeµ, (2.6) (2.5) (2.6) (2.6) where δxµ := ξµ is a vector ﬁeld so that Lξ is the Lie derivative LξXµν = ξρ∂ρXµν −∂ρξµXρν + ∂νξρXµρ, (2.7) (2.7) that generates inﬁnitesimal diﬀeomorphisms of the zweibeine and η = η(x0, x1) is a local Galilean boost. 2.2 Flat Newton-Cartan spacetime JHEP04(2021)186 An important special case is, of course, ﬂat Newton-Cartan spacetime [44, 71]. For suitable coordinates x0 = t, x1 = x it is given by τµ = δt µ, eµ = δx µ, vµ = −δµ t , eµ = δµ x, mµ = ∂µθ, (2.10) (2.10) where θ is an arbitrary function, i.e. mµ is a pure gauge. The residual coordinate transfor- mations are exactly global Galilean transformations t′ = t + a, (2.11) x′ = x + vt + b, (2.12) θ′(t′, x′) = θ(t, x) −1 2v2t + vx, (2.13) (2.11) (2.13) where we have the parameters v for the Galilean boost, and a, b for the translations. In particular we may choose θ = constant, but it will regain spacetime dependence after doing a boost. where we have the parameters v for the Galilean boost, and a, b for the translations. In particular we may choose θ = constant, but it will regain spacetime dependence after doing a boost. 2.1 Geometric content Contrary to the relativistic case, the boost does not aﬀect all the compo- nents in the same way — the hallmark of non-relativistic physics. We see that τµ, eµ indeed transform as tensors since they are invariant under local Galilean transformations. The general dimensional case where spatial rotations also enter can be found in appendix A.1. A generalization of the Newtonian potential Φ also arises. The principle of covariance tells us that it must be obtained as a projection of a gauge ﬁeld. Indeed one ﬁnds that it is given by Φ = −vµmµ, (2.8) (2.8) where mµ is a U(1) gauge ﬁeld known as the mass (or particle number) gauge ﬁeld. mµ transforms as δmµ = Lξmµ + η eµ + ∂µσ, (2.9) (2.9) – 4 – where σ is a U(1) transformation parameter. From a more group theoretical perspective, mµ is the gauge connection associated with the central charge of the Bargmann group. The Bargmann group is the non-trivial central extension of the Galilean group, and the extra generator corresponds to mass or particle number conservation [28]. We review these groups in appendix B. In other words, the geometry we have described above is the result of gauging the Bargmann group, a useful approach that has been studied in for example [25, 69, 70]. This also makes it clear that mµ is an integral part of the geometry. 2.3 Covariant derivatives and matter actions If we want to geometrize a given non-relativistic ﬁeld theory, we must use Galilean covariant derivatives for the theory to be compatible with local Galilei transformations. Analogous to the relativistic case, we may introduce a covariant derivative ∇µ with a Galilean or Newton-Cartan aﬃne connection Γρ µν and a spin connection ωµAB. The construction is similar to Lorentzian geometry, with the main diﬀerence being that the symmetry of the tangent and frame bundles is Galilean [72–75]. For this paper, it is suﬃcient to restrict to scalar ﬁelds φ where it, of course, reduces to the partial derivative, ∇µφ = ∂µφ. The general case is discussed further in appendix A.2. Besides the local Galilean symmetry, the scalar ﬁelds also transform under U(1) mass or particle number symmetry. To not break the particle number symmetry, we must use a U(1) covariant derivative. This is naturally deﬁned by taking Dµ = ∂µ + mmµ, (2.14) (2.14) where m is the mass of the ﬁeld. A temporal derivative is then formed in a covariant way as ∼vµDµ and a spatial derivative as ∼eµDµ. Notice that the ﬁrst transform under a local Galilean boost, which must be compensated by other terms. – 5 – Given a matter theory with ﬁeld ϕ coupled to a Newton-Cartan geometry, we can write its Bargmann invariant action S and Lagrangian L as S [ϕ, τ, e, m] = Z M d2xe L [ϕ, τ, e, m] , (2.15) (2.15) where we have the Galilean invariant measure e := det(τµ, eµ). (2.16) (2.16) All dependence on mµ must be through the covariant derivative. JHEP04(2021)186 In general, it is not an easy task to form Lagrangians that respect both boost and particle number symmetries [43, 45, 76]. One way where this is guaranteed is to consider null reductions of relativistic theories: Any 3d ﬁeld theory on a Lorentzian background with a null Killing vector can be null-reduced to a 2d ﬁeld theory on the type of Newton- Cartan background we have considered here. In particular, Schrödinger-type models are obtained by null reductions of Klein-Gordon-type actions. In appendix C, we review this procedure in more detail. 2.4 Matter currents We can deﬁne three covariantly conserved currents of the action (2.15) as the response to the variation of the background geometry: δbgdS [ϕ, τ, e, m] := Z M d2x e (Eµδτµ + Pµδeµ + J µδmµ) (2.17) (2.17) or equivalently or equivalently Eµ := e−1 δS δτµ , Pµ := e−1 δS δeµ , J µ := e−1 δS δmµ , (2.18) (2.18) where here Eµ is the energy current, Pµ the momentum current and J µ the mass current. These three currents are of fundamental importance to our constructions below. We stress again that the mass current is always present in a Bargmann invariant theory. In fact, breaking the U(1) symmetry would lead to drastic consequences. The particles’ mass would no longer be well-deﬁned, and the non-relativistic dispersion relation E = P 2/(2m) would not hold. Such a situation is rather unphysical. In quantum mechanics, we would furthermore have that the states are not localizable [28]. In conclusion, we better not break the Bargmann symmetry to Galilei if we want to avoid odd physics. The diﬀeomorphisms of the background ﬁelds (2.5)–(2.6) give the covariant conserva- on laws of the currents: 0 = ∂µEµ+ e−1∂µe Eµ, 0 = ∂µPµ+ e−1∂µe Pµ, 0 = ∂µJ µ+ e−1∂µe J µ. (2.19) (2.19) These can be rewritten in terms of the covariant derivative ∇µ using e−1∂µe = Γρ µρ for any Newton-Cartan connection Γρ µν. We then obtain for Cµ = {Eµ, Pµ, J µ} 0 = ∇µCµ + 2Γρ [µρ]Cµ, (2.20) (2.20) – 6 – – 6 – where 2Γρ [µρ] is the torsion of the chosen connection. Unlike the Levi-Civita connection, Newton-Cartan connections are naturally torsionful and non-metric compatible [75]. More discussion can be found in appendix A.2. where 2Γρ [µρ] is the torsion of the chosen connection. Unlike the Levi-Civita connection, Newton-Cartan connections are naturally torsionful and non-metric compatible [75]. More discussion can be found in appendix A.2. Finally, as the ﬁelds are not manifestly boost-invariant, a Galilean boost relates Pµ and J µ through the on-shell Ward identity Pµτµ = −J µeµ, (2.21) (2.21) which shows that Pµ should be thought of as the stress-mass current. This relation is well-known for non-relativistic theories on ﬂat spacetimes. Here it shows up as relations between the Noether currents after a simplifying redeﬁnition [77]. JHEP04(2021)186 3 Bilinear deformations and Newton-Cartan gravity This section deﬁnes three fundamental bilinear deformations for non-relativistic QFTs and derives the corresponding gravity formulations. As reviewed in the previous section, any 2d non-relativistic Bargmann QFT has three fundamental symmetries, corresponding to mass, momentum, and energy conservation. The conserved currents are denoted by J µ, Pµ and Eµ, respectively, as reviewed in the previous section. We can choose any two of them and construct a bilinear operator. For instance, the TT operator corresponds to the choice O2,1 = e ϵµνEµPν = e det(Eµ, Pν), (3.1) (3.1) where ϵµν is the Levi-Civita symbol. The bilinear operator which triggers the hard rod deformation is O0,1 = e ϵµνJ µPν. The remaining bilinear operator is O0,2 = e ϵµνJ µEν. We shall call the corresponding deformation the JE deformation. The three bilinear defor- mations are deﬁned by dSλ dλ = − Z d2x e Oa,b(x), (3.2) (3.2) here Sλ is the deformed action and (a, b) = (0, 1), (0, 2), (2, 1). where Sλ is the deformed action and (a, b) = (0, 1), (0, 2), (2, 1). where Sλ is the deformed action and (a, b) = (0, 1), (0, 2), (2, 1). To derive the gravity formulation, we use the fact that the three conserved currents can be written as the variations of the action as deﬁned through (2.18). Using these relations, the deﬁnition (3.2) can be seen as equations for the classical action. Let us explain this point more explicitly by the TT deformation. Consider the deformed partition function Zλ = Z Dφ e−Sλ[φ]. (3.3) (3.3) From (3.2), we have dZλ dλ = Z Dφ −dSλ[φ] dλ e−Sλ[φ] = bD2,1Zλ, (3.4) (3.4) where where bD2,1 = Z d2x ϵµν : δ δτµ(x) δ δeν(x) : . (3.5) (3.5) – 7 – Here the normal ordering means we subtract the contribution of the term proportional to δ2S/δτµδeν.3 Similar equations can be derived for the other two deformations. Notice that (3.4) is similar to a diﬀusion equation. We can solve it formally by a heat kernel-like approach, which will be discussed shortly. The formal solution gives us the gravity action immediately. This approach has been applied in the relativistic QFTs in [78–80]. 3.1 Simple examples 3.2 The bilinear deformations 3.2 The bilinear deformations The bilinear deformations We can apply the same method to solve the ﬂow equation of the bilinear deformations (3.2). Let us consider the TT deformed partition function We can apply the same method to solve the ﬂow equation of the bilinear deformations (3.2). Let us consider the TT deformed partition function We can apply the same method to solve the ﬂow equation of the bilinear deformations (3.2). Let us consider the TT deformed partition function ∂λZλ[τµ, eµ] = bD2,1Zλ[τµ, eµ]. (3.14) (3.14) Following the same steps, we arrive at the following formal solution Zλ[τµ, eµ] = Z D˜τµD˜eµ exp −1 λ Z d2xϵµν(τµ −˜τµ)(eν −˜eν) Z0[˜τµ, ˜eµ], (3.15) (3.15) JHEP04(2021)186 where as before we neglected the prefactors from the Gaussian integral. From (3.15), we can extract the deformed classical action STT λ [τµ, eµ; ˜τµ, ˜eµ\|φ] = 1 λ Z d2x ϵµν(τµ −˜τµ)(eν −˜eν) + S0[˜τµ, ˜eµ\|φ]. (3.16) (3.16) Here S0 is the undeformed action on the background described by the auxiliary zweibein (˜τµ, ˜eµ). The other term can be interpreted as the non-relativistic gravity action, which couples to the undeformed theory. Notice that the gravity action takes the same form as its relativistic counterpart [14], but it is also manifestly Galilean invariant under (2.5)–(2.6). Similarly, for the hard rod and JE deformations, we ﬁnd the deformed classical actions SHR λ [mµ, eµ; ˜mµ, ˜eµ\|φ] = 1 λ Z d2x ϵµν(mµ −˜mµ)(eν −˜eν) + S0[ ˜mµ, ˜eµ\|φ] (3.17) (3.17) and SJE λ [mµ, τµ; ˜mµ, ˜τµ\|φ] = 1 λ Z d2x ϵµν(mµ −˜mµ)(τν −˜τν) + S0[ ˜mµ, ˜τµ\|φ]. (3.18) (3.18) Notice that for these two deformations, the gravity action involve both the zweibein and the U(1) gauge ﬁeld, similar to those of JTa deformed QFTs [4, 80]. Since our derivation of the deformed action is somewhat heuristic, let us now verify that the deformed actions indeed satisfy the deﬁnition of the bilinear deformations (3.2). We consider the TT deformation as an example. The proof for the other two cases is similar. The action (3.16) depends on both the zweibein (τµ, eµ) and the auxiliary zweibein (˜τµ, ˜eµ). To obtain the TT deformed classical action, we need to integrate out the auxiliary zweibein (˜τµ, ˜eµ). In practice, this means ﬁnding the saddle-point solution for (˜τµ, ˜eµ) and plug it back in. We denote the saddle-point solution by ˜τ ⋆ µ and ˜e⋆ µ. 3.1 Simple examples As a warm-up, we consider two simple examples. The ﬁrst one is the 1d heat equation ∂tf(t, x) = ∂2 xf(t, x), (3.6) (3.6) JHEP04(2021)186 JHEP04(2021)186 which can be solved in a few steps: First, we can write which can be solved in a few steps: First, we can write f(t, x) = et∂2 xf(0, x). (3.7) (3.7) Secondly, rewrite Secondly, rewrite f(0, x) = Z dy δ(x −y)f(0, y) = 1 2π Z dy Z dp eip(x−y)f(0, y). (3.8) (3.8) Finally, plug (3.8) into the right-hand side of (3.7) and integrate out p. We obtain f(t, x) = 1 2 √ πt Z dy e−1 4t(x−y)2f(0, y). (3.9) (3.9) This is nothing but the heat kernel solution. This is nothing but the heat kernel solution. g Next, we consider a slightly more non-trivial equation which involves functio- nal derivatives4 Next, we consider a slightly more non-trivial equation which involves functio- nal derivatives4 ∂tZt[φ] = Z d2x : δ δφ(x) δ δφ(x) : Zt[φ]. (3.10) (3.10) This equation can be solved similarly. First, we have This equation can be solved similarly. First, we have Zt[φ] = exp t Z d2x : δ δφ(x) δ δφ(x) : Z0[φ]. (3.11) (3.11) Secondly, rewrite Z0[φ] = Z Dϕ δ(φ −ϕ)Z0[ϕ] ∝ Z Dϕ Z DJ e R d2xJ(x)[φ(x)−ϕ(x)]Z0[ϕ], (3.12) Z0[φ] = Z Dϕ δ(φ −ϕ)Z0[ϕ] ∝ Z Dϕ Z DJ e R d2xJ(x)[φ(x)−ϕ(x)]Z0[ϕ], (3.12) (3.12) where δ(φ −ϕ) is the functional delta-function in the proper sense and ϕ is an auxiliary scalar ﬁeld to be integrated over. Plugging into (3.11) and integrating out J(x), we obtain Zt[φ] ∝ Z Dϕ e−1 4t R d2x[φ(x)−ϕ(x)]2Z0[ϕ], (3.13) (3.13) where we have neglected the prefactors which are not important for us. 3More explicitly, we have : δ2 δτµ(x)δeν(x)e−Sλ := δ2 δτµ(x)δeν(x)e−Sλ + δ2Sλ δτµ(x)δeν(x)e−Sλ. 4The interpretation of normal ordering is the same as (3.5). – 8 – 3.2 The bilinear deformations We will show that STT[τµ, eµ\|φ] := STT λ [τµ, eµ; ˜τ ⋆ µ, ˜e⋆ µ\|φ] satisﬁes the deﬁnition of the TT deformation. Taking variation of (3.16) with respect to ˜τµ and ˜eµ, we obtain the saddle-point equations τµ = ˜τ ⋆ µ + λ ˜e ϵµν Pν 0 , eµ = ˜e⋆ µ −λ ˜e ϵµνEν 0 , (3.19) (3.19) where P0 and E0 are the undeformed currents. On the other hand, taking variations with respect to τµ and eµ lead to the deﬁnitions of the deformed currents δSTT[τµ, eµ\|φ] δτµ = e Eµ, δSTT[τµ, eµ\|φ] δeµ = e Pµ, (3.20) (3.20) – 9 – – 9 – which can be written as which can be written as which can be written as τµ = ˜τµ + λ e ϵµν Pν, eµ = ˜eµ −λ e ϵµνEν. (3.21) (3.21) Here Eµ and Pµ are the deformed currents. Taking derivative of STT with respect to λ, we have Here Eµ and Pµ are the deformed currents. Taking derivative of STT with respect to λ, we have dSTT dλ = −1 λ2 Z d2x ϵµν(τµ −˜τ ⋆ µ)(eν −˜e⋆ ν) + δSTT δ˜τ ⋆µ ∂˜τ ⋆ µ ∂λ + δSTT δ˜e⋆µ ∂˜e⋆ µ ∂λ . (3.22) (3.22) From the deﬁnition of saddle-point equation, JHEP04(2021)186 δSTT δ˜τ ⋆µ = δSTT δ˜e⋆µ = 0, (3.23) (3.23) and therefore the last two terms in (3.22) vanish. Using (3.21), we have dSTT dλ = − Z d2x e2 ϵµνEµPν = − Z d2x e O2,1, (3.24) (3.24) which is precisely (3.2). The generalization to the other two cases is straightforward. 4 Deformed classical Lagrangians In this section, we derive the deformed classical Lagrangian for the Schrödinger model with a generic potential for all three bilinear deformations. The same result can be derived from two diﬀerent approaches. The ﬁrst one is the direct approach where one starts from the deﬁnition (3.2) and work out the deformed Lagrangian order by order in λ; the other approach exploits the gravity interpretation we developed in the previous section and gives closed-form expressions for the deformed Lagrangians. The fact that these two quite dif- ferent methods lead to the same deformed Lagrangians can be seen as a non-trivial test of our proposal. 4.1 Schrödinger model and conserved currents (4.5) Eµ = i 2δµ t φ∂tφ† −φ†∂tφ −2iL + δµ x ∂xφ†∂tφ + ∂xφ∂tφ† , Pµ = i 2δµ t φ∂xφ† −φ†∂xφ + δµ x L + 2∂xφ∂xφ† , 1 i J µ = −1 2δµ t φφ† + i 2δµ x φ†∂xφ −φ∂xφ† . (4.5) (4.5) In general curved background, the currents can be calculated via (2.18), leading to Eµ = i 2vµvρ φDρφ† −φ†Dρφ −eµeρvσ Dρφ†Dσφ + Dσφ†Dρφ −vµ L, Pµ = i 2vµeρ φ†Dρφ −φDρφ† + eµeρeσ Dρφ†Dσφ + Dσφ†Dρφ + eµ L, J µ = 1 2vµ φφ† + i 2eµeν φ†Dνφ −φDνφ† . (4 Eµ = i 2vµvρ φDρφ† −φ†Dρφ −eµeρvσ Dρφ†Dσφ + Dσφ†Dρφ −vµ L, Pµ = i 2vµeρ φ†Dρφ −φDρφ† + eµeρeσ Dρφ†Dσφ + Dσφ†Dρφ + eµ L, (4.6) J µ = 1 2vµ φφ† + i 2eµeν φ†Dνφ −φDνφ† . (4.6) It is easy to check that on ﬂat spacetime as described in section 2.2, (4.6) reduces to (4.5). 4.1 Schrödinger model and conserved currents Let us ﬁrst deﬁne the Schrödinger model and its conserved currents. In curved space, the action is given by SSch = Z d2x e i 2vµ(φDµφ† −φ†Dµφ) −eµeνDµφ†Dνφ −V (\|φ\|) , (4.1) (4.1) where φ is a complex scalar ﬁeld and V (\|φ\|) is any potential that does not depend on the metric nor on the covariant derivative (2.14) with m = 1/2. Furthermore, we require that the potential is invariant under Hermite conjugation. Taking V (\|φ\|) = 0 leads to the non- relativistic free boson. One slightly more non-trivial example is the Lieb-Liniger model, or the non-linear Schrödinger model where we take V (\|φ\|) = c φ†φ†φφ, with c being the coupling constant. One nice feature of the Schrödinger model is that it can be obtained from a relativistic 3d Klein-Gordon type theory. This is described in detail in appendix C. – 10 – In ﬂat spacetime, we recover the familiar Schrödinger action In ﬂat spacetime, we recover the familiar Schrödinger action S = Z d2x L = Z d2x i 2(φ†∂tφ −φ∂tφ†) −∂xφ†∂xφ −V (\|φ\|) . (4.2) (4.2) Now we discuss the symmetries of the action (4.2). Spacetime translation invariance leads to a conserved local Noether stress-energy tensor T µν. In terms of the Lagrangian, T µν = ∂L ∂(∂µφ)∂νφ + ∂L ∂(∂µφ†)∂νφ† −L δµ ν . (4.3) (4.3) The energy and momentum currents are identiﬁed with Eµ = T µt and Pµ = T µx, respec- tively. In addition, there is a global U(1) symmetry φ →eiθφ which is related to the conservation of mass. The Noether current is given by The energy and momentum currents are identiﬁed with Eµ = T µt and Pµ = T µx, respec- tively. In addition, there is a global U(1) symmetry φ →eiθφ which is related to the conservation of mass. The Noether current is given by JHEP04(2021)186 J µ = i 2 ∂L ∂(∂µφ)φ − ∂L ∂(∂µφ†)φ† ! . (4.4) (4.4) The corresponding conserved charge is the total mass of the system. The three conserved currents written explicitly are Eµ = i 2δµ t φ∂tφ† −φ†∂tφ −2iL + δµ x ∂xφ†∂tφ + ∂xφ∂tφ† , Pµ = i 2δµ t φ∂xφ† −φ†∂xφ + δµ x L + 2∂xφ∂xφ† , J µ = −1 2δµ t φφ† + i 2δµ x φ†∂xφ −φ∂xφ† . 4.2 Deformed Lagrangian I. The direct approach We are now ready to derive the deformed Lagrangians for the three bilinear deformations. In this subsection, we perform the calculation using the direct approach. This was the ﬁrst approach to derive the deformed Lagrangian in the relativistic case (see for example [2, 81]). In this approach, one performs a formal expansion of the deformed Lagrangian Lλ = ∞ X k=0 Lk λk (4.7) (4.7) and calculates Lk order by order using the deﬁnition. This can always be worked out explicitly up to certain orders in λ. By observing the patterns of Lk, one can usually make – 11 – an ansatz for the deformed Lagrangian and turn the deﬁnition into a diﬀerential equation, which can be solved and gives the deformed Lagrangian. This strategy works fairly well for the hard rod deformation because the deformed Lagrangian takes a compact form, and it is relatively easy to guess the general pattern. However, for TT and JE deformations, the closed-form deformed Lagrangians are quite complicated, as we will see shortly. Therefore it is hard to see the patterns in these cases. Nevertheless, we can work out the results up to relatively high orders as perturbative data, which can be checked against the closed-form expressions obtained from other approaches. The hard rod deformation. Let us start with the simplest case, namely the hard rod deformation. We work in a ﬂat spacetime. The deﬁnition is JHEP04(2021)186 dLHR λ dλ = −ϵµνJ µPν. (4.8) (4.8) We expand both the Lagrangian and current densities in λ We expand both the Lagrangian and current densities in λ Lλ = ∞ X n=0 Lnλn, J µ = ∞ X n=0 Jµ nλn, Pµ = ∞ X n=0 P µ n λn. (4.9) (4.9) Plugging into (4.8), we obtain the following recursion relation Ln+1 = − 1 n + 1 n X k=0 ϵµνJµ k P ν n−k, (4.10) (4.10) where Jµ k and T µ k are deﬁned via Lk: where Jµ k and T µ k are deﬁned via Lk: where Jµ k and T µ k are deﬁned via Lk: Jµ k := i ∂Lk ∂(∂µφ)φ − ∂Lk ∂(∂µφ†)φ† ! , P µ k := ∂Lk ∂(∂µφ)∂xφ + ∂Lk ∂(∂µφ†)∂xφ† −Lk δµ x. (4.11) (4.11) Using (4.11), (4.10) and the initial condition L0 = L in (4.2), we can calculate Lk order by order. 4.2 Deformed Lagrangian I. The direct approach The ﬁrst few orders are given by L1 = −φφ† 2 ! L + φxφ† x −1 4 φ2(φ† x)2 + φ2 x(φ†)2 , L2 = −φφ† 2 !2 L + φxφ† x + 1 16φφ†(φ† xφ −φ†φx)2, L3 = −φφ† 2 !3 L + φxφ† x −1 32(φφ†)2(φ† xφ −φ†φx)2. (4.12) (4.12) where we have deﬁned the shorthand notations where we have deﬁned the shorthand notations φt := ∂tφ, φx := ∂xφ, φ† t := ∂tφ†, φ† x := ∂xφ†. (4.13) (4.13) – 12 – – 12 – Working out a few more orders, we can ﬁnd the pattern Ln = −φφ† 2 !n (L + φxφ† x) + (−1)n 2n+2 (φφ†)n−1(φ† xφ −φ†φx), n ≥2. (4.14) (4.14) The full deformed Lagrangian is then given by The full deformed Lagrangian is then given by LHR λ = 1 2 + λφφ† 2L −λ 8 (4 + λφφ†)(φ† xφ + φxφ†)2 . (4.15) (4.15) The TT deformation. Now we consider the TT deformation whose deﬁnition is JHEP04(2021)186 dLλ dλ = −ϵµνEµPν. (4.16) (4.16) Similarly, we expand the Lagrangian and the currents in λ Similarly, we expand the Lagrangian and the currents in λ Lλ = ∞ X n=0 Ln λn, Eµ = ∞ X n=0 Eµ nλn, Pµ = ∞ X n=0 P µ n λn, (4.17) (4.17) which leads to a similar recursion relation which leads to a similar recursion relation Ln+1 = − 1 n + 1 n X k=0 ϵµνEµ k P ν n−k, (4.18) Ln+1 = − 1 n + 1 n X k=0 ϵµνEµ k P ν n−k, (4.18) (4.18) where where where µ ∂Lk ∂Lk † µ where Eµ k := ∂Lk ∂(∂φ)∂tφ + ∂Lk ∂(∂φ†)∂tφ† −Lkδµ t (4.19) Eµ k := ∂Lk ∂(∂µφ)∂tφ + ∂Lk ∂(∂µφ†)∂tφ† −Lkδµ t (4.19) (4.19) and P µ k is given in (4.11). The ﬁrst few orders are L0 = L and and P µ k is given in (4.11). 4.2 Deformed Lagrangian I. The direct approach The ﬁrst few orders are L0 = L and L1 = 1 2 iφφtφ†2 x −iφ†φ† tφ2 x + φ†2 x φ2 x + i 2 φ†φt −φ† tφ V −V2, L1 = 1 2 iφφtφ†2 x −iφ†φ† tφ2 x + φ†2 x φ2 x + i 2 φ†φt −φ† tφ V −V2, L2 = −V3 + i 2 φ†φt −φφ† t V2 −φ†2 x φ2 x V −2φ3 xφ†3 x + iφxφ† x φ† tφ2 xφ† −φtφ†2 x φ −1 2φtφ† tφxφ† xφφ† + i 2φ2 xφ†2 x φtφ† −φ† tφ + 1 4φtφ† t φ†2 x φ2 + φ2 xφ†2 . (4.20) 2 2 L2 = −V3 + i 2 φ†φt −φφ† t V2 −φ†2 x φ2 x V −2φ3 xφ†3 x + iφxφ† x φ† tφ2 xφ† −φtφ†2 x φ −1 2φtφ† tφxφ† xφφ† + i 2φ2 xφ†2 x φtφ† −φ† tφ + 1 4φtφ† t φ†2 x φ2 + φ2 xφ†2 . (4.2 (4.20) We see that the second-order result is already quite lengthy. Higher-order terms are more complicated, and it is hard to see the general pattern. Nevertheless, we can derive a closed-form result from the gravity approach. he JE deformation. This deformation is deﬁned using the mass and energy currents The JE deformation. This deformation is deﬁned using the mass and energy currents dLJE λ dλ = −ϵµνJ µEν. (4.21) (4.21) We expand the Lagrangian and currents in λ as before, which leads to the following recur- sion relation n We expand the Lagrangian and currents in λ as before, which leads to the following recur- sion relation LJE n+1 = − 1 n + 1 n X k=0 ϵµνJµ k Eν n−k, (4.22) (4.22) – 13 – – 13 – where Jµ k and Eν k are deﬁned via Lk in (4.11) and (4.19). The ﬁrst few Lk are given by L0 = L and where Jµ k and Eν k are deﬁned via Lk in (4.11) and (4.19). 4.2 Deformed Lagrangian I. The direct approach The ﬁrst few Lk are given by L0 = L and L1 = i 2 φxφ† −φ† xφ V + i 2φxφ† x φxφ† −φ† xφ −1 2φφ† φtφ† x + φ† tφx , L2 = −1 4φφ†V2 + 1 4 φ2φ†2 x + φ†2φ2 x −4φφ†φxφ† x + iφφ† φtφ† −φ† tφ V + 1 4φxφ† x φ2φ†2 x + φ†2φ2 x −1 4φtφ† tφ2φ†2 −3 4φ2 xφ†2 x φφ† + i 2φxφ† xφφ† φtφ† −φ† tφ −i 4φφ† φtφ†2 x φ −φ† tφ2 xφ† . (4.23) (4.23) JHEP04(2021)186 Like the TT deformation, the results get more involved at higher orders, and the pattern for a closed-form expression for Ln is not obvious. 4.3 Deformed Lagrangian II. The gravity approach In this subsection, we present another approach to compute the deformed classical La- grangian. This approach exploits the gravity actions (3.16), (3.17) and (3.18). We take the TT deformation as an example. Starting from (3.16), we ﬁx the zweibein τµ, eµ in the ﬂat gauge τµ = δt µ and eµ = δx µ described in section 2.10. Then we ﬁnd the saddle points ˜τ ⋆ µ and ˜e⋆ µ and plug back in the action. The deformed action is given by STT[δt µ, δx µ; ˜τ ⋆ µ, ˜e⋆ µ\|φ]. This approach has been applied to the relativistic case in [18]. The deformed actions for the other two deformations can be obtained similarly. The main diﬀerence is that the ﬂat limit of the U(1) gauge ﬁeld is a pure gauge mµ = ∂µθ. In this subsection, we can simply take mµ = 0 by gauge ﬁxing. The hard rod deformation. We consider hard rod deformation ﬁrst. The saddle-point equation obtained from (3.17) is 0 = ˜mµ + λ ˜e ϵµν Pν 0 , δx µ = ˜eµ −λ ˜e ϵµν J ν 0 . (4.24) (4.24) where Pν 0 and J ν 0 are given in (4.6). There is a unique solution to this equation, which is given by ˜m⋆ t = −λ 2 + λφφ† 1 8λ φφ† x + φ†φx 2 4 + λφ†φ + 2 L0 + 2φxφ† x , ˜m⋆ x = iλ 2 + λφφ† φ† xφ −φxφ† , ˜e⋆ t = iλ 2 + λφφ† φ† xφ −φxφ† , ˜e⋆ x = 2 2 + λφφ† . (4.25) (4.25) Plugging these into SHR λ [0, δx µ; ˜m⋆ µ, ˜e⋆ µ\|φ] reproduces precisely the deformed Lagrangian given in (4.15). Notice that the deformation gives a non-trivial Newtonian potential Φ⋆= ˜m⋆ t . – 14 – – 14 – The TT deformation. Now we consider the TT deformation. The saddle-point equa- tion reads δt µ = ˜τµ + λ ˜e ϵµν Pν 0 , δx µ = ˜eµ −λ ˜e ϵµν Eν 0 . (4.26) (4.26) These equations can also be solved explicitly. There are several solutions to this equation; we choose the one that is regular in the λ →0 limit. However, the solution is too involved to be presented here explicitly. It can be found in the ancillary notebook. 4.3 Deformed Lagrangian II. The gravity approach Plugging in STT λ [δt µ, δx µ; ˜τ ⋆ µ, ˜e⋆ µ\|φ], we obtain the following expression for the deformed Lagrangian These equations can also be solved explicitly. There are several solutions to this equation; we choose the one that is regular in the λ →0 limit. However, the solution is too involved to be presented here explicitly. It can be found in the ancillary notebook. Plugging in STT λ [δt µ, δx µ; ˜τ ⋆ µ, ˜e⋆ µ\|φ], we obtain the following expression for the deformed Lagrangian LTT λ = −F1 + 2√F2 4λ(1 −λV), (4.27) (4.27) JHEP04(2021)186 where F1 = iλ φ† tφ−φtφ† +4λV−2, F2 = λ4φφ† φ† tφx−φtφ† x V−λ3 φ† tφx−φtφ† x 2iV φ† xφ+φxφ† +φφ† φ† tφx−φtφ† x +λ2 1 2φtφ† tφφ†−4φxφ† xV−1 4 φ†2 t φ2+φ2 t φ†2 +2i(φ† tφx−φtφ† x) φ† xφ+φxφ† +λ 4φ† xφx+i φ† tφ−φtφ† +1. (4.28) (4.28) As we can see, the result is highly non-trivial. Performing a perturbative expansion in λ to high powers, we can check that the results match what we obtained from the direct approach (4.20). The JE deformation. Finally, we consider the JE deformation. The saddle-point equa- tions read tions read 0 = ˜mµ + λ ˜e ϵµν Eν 0 , δt µ = ˜τµ −λ ˜e ϵµν J ν 0 . (4.29) (4.29) The solution can be found but is again rather involved to be written down. There is a unique solution which is regular in the λ →0 limit. Plugging this solution to SJE λ [0, δt µ; ˜m⋆ µ, ˜τ ⋆ µ\|φ], we obtain the deformed Lagrangian The solution can be found but is again rather involved to be written down. There is a unique solution which is regular in the λ →0 limit. Plugging this solution to SJE λ [0, δt µ; ˜m⋆ µ, ˜τ ⋆ µ\|φ], we obtain the deformed Lagrangian LJE λ = G1 + √G2 2λ2φ†φ , (4.30) (4.30) where where G1 = 2iλ(φ† xφ −φxφ†) −4, (4.31) G2 = −λ4φ2φ†2 φ† tφ + φtφ† −4λ3φφ†(φ† tφ + φtφ†) −λ2 16φφ†V −4[φ†2 x φ2 + φ2 xφ†2 + 2φxφ† xφφ† + 2iφφ†(φ† tφ −φtφ†)] + 16i(φxφ† −φφ† x) + 16. (4.32) (4.31) (4.32) + 16i(φxφ† −φφ† x) + 16. 4.3 Deformed Lagrangian II. The gravity approach (4.32) We can check explicitly that the perturbative expansion of (4.30) match the results from the direct approach. – 15 – – 15 – 5 Dynamical coordinates and gauge ﬁelds 5 In the relativistic case, TT deformation can be seen as a dynamical or ﬁeld-dependent change of coordinates [13, 20, 21]. However, such an interpretation is only valid on-shell (for a more detailed discussion, see [18]), it has several important applications. At the classical level, the dynamical change of coordinates gives yet another way to derive the classical deformed Lagrangian [82] as well as ﬁnding solutions to the deformed equation of motion and analyze the deformed classical symmetries [83]. At the quantum level, it can be used to derive the deformed S-matrix [13], at least in ﬂat spacetime. In this section, we will show that TT deformation of non-relativistic QFTs also has such an interpretation. For the other two bilinear deformations, which involve the current J µ, the interpretation is also interesting. In addition to the change of coordinates, one also needs to make a dynamical change of the U(1) gauge ﬁeld mµ. More explicitly, in ﬂat spacetime, the undeformed gauge ﬁeld is a pure gauge mµ = ∂µθ. Under the bilinear deformations, we have θ 7→Θ where Θ is ﬁeld dependent. Such interpretations ﬁrst appear in the J ¯T and JTa deformations of relativistic QFTs [3, 4].5 In what follows, we will ﬁrst give the proposals of the dynamical change of coordinates and gauge ﬁelds and then apply them to ﬁnd the deformed Lagrangians, which match our previous results. Finally, we apply them to ﬁnd the deformed quantum S-matrix. JHEP04(2021)186 5Here we mean the undeformed theory is relativistic. The deformed theory is, of course, no longer Lorentz invariant. 5.1 The dynamical coordinates Again we consider the saddle-point equation of the gravity action and take the auxiliary ﬁelds in the ﬂat gauge ˜mµ = ∂µθ and ˜eµ = δx µ, which leads to JHEP04(2021)186 mµ = ∂µθ + λ ϵµνPν 0 , eµ = δx µ −λϵµνJ ν 0 , (5.4) (5.4) where Pν 0 and J ν 0 are the ﬂat space currents (4.5) with the modiﬁcation where Pν 0 and J ν 0 are the ﬂat space currents (4.5) with the modiﬁcation ∂µφ 7→Dµφ = ∂µ + i 2∂µθ φ, ∂µφ† 7→Dµφ† = ∂µ −i 2∂µθ φ†. (5.5) (5.5) In the TT deformation, we can simply take θ = 0 by gauge ﬁxing. For the hard rod and JE deformations, we keep the pure gauge term explicitly for later convenience. Now we need to make a proper interpretation of (5.4). We propose that it deﬁnes a change of coordinate (t, x) 7→(t, X) together with a change of gauge θ 7→Θ as follows ∂µΘ = ∂µθ + λ ϵµνPν 0 , ∂µX = δx µ −λϵµνJ ν 0 . (5.6) (5.6) Notice that here only the spatial coordinate is transformed; the temporal direction is left invariant. This is very natural since eµ is related to the spatial direction. Similarly, to make such interpretations we need to check the consistency relations ∂µ∂νΘ = ∂ν∂µΘ and ∂µ∂νT = ∂ν∂µT which follow from the conservation equations of the currents. Notice that here only the spatial coordinate is transformed; the temporal direction is left invariant. This is very natural since eµ is related to the spatial direction. Similarly, to make such interpretations we need to check the consistency relations ∂µ∂νΘ = ∂ν∂µΘ and ∂µ∂νT = ∂ν∂µT which follow from the conservation equations of the currents. The JE deformation. This case is similar to the hard rod deformation. The relevant equations are mµ = ∂µθ + λ ϵµνEν, τµ = δt µ −λϵµνJ ν. (5.7) (5.7) We propose that this corresponds to the coordinate transformation (t, x) 7→(T, x) together with the gauge transformation θ 7→Θ We propose that this corresponds to the coordinate transformation (t, x) 7→(T, x) together with the gauge transformation θ 7→Θ ∂µΘ = ∂µθ + λ ϵµνEν, ∂µT = δt µ −λϵµνJ ν. (5.8) (5.8) 5.1 The dynamical coordinates There are diﬀerent ways to ﬁnd the dynamical coordinates and gauge ﬁelds [3, 13, 18, 20, 22], one of which is provided by the gravity formulation. The TT deformation. Let us ﬁrst discuss TT deformation. From the gravity formu- lation, we derive the saddle-point equations for the auxiliary ﬁelds ˜τµ, ˜eµ (3.21). In the previous section, we ﬁx τµ = δt µ, eµ = δx µ in the ﬂat gauge and solve for ˜τµ, ˜eµ. Alternatively, we could ﬁx ˜τµ = δt µ, ˜eµ = δx µ in (3.21), which leads to the following equations τµ = δt µ + λϵµνPν 0 , eµ = δx µ −λϵµνEν 0 , (5.1) (5.1) where Pν 0 and Eν 0 are the currents in ﬂat space (4.5). These equations are no longer saddle-point equations for ˜τµ and ˜eµ. Instead, following the intuition from the relativistic case, we can interpret them as deﬁning a change of coordinates from (x1, x2) = (t, x) to (X1, X2) = (T, X) by setting τµ = ∂µX1 and eµ = ∂µX2 in (5.1). Therefore, the dynamical change of coordinates (t, x) 7→(T, X) is deﬁned by ∂µT = δt µ + λϵµνPν 0 , ∂µX = δx µ −λϵµνEν 0 . (5.2) (5.2) One important comment is that, to interpret τµ and eµ as derivatives of the new coordinates, they need to satisfy the consistency condition ∂µ∂νXa = ∂ν∂µXa. This is guaranteed by 5Here we mean the undeformed theory is relativistic. The deformed theory is, of course, no longer Lorentz invariant. – 16 – the conservation of the currents. For example, we need to check that ∂µ∂νT = ∂ν∂µT. Using (5.2), we have ∂µ∂νT −∂ν∂µT = ∂µϵναPα 0 −∂νϵµαPα 0 = −ϵµν∂αPα 0 , (5.3) (5.3) which is zero due to the conservation equation ∂αPα 0 = 0. Notice that the conservation is valid only on-shell, namely when the fundamental ﬁelds satisfy equations of motion. This implies the dynamical change of coordinate interpretation is an on-shell statement. The hard rod deformation. Now, we consider the hard rod deformation. 5.2 Deformed Lagrangian III. Dynamical coordinates In this subsection, we derive the deformed Lagrangians using the dynamical coordinates and gauge ﬁelds as a non-trivial check of our proposals (5.2), (5.6), (5.8). The derivation for the TT deformation is similar to the relativistic case. The generalizations to the other two deformations are new results. The calculation involves two steps. In the ﬁrst step, we ﬁnd the quantities ∂µXa and ∂µΘ in terms of fundamental ﬁelds and their derivatives. In the second step, we perform a change of coordinates/gauge ﬁeld of the original Lagrangian to the new coordinates/gauge ﬁeld and plug in the quantities that we found in the ﬁrst step. This leads to the deformed Lagrangian in the new coordinates/gauge ﬁeld. – 17 – The TT deformation. We ﬁrst discuss how to obtain the Jacobian ∂µXa. Notice that the right hand side of (5.2) is given in terms of fundamental ﬁelds and their derivatives (4.5). We rewrite the derivatives of the ﬁelds by the chain rule ∂µφ = ∂Xaφ ∂µXa and ∂µφ† = ∂Xaφ† ∂µXa. Then (5.2) becomes an equation for ∂µXa, which can be solved explicitly in terms of φ, φ† and ∂Xaφ, ∂Xaφ†. The solution is considerably more complicated than the relativistic case and can be found in the ancillary notebook. After obtaining the Jacobian ∂µXa, we plug it into After obtaining the Jacobian ∂µXa, we plug it into LTT λ = 1 det(∂µXa) (L(T, X) + λ ϵµνEµ 0 Pν 0 ) . (5.9) (5.9) JHEP04(2021)186 The determinant det(∂µXa) comes from the change of the integration measure d2x → det(∂µXa)d2X in the action. The quantities in the bracket are written in terms of ∂Xaφ, ∂Xaφ† by the chain rule. After plugging in the explicit forms of the Jacobian, we obtain the deformed Lagrangian (4.27) in the new coordinates. The hard rod deformation. Let us ﬁrst explain how to obtain the quantities ∂µΘ and ∂µX. Writing out the ﬁrst equation of (5.6) explicitly ∂tΘ = iλ 2 Dtφ φ† −φ Dtφ† + λ DxφDxφ† −λ V, (5.10) ∂xΘ = iλ 2 Dxφ φ† −φ Dxφ† , (5.10) where where Dµφ = ∂µφ + i 2∂µΘ φ, Dµφ† = ∂µφ† −i 2∂µΘ φ†. (5.11) (5.11) Plugging into (5.10), we can solve for ∂µΘ in terms of the fundamental ﬁelds and their derivatives. The explicit results can be found in the ancillary ﬁle. 5.2 Deformed Lagrangian III. Dynamical coordinates To rewrite the second equation (5.13) explicitly, we perform the change of coordinate (t, x) 7→(t, X) where the new spacial coordinate X depends on t and x. We have the following chain rule ∂tφ(t, x) = ∂tφ(t, X) + ∂xX ∂Xφ(t, X), ∂xφ(t, x) = ∂xX ∂Xφ(t, X). (5.12) (5.12) The second equation then can be written as ∂xX = 1 + λ 2 φφ†, ∂tX = iλ 2 ∂xX DXφφ† −φDXφ† , (5.13) (5.13) where where DXφ = ∂Xφ + i 2∂XΘ φ, DXφ† = ∂Xφ† −i 2∂XΘ φ†. (5.14) (5.14) Here ∂XΘ = ∂xΘ/∂xX. We can solve (5.13) explicitly, which leads to Here ∂XΘ = ∂xΘ/∂xX. We can solve (5.13) explicitly, which leads to ∂tX = iλ 2 ∂Xφ φ† −φ ∂Xφ† , ∂xX = 1 + λ 2 φφ†. (5.15) (5.15) – 18 – After obtaining ∂µΘ, ∂µX in terms of φ, ∂tφ, ∂Xφ and their conjugates, we plug into After obtaining ∂µΘ, ∂µX in terms of φ, ∂tφ, ∂Xφ and their conjugates, we plug into 1 ∂xX (L(Θ, X) + λϵµνJ µ 0 Pν 0 ) , (5.16) (5.16) where ∂xX comes from the change of integration measure dtdx 7→dtdX. In the bracket, we replace the partial derivative by the covariant counterparts (5.11) and perform the change of coordinate from (t, x) to (t, X). Going through these steps, we ﬁnd the deformed Lagrangian (4.15). The JE deformation. This case is similar to the hard rod deformation, and thus we will be brief. The chain rule for the change of coordinate (t, x) 7→(T, x) is now JHEP04(2021)186 ∂tφ(t, x) = ∂tT ∂T φ(T, x), ∂xφ(t, x) = ∂xφ(T, x) + ∂xT ∂T φ(T, x). (5.17) (5.17) After ﬁnding the solution of ∂µΘ, ∂µT, we plug into After ﬁnding the solution of ∂µΘ, ∂µT, we plug into 1 ∂tT (L0(Θ, T) + λϵµνJ µ 0 Eν 0 ) . (5.18) (5.18) This reproduces (4.30). 5.3 Deformed quantum S-matrices As another application for the dynamical coordinates/gauge ﬁeld, we derive the deformed S- matrix in this section. The main steps parallel the derivations for the relativistic case [4, 13], adapted to the non-relativistic settings. The deformed S-matrix for non-relativistic QFTs have been derived using other methods, see [8, 9]. It is shown that the eﬀect of solvable bilinear deformations on the S-matrix is by multiplying a CDD like phase factor. We will conﬁrm these results from the dynamical coordinate point of view. TT deformation. To deﬁne the S-matrix, we need the notion of asymptotic states. Let us consider the asymptotic in-states. In the far past t →−∞, the ﬁelds are free and allow the following mode expansion φin = Z dp √ 2πain(p) e−itωp+ixp−imθ, φ† in = Z dp √ 2πa† in(p) eitωp−ixp+imθ, (5.19) (5.19) where ωp = p2. Several remarks are in order. Firstly, notice that the mode expansion of each ﬁeld involves only one type of ladder operators instead of both. This is due to the non-relativistic nature of the QFTs under consideration. Secondly, we included a background gauge potential θ in the mode expansion, which can in principle be absorbed as a normalization of the ﬁeld. Here we put it explicitly because, under the hard rod and JE deformations, we have the additional transformation θ 7→Θ, which has non-trivial eﬀects. It is, therefore, convenient to include them in the ﬁrst place. The Fourier modes in the expansion of φin satisﬁes the free Schrödinger equation iDtφ = −D2 xφ, Dµφ = (∂µ + im∂µθ)φ, (5.20) (5.20) – 19 – where m can be seen as the mass of the particle. In the previous sections, we have put m = 1/2, here it is more convenient to leave it generic. where m can be seen as the mass of the particle. In the previous sections, we have put m = 1/2, here it is more convenient to leave it generic. Under TT deformation, we are equivalently putting the theory on the new coordi- nates (T, X). Therefore it is more natural to perform the mode expansion in terms of the new coordinates φin = Z dp √ 2πAin(p) e−iωpT+ipX+imθ, φ† in = Z dp √ 2πA† in(p) eiωpT−ipX−imθ. (5.21) (5.21) Notice that for the TT deformation, the gauge ﬁeld is left untouched. 6There is a relative sign between the two terms in the exponent since in our deﬁnition of Eµ, the eigenvalue of E0 is −P i p2 i . 5.3 Deformed quantum S-matrices Comparing the two expansions (5.19) and (5.21), we ﬁnd that the two sets of modes are related by Comparing the two expansions (5.19) and (5.21), we ﬁnd that the two sets of modes are related by JHEP04(2021)186 A† in(p) = a† in(p) eiωp∆T−ip∆X, (5.22) (5.22) where ∆T = t−T and ∆X = x−X. From the deﬁnition of the dynamical coordinates (5.2), we have where ∆T = t−T and ∆X = x−X. From the deﬁnition of the dynamical coordinates (5.2), we have ∂µ(∆T) = −λϵµνPν 0 , ∂µ(∆X) = +λϵµνEν 0 . (5.23) (5.23) In the far past t →−∞we can integrate along the spacial direction and obtain In the far past t →−∞we can integrate along the spacial direction and obtain ∆T(x) = const1 + λ Z x −∞ P0(x′)dx′, ∆X(x) = const2 −λ Z x −∞ E0(x′)dx′. (5.24) (5.24) We introduce the notations P<(x) = Z x −∞ P0(x′)dx′, P>(x) = Z ∞ x P0(x′)dx′, (5.25) (5.25) where P < (x) and P>(x) measure the total momentum to the left and right of x. We deﬁne E<(x) and E>(x) similarly. The integration constants in (5.24) can be chosen arbitrarily. For convenience, we follow [4, 13] and choose the constants in a parity symmetric way const1 = −λ 2 Z ∞ −∞ P0(x) dx, const2 = +λ 2 Z ∞ −∞ E0(x) dx. (5.26) (5.26) We then have ∆T(x) = −λ 2 (P>(x) −P<(x)) , ∆X(x) = −λ 2 (E<(x) −E>(x)). (5.27) (5.27) In the inﬁnite past, the spatial ordering of the particles is equivalent to their momentum ordering, which is special to 1+1 dimensional physics. Keeping this in mind, we have6 A† in(pk) = a† in(pk) × exp  −iλ 2   k−1 X j=1 (ejpk −pjek) + N X j=k+1 (ekpj −ejpk)    , (5.28) (5.28) 6There is a relative sign between the two terms in the exponent since in our deﬁnition of Eµ, the eigenvalue of E0 is −P i p2 i . – 20 – – 20 – where ek = ωpk = p2 k is the energy of the k-th particle. As a result, the deformed and undeformed in-states are related by a phase factor where ek = ωpk = p2 k is the energy of the k-th particle. 5.3 Deformed quantum S-matrices As a result, the deformed and undeformed in-states are related by a phase factor \|{pj}in⟩λ = exp  −iλ X j<k (ejpk −pjek)  \|{pj}in⟩0, (5.29) (5.29) which takes the same form as in the relativistic case. A similar analysis can be done for the out-states. Therefore, we conclude under TT deformation, the S-matrix is deformed in the same way as in relativistic QFT STT λ ({pi}, {¯pj}) = e−iλP j<k(ejpk−pjek)e−iλP j<k(¯ej ¯pk−¯pj¯ek) S0 ({pi}, {¯pj}) , (5.30) JHEP04(2021)186 JHEP04(2021)186 (5.30) where {pi} and {¯pj} are the momenta for the in- and out-states. where {pi} and {¯pj} are the momenta for the in- and out-states. The hard rod deformation. Now we consider the hard rod deformation. The mode expansion in the dynamical coordinates and gauge ﬁeld is given by The hard rod deformation. Now we consider the hard rod deformation. The mode expansion in the dynamical coordinates and gauge ﬁeld is given by φin = Z dp √ 2πAin(p)e−iωpt+ipX+imΘ, φ† in = Z dp √ 2πA† in(p)eiωpt−ipX−imΘ. (5.31) (5.31) Notice that the time coordinate is unchanged in this case, but the gauge θ is changed. The two sets of modes are thus related by A† in(p) = a† in(p)e−ip∆X−im∆Θ, (5.32) (5.32) where ∆Θ = θ −Θ. From (5.6), we ﬁnd where ∆Θ = θ −Θ. From (5.6), we ﬁnd where ∆Θ = θ −Θ. From (5.6), we ﬁnd ∂µ(∆X) = +λϵµνJ ν, ∂µ(∆Θ) = −λϵµνPν. (5.33) (5.33) Integrating these equations along the spacial direction in the asymptotic past as before, we obtain ∆X(x) = −λ 2 (M<(x) −M>(x)), ∆Θ(x) = −λ 2 (P>(x) −P<(x)), (5.34) (5.34) where M<(x) and M>(x) are the total mass to the left and right of x, respectively. Similar considerations like before lead to the following deformed S-matrix SHR λ ({pi}, {¯pj}) = e+iλP j<k(mjpk−pjmk)e+iλP j<k( ¯mj ¯pk−¯pj ¯mk) S0 ({pi}, {¯pj}) , (5.3 (5.35) where {mk} and { ¯mk} are the masses of the particles of the in- and out-states. For simple theories with only one type of particle, we have mk = ¯mk = m. Let us comment on an important diﬀerence between the hard rod and TT deformation. In the hard rod case, the temporal direction is not modiﬁed. Therefore the non-locality only occurs in the spatial direction. Alternatively, we can integrate the ﬁrst equation of (5.33) by taking the integration constant to be zero. 5.3 Deformed quantum S-matrices This leads to X(x) = x −λ Z x −∞ J 0(x′)dx′. (5.36) (5.36) – 21 – – 21 – Let us assume for simplicity that there is only one type of particle with mass m and take λ > 0. In this case, we have X(x) = x −(λm) × {number of particles to the left of x}. (5.37) X(x) = x −(λm) × {number of particles to the left of x}. (5.37) X(x) = x −(λm) × {number of particles to the left of x}. (5.37) (5.37) As alluded in the introduction, this new coordinate has an intuitive physical interpreta- tion [8, 9]. Suppose instead of considering a collection of point particles that we consider hard rods of size mλ. The new coordinate X(x) is measuring the free space between the rods to the left of x. The phase factor in the deformed S-matrix precisely takes into account the fact that the ‘particle’ now has a ﬁnite size. Therefore, the hard rod deformation makes the point particles to ﬁnite-sized hard rods, which is the origin of its name. For λ < 0, the interpretation is that the distance between the particles is increased. JHEP04(2021)186 For TT deformation, similar interpretation applies to the change of coordinates in the spacial direction x →X. Namely, particles become hard rods under the deformation. The size of each rod is proportional to the energy of the rod. On the other hand, for TT deformation, the temporal coordinate is also transformed t →T. Therefore the non- locality is also in the time direction. For JE deformation, only the temporal coordinate is transformed. The physical interpretation for the change of coordinates in the temporal direction seems to be more subtle. This is also reﬂected in the fact that the deformed Lagrangians of the TT and JE deformations are much more complicated than the hard rod deformed one. The JE deformation. Finally, we consider the JE deformation. The mode expansion in the new coordinates and background gauge ﬁeld is φ† in = Z dp √ 2πA† ineiωpT−ipx−imΘ. (5.38) φin = Z dp √ 2πAine−iωpT+ipx+imΘ, φ† in = Z dp √ 2πA† ineiωpT−ipx−imΘ. (5.38) (5.38) The two sets of modes are related by A† in(p) = ain(p) eiωp∆T−im∆Θ. (5.39) (5.39) Using (5.8), we have Using (5.8), we have ∂µ(∆T) = λϵµνJ ν, ∂µ(∆Θ) = −λϵµνEν. (5.40) ∂µ(∆T) = λϵµνJ ν, ∂µ(∆Θ) = −λϵµνEν. 6 Conclusions and discussions We have shown that for non-relativistic QFTs, three fundamental solvable bilinear defor- mations can be deﬁned: TT, hard rod, and JE. These deformations can be interpreted as coupling the undeformed QFT to speciﬁc Newton-Cartan geometries. The gravity formula- tions oﬀer us a geometrical perspective for such deformations and provide us with powerful tools to compute important physical quantities. Classically, we computed the deformed Lagrangians in closed forms for the Schrödinger model with a generic potential. Quantum mechanically, we derived the deformed quantum S-matrices for the three deformations. There are many future directions one can pursue based on the current work. One immediate question is the quantization of the Newton-Cartan gravities in this paper. This question is of great conceptual importance and may shed light on the relativistic case as well. Technically quantizing Newton-Cartan gravity can be anticipated to be more tractable because of a preferred time foliation [61, 62]. Furthermore, it gives a new route to reach relativistic quantum gravity by considering relativistic corrections after quantization [84, 85]. One exciting and concrete question in the non-relativistic context is the hard rod deformation of the free boson. On the one hand, it is shown that quantum mechanically, the deformed theory describes a collection of free hard rods [8, 9], interacting only when they touch each other. This quantum mechanical model has been known for a few decades and has been studied extensively (see for example [86–88]). On the other hand, our work gives a rather diﬀerent, although not completely unexpected, formulation of the model — coupling the free boson to a non-relativistic gravity theory. This paper has mainly focused on the classical aspects of the gravity formulation, although we also derived the deformed S-matrix. It is interesting to study the quantum aspects of the gravity theory and make more direct contacts to the quantum hard rod model. JHEP04(2021)186 In the relativistic case, the topological gravity theory that appears is similar to Jackiw- Teitelboim gravity. Recently non-relativistic formulations of Jackiw-Teitelboim (JT) grav- ity have been studied [67, 68]. It would be interesting to explore the connection with the gravity approach further. Through the JT/SYK correspondence, this relates to the non- relativistic regime of the Sachdev-Ye-Kitaev (SYK) model on the boundary. This could also provide a path to quantizing the geometry through the relation to the BF formalism. Another interesting direction is to explore the relations with TT deformations of rel- ativistic theories. 5.3 Deformed quantum S-matrices (5.40) (5.40) Integrating along the spacial direction and choosing the parity symmetric integration con- stants, we ﬁnd in the asymptotic past ∆T(x) = −λ 2 (M<x −M>x), ∆Θ(x) = −λ 2 (E>x −E<x). (5.41) (5.41) It follows that the deformed S-matrix is given by SJE λ ({pi}, {¯pj}) = e−iλP j<k(mjek−mkej)e−iλP j<k( ¯mj¯ek−¯mk¯ej) S0 ({pi}, {¯pj}) . (5.42) SJE λ ({pi}, {¯pj}) = e−iλP j<k(mjek−mkej)e−iλP j<k( ¯mj¯ek−¯mk¯ej) S0 ({pi}, {¯pj}) . (5.42) (5.42) We found that the deformed S-matrices for the three bilinear deformations have similar structures. In fact, this structure holds in more general situations. For integrable theories, one can construct similar solvable bilinear deformations using the higher conserved currents. They lead to similar CDD factors, as has been shown in [9]. We found that the deformed S-matrices for the three bilinear deformations have similar structures. In fact, this structure holds in more general situations. For integrable theories, one can construct similar solvable bilinear deformations using the higher conserved currents. They lead to similar CDD factors, as has been shown in [9]. – 22 – 6 Conclusions and discussions It is well-known that a non-relativistic QFT in D dimension can be obtained from relativistic QFTs in at least two ways: The ﬁrst one is by a null reduction of a D + 1-dimensional QFT, which is discussed in more detail in appendix C; the other is by performing a 1/c expansion of a D-dimensional relativistic QFT where c is the speed of light. In the context of solvable deformations, both relations are interesting to explore. From the null reduction perspective, the Schrödinger model we considered in this paper can be obtained by a 3d Klein-Gordon model with a generic potential. The three fundamental currents J µ, Pµ and Eµ are diﬀerent components of the stress-energy tensor of the 3d theory. It would be fascinating to see whether we can ‘uplift’ the deformations we deﬁned in this paper to the 3d theory in some proper sense. This might give a concrete clue for deﬁning a TT like deformation for QFTs in higher dimensions, at least in 3d. – 23 – From the 1/c expansion perspective, we can start with a relativistic QFT, TT deform it, and perform the 1/c expansion. If such a procedure is well-deﬁned, it gives another way to deﬁne a TT deformed theory. As an interesting example, it is known that the Lieb- Liniger model can be obtained from the Sinh-Gordon theory by taking the non-relativistic limit [89, 90]. The TT deformation of the Sinh-Gordon model has been studied in [2, 6]. Therefore, it is interesting to take the 1/c expansion of the deformed Sinh-Gordon model and compare it with what we obtained in the current work. We expect the results to be rather diﬀerent since it is known that the 1/c expansion is typically related to the Type II Newton-Cartan gravity, which is brieﬂy studied in appendix A.1.2. It would be interesting to clarify the details and generalize to other theories. JHEP04(2021)186 Yet another interesting direction is to study the holographic dual of the deformed non-relativistic theories. In the relativistic case, there have been several proposals for the holographic dictionary [21, 91–93]. It would be interesting to see how these proposals are generalized to the non-relativistic cases. We emphasize that the hard rod and JE deformations are new in the non-relativistic contexts, and it would be exciting to see their holographic interpretations. Acknowledgments YJ would like to thank Yang Zhang for initiating the collaboration with JX. The work of DH is supported by the Swiss National Science Foundation through the NCCR SwissMAP. We would like to thank Paolo Ceschin, Riccardo Conti and especially Roberto Tateo for helpful correspondences. 6 Conclusions and discussions To explore the holographic dictionary, it is necessary ﬁrst to study the deformed theories coupled to non-relativistic conformal Type I Newton-Cartan geometry, which has local Schrödinger symmetry [32, 40, 44, 94]. Such theories exhibit a larger symmetry and should be under more analytical control. As a result, more physical quantities can be computed. It would be interesting to compute physical quantities such as the spectrum [1, 2], partition functions [95–100], and correlation functions [22, 101–107] more explicitly in these cases. Finally, it is known that the TT deformation has a deep connection with string theory, see for example [108–116]. It would be interesting to explore similar connections between our three bilinear deformations and the non-relativistic string theories [56–63]. Some of these questions have been investigated recently in [117] for the TT deformation. We believe our results in this paper will be helpful to pursue this direction further. Note added. While ﬁnishing this paper, we became aware of the paper [118], which has some partial overlap with our work. In particular, the dynamical change of coordinates and the deformed classical Lagrangian of the TT deformation are derived independently. A Review of D-dimensional Newton-Cartan geometry In this appendix, we review Newton-Cartan geometry in general D = d + 1 dimensions, of which there are actually two related but distinct types. We will study the signiﬁcance of torsion and non-metricity in the connections, where the situation is fundamentally diﬀerent – 24 – from Lorentzian geometry. Finally, we will study matter theories on these geometries, their currents, and on-shell Ward identities. from Lorentzian geometry. Finally, we will study matter theories on these geometries, their currents, and on-shell Ward identities. 7In section 2 we considered eµ as the fundamental object instead of hµν, but this was only because in D = 2 we have that since hµν is of rank 1, it factorises as hµν = eµeν. D = 2 we have that since hµν is of rank 1, it factorises as hµν = eµeν. 7In section 2 we considered eµ as the fundamental object instead of hµ A.1 Newton-Cartan geometries The vielbeine transforms under local Galilean transformations as Flat spatial indices can be raised and lowered at will with δab and δab, but the same is not true for the zero indices. The vielbeine transforms under local Galilean transformations as δτµ = Lξτµ, (A.11) δea µ = Lξea µ + λabeb µ + λaτµ, (A.12) δvµ = Lξvµ + eµ aλa, (A.13) δeµ b = Lξeµ b + λbaeµ a, (A.14) (A.14) where λab is a local spatial rotation and λa = eµaλµ is a local Galilean boost. We review the Galilei algebra and other non-relativistic algebras in appendix B. The elemental Newton- Cartan geometry and its curvature tensors can also elegantly be obtained as the gauging of the Galilei algebra [70]. JHEP04(2021)186 Depending on what extra ﬁelds there are present, we can deﬁne two distinct types of Newton-Cartan geometry, whose properties we review below.8 9One can also do the more general 1/c expansion, which introduces several new ﬁelds, see [127, 128]. A.1.1 Type I Type I Newton-Cartan geometry is the type we have considered in the main text, where the local frame bundle symmetry is the Bargmann group [25, 69, 121, 122]. We have here that the extra ﬁeld is the U(1) gauge ﬁeld mµ. It is closely related to the Newtonian potential with Φ = −vµmµ being the Newtonian potential. It transforms as δmµ = Lξmµ + λaea µ + ∂µσ, (A.15) (A.15) where σ is an arbitrary function. The geometry can likewise be obtained by gauging the Bargmann algebra [25]. It can also conveniently be embedded in a D + 1 dimensional Lorentzian spacetime with a null Killing vector as we will review in appendix C. It is not the geometry that describes Newtonian gravity in a covariant formulation unless dτ = 0, but it has other applications as advertised in the main text. See [27, 123] for more details. A.1 Newton-Cartan geometries Newton-Cartan geometry essentially consists of the metrics7 τµ, hµν satisfying τµhµν = 0 (A.1) (A.1) and their projective inverses −vµ, hµν [23, 24]. τµ is called the clock-form and gives the local direction of time. The total time elapsed for an observer following her world-line γ between two events A and B is JHEP04(2021)186 ∆tAB = Z γ τ = Z tB tA τµ (x(t)) ˙xµ(t)dt, (A.2) (A.2) which need not be the same as that of another observer with worldline γ′ between the same events because of local time-dilation. On the other hand, hµν is the inverse spatial metric, which is degenerate and of corank 1 because of τµhµν = 0. The metrics satisfy the completeness relations −τνvµ + hµρhρν = δµ ν , (A.3) (A.3) and they transform as and they transform as δτµ = Lξτµ, (A.4) δhµν = Lξhµν + τµλν + τνλµ, (A.5) δvµ = Lξvµ + hµνλν, (A.6) δhµν = Lξhµν, (A.7) (A.7) where λµ satisfying vµλµ = 0 is the Galilean boost parameter and ξµ is a diﬀeomorphism generating vector ﬁeld as in (2.7). τµ, hµν are thus tensorial, while vµ, hµν transform under Galilean boosts, corresponding to ambiguity in deﬁning them as proper inverses. We can deﬁne the (inverse) vielbeine with Galilean frame bundle covariance as EA µ = τµ, ea µ , Eµ A = (−vµ, eµ a) , (A.8) (A.8) where the spatial vielbeine now gets a spatial frame index a, b, . . . = {1, . . . , d = D −1}. Written out in components the completeness relations are τµeµ b = 0, τµvµ = −1, ea µeµ b = δa b , ea µvµ = 0, −τµvν + ea µeν a = δν µ. (A.9) The spatial metrics are related to the vielbeine as hµν := δabeµ aeν b, hµν := δabea µeb ν. (A.10) (A.10) 7In section 2 we considered eµ as the fundamental object instead of hµν, but this was only because in D = 2 we have that since hµν is of rank 1, it factorises as hµν = eµeν. – 25 – – 25 – Flat spatial indices can be raised and lowered at will with δab and δab, but the same is not true for the zero indices. y g g , p [ , ] 9One can also do the more general 1/c expansion, which introduces several new ﬁelds, see [127, 128]. 9One can also do the more general 1/c expansion, which introduces several new ﬁe A.1.2 Type II Type II Newton-Cartan geometry is the geometry that arises in the large speed of light c expansion of general relativity when expanded in 1/c2 in a covariant fashion [27, 124– 126]. In addition to the fundamental Newton-Cartan metrics τµ, vµ, hµν, hµν, which are leading order, there are also two next-to-leading order gauge ﬁelds mµ and ¯Φµν. The mµ here is not a U(1) gauge ﬁeld as it has additional terms proportional to dτ compared to (A.15); it is a so-called torsional-U(1) gauge ﬁeld. The transformation properties of mµ only coincides with its Type I cousin when dτ = 0. The ﬁelds all arise from the large speed of light expansion9 in 1/c2 of any D-dimensional Lorentzian metric, which can be expanded systematically as gµν = −c2τµτν + ¯hµν + c−2 ¯Φµν + O c−4 , gµν = hµν −c−2 ˆvµˆvν + hµρhνσ ¯Φρσ + O c−4 , (A.16) (A.16) 8There is also many other interesting extended geometries, see for example [119, 120]. 9One can also do the more general 1/c expansion, which introduces several new ﬁelds, see [127, 128]. 8There is also many other interesting extended geometries, see for example [119, 120]. 9One can also do the more general 1/c expansion, which introduces several new ﬁelds, see [127, 128]. – 26 – where10 ¯hµν := hµν −2τ(µmν), (A.17) ˆΦ := −vµmµ + 1 2hµνmµmν, (A.18) ˆvµ := vµ −hµνmν, (A.19) ˆvµ := vµ −hµνmν, (A.19) (A.19) all are invariant under local Galilean boosts and satisfy the completeness relation [70, 71] all are invariant under local Galilean boosts and satisfy the completeness relation [70, 71] τ ˆvµ + hµρ¯h δµ (A 20) (A.20) (A.20) −τνˆvµ + hµρ¯hρν = δµ ν . (A.20) these, it is easy to form objects that are guaranteed to be Galilean boost invariant. ith these, it is easy to form objects that are guaranteed to be Galilean boost invariant. JHEP04(2021)186 The Lorentzian metric can, in principle, be expanded to any order. The gauge transfor- mations of the next-to-leading order ﬁelds mµ and ¯Φµν can be traced back to be the result of subleading diﬀeomorphisms. This is the geometry that describes Newtonian gravity in a covariant formulation. When dτ = 0, ¯Φµν decouples and the two geometries coincides, but they are distinct with diﬀerent couplings to matter. Relativistic matter ﬁelds can be expanded in 1/c2 in a similar spirit. A.1.2 Type II One can then also expand the Lagrangian of the matter ﬁelds coupled to Lorentz geometry systematically order-by-order. The higher the order, the more relativistic eﬀects are taken into account. From now on, we focus on Type I Newton-Cartan geometry, although many statements are identical or have closely related equivalents in Type II. We refer the reader to for example [27, 123, 126, 127, 129, 130] for more information and applications. 10These ﬁelds can likewise be deﬁned in Type I Newton-Cartan geometry, where they are also boost invariant, but not U(1) invariant. A.2 Connections and curvatures (A.24) (A.24) As can be seen from (A.2), all observers agree on the time interval between two events independent of their worldline as the closedness of τµ implies H τ = 0. As can be seen from (A.2), all observers agree on the time interval between two events independent of their worldline as the closedness of τµ implies H τ = 0. τ ∧dτ = 0 τ ∧dτ = 0 τ ∧dτ = 0. This is known as twistless torsional Newton-Cartan geometry (TTNC). In general, we have H τ ̸= 0, so observers experience local time dilation. However, there is a foliation of spacetime into spatial hypersurfaces of simultaneity as is guaranteed locally by the Frobenius theorem. We can, without loss of generality, write τ ∧dτ = 0 τ ∧dτ = 0 τ ∧dτ = 0. This is known as twistless torsional Newton-Cartan geometry (TTNC). In general, we have H τ ̸= 0, so observers experience local time dilation. However, there is a foliation of spacetime into spatial hypersurfaces of simultaneity as is guaranteed locally by the Frobenius theorem. We can, without loss of generality, write JHEP04(2021)186 τµ = e−Ψ∂µT, (A.25) (A.25) where Ψ = Ψ(x) is known as the Luttinger potential measuring the local time dilation and T = T(x) is the time-function, which can also be taken as a coordinate. where Ψ = Ψ(x) is known as the Luttinger potential measuring the local time dilation and T = T(x) is the time-function, which can also be taken as a coordinate. τ ∧dτ ̸= 0 τ ∧dτ ̸= 0 τ ∧dτ ̸= 0. This is known as general torsional Newton-Cartan geometry (TNC). It is acausal because locally any two points can be connected with space-like curves, i.e., one with tangent vectors τµ ˙xµ = 0 [131]. For our purposes of coupling a ﬁeld theory to a background geometry, this is the relevant one: Only in this case can we do completely arbitrary variations as τµ is unconstrained. τ ∧dτ ̸= 0 τ ∧dτ ̸= 0 τ ∧dτ ̸= 0. This is known as general torsional Newton-Cartan geometry (TNC). It is acausal because locally any two points can be connected with space-like curves, i.e., one with tangent vectors τµ ˙xµ = 0 [131]. A.2 Connections and curvatures To form diﬀeomorphic invariant actions, we must introduce covariant derivatives ∇µ. It is natural to require the covariant conservation of the metrics through 0 = ∇ρτµ = ∂ρτµ −Γλ ρµτλ, (A.21) 0 = ∇ρhµν = ∂ρhµν + Γµ ρλhλν + Γν ρλhµλ, (A.22) (A.21) (A.22) (A.21) (A.22) where Γρ µν is the aﬃne connection. Any connection that satisﬁes this is a Newton-Cartan metric-compatible connection [72, 73, 75]. Notice that this does not imply that vµ and hµν are covariantly constant because of the degenerate metric structure. Equation (A.21) implies that the temporal part of torsion of a Newton-Cartan connection is always ﬁxed to be (dτ)µν = 2∂[µτν] = τρΓρ [µν]. (A.23) (A.23) There is no equivalent of the Levi-Civita connection because there is no unique solution to requiring torsionlessness and metricity. Contrary to Lorentzian connections, we can see from (A.21) that requiring a torsionless connection actually puts a constraint on the temporal vielbein. This serves as another indication that both torsion and non-metricity are natural features of Newton-Cartan geometry, a statement that can be made precise by studying Galilean frame bundles [75]. Depending on the properties of (dτ)µν we may subdivide Newton-Cartan geometry into three diﬀerent classes: There is no equivalent of the Levi-Civita connection because there is no unique solution to requiring torsionlessness and metricity. Contrary to Lorentzian connections, we can see from (A.21) that requiring a torsionless connection actually puts a constraint on the temporal vielbein. This serves as another indication that both torsion and non-metricity are natural features of Newton-Cartan geometry, a statement that can be made precise by studying Galilean frame bundles [75]. Depending on the properties of (dτ)µν we may subdivide Newton-Cartan geometry into three diﬀerent classes: – 27 – dτ = 0 dτ = 0 dτ = 0. This is torsionless Newton-Cartan geometry. In this class, there exists a notion of absolute time t as we have (up to topological obstructions) dτ = 0 dτ = 0 dτ = 0. This is torsionless Newton-Cartan geometry. In this class, there exists a notion of absolute time t as we have (up to topological obstructions) dτ = 0 dτ = 0 dτ = 0. This is torsionless Newton-Cartan geometry. In this class, there exists a notion of absolute time t as we have (up to topological obstructions) (dτ)µν = 0 =⇒ τµ = ∂µt. A.2 Connections and curvatures In components, this gives Rµνσρ := −∂µΓρ νσ + ∂νΓρ µσ −Γρ µλΓλ νσ + Γρ νλΓλ µσ. (A.35) The Ricci tensor can also always be deﬁned as The Ricci tensor can also always be deﬁned as Rµν := Rµρνρ, (A.36) (A.36) and as the Newton-Cartan connections are both torsionful and non-metric, the antisym- metric part is in general non-zero. and as the Newton-Cartan connections are both torsionful and non-metric, the antisym- metric part is in general non-zero. A.2 Connections and curvatures For our purposes of coupling a ﬁeld theory to a background geometry, this is the relevant one: Only in this case can we do completely arbitrary variations as τµ is unconstrained. The canonical choice for a connection is ˇΓλ µν := −vλ∂µτν + 1 2hλσ (∂µhνσ + ∂νhµσ −∂σhµν) , (A.26) (A.26) which has both torsion 2ˇΓλ [µν] = −2vλ∂[µτν] (A.27) ˇ 2ˇΓλ [µν] = −2vλ∂[µτν] (A.27) τν = ˇ∇µhνρ = 0 we have (A.27) and non-metricity, as besides ˇ∇µτν = ˇ∇µhνρ = 0 we have ˇ∇µvν = 1 2hνρLvhρµ, ˇ∇µhνρ = τ(νLvhρ)µ, (A.28) (A.28) where Lv is the Lie derivative along the ﬂow of vµ. The connection (A.26) is U(1) invariant as it is only built from the vielbeine, but it transforms under local Galilean transforma- tions. This is in contrast with the Levi-Civita connection, which is invariant under local Lorentzian transformations. It is still, in a sense, the closest we get to a “Levi-Civita connection" in NC geometry: It has the minimal torsion allowed as the spatial torsion 2ea λˇΓλ [µν] = 0 is zero. [µ ] Another natural connection to work with is the manifestly boost invariant connection ¯Γλ µν := −ˆvλ∂µτν + 1 2hλσ ∂µ¯hνσ + ∂ν¯hµσ −∂σ¯hµν , (A.29) (A.29) where the boost invariant ﬁelds ˆvλ, ¯hµν are given by (A.17), (A.19). The connection is torsionful with 2¯Γλ [µν] = −2ˆvλ∂[µτν] (A.30) (A.30) – 28 – – 28 – and non-metricity, as besides ¯∇µτν = ¯∇µhνρ = 0 we have and non-metricity, as besides ¯∇µτν = ¯∇µhνρ = 0 we have and non-metricity, as besides ¯∇µτν = ¯∇µhνρ = 0 we have ¯∇ρvµ = 1 2hµλLˆv¯hρλ −hµλ τρ∂λ ˆΦ −ˆΦ(dτ)ρλ , (A.31) ¯∇ρ¯hµν = τ(µLˆv¯hν)ρ + 2ˆΦ(dτ)ρ(µτν) −2τµτν∂ρ ˆΦ −2τρτ(µ∂ν) ˆΦ. (A.32) (A.31) (A.32) The connection depends on mµ linearly through (A.17)–(A.19) and is thus not U(1) in- variant: It is not possible to write down a form where both symmetries are manifest simultaneously using just Type I ﬁelds. One thus needs to work harder to guarantee the Bargmann symmetry of ﬁeld theories; see [71, 76] for more comments. JHEP04(2021)186 Whatever choice of connection one makes, we can deﬁne a Riemann curvature the usual way through the commutator of the covariant derivatives [123]: [∇µ, ∇ν] Xσ = RµνσρXρ −2Γρ [µν]∇ρXσ, (A.33) [∇µ, ∇ν] Xρ = −RµνσρXσ −2Γσ [µν]∇σXρ, (A.34) where Xµ as a vector and Xµ a covector. A.3 Matter currents of ﬁeld theories on Newton-Cartan backgrounds Diﬀerent choices for what set of ﬁelds one varies exist and each choice deﬁnes a diﬀer- ent set of currents as the response to said variations. Using the boost invariant ﬁelds ˆvµ, hµν, ˆΦ one will get a set of currents with all indices down, with manifest boost invari- ance [43, 45, 71, 132]. The canonical set of currents, in the sense that they in ﬂat gauge are the Noether cur- rents and all spacetime indices are raised, is deﬁned as the response to varying τµ, ea µ, mµ: δbgdS [ϕ, τ, e, m] := Z M dDx e Eµδτµ + Pµaδea µ + J µδmµ , (A.37) (A.37) where e := det(τµ, ea µ) is the measure, or equivalently where e := det(τµ, ea µ) is the measure, or equivalently Eµ := e−1 δS δτµ , Pµa := e−1 δS δeaµ , J µ := e−1 δS δmµ , (A.38) (A.38) where here Eµ is the energy current, Pµa the momentum current and J µ the mass current. A Galilean boost relates Pµa and J µ through the on-shell Ward identity Pµaτµ = −J µeµa, (A.39) (A.39) – 29 – – 29 – which shows that Pµa should be thought of as the stress-mass current. In D > 2, we also have a rotational on-shell Ward identity which shows that Pµa should be thought of as the stress-mass current. In D > 2, we also have a rotational on-shell Ward identity 0 = eµ[aPµ b], (A.40) (A.40) telling us that the spatial components of the momentum current are symmetric, also well- known from studying the Noether currents in ﬂat spacetime. Diﬀeomorphism invariance of the Lagrangian guarantees that the currents satisﬁes the following covariant conservation equations: JHEP04(2021)186 = ∂µEµ + e−1∂µe Eµ, 0 = ∂µPµa + e−1∂µe Pµa, 0 = ∂µJ µ + e−1∂µe J µ, (A.41) (A.41) which can be written as a covariant Newton-Cartan derivative using for example the con- nections (A.26) or (A.29) if one wants to make covariance explicit, using the formula e−1∂µ (eXµ) = ∇µXµ + 2Γρ [µρ]Xµ, (A.42) (A.42) for any vector ﬁeld Xµ. for any vector ﬁeld Xµ. B Non-relativistic groups The hallmark of non-relativistic groups is that there is a notion of absolute time in the sense that there is no boost rescaling the time coordinate [49, 69]. This is exactly not the case for the Poincaré group ISO(1, d) = SO(1, d) ⋉R1,d because of its Lie algebra commutator [J0j, Pk] = −δjkH, (B.1) (B.1) here J0j is a Lorentz boost, Pk a spatial momentum and H the Hamiltonian generator. The simplest non-relativistic group is the d dimensional Euclidean group ISO(d) since there is no time direction. Its Lie algebra generators consist of spatial momenta Pi and spatial rotation generators Jij = −Jji. The non-zero commutation relations are [Jij, Pk] = δikPj −δjkPi, (B.2) [Jij, Jkl] = δikJjl −δjkJil −δilJjk + δjlJik. (B.3) (B.2) (B.3) (B.2) (B.3) When including the Hamiltonian H and non-relativistic Galilean boosts generated by Gi, we get the Lie algebra of the Galilei group Gal(d) = SO(d) ⋉Rd ⋉R1,d, which has some additional non-zero commutators given by [H, Gi] = Pi, (B.4) [Jij, Gk] = δikGj −δjkGi. (B.5) (B.4) (B.5) (B.4) (B.5) The Galilei algebra can be obtained by İnönü-Wigner contracting the Poincaré algebra [133]. The Galilei algebra can be obtained by İnönü-Wigner contracting the Poincaré algebra [133]. – 30 – – 30 – The Galilei group’s central extension is non-trivial and of special interest and is called the Bargmann group Barg(d) = SO(d) ⋉Rd ⋉ R1,d ⊗U(1) [28]. Its Lie algebra has, in addition to the above non-zero commutation relations, a new non-zero one given by [Pi, Gj] = Nδij, (B.6) (B.6) where N is a central charge corresponding to particle number or mass. The algebra can be obtained from a null reduction of the Poincaré algebra in D + 1 dimensions as well, which lays the foundations for the next section. where N is a central charge corresponding to particle number or mass. The algebra can be obtained from a null reduction of the Poincaré algebra in D + 1 dimensions as well, which lays the foundations for the next section. There are also conformal extensions like the Galilean conformal algebra [134–136] and the Schrödinger algebra, which has an anisotropic scaling [94, 137, 138]. Both allow for inﬁnite-dimensional Virasoro-like extensions [139]. JHEP04(2021)186 C Null reductions In this appendix, we review how to deﬁne the null reduction of D+1 dimensional Lorentzian geometries with a null Killing vector to obtain a D dimensional Type I Newton-Cartan geometry. We also study how to derive Bargmann invariant matter theories from the null reduction of relativistic ones. Lorentzian spacetimes with a null Killing vector C.1 Lorentzian spacetimes with a null Killing vector The Type I NC ﬁelds ﬁt into the null reduction of D + 1-dimensional Lorentzian metric gMN with M = (µ, u) and N = (ν, u) [39, 140, 141]. Any Lorentzian metric with a null isometry ∂u can be written in adapted coordinates as gMN = gµν gµu guν guu ! = ¯hµν τµ τν 0 ! , (C.1) gMN = gµν gµu guν guu ! = hµν −ˆvµ −ˆvν 2ˆΦ ! , (C.2) (C.1) (C.2) where the Galilean boost invariant objects ¯hµν, ˆvν, ˆΦ are deﬁned in (A.17)–(A.19). Notice that we have the constraint guu = 0, which means that we cannot do variations with respect to guu without leaving the null hypersurface. Thus, we can not obtain the current corresponding to guu in the null reduced theory, but all remaining ones are available to us. We can, of course, also decompose the Lorentzian metric in terms of vielbeine as gMN = η ˆ A ˆBE ˆ A ME ˆB N, (C.3) gMN = η ˆ A ˆBE ˆ A ME ˆB N, (C.3) where the D + 1-dimensional Lorentzian vielbeine with ˆA = (A, u) = (0, a, u) and ˆB = (B, u) = (0, b, u) can be written as where the D + 1-dimensional Lorentzian vielbeine with ˆA = (A, u) = (0, a, u) and ˆB = (B, u) = (0, b, u) can be written as E ˆ A M = τµ eµa −mµ 0 0 1 ! , EM ˆ A = −vµ eµa 0 −mµvµ mµeµa 1 ! , (C.4) (C.4) where the inverse vielbein is solved such that the completeness relation holds. – 31 – These formulae are useful for null reducing relativistic theories on Lorentzian back- grounds to obtain non-relativistic theories on Type I NC backgrounds, as we shall now see an example of. C.2 Klein-Gordon theory We here want to obtain the Schrödinger model on a general Newton-Cartan background by performing a null reduction of the Klein-Gordon model [40, 43]. Our stating point is the complex Klein-Gordon scalar ﬁeld Ψ (t, x, u) with potential V (\|Ψ\|) coupled to Lorentzian geometry as JHEP04(2021)186 JHEP04(2021)186 ˆSKG [Ψ, g] = Z dD+1x√−g −gMN∂MΨ†∂NΨ −V (\|Ψ\|) . (C.5) (C.5) Since we want a Bargmann scalar of mass m, the higher-dimensional ﬁeld must be taken to be of the form Ψ (t, x, u) = e+imuφ (t, x) . (C.6) (C.6) Using the null reduction of the metric (C.2), we can easily perform the null reduction decomposing the metric and taking derivatives of the scalar ﬁeld. The result is Using the null reduction of the metric (C.2), we can easily perform the null reduction decomposing the metric and taking derivatives of the scalar ﬁeld. The result is SSchr = Z dDxe imvµφDµφ† −imvνφ†Dνφ −hµνDµφ†Dνφ −V (\|φ\|) , (C.7) (C.7) where we have deﬁned the U(1)-covariant derivative Dµφ := ∂µφ + immµφ. (C.8) (C.8) Upon setting m = 1/2 and D = 2 one obtains the action (4.1) considered in the main text. In addition to local Galilean symmetry, we have U(1) symmetry under Upon setting m = 1/2 and D = 2 one obtains the action (4.1) considered in the main text. In addition to local Galilean symmetry, we have U(1) symmetry under φ (x) →e−imσ(x)φ (x) (C.9) mµ →mµ + ∂µσ (x) . (C.10) (C.9) (C 10) (C.9) (C.9) (C.10) (C.10) The relation to the relativistic Hilbert energy-momentum tensor found by varying the D + 1-dimensional metric gMN T MN Hil := 1 2 √−g δS δgMN , (C.11) (C.11) is is Eµ = T µu Hil −T µν Hilmν, (C.12) Pµa = T µν Hileµa, (C.13) J µ = −T µν Hilτν. (C.14) J µ = −T µν Hilτν. (C.14) (C.14) This decomposition holds in general and can also be used to show how the lower-dimensional currents can be used to assembly the relativistic energy-momentum tensor. – 32 – Open Access. 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https://openalex.org/W2935326406	https://europepmc.org/articles/pmc6404334?pdf=render	English	null	GSEPD: a Bioconductor package for RNA-seq gene set enrichment and projection display	BMC bioinformatics	2,019	cc-by	4,859	GSEPD: a Bioconductor package for RNA- seq gene set enrichment and projection display Karl Stamm1,2, Aoy Tomita-Mitchell2 and Serdar Bozdag1* Karl Stamm1,2, Aoy Tomita-Mitchell2 and Serdar Bozdag1* tal signal or sample mishandling may generate outli that perturb the experimental signal in ways unnotic by the investigator. * Correspondence: serdar.bozdag@marquette.edu 1Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee, WI, USA Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Open Access Abstract Background: RNA-seq, wherein RNA transcripts expressed in a sample are sequenced and quantified, has become a widely used technique to study disease and development. With RNA-seq, transcription abundance can be measured, differential expression genes between groups and functional enrichment of those genes can be computed. However, biological insights from RNA-seq are often limited by computational analysis and the enormous volume of resulting data, preventing facile and meaningful review and interpretation of gene expression profiles. Particularly, in cases where the samples under study exhibit uncontrolled variation, deeper analysis of functional enrichment would be necessary to visualize samples’ gene expression activity under each biological function. Results: We developed a Bioconductor package rgsepd that streamlines RNA-seq data analysis by wrapping commonly used tools DESeq2 and GOSeq in a user-friendly interface and performs a gene-subset linear projection to cluster heterogeneous samples by Gene Ontology (GO) terms. Rgsepd computes significantly enriched GO terms for each experimental condition and generates multidimensional projection plots highlighting how each predefined gene set’s multidimensional expression may delineate samples. Conclusions: The rgsepd serves to automate differential expression, functional annotation, and exploratory data analyses to highlight subtle expression differences among samples based on each significant biological function. Keywords: RNA-Seq, Transcriptome, Gene ontology, Differential gene expression, Clustering, Visualiza eq, Transcriptome, Gene ontology, Differential gene expression, Clustering, Visualization, Bioconductor Stamm et al. BMC Bioinformatics (2019) 20:115 https://doi.org/10.1186/s12859-019-2697-5 Stamm et al. BMC Bioinformatics (2019) 20:115 https://doi.org/10.1186/s12859-019-2697-5 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background there is an available reference genome or by building a transcriptome assembly de novo [9, 10]. DGE analysis is performed to compute statistically significant differen- tially expressed (DE) genes using tools such as DESeq2 [3], edgeR [11], limma [12] and Cufflinks [2]. DGE ana- lysis could result thousands of genes, thus to better characterize the underlying biological functions of the DE genes, functional enrichment analysis is performed using tools such as GOSeq [8] and SeqGSEA [13]. RNA-seq is a revolutionary technology to measure genome-wide gene expression of biological samples at high resolution by sequencing messenger RNA (mRNA) molecules [1]. Common usages of RNA-Seq technology are computing transcription abundances [2], finding dif- ferentially expressed genes between two or more groups [3], de novo transcriptome assembly [4, 5] and finding novel genes and splicing patterns [6]. Among these usages, differential gene expression (DGE) analysis followed by functional enrichment is a common workflow in gene expression studies [2, 7, 8]. However, particularly when biological samples are not well separated (e.g., mammalian tissue or human disease samples are often heterogeneous or heterocellular), a direct two-group DGE analysis can result in unmanage- able lists of DE genes with uncertain significance [14]. Furthermore, batch effects may obscure the experimen- tal signal or sample mishandling may generate outliers that perturb the experimental signal in ways unnoticed by the investigator. After RNA-seq reads are generated using a sequencing instrument, gene expression abundance is estimated by mapping the sequencing reads to a reference genome if * Correspondence: serdar.bozdag@marquette.edu 1Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee, WI, USA Full list of author information is available at the end of the article Page 2 of 6 Stamm et al. BMC Bioinformatics (2019) 20:115 Stamm et al. BMC Bioinformatics (2019) 20:115 Page 2 of 6 In these scenarios, list of DE genes and even signifi- cantly enriched biological processes would be hard to in- terpret for biologists. Alternatively, after computing significantly enriched biological processes, samples could be visualized based on their activity for each of these biological processes. Per biological process visualization would enable biologists to have a deeper understanding of the samples’ activity with respect to each significant biological process. genes in that GO term. In order to study if samples segre- gate in their original groups with respect to a particular GO term, GSEPD performs clustering of samples based on the expression of all genes in a significantly enriched GO term. GSEPD can also incorporate non-tested sam- ples (i.e., samples that are not in the predefined groups) in clustering to enable investigators label unclassified or in- determinate samples by their expression profiles among GO terms relevant to the experiment. To streamline the analysis of RNA-seq datasets to achieve the aforementioned goals, we developed a soft- ware toolkit GSEPD (gene set enrichment and projection display). GSEPD produces DE gene lists, significantly enriched gene ontology (GO) terms, and importantly their cross-product: a mapping of which genes are per- turbed within each GO term, and how genes associated with those terms define the samples’ expression profiles in the context of the other RNA-Seq samples. GSEPD provides various plots and tables to summarize the re- sults and give its users a comprehensive outlook of the underlying RNA-seq data. GO term-based clustering of samples is performed by using k-means clustering where k = 2. Briefly, for a given GO term with N genes, each sample is represented as an N-dimensional vector of expression of all genes in the GO term. To avoid broad GO terms associated with thousands of genes, only GO terms with less than m (m = 31 by de- fault) genes are evaluated by GSEPD for clustering. g y g To assess the quality of the clustering outcome, a val- idity score called V-measure [16] is computed. The V-measure computes the concordance between cluster assignments and actual class labels of the samples. The V-measure of a clustering is the harmonic mean of the cluster’s homogeneity and completeness. Implementation In order to assess the significance of V-measure score, GSEPD computes an empirical p-value for each GO term-based clustering by permuting sample group labels (i.e., class labels) and re-calculating the V-measure. The p-value is the proportion of random assignments that achieve a higher V-measure. By default, GSEPD per- forms adaptive permutation up to 400 times to resolve segregation by p < 0.01. We built GSEPD as a Bioconductor package named rgsepd to ensure that it is readily available, simple to install, and bundled with both test data and documenta- tion. The system architecture of GSEPD is shown in Fig. 1. The interface to GSEPD is a short list of R com- mands and all the functions are fully automatic after providing the input data as a matrix. GSEPD generates all tables and figures for the input data within minutes. GSEPD requires two types of input data to run: the multisample RNA-seq raw counts matrix and sample in- formation matrix. Input should be loaded as a matrix in R with RefSeq ID numbers as row and sample identifiers as column names. The sample information matrix is used to link sample identifiers with test conditions and short labels (for plotting into figures). Given input data, GSEPD automatically computes DE genes between two groups with default parameters of DESeq2, adjusted if necessary for small sample counts [3]. GSEPD also uti- lizes GOSeq [8] for GO term enrichment analysis, once each for downregulated, upregulated and all genes in the DE gene list. GSEPD visualizes significant GO terms in scatter plots and subspace principle component analysis (PCA) figures to allow further exploration of the results by the user. Vector projection of samples is performed based on gene set of the GO term to score each sample’s similarity to the centroid of each group and to highlight any outlier sam- ples for the gene set. In order to assess the concordance between group label of a sample and its localization in the clustering, GSEPD performs vector projection. First, we define the mean expression of the GO term gene set in samples of each group as the centroid of the group, and define an axis connecting both group centroids where one of the centroids is chosen as the origin in a N-dimensional Euclidean space (Fig. 2). Each sample is projected on this axis to compute two scores named alpha and beta. A cluster’s homogeneity is computed based on the entropy of class labels within the cluster, i.e., maximum homogeneity is achieved when all members of the cluster belongs to the same cluster. The completeness of a cluster is computed based on what percent of members of a class are assigned to the cluster. A cluster would have maximum completeness if it has all members of a class. In ideal cases, clusters should be homogenous and complete. We demonstrated the usage of GSEPD on a time series dataset of H1ESC cell lines [15]. GSEPD computed DE genes and significantly enriched GO terms between two time points, and clustered samples from all time points based on their activity in each significant GO term. GSEPD is implemented as a Bioconductor package named rgsepd and freely available under GPL-3 license. Implementation One of the novel features of GSEPD is to focus on each significantly enriched GO term and assess how samples are segregated with respect to the expression of Stamm et al. BMC Bioinformatics (2019) 20:115 Page 3 of 6 Fig. 1 Systems Architecture diagram of the components of the GSEPD system, with major sections in red outlines. Blue items indicate automated systems. An experiment starts at the upper left, with the Sequencing Facility where the tissue samples are converted to gene expression quantification through sequencing and processing external to GSEPD. The user then creates a table of count data and defines the sample metadata and conditions to be compared (lower left, green items indicate user inputs). Across the top are External Resources, where functional annotation databases are curated by third parties and plug in to the rgsepd software package. The R code wraps subprocesses for differential expression, set enrichment, and set based projection scoring. The orange cylinder of sample data indicates a normalization produced by DESeq2 with useful expression measurements. Within the Projection Engine box are small diagrams of the integral vector projections and clustering analyses Fig. 1 Systems Architecture diagram of the components of the GSEPD system, with major sections in red outlines. Blue items indicate automated systems. An experiment starts at the upper left, with the Sequencing Facility where the tissue samples are converted to gene expression quantification through sequencing and processing external to GSEPD. The user then creates a table of count data and defines the sample metadata and conditions to be compared (lower left, green items indicate user inputs). Across the top are External Resources, where functional annotation databases are curated by third parties and plug in to the rgsepd software package. The R code wraps subprocesses for differential expression, set enrichment, and set based projection scoring. The orange cylinder of sample data indicates a normalization produced by DESeq2 with useful expression measurements. Within the Projection Engine box are small diagrams of the integral vector projections and clustering analyses groups and can help assess how samples from other groups “behave” for a given GO term. The alpha score is the distance between projected point on the axis to the origin and the beta score is the Euclidean distance between the sample and the projected point in the axis (Fig. 2). Implementation Beta score mea- sures the goodness of fit and flag samples which do not fit the linear assumptions of the two-group compari- son performed by DESeq2 whereas alpha score is used to measure the confidence of the cluster assignments. Alpha and beta scores are computed for samples from other GO term-based clustering and vector projection is per- formed for each significant GO term with gene sets ≤m, creating an alpha and beta score for each sample and GO term pair. GSEPD produces heatmaps of gene ex- pression for DE genes, heatmaps of alpha scores for sig- nificant GO terms, multi-panel scatterplots of genes in significant GO terms, PCA plots of samples and tables. All thresholds and parameters are configurable before runtime, and configurable output folders and formulaic file naming conventions ensure easy reproducibility or automated parameter sweeps. A tutorial and explan- ation of all outputs are available within the package vignette/manuals. Fig. 2 Vector Projection Illustration. With the origin at the cross, vector AP is projected onto vector AB, yielding the green projection. In GSEPD, the point A is the centroid of class A, and point B is the centroid of class B. Point P is any one sample. The green vector is the alpha score and the black perpendicular line from point P is the beta score Results and discussion We run GSEPD on a time series dataset (five time points with two replicates) along the differentiation of H1ESC cells into cardiomyocytes (NCBI SRA accession number SRP048993) [15]. We used GSEPD to compare samples of day 3 and 5, which is a critical turning point between early tissue development and heart muscle precursors [15]. Pairwise comparison of all time points revealed that time points day 3 and day 5 had the fewest DE genes (3279 genes with p < 0.05, comprising 2214 GO terms with p < 0.05, 1073 of which were found to cluster sam- ples with a significant V-measure score p < 0.01). Fig. 2 Vector Projection Illustration. With the origin at the cross, vector AP is projected onto vector AB, yielding the green projection. In GSEPD, the point A is the centroid of class A, and point B is the centroid of class B. Point P is any one sample. The green vector is the alpha score and the black perpendicular line from point P is the beta score The heatmap of alpha scores (HMA) plot is shown in Fig. 3. The HMA plot can visualize if any sample “behave” Stamm et al. BMC Bioinformatics (2019) 20:115 Page 4 of 6 Fig. 3 GSEPD Results from the H1ESC Study. The H1ESC dataset is evaluated with GSEPD’s Alpha/Beta scores. Notes along the bottom are a coded sample identifier ending in the time point name D3 for day 3, D1 for day 1, and so on. This figure shows GO terms with significant segregation between day 3 (green) and day 5 (red). GSEPD was instructed via input parameter to display only the top 8 results. The color bar across the top indicates which samples were part of the DESeq2 contrast, here day 3 in green versus day 5 in red, with black denoting non-tested samples background knowledge, the user of GSEPD can thus extract pathway activation knowledge from RNA-seq count data. similar to its own group or some other group. For in- stance, for the GO term “cardiac atrium morphogenesis,” the day 3 samples are unique (i.e., bright green), the day 0 and the day 1 samples have average alpha scores (i.e., faded gray) with the day 1 samples are slightly more simi- lar to the day 3 samples, while the samples from later days (i.e., day 8 and 14) behave quite similar to the day 5 samples. Results and discussion Labels D5x2 and D3x2 indicate class centroids of the comparison of two samples of day 5 versus two samples of day 3. The small point labels are specified by the user as each sample’s “shortname,” a parameter given to GSEPD of results, GSEPD was implemented as an open source Bio- conductor package. By utilizing the GO hierarchy through GOSeq, GSEPD can quickly identify significantly enriched GO terms with DE genes computed by DESeq2. Further- more, GSEPD can visualize how each sample behaves with regard to each significant GO term. Byproducts including sample PCA figures save time and effort and can identify sample batch effects that may confound analyses and be obscured by rudimentary differential expression produced by other pipelines. dimensional space (although they do not appear per- pendicular in the two-gene subspace), indicating samples of day 0 and day 1 fall between the clusters of the day 3 and the day 5 samples and whereas the day 8 and the day 14 samples are clustered with the day 5 samples for this GO term. Results and discussion GSEPD also extracts significant GO terms into multi-page scatterplots of genes showing orthogonal views of samples on the high-dimensional clusters. For instance, for the “cardiac atrium morphogenesis” a 28-gene GO term in the HMA figure (Fig. 3), a sample scatterplot between PITX2 and NOTCH1 is shown in Fig. 4. In this scatterplot PITX2 is shown downregulated in class day 3 (green) versus class day 5 (red), whereas NOTCH1 is upregulated by 1.5 units of logged normalized counts. Colored lines (corre- sponding to cells of the heatmap in Fig. 3) are perpendicular to the thick black axis in the 28- The results in the HMA plot also show that the day 3 samples were unique in GO terms “mesodermal cell fate specification”, “mesodermal cell fate commit- ment”, “negative regulation of cell fate commitment”, and “regulation of mesoderm development,” suggest- ing a unique spike of gene activation that deactivated on all other time points. With no biological systems Stamm et al. BMC Bioinformatics (2019) 20:115 Page 5 of 6 Fig. 4 Scatterplot of Two Genes. Corresponding to ‘atrial cardiac muscle tissue development’ GO term in Fig. 3, this diagram is one part of generated file GSEPD.D3x2.D5x2.GO0003209.pdf (first two genes). Points as triangles, circles, and crosses correspond to the input samples. Solid dots indicate the projection coordinate. Labels D5x2 and D3x2 indicate class centroids of the comparison of two samples of day 5 versus two samples of day 3. The small point labels are specified by the user as each sample’s “shortname,” a parameter given to GSEPD Fig. 4 Scatterplot of Two Genes. Corresponding to ‘atrial cardiac muscle tissue development’ GO term in Fig. 3, this diagram is one part of generated file GSEPD.D3x2.D5x2.GO0003209.pdf (first two genes). Points as triangles, circles, and crosses correspond to the input samples. Solid dots indicate the projection coordinate. Labels D5x2 and D3x2 indicate class centroids of the comparison of two samples of day 5 versus two samples of day 3. The small point labels are specified by the user as each sample’s “shortname,” a parameter given to GSEPD Fig. 4 Scatterplot of Two Genes. Corresponding to ‘atrial cardiac muscle tissue development’ GO term in Fig. 3, this diagram is one part of generated file GSEPD.D3x2.D5x2.GO0003209.pdf (first two genes). Points as triangles, circles, and crosses correspond to the input samples. Solid dots indicate the projection coordinate. Competing interests h h d l h The authors declare that they have no competing interests. Funding 12. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. 12. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. This study is funded by the Department of Surgery at the Medical College of Wisconsin. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. 13. Wang X, Cairns MJ. SeqGSEA: a Bioconductor package for gene set enrichment analysis of RNA-Seq data integrating differential expression and splicing. Bioinformatics. 2014;30(12):1777–9. Acknowledgements Not applicable 11. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40. Availability of data and materials The datasets generated and/or analyzed during the current study are available at project website (https://bioconductor.org/packages/release/bioc/html/ rgsepd.html). 14. Ramsköld D, Wang ET, Burge CB, Sandberg R. An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data. PLoS Comput Biol. 2009;5(12):e1000598. 15. Kim MS, Horst A, Blinka S, Stamm K, Mahnke D, Schuman J, Gundry R, Tomita-Mitchell A, Lough J. Activin-a and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis. PLoS One. 2015;10(2):e0118670. Conclusions Hirschberg, "V-Measure: A Conditional Entropy-Based External Cluster Evaluation Measure", in EMNLP-CoNLL, 2007, 7, no., p. . 410–420"V- Measure: A Conditional Entropy-Based External Cluster Evaluation Measure", in EMNLP-CoNLL, 7, no., 2007, p. . 410–420. Availability and requirements 4. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52. GSEPD is implemented as a Bioconductor package named rgsepd and freely available under GPL-3 license for aca- demic and non-academic usage. The Bioconductor system will install required additional packages DESeq2, GOSeq, and the GO databases, available to any Mac, Linux, and Windows PC. Generating the input matrix will require other tools. Description of the 13 types of figures and 12 types of tables generated by each comparison run are available in the bundled package manuals. Instructions, manuals, and sample data are available in the online help files and the project website at https://bioconductor.org/ packages/release/bioc/html/rgsepd.html. 5. Schulz MH, Zerbino DR, Vingron M, Birney E. Oases: robust de novo RNA- seq assembly across the dynamic range of expression levels. Bioinformatics. 2012;28(8):1086–92. 6. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5. 7. Williams CR, Baccarella A, Parrish JZ, Kim CC. Empirical assessment of analysis workflows for differential expression analysis of human samples using RNA- Seq. BMC Bioinformatics. 2017;18(1):38. 7. Williams CR, Baccarella A, Parrish JZ, Kim CC. Empirical assessment of analysis workflows for differential expression analysis of human samples using RNA- Seq. BMC Bioinformatics. 2017;18(1):38. 8. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11(2):R14. 8. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11(2):R14. 9. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36. 9. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Consent for publication Not applicable Consent for publication Not applicable Author details 1 1Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee, WI, USA. 2Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, USA. 1Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee, WI, USA. 2Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, USA. 4. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52. Authors’ contributions KS devised, designed and implemented the tool, wrote and revised the manuscript. AM provided data and architectural support. SB provided strategic guidance during design and implementation of the tool, wrote and revised the manuscript. All authors read and approved the final manuscript. 16. A. Rosenberg and J. Hirschberg, "V-Measure: A Conditional Entropy-Based External Cluster Evaluation Measure", in EMNLP-CoNLL, 2007, 7, no., p. . 410–420"V- Measure: A Conditional Entropy-Based External Cluster Evaluation Measure", in EMNLP-CoNLL, 7, no., 2007, p. . 410–420. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate N li bl Ethics approval and consent to participate Not applicable 11. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40. 12. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. 5. Schulz MH, Zerbino DR, Vingron M, Birney E. Oases: robust de novo RNA- seq assembly across the dynamic range of expression levels. Bioinformatics. 2012;28(8):1086–92. 6. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5. 10. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1):15–21. Conclusions GSEPD is a user-friendly RNA-seq analysis toolkit. To en- able rapid and simple installation and ensure reproducibility Page 6 of 6 Page 6 of 6 Page 6 of 6 Stamm et al. BMC Bioinformatics (2019) 20:115 4. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52. 5. Schulz MH, Zerbino DR, Vingron M, Birney E. Oases: robust de novo RNA- seq assembly across the dynamic range of expression levels. Bioinformatics. 2012;28(8):1086–92. 6. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5. 7. Williams CR, Baccarella A, Parrish JZ, Kim CC. Empirical assessment of analysis workflows for differential expression analysis of human samples using RNA- Seq. BMC Bioinformatics. 2017;18(1):38. 8. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11(2):R14. 9. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36. 10. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1):15–21. 11. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40. 12. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. 13. Wang X, Cairns MJ. SeqGSEA: a Bioconductor package for gene set enrichment analysis of RNA-Seq data integrating differential expression and splicing. Bioinformatics. 2014;30(12):1777–9. 14. Ramsköld D, Wang ET, Burge CB, Sandberg R. An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data. PLoS Comput Biol. 2009;5(12):e1000598. 15. Kim MS, Horst A, Blinka S, Stamm K, Mahnke D, Schuman J, Gundry R, Tomita-Mitchell A, Lough J. Activin-a and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis. PLoS One. 2015;10(2):e0118670. 16. A. Rosenberg and J. Abbreviations D D ff l DE: Differential expressed; GO: Gene Ontology; HMA: Heatmap of alpha scores; mRNA: messenger RNA; PCA: Principal Component Analysis 10. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1):15–21. 10. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1):15–21. 7. Williams CR, Baccarella A, Parrish JZ, Kim CC. Empirical assessment of analysis workflows for differential expression analysis of human samples using RNA- Seq. BMC Bioinformatics. 2017;18(1):38. 13. Wang X, Cairns MJ. SeqGSEA: a Bioconductor package for gene set enrichment analysis of RNA-Seq data integrating differential expression and splicing. Bioinformatics. 2014;30(12):1777–9. References 1. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2008;10(1):57–63. 2. Garber M, Grabherr MG, Guttman M, Trapnell C. Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods. 2011;8(6):469–77. 3. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.
https://openalex.org/W2069524150	https://wwwnc.cdc.gov/eid/article/12/10/pdfs/06-0411.pdf	English	null	Active Cytomegalovirus Infection in Patients with Septic Shock	Emerging infectious diseases	2,006	cc-by	4,085	Patients and Methods Cytomegalovirus (CMV) is a human β-herpesvirus that has high seroprevalence in adults. CMV disease predomi- nantly occurs as an opportunistic infection in patients with severe immunosuppression and rarely occurs in immuno- competent patients (2). Clinical diagnosis of CMV disease, without the use of virus diagnostics, is hampered by the fact that the clinical signs and symptoms are not very spe- cific. Patients in intensive care units (ICUs) are rarely monitored for active CMV infection; therefore, the devel- opment of active CMV infection remains unrecognized in most critically ill patients. Active Cytomegalovirus Infection in Patients with Septic Shock Lutz von Müller,* Anke Klemm,* Manfred Weiss,* Marion Schneider,* Heide Su Nilgün Durmus,* Walter Hampl,* and Thomas Mertens* Cytomegalovirus (CMV) is a pathogen of emerging importance for patients with septic shock. In this prospec- tive study, 25 immunocompetent CMV-seropositive patients with septic shock and an intensive care unit stay of >7 days were monitored by using quantitative pp65-antigenemia assay, shell vial culture, and virus isolation. Within 2 weeks, active CMV infection with low-level pp65-antigenemia (median 3 positive/5×105 leukocytes) developed in 8 (32%) patients. Infection was controlled within a few weeks (medi- an 26 days) without use of antiviral therapy. Duration of intensive care and mechanical ventilation were significant- ly prolonged in patients with active CMV infection. CMV reactivation was associated with concomitant herpes sim- plex virus reactivation (p = 0.004). The association between active CMV infection and increased illness could open new therapeutic options for patients with septic shock. Future interventional studies are required. receiving immunosuppressive therapy; however, the inci- dence of active CMV infection is controversial (3,4), and not all centers detected active CMV infections in these patients (5–7). Among critically ill patients, the highest incidence of active CMV infection was in patients with septic shock (3). The causality of sepsis, consecutive CMV reactivation, and CMV-associated pulmonary disease is supported by a mouse model of murine CMV reactivation after cecal ligation and puncture (8,9). Many factors could stimulate CMV reactivation in patients with septic shock; e.g., proinflammatory cytokines (10,11), transient immune paralysis (compensatory antiinflammatory response syn- drome) (12), and drugs (13). This pilot study investigated the incidence and the nat- ural course of active CMV infection in patients with septic shock and different strategies for CMV monitoring. The study may stimulate future interventional trials aimed at preventing CMV-associated illness of patients with septic shock. S epsis and septic shock are defined as a clinical syn- drome with severe inflammatory response (1). Despite the availability of antimicrobial, antifungal, and supportive therapies, septic shock is fatal for about one third of patients. S * University Hospital Ulm, Ulm, Germany Clinical Data The following values were regularly recorded: body temperature, heart rate, blood pressure, respiratory rate, need for mechanical ventilation, oxygen supply (FiO2), urinary output, hemodiafiltration, partial pressure of oxy- gen in arterial blood, pH, leukocyte count, platelet count, serum bilirubin, aspartate aminotransferase (AST), C-reac- tive protein, and serum creatinine. The severity of organ failure over time was recorded by monitoring the most rel- evant organ functions (pulmonary, cardiovascular, hemato- logic, hepatic) and using the Sepsis-related Organ Failure Assessment Score (SOFA) (16). Impairment of the central nervous system was not evaluated (Glasgow Coma Scale) because most patients received sedatives. Active CMV Infection During the first 2 weeks after onset of septic shock, active CMV infection was detected by sensitive quantita- tive pp65-antigenemia assay in 8 (32%) patients (15). Active CMV infection was also detected by shell vial cul- ture in 4 of these patients (in bronchial aspirate for 3 patients and in urine for 1). For 1 patient for whom shell vial culture in bronchial aspirate was positive, shell vial culture was also positive in leukocytes. Initial detection of active CMV infection was delayed when using shell vial culture (detected 1, 11, 20, and 21 days after onset of sep- tic shock) compared with pp65-antigenemia in the same patients (0, 7, 10, and 14 days). Overall, pp65-antigenemia was low (median 3 positive/5×105 leukocytes; range 1–17) and became nondetectable with no antiviral therapy (medi- an 26 days after onset of active CMV infection; range 1–61 days). One patient died while CMV infection was still active. Leukocytes, bronchial aspirate, and urine were investi- gated by shell vial culture and viral isolation with human embryonic lung fibroblasts. Three days after infection, shell vial cultures were fixed with methanol and analyzed by IFA (anti-CMV immediate early antibody, Argene Biosoft, Viva Diagnostika; FITC-conjugated goat anti- mouse IgG, Dianova). Phase contrast microscopy was used to analyze viral isolation for >6 weeks. Cytopathic effects of various viruses were confirmed by using viral typing with IFA and monoclonal antibodies. At the initial evaluation, the following antibodies were determined semiquantitatively by using ELISA (Medac, Hamburg, Germany): CMV IgG, CMV IgM, and herpes simplex virus (HSV) IgG. Patients with antibody indices >1 were considered antibody positive. CMV IgM antibodies were found in 2 (25%) of 8 patients with and 2 (12%) of 17 patients without active CMV infection, a difference that was not significant. Also the quantitative levels of CMV IgG and IgM antibodies did not differ between groups with and without active CMV infection (Table). Patients Among 375 patients in ICU, 38 consecutive patients with septic shock were eligible, but 13 were excluded because of CMV seronegativity (n = 5), immunosuppres- sive therapy (n = 2), or ICU stay <7 days (n = 6). Thus, 25 CMV-seropositive patients with septic shock and an ICU stay >7 days were enrolled in the study. Statistics Statistical analysis was performed by using nonpara- metric tests (Fisher exact test, Mann-Whitney U test) and GraphPad Prism 3.02 software (GraphPad Software, San Diego, CA, USA). Significance level was set at p = 0.05. Virologic Examinations CMV monitoring was performed twice during the first week of the study and once a week thereafter until the patient was discharged from ICU. Quantitative pp65-anti- genemia assay; shell vial culture; and viral isolation in leukocytes, urine, and bronchial aspirate were performed as previously described (15). Briefly, pp65 antigenemia was determined in blood collected in EDTA tubes and sub- jected to dextran sedimentation (1% dextran in phosphate- buffered saline). Duplicates of 5×105 leukocytes were placed onto glass slides, and the pp65 antigen-positive cells were evaluated by immunofluorescence assay (IFA) by using a mixture of 2 monoclonal mouse anti-pp65 anti- bodies (20:1; Virion, Rüschlikon, Switzerland; Argene Biosoft, Viva Diagnostika, Hürth, Germany) and goat anti- mouse immunoglobulin (Ig) G (Dianova, Hamburg, Germany) conjugated with fluorescein isothiocyanate (FITC). Patients For 9 consecutive months, patients in the anesthesio- logic ICU, University Hospital Ulm, Ulm, Germany, who had septic shock, were monitored for active CMV infec- tion. We did not monitor patients who underwent splenec- tomy, transplantation patients, or patients receiving immunosuppressive therapy. Also, patients who had been in ICU <7 days were excluded because CMV reactivation and CMV-associated illness were expected to develop with a time delay. To define septic shock, we used the criteria established by the American College of Chest Physicians/Society of Critical Care Medicine (14). Clinicians were not made aware of virologic results. To avoid exogenous CMV infections, transfusions were limit- ed to filtered leukocyte-reduced blood products. The study During recent years, CMV has been discussed as an emerging pathogen in critically ill patients who are not 1517 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 RESEARCH was approved by the local ethics committee and was in accordance with the Helsinki Declaration; informed con- sent was obtained. Active Cytomegalovirus Infection and Septic Shock Active Cytomegalovirus Infection and Septic Shock groups, and catecholamine treatment for cardiovascular dysfunction was similar for both groups. Other Viral Infections All 25 patients were HSV seropositive; viral isolation in bronchial aspirate showed reactivation of HSV in 8 (32%) patients, thereby showing for the first time that HSV and CMV reactivation were associated (p = 0.004, Table) and occurred simultaneously (Figure). Active HSV infections developed without skin or mucosal rash (occult HSV infection). Because of the low number of cases, the clinical outcome of patients with active CMV, HSV, or CMV/HSV coinfection could not be further differentiated. Viral isolation in bronchial aspirate and urine did not detect additional opportunistic viral infections such as polyoma BK virus and exogenous viral infections such as adenovirus, respiratory syncytial virus, and parainfluenza- virus in any patient. Overall, the severity of sepsis-related failure of multi- ple organs, determined by SOFA score (16), did not differ between patients with and without active CMV infection; however, patients with active CMV infection required mechanical ventilation and ICU therapy for a longer time than did patients without active CMV infection (p = 0.0025) (Table). Although mortality rates were not signifi- cantly different between patients with and without active CMV infection (63% vs 35%; p > 0.05), the deaths occurred later (median 44 days after onset of septic shock, range 24–72 days) for patients with active CMV infection than for patients without (median 21 days, range 14–35 days) (p = 0.03). The clinical course of patients with positive CMV shell vial culture in bronchial aspirate was associated with the longest duration of mechanical ventilation (47, 50, and 80 days) and of ICU stay (50, 71, and 87 days); however, because of the low number of cases, statistical analysis was not performed. Characteristics of Patients with and without Active CMV Infection Patient characteristics such as age, sex, primary dis- ease, and severity of organ failure at time of entry into the study did not differ between patients with and without active CMV infection (Table). Hydrocortisone (200 mg/day) was given to patients in both groups; no differ- ences between groups were noted in body temperature, leukocyte count, platelet count, serum creatinine level, serum bilirubin level, AST level, and C-reactive protein level. Systemic infection by gram-positive and gram- negative microorganisms was detected equally in both 1518 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 Discussion CMV reactivation occurred during the first 2 weeks after onset of septic shock (median 7 days) and was asso- ciated with HSV reactivation, which occurred during the same peri- od. The incidence of active HSV infection was different between groups 1 and 2 (75% vs 12%; p = 0.004). Active CMV infection was detected first by CMV pp65 antigenemia; active HSV infection was detected by virus isolation of bronchial aspirates. Figure. First detection of cytomegalovirus (CMV) and herpes sim- plex virus (HSV) reactivation after onset of septic shock. Incidence of active CMV and HSV infection is shown for patients with active CMV infection (group 1; n = 8) and without active CMV infection (group 2; n = 17). CMV reactivation occurred during the first 2 weeks after onset of septic shock (median 7 days) and was asso- ciated with HSV reactivation, which occurred during the same peri- od. The incidence of active HSV infection was different between groups 1 and 2 (75% vs 12%; p = 0.004). Active CMV infection was detected first by CMV pp65 antigenemia; active HSV infection was detected by virus isolation of bronchial aspirates. Figure. First detection of cytomegalovirus (CMV) and herpes sim- plex virus (HSV) reactivation after onset of septic shock. Incidence of active CMV and HSV infection is shown for patients with active CMV infection (group 1; n = 8) and without active CMV infection (group 2; n = 17). CMV reactivation occurred during the first 2 weeks after onset of septic shock (median 7 days) and was asso- ciated with HSV reactivation, which occurred during the same peri- od. The incidence of active HSV infection was different between groups 1 and 2 (75% vs 12%; p = 0.004). Active CMV infection was detected first by CMV pp65 antigenemia; active HSV infection was detected by virus isolation of bronchial aspirates. As expected, quantitative analysis of CMV IgG and IgM antibodies could not discriminate between patients with and without active CMV infection. CMV IgG anti- bodies were analyzed to identify patients with previous CMV infection (CMV-seropositive patients); however, diagnosis of active CMV infection by detection of CMV IgM antibodies or rising CMV antibody titers are no longer recommended when sensitive CMV monitoring by pp65- antigenemia assay or CMV PCR are available because the information they provide is limited. Discussion The clinical role of active CMV infection in patients with septic shock is an area of ongoing discussion (4). We demonstrated that active CMV infection is associated with prolonged ventilation time and ICU stay. Ventilation time and ICU stay were more prolonged in a subgroup of patients for whom shell vial culture in bronchial aspirate was positive. CMV infection was associated with pul- monary disease despite low pp65 antigenemia and self- limiting CMV infection. We suppose that immunopathologic mechanisms could contribute to CMV- associated illness (22) in addition to direct cytopathic effects of the infection (20). Association of tumor necrosis factor and pulmonary immunopathologic features of active CMV infection was recently confirmed in a mouse model showing murine CMV reactivation after cecal ligation and puncture (9). recognized in such patients because critically ill patients are not routinely monitored for CMV infection. CMV reactivation developed in one third of our patients within 2 weeks of onset of septic shock, as has been found in studies using a similar prospective study design (3,11). Diagnostic assays of different sensitivity, different patient groups, and study designs could account for discrepant results obtained by other groups (5,10). Thus, onset of active CMV infection was detected later in the retrospective studies (4,17,18). Active CMV infection in patients with septic shock was characterized by a low viral load and resolved within a few weeks without antiviral therapy. We hypothesize that upon CMV reactivation, patients with septic shock could mount a protective antiviral immune response, which was differ- ent from the immune response of most patients after trans- plantations (19); however, this hypothesis remains to be confirmed. Deaths occurred later for patients with active CMV infection than for those without active CMV infection. This finding could be the consequence of CMV-associated disease, as has been suggested (17). Although our study was not designed to clarify the causality between active CMV infection and increased illness, we argue that active CMV infection increases illness and not vice versa. In the mouse model of CMV reactivation, the causality between sepsis, CMV reactivation, and pulmonary disease has already been shown (9). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 Discussion While CMV is well known as a cause of serious illness in immunosuppressed patients, it is now being discussed as a pathogen of emerging importance for patients with septic shock. Generally, active CMV infection is not Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 1519 1519 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 RESEARCH Figure. First detection of cytomegalovirus (CMV) and herpes sim- plex virus (HSV) reactivation after onset of septic shock. Incidence of active CMV and HSV infection is shown for patients with active CMV infection (group 1; n = 8) and without active CMV infection (group 2; n = 17). CMV reactivation occurred during the first 2 weeks after onset of septic shock (median 7 days) and was asso- ciated with HSV reactivation, which occurred during the same peri- od. The incidence of active HSV infection was different between groups 1 and 2 (75% vs 12%; p = 0.004). Active CMV infection was detected first by CMV pp65 antigenemia; active HSV infection was detected by virus isolation of bronchial aspirates. RESEARCH sitive assays for patients with septic shock, which was shown here in that shell vial culture in blood cells detect- ed only 1 patient with active CMV infection. Less sensitive assays could have been also the problem of studies that failed to detect active CMV infection in critically ill patients (5–7). We assume that assays with sensitivity sim- ilar to that of our pp65-antigenemia assay (e.g., CMV PCR of blood cells) may be equally used for CMV monitoring of patients with septic shock, considering the results of patients who had received transplants (3,11,15). Shell vial culture was more likely to detect active CMV infection in bronchial aspirate than in urine or blood cells. Pulmonary CMV infection may be relevant for patients with septic shock (8,20). Shell vial culture of urine was rarely positive for CMV in patients with septic shock, a finding which differed for patients having received a kid- ney transplant (21). Figure. First detection of cytomegalovirus (CMV) and herpes sim- plex virus (HSV) reactivation after onset of septic shock. Incidence of active CMV and HSV infection is shown for patients with active CMV infection (group 1; n = 8) and without active CMV infection (group 2; n = 17). References 1. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med. 2003;31:1250–6. 2. Pass RF. Cytomegalovirus. In: Knipe DM, Howley PM, editors. Fields virology. Philadelphia: Lippincott Williams & Wilkins; 2001. p. 2675–2705. 3. Heininger A, Jahn G, Engel C, Notheisen T, Unertl K, Hamprecht K. Human cytomegalovirus infections in nonimmunosuppressed criti- cally ill patients. Crit Care Med. 2001;29:541–7. 4. Jaber S, Chanques G, Borry J, Souche B, Verdier R, Perrigault PF, et al. Cytomegalovirus infection in critically ill patients: associated fac- tors and consequences. Chest. 2005;127:233–41. 5. Stephan F, Meharzi D, Ricci S, Fajac A, Clergue F, Bernaudin JF. Evaluation by polymerase chain reaction of cytomegalovirus reacti- vation in intensive care patients under mechanical ventilation. Intensive Care Med. 1996;22:1244–9. 6. Desachy A, Ranger-Rogez S, Francois B, Venot C, Traccard I, Gastinne H, et al. Reactivation of human herpesvirus type 6 in multi- ple organ failure syndrome. Clin Infect Dis. 2001;32:197–203. 7. Razonable RR, Fanning C, Brown RA, Espy MJ, Rivero A, Wilson J, et al. Selective reactivation of human herpesvirus 6 variant a occurs in critically ill immunocompetent hosts. J Infect Dis. 2002;185:110–3. 8. Cook CH, Zhang Y, McGuinness BJ, Lahm MC, Sedmak DD, Ferguson RM. Intra-abdominal bacterial infection reactivates latent pulmonary cytomegalovirus in immunocompetent mice. J Infect Dis. 2002;185:1395–400. After organ transplants, CMV-associated illness and death could be reduced by early antiviral therapy; however, delayed therapy has been less effective (2). Anecdotal reports show that critically ill patients with already estab- lished CMV organ disease may not benefit from antiviral therapy (3,4,20). The effect of preemptive antiviral therapy or antiviral prophylaxis has not been tested so far in patients with septic shock; however, in the mouse model, prophylac- tic treatment with ganciclovir prevented murine CMV reac- tivation and CMV-associated pulmonary fibrosis (9). 9. Cook CH, Zhang Y, Sedmak DD, Martin LC, Jewell S, Ferguson RM. Pulmonary cytomegalovirus reactivation causes pathology in immunocompetent mice. Crit Care Med. 2006;34:842–9. 10. Docke WD, Prosch S, Fietze E, Kimel V, Zuckermann H, Klug C, et al. Cytomegalovirus reactivation and tumour necrosis factor. Lancet. 1994;343:268–9. 11. Kutza AST, Muhl E, Hackstein H, Kirchner H, Bein G. High inci- dence of active cytomegalovirus infection among septic patients. Clin Infect Dis. 1998;26:1076–82. 12. Bone RC. Sir Isaac Newton, sepsis, SIRS, and CARS. Crit Care Med. 1996;24:1125–8. 13. Active Cytomegalovirus Infection and Septic Shock Recently, reactivations of HSV and human herpesvirus 6 have been reported in critically ill patients (7,23). We demonstrated for the first time an association between active HSV and CMV infection (p = 0.004). HSV was iso- lated from bronchial aspirate in the absence of skin and mucosal lesions, whereas other herpesviruses, such as varicella-zoster virus, could not be isolated. The coinci- dence of HSV and CMV reactivation during the first 2 weeks of septic shock suggests a common trigger mecha- nism for herpesvirus reactivations. In future studies, more sensitive assays (e.g., PCR) may be used to analyze the incidence of other occult herpesvirus reactivations. Reactivation of polyoma BK virus, which commonly caus- es opportunistic infection after transplantation, was not detected by virus isolation. This finding leads to the hypothesis that the conditions that stimulate polyomavirus reactivation and those that stimulate CMV and HSV reac- tivation may differ. The absence of exogenous viral infec- tion (e.g., adenovirus, respiratory syncytial virus, parainfluenzavirus) strengthens the suggestion that exoge- nous nosocomial viral infections are uncommon in patients in ICUs (24). Thus, monitoring for viral infections could focus on endogenous herpesvirus reactivations in patients with septic shock. Immunosuppression and proinflamma- tory cytokines, drugs, or combinations are presumed to be involved in herpesvirus reactivations; however, the exact mechanisms are still elusive for patients with septic shock (13,25). Dr von Müller is a senior researcher in the Department of Virology, University Hospital Ulm, Ulm, Germany. He special- izes in pediatrics, microbiology, and epidemiology of infectious diseases. His main research interest is clinical virology and the role of antiviral immunity. References Prosch S, Wendt CE, Reinke P, Priemer C, Oppert M, Kruger DH, et al. A novel link between stress and human cytomegalovirus (HCMV) infection: sympathetic hyperactivity stimulates HCMV activation. Virology. 2000;272:357–65. Despite the low patient number in this and previous studies, we suggest that CMV is a pathogen of emerging importance that can no longer be ignored for patients with septic shock. Thus, interventional studies aimed at pre- venting CMV-associated illness in patients with septic shock are needed. 14. American College of Chest Physicians/Society of Critical Care Medicine consensus conference: definitions for sepsis and organ fail- ure guidelines for the use of innovative therapies in sepsis. Crit Care Med. 1992;20:864–74. 15. von Müller L, Hampl W, Hinz J, Meisel H, Reip A, Engelmann E, et al. High variability between results of different in-house tests for cytomegalovirus (CMV) monitoring and a standardized quantitative plasma CMV PCR assay. J Clin Microbiol. 2002;40:2285–7. Discussion In a previous study we compared different assays for CMV monitoring of patients with organ transplants and demonstrated equal sensitivity of our pp65 antigenemia assay and CMV PCR of blood cells but lower sensitivity of shell vial culture, CMV PCR in plasma, and CMV mRNA detection by nucleic acid sequence-based amplification (15). Because of low viral loads, the incidence of active CMV infection could be easily underestimated by less sen- 1520 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 Address for correspondence: Thomas Mertens, Department of Virology, University Hospital Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany; email: thomas.mertens@uniklinik-ulm.de University Hospital Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany; email: thomas.mertens@uniklinik-ulm.de Address for correspondence: Thomas Mertens, Department of Virology, Acknowledgment We thank everyone involved in diagnosis and treatment of the critically ill patients for their excellent assistance. 16. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonca A, Bruining H, et al. The SOFA (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. Intensive Care Med. 1996;22:707–10. This study was supported by Roche Diagnostics, Mannheim, Germany. The funding source was not involved in the study design, collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the manu- script for publication. 17. Domart Y, Trouillet JL, Fagon JY, Chastre J, Brun-Vezinet F, Gibert C. Incidence and morbidity of cytomegaloviral infection in patients with mediastinitis following cardiac surgery. Chest. 1990;97:18–22. 1521 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 RESEARCH 23. Cook CH, Martin LC, Yenchar JK, Lahm MC, McGuinness B, Davies EA, et al. Occult herpes family viral infections are endemic in criti- cally ill surgical patients. Crit Care Med. 2003;31:1923–9. 18. Cook CH, Yenchar JK, Kraner TO, Davies EA, Ferguson RM. Occult herpes family viruses may increase mortality in critically ill surgical patients. Am J Surg. 1998;176:357–60. 19. Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker AF, Griffiths PD. Application of viral-load kinetics to identify patients who devel- op cytomegalovirus disease after transplantation. Lancet. 2000;355:2032–6. 24. Daubin C, Vincent S, Vabret A, du Cheyron D, Parienti JJ, Ramakers M, et al. Nosocomial viral ventilator-associated pneumonia in the intensive care unit: a prospective cohort study. Intensive Care Med. 2005;31:1116–22. 20. Papazian L, Thomas P, Bregeon F, Garbe L, Zandotti C, Saux P, et al. Open-lung biopsy in patients with acute respiratory distress syn- drome. Anesthesiology. 1998;88:935–44. 25. Soderberg-Naucler C, Fish KN, Nelson JA. Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell. 1997;91:119–26. 21. Brennan DC. Cytomegalovirus in renal transplantation. J Am Soc Nephrol. 2001;12:848–55. Address for correspondence: Thomas Mertens, Department of Virology, 22. Barry SM, Johnson MA, Janossy G. Cytopathology or immunopathology? The puzzle of cytomegalovirus pneumonitis revisited. Bone Marrow Transplant. 2000;26:591–7. University Hospital Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany; email: thomas.mertens@uniklinik-ulm.de 1522 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006 Search past issues Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 10, October 2006
https://openalex.org/W3006569505	https://repositorium.sdum.uminho.pt/bitstream/1822/64096/1/20257-molecules-25-00795-v2.pdf	English	null	Bioorthogonal Labeling Reveals Different Expression of Glycans in Mouse Hippocampal Neuron Cultures during Their Development	Molecules/Molecules online/Molecules annual	2,020	cc-by	9,125	ceived: 9 January 2020; Accepted: 10 February 2020; Published: 12 February 2020 Received: 9 January 2020; Accepted: 10 February 2020; Published: 12 February 2020 Abstract: The expression of diﬀerent glycans at the cell surface dictates cell interactions with their environment and other cells, being crucial for the cell fate. The development of the central nervous system is associated with tremendous changes in the cell glycome that is tightly regulated. Herein, we have employed biorthogonal Cu-free click chemistry to image temporal distribution of diﬀerent glycans in live mouse hippocampal neurons during their maturation in vitro. We show development-dependent glycan patterns with increased fucose and decreased mannose expression at the end of the maturation process. We also demonstrate that this approach is biocompatible and does not aﬀect glycan transport although it relies on an administration of modiﬁed glycans. The applicability of this strategy to tissue sections unlocks new opportunities to study the glycan dynamics under more complex physiological conditions. Keywords: glycosylation; biorthogonal chemistry; neuronal development; imaging molecules molecules molecules Molecules 2020, 25, 795; doi:10.3390/molecules25040795 Bioorthogonal Labeling Reveals Diﬀerent Expression of Glycans in Mouse Hippocampal Neuron Cultures during Their Development Diana Soares da Costa 1,2,* , João C. Sousa 2,3 , Sandro Dá Mesquita 2,3,† , Nevena I. Petkova-Yankova 1,2 , Fernanda Marques 2,3, Rui L. Reis 1,2,4, Nuno Sousa 2,3 and Iva Pashkuleva 1,2,* Diana Soares da Costa 1,2,* , João C. Sousa 2,3 , Sandro Dá Mesquita 2,3,† , Nevena I. Petkova-Yankova 1,2 , Fernanda Marques 2,3, Rui L. Reis 1,2,4, Nuno Sousa 2,3 and Iva Pashkuleva 1,2,* 1 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Barco, 4805-017 Guimarães, Portugal; nipetkova@chem.uni-soﬁa.bg (N.I.P.-Y.); rgreis@i3bs.uminho.pt (R.L.R.) 1 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Barco, 4805-017 Guimarães, Portugal; nipetkova@chem.uni-soﬁa.bg (N.I.P.-Y.); rgreis@i3bs.uminho.pt (R.L.R.) g p g g p 2 Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal; jcsousa@med.uminho.pt (J.C.S.); sd8tf@virginia.edu (S.D.M.); fmarques@med.uminho.pt (F.M.); njcsousa@med.uminho.pt (N.S.) 3 ICVS/3B’s—PT Government Associate Laboratory, 4710-057 Braga, Guimarães, Portugal 4 / y, g , , g 4 The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, Barco, 4805-017 Guimarães, Portugal p g * Correspondence: diana.costa@i3bs.uminho.pt (D.S.d.C.); pashkuleva@i3bs.uminho.pt (I.P.) p p ( ); p p ( ) † Present Address: Center for Brain Immunology and Glia, Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA. † Present Address: Center for Brain Immunology and Glia, Department of Neuroscience, School of Medi esent Address: Center for Brain Immunology and Glia, Department of Neuroscience, School of Medicine, niversity of Virginia, Charlottesville, VA 22908, USA. gy , p , University of Virginia, Charlottesville, VA 22908, USA. University of Virginia, Charlottesville, VA 22908, USA. Academic Editor: Vito Ferro Academic Editor: Vito Ferro Received: 9 January 2020; Accepted: 10 February 2020; Published: 12 February 2020 molecules molecules 1. Introduction Glycans displayed at the cell surface determine the cell interactome and fate [1,2]. In the nervous system, glycoconjugates play central roles in development, regeneration and synaptic plasticity [3,4]. They participate in the formation of a complex molecular network (both at the cell surface and in the extracellular matrix) that mediates recognition processes and triggers speciﬁc pathways. Their fundamental role in the central nervous system (CNS) is evidenced by the neuropathological and psychomotor incapacities of patients with congenital glycosylation diseases [5–7]. The structural diversity of glycans provides a myriad of possible combinations that allow ﬁne regulation of the cell interactome: glycosylation patterns are cell-speciﬁc and in the brain, they are tightly regulated during diﬀerent development stages [4,8]. Therefore, approaches that allow to www.mdpi.com/journal/molecules Molecules 2020, 25, 795; doi:10.3390/molecules25040795 2 of 12 Molecules 2020, 25, 795 monitor, control and modify glycan expression on cells are powerful tools to understand and regulate the cell fate. The roles of speciﬁc proteins in dynamic cellular processes are typically elucidated by using monoclonal antibodies and genetic ﬂuorescent protein fusions. Analytical techniques for glycan proﬁling are not as straightforward as those for proteins because glycans biosynthesis is not genetically encoded and glycans structure can vary in function of the cellular environment. Therefore, imaging and quantitative analysis are extremely diﬃcult to perform using conventional biochemical methods [9]. Bioorthogonal chemistry was introduced by Bertozzi in the early 2000s and allows biomolecule tracking in living systems without disturbing the natural biochemical processes [9,10]. In particular, metabolic glycoengineering (MGE) is a powerful method that overcomes the aforementioned limitations in the glycan analysis [11]. MGE allows the study of the cell glycorepertoire via introduction of unnatural sugars, so-called biorthogonal reporters, into native glycans without detriment of cell physiology due to the promiscuity of the enzymes of the Roseman–Warren pathway [12]. The use of azides and alkynes as reactive moieties in biorthogonal reporters has fostered advances in glycan imaging [13]. Both functional groups are small, biologically inert, and capable of reacting selectively with exogenous agents bearing complementary functionality at physiological pH, thus, allowing the labeling of the biomolecule with ﬂuorophores or aﬃnity tags [14]. Azide groups can react with the complementary phosphines via the Staudinger ligation [15,16], linear alkynes via the Cu-catalyzed azide–alkyne cycloaddition (CuAAC) [16,17], and cyclooctynes via the strain-promoted azide–alkyne cycloaddition (SAAC), also termed “Cu-free click chemistry” [16–20]. 1. Introduction MGE has been successfully applied for in situ imaging of glycans in diﬀerent cell lines and living organisms [21–28]. Despite the crucial role of glycans during neuronal development, the metabolic labeling and imaging of glycan structures in primary neurons have been reported only recently using CuAAC protocol [29]. Herein, we describe an alternative Cu-free protocol applied to mouse hippocampal neuron cultures [15,30]. The main advantage of using Cu-free click chemistry is to evade the copper-associated toxicity in biological systems [19]. We used three diﬀerent reporters, namely azido-modiﬁed N-acetyl derivatives of mannosamine and glucosamine and azido-modiﬁed fucose, in combination with biorthogonal coupling with a labeled cyclooctyne derivative to image the expression of glycans during diﬀerent stages of the neuron development. We demonstrate that this protocol is a fast and reliable approach for in situ characterization of glycans in neurons. 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures Furthermore, the choice for the 7 and 14 days was made in order to avoid overcrowded cultures because: i) it would be harder to image individual cells as well as cell to ll ii) ll ld li b h h h l i h i l Figure 1. Immunofluorescence images of neuroprogenitor cells after different culture times: (a) 1 day, (b) 3 days and (c) 14 days. β actin is represented in red (a, b); βIII Tubulin in green (a–c) and glial fibrillary acidic protein (GFAP) in red (c). Figure 1. Immunoﬂuorescence images of neuroprogenitor cells after diﬀerent culture times: (a) 1 day, (b) 3 days and (c) 14 days. β actin is represented in red (a,b); βIII Tubulin in green (a–c) and glial ﬁbrillary acidic protein (GFAP) in red (c). Figure 1. Immunofluorescence images of neuroprogenitor cells after different culture times: (a) 1 day, (b) 3 days and (c) 14 days. β actin is represented in red (a, b); βIII Tubulin in green (a–c) and glial fibrillary acidic protein (GFAP) in red (c). Figure 1. Immunoﬂuorescence images of neuroprogenitor cells after diﬀerent culture times: (a) 1 day, (b) 3 days and (c) 14 days. β actin is represented in red (a,b); βIII Tubulin in green (a–c) and glial ﬁbrillary acidic protein (GFAP) in red (c). At day 7 and 14, cultures were supplemented with the unnatural metabolic precursors containing azido groups. Cells were allowed to metabolize the supplemented carbohydrates for 24 h and then the labelled cyclooctyne was introduced to initiate the click reaction. We tested different reaction conditions and found that for the studied cell cultures cyclooctyne concentrations of 50 µM and reaction time of 1 h gave the optimal output. The supplementation of the azidocarbohydrates resulted in different fluorescence intensity At day 7 and 14, cultures were supplemented with the unnatural metabolic precursors containing azido groups. Cells were allowed to metabolize the supplemented carbohydrates for 24 h and then the labelled cyclooctyne was introduced to initiate the click reaction. We tested diﬀerent reaction conditions and found that for the studied cell cultures cyclooctyne concentrations of 50 µM and reaction time of 1 h gave the optimal output. The supplementation of the azidocarbohydrates resulted in different fluorescence intensity among the tested carbohydrates and culture times. 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures Unlike the other biomacromolecules (polynucleotides and proteins), carbohydrates are not a genetic product. Thus, unnatural metabolic precursors can be interspersed in the carbohydrate biosynthetic pathways [31,32]. The incorporation of unnatural monosaccharides bearing reactive functional groups into cell-surface glycoconjugates provides a scenario in which the glycan can be further elaborated with an exogenously delivered imaging reagent [31]. The selectivity and rate of the reaction between the imaging agent and the incorporated carbohydrate determine the success of this strategy. Thus, the choice of the functional group/labelling reaction is crucial [20]. Among diﬀerent possibilities, the copper-free azide–alkyne cycloaddition and the Staudinger ligation are better options for studies involving living cells and organisms. Herein, we have selected the copper-free azide–alkyne cycloaddition as the product stability and reaction rate of the Staudinger ligation can be compromised under in vivo conditions [33]. We prepared N-azidoacetylmannosamine (ManNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose following previously described procedures (Scheme S3) [10–36]. The membrane penetration of unnatural metabolites is a key point of this approach and thus, the compounds were peracetylated to improve their uptake by the cells. The obtained unnatural metabolic precursors were feed into the culture medium of mouse hippocampal cells. To select the time points at which the supplementation would be performed, we ﬁrst studied the diﬀerentiation of neuroprogenitor cells into neurons by immunoﬂuorescence with 3 of 12 Molecules 2020, 25, 795 key neuron markers (β actin, βIII Tubulin) and glial ﬁbrillary acidic protein (GFAP) as an astrocyte marker. We observed a diﬀerentiation process up to 14 days (Figure 1). At this time point mainly neurons (Figure 1c, βIII Tubulin staining in green) and very few astrocytes are visible (Figure 1c, GFAP labeling in red) and thus, we selected it as an end point for our experiments. Because at day 3 very few cells were positive for βIII Tubulin (Figure 1b), we selected day 7 as an intermediate timepoint for metabolic labeling. Furthermore, the choice for the 7 and 14 days was made in order to avoid overcrowded cultures because: i) it would be harder to image individual cells as well as cell to cell contact; ii) more cells could split between them the sugars thus lowering the signal. first studied the differentiation of neuroprogenitor cells into neurons by immunofluorescence with key neuron markers (β actin, βIII Tubulin) and glial fibrillary acidic protein (GFAP) as an astrocyte marker. 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures We observed a differentiation process up to 14 days (Figure 1). At this time point mainly neurons (Figure 1c, βIII Tubulin staining in green) and very few astrocytes are visible (Figure 1c, GFAP labeling in red) and thus, we selected it as an end point for our experiments. Because at day 3 very few cells were positive for βIII Tubulin (Figure 1b), we selected day 7 as an intermediate timepoint for metabolic labeling. Furthermore, the choice for the 7 and 14 days was made in order to avoid overcrowded cultures because: i) it would be harder to image individual cells as well as cell to ll ii) ll ld li b h h h l i h i l key neuron markers (β actin, βIII Tubulin) and glial ﬁbrillary acidic protein (GFAP) as an astrocyte marker. We observed a diﬀerentiation process up to 14 days (Figure 1). At this time point mainly neurons (Figure 1c, βIII Tubulin staining in green) and very few astrocytes are visible (Figure 1c, GFAP labeling in red) and thus, we selected it as an end point for our experiments. Because at day 3 very few cells were positive for βIII Tubulin (Figure 1b), we selected day 7 as an intermediate timepoint for metabolic labeling. Furthermore, the choice for the 7 and 14 days was made in order to avoid overcrowded cultures because: i) it would be harder to image individual cells as well as cell to cell contact; ii) more cells could split between them the sugars thus lowering the signal. first studied the differentiation of neuroprogenitor cells into neurons by immunofluorescence with key neuron markers (β actin, βIII Tubulin) and glial fibrillary acidic protein (GFAP) as an astrocyte marker. We observed a differentiation process up to 14 days (Figure 1). At this time point mainly neurons (Figure 1c, βIII Tubulin staining in green) and very few astrocytes are visible (Figure 1c, GFAP labeling in red) and thus, we selected it as an end point for our experiments. Because at day 3 very few cells were positive for βIII Tubulin (Figure 1b), we selected day 7 as an intermediate timepoint for metabolic labeling. 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures At day 7, highest intensity is visible for the cells supplemented with Ac4ManAz (Figure 2a), lower for the Ac4GlcAz supplemented cultures (Figure 2b) and a faint signal for Ac4FucAz that reveals less fucose at this stage (Figure 2c). Fluorescence is visible along the cell body but also throughout the dendrites for cells supplemented with mannose and glucose analogs (Figure 2). The supplementation of the azidocarbohydrates resulted in diﬀerent ﬂuorescence intensity among the tested carbohydrates and culture times. At day 7, highest intensity is visible for the cells supplemented with Ac4ManAz (Figure 2a), lower for the Ac4GlcAz supplemented cultures (Figure 2b) and a faint signal for Ac4FucAz that reveals less fucose at this stage (Figure 2c). Fluorescence is visible along the cell body but also throughout the dendrites for cells supplemented with mannose and glucose analogs (Figure 2). At day 14, when the cell culture is mostly composed by diﬀerentiated cells (Figure 3a2–d2) the glycosylation pattern is diﬀerent: incorporation of fucose increases and matches mannose and glucose derivative levels in neuronal glycoproteins (Figure 3a1–d1). These results indicate a decrease in the mannose and glucose derivative incorporation during the diﬀerentiation process and increase in the glycoprotein fucosylation. Sialylation (and more speciﬁcally polysialylation) and fucosylation are major post-translational modiﬁcations occurring in carbohydrate-carrying molecules, e.g., proteins, in the nervous system. These post-translational functionalizations are related with proliferation, migration and diﬀerentiation of neural progenitors [4]. Higher expression of mannose/glucose-containing glycoproteins at day 7 might indicate abundant sialylation as either mannose or glucose can be metabolized by cells to sialic acid [36]. Polysialylated Neural Cell Adhesion Molecule (NCAM) is associated with neuritogenesis and neurite outgrowth of hippocampal neurons in culture. The fact that these processes are very intensive within the ﬁrst days of culture [29] can explain the results obtained with Ac4ManNAz and Ac4GlcNAz incorporation (Figure 2). While polysialylation gradually decreases [37], fucosylation increases with neuronal maturation [38]. Fucosylated glycoproteins are involved in neuronal communication. Their expression changes extensively during the course of neuronal development in mouse hippocampal tissue and during maturation of neurons in culture [38]. These previous results agree with our ﬁnding that fucose becomes more abundant with diﬀerentiation of neuroprogenitor cells (Figure 3). 4 of 12 12 Molecules 2020, 25, 795 Molecules 2020 25 x FO Figure 2. 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures Metabolic labeling (green) of hippocampal cell cultures at day 7: cultures supplemented with peracetylated azido-functionalized (a) mannose (Ac4ManAz), (b) glucose (Ac4GlcAz) and (c) fucose (Ac4FucAz) followed by the click imaging molecule dibenzylcyclooctyne‐Fluor 488/561 (DBCO-488/561, 50 µM) (d) Control conditions: (d1) cell culture without supplementation, (d2) culture supplemented only with the DBCO-488/561 (50 µM), (d3) culture supplemented with unmodified mannose and DBCO-488/561 (50 µM). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and are shown in blue. (e) The graphic represents fluorescence intensity measured with Fiji software (v1.50e) using representative images for the different conditions Figure 2. Metabolic labeling (green) of hippocampal cell cultures at day 7: cultures supplemented with peracetylated azido-functionalized (a) mannose (Ac4ManAz), (b) glucose (Ac4GlcAz) and (c) fucose (Ac4FucAz) followed by the click imaging molecule dibenzylcyclooctyne-Fluor 488/561 (DBCO-488/561, 50 µM) (d) Control conditions: (d1) cell culture without supplementation, (d2) culture supplemented only with the DBCO-488/561 (50 µM), (d3) culture supplemented with unmodiﬁed mannose and DBCO-488/561 (50 µM). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and are shown in blue. (e) The graphic represents ﬂuorescence intensity measured with Fiji software (v1.50e) using representative images for the diﬀerent conditions. Figure 2. Metabolic labeling (green) of hippocampal cell cultures at day 7: cultures supplemented with peracetylated azido-functionalized (a) mannose (Ac4ManAz), (b) glucose (Ac4GlcAz) and (c) fucose (Ac4FucAz) followed by the click imaging molecule dibenzylcyclooctyne‐Fluor 488/561 (DBCO-488/561, 50 µM) (d) Control conditions: (d1) cell culture without supplementation, (d2) culture supplemented only with the DBCO-488/561 (50 µM), (d3) culture supplemented with unmodified mannose and DBCO-488/561 (50 µM). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and are shown in blue. (e) The graphic represents fluorescence intensity measured with Fiji software (v1.50e) using representative images for the Figure 2. Metabolic labeling (green) of hippocampal cell cultures at day 7: cultures supplemented with peracetylated azido-functionalized (a) mannose (Ac4ManAz), (b) glucose (Ac4GlcAz) and (c) fucose (Ac4FucAz) followed by the click imaging molecule dibenzylcyclooctyne-Fluor 488/561 (DBCO-488/561, 50 µM) (d) Control conditions: (d1) cell culture without supplementation, (d2) culture supplemented only with the DBCO-488/561 (50 µM), (d3) culture supplemented with unmodiﬁed mannose and DBCO-488/561 (50 µM). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and are shown in blue. (e) The graphic represents ﬂuorescence intensity measured with Fiji software (v1.50e) using representative images for the diﬀerent conditions. 5 of 12 of 12 Molecules 2020, 25, 795 Molecules 2020 25 x F Figure 3. different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters Sialylation (and more specifically polysialylation) and fucosylation are major post-translational modifications occurring in carbohydrate-carrying molecules, e.g. proteins, in the nervous system. These post-translational functionalizations are related with proliferation, migration and differentiation of neural progenitors [4]. Higher expression of mannose/glucose-containing glycoproteins at day 7 might indicate abundant sialylation as either mannose or glucose can be metabolized by cells to sialic acid [36]. Polysialylated Neural Cell Adhesion Molecule (NCAM) is associated with neuritogenesis and neurite outgrowth of hippocampal neurons in culture. The fact that these processes are very intensive within the first days of culture [29] can explain the results Glycosylation is incomparably crucial and, therefore, tightly controlled in neurons [4]. The formation of glycan linkage is catalyzed by highly selective glycosyltransferases (GT) with speciﬁcity both for substrate and donor nucleotide carbohydrate. The results described above showed that neuronal GT tolerate the use of unnatural azido adducts. We have used low concentration to avoid changes in the machinery used by the cell to transport and modify these molecules. To conﬁrm that the carbohydrate transporters and transferases are not aﬀected by these unnatural molecules, we performed RT-PCR analysis for the expression level of the respective genes (Figures 4–6). Molecules 2020, 25, x FOR PEER REVIEW 6 of 12 molecules. To confirm that the carbohydrate transporters and transferases are not affected by these unnatural molecules, we performed RT-PCR analysis for the expression level of the respective genes (Figures 4–6) obtained with Ac4ManNAz and Ac4GlcNAz incorporation (Figure 2). While polysialylation gradually decreases [37], fucosylation increases with neuronal maturation [38]. Fucosylated glycoproteins are involved in neuronal communication. Their expression changes extensively during the course of neuronal development in mouse hippocampal tissue and during maturation of neurons in culture [38]. These previous results agree with our finding that fucose becomes more abundant with differentiation of neuroprogenitor cells (Figure 3). 2.2. Gene Expression Levels of Carbohydrate Transporters Glycosylation is incomparably crucial and, therefore, tightly controlled in neurons [4]. The formation of glycan linkage is catalyzed by highly selective glycosyltransferases (GT) with specificity both for substrate and donor nucleotide carbohydrate. The results described above showed that neuronal GT tolerate the use of unnatural azido adducts. We have used low concentration to avoid changes in the machinery used by the cell to transport and modify these Figure 4. Expression of genes coding protein-O-mannosyltransferases (Pomt1 and Pomt2) after 7 and 14 days in culture (7D and 14D). Figure 4. 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures (e) The graphic represents ﬂuorescence intensity measured with Fiji software (v1.50e) using representative images for the diﬀerent conditions. Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters 2.1. Metabolic Labelling of Mouse Hippocampal Neuronal Cultures Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, (b1) Ac4GlcAz and (c1) Ac4FucAz followed by DBCO-488/561 (50 µM); (a2–c2) immunostaining of βIII tubulin for the same cultures (red) and nuclei with DAPI (blue); (d1,2) control samples supplemented with unmodified mannose and DBCO-488/561 (50 µM). (e) The graphic represents fluorescence intensity measured with Fiji software (v1.50e) using representative images for the different conditions. Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, (b1) Ac4GlcAz and (c1) Ac4FucAz followed by DBCO-488/561 (50 µM); (a2–c2) immunostaining of βIII tubulin for the same cultures (red) and nuclei with DAPI (blue); (d1,2) control samples supplemented with unmodiﬁed mannose and DBCO-488/561 (50 µM). (e) The graphic represents ﬂuorescence intensity measured with Fiji software (v1.50e) using representative images for the diﬀerent conditions. Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, (b1) Ac4GlcAz and (c1) Ac4FucAz followed by DBCO-488/561 (50 µM); (a2–c2) immunostaining of βIII tubulin for the same cultures (red) and nuclei with DAPI (blue); (d1,2) control samples supplemented with unmodified mannose and DBCO-488/561 (50 µM). (e) The graphic represents fluorescence intensity measured with Fiji software (v1.50e) using representative images for the Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, (b1) Ac4GlcAz and (c1) Ac4FucAz followed by DBCO-488/561 (50 µM); (a2–c2) immunostaining of βIII tubulin for the same cultures (red) and nuclei with DAPI (blue); (d1,2) control samples supplemented with unmodiﬁed mannose and DBCO-488/561 (50 µM). (e) The graphic represents ﬂuorescence intensity measured with Fiji software (v1.50e) using representative images for the diﬀerent conditions. Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, (b1) Ac4GlcAz and (c1) Ac4FucAz followed by DBCO-488/561 (50 µM); (a2–c2) immunostaining of βIII tubulin for the same cultures (red) and nuclei with DAPI (blue); (d1,2) control samples supplemented with unmodified mannose and DBCO-488/561 (50 µM). (e) The graphic represents fluorescence intensity measured with Fiji software (v1.50e) using representative images for the Figure 3. Hippocampal cells after 14 days in culture: (a1–c1) metabolic labelling of (a1) Ac4ManAz, (b1) Ac4GlcAz and (c1) Ac4FucAz followed by DBCO-488/561 (50 µM); (a2–c2) immunostaining of βIII tubulin for the same cultures (red) and nuclei with DAPI (blue); (d1,2) control samples supplemented with unmodiﬁed mannose and DBCO-488/561 (50 µM). different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters The concentration used in this study (50 µM) is far below this range and therefore it is not expected to deleteriously affect neurons. However, the cell culture was performed in conditions of hyperglycemia (glucose concentration in Neurobasal A medium, Invitrogen is 25 mM), i.e., we used a high glucose concentration. An additional increase in the glucose concentration (by addition of azido modified glucose, Figure 6) can cause stress and be the reason for the increased expression of Glut1, as reported for adult neural stem cells [41]. ules 2020, 25, x FOR PEER REVIEW ion. Disruption of the normal glucose homeostasis is the pathophysiological cause fo disorders [39]. In euglycemic condition, glucose concentration in the plasma is arou and this corresponds to brain levels of approximately 1–2.5 mM [40]. The concentration study (50 µM) is far below this range and therefore it is not expected to deleteriousl ons. However, the cell culture was performed in conditions of hyperglycemia ( entration in Neurobasal A medium, Invitrogen is 25 mM), i.e., we used a high entration. An additional increase in the glucose concentration (by addition of azido m Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D d 14D) * 0 05 i di i i ll i ifi diff Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D and 14D). * p < 0.05 indicates a statistically signiﬁcant diﬀerence. lucose, Figure 6) can cause stress and be the reason for the increased expression of Glut1, as eported for adult neural stem cells [41]. and 14D). p < 0.05 indicates a statistically significant difference. The significant change in Glut1 expression indicates that brain cells are highly responsive and nsitive to glucose fluctuations. Mammalian brain cells use glucose as a main source of energy; refore, they depend on the tight regulation of glucose metabolism for proper physiological brain Figure 6. Fucose transporter and transferases gene expression after 7 and 14 days in culture (7D and 14D) Figure 6. Fucose transporter and transferases gene expression after 7 and 14 days in culture (7D and 14D). Figure 6. Fucose transporter and transferases gene expression after 7 and 14 days in culture (7D and 14D) Figure 6. Fucose transporter and transferases gene expression after 7 and 14 days in culture (7D and 14D). 3. Materials and Methods 3.1. different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters The significant change in Glut1 expression indicates that brain cells are highly responsive and sitive to glucose fluctuations. Mammalian brain cells use glucose as a main source of energy; efore, they depend on the tight regulation of glucose metabolism for proper physiological brain Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D and 14D). * p < 0.05 indicates a statistically signiﬁcant diﬀerence. Molecules 2020, 25, x FOR PEER REVIEW 7 of 12 unction. Disruption of the normal glucose homeostasis is the pathophysiological cause for many brain disorders [39]. In euglycemic condition, glucose concentration in the plasma is around 5–8 mM, and this corresponds to brain levels of approximately 1–2.5 mM [40]. The concentration used in his study (50 µM) is far below this range and therefore it is not expected to deleteriously affect neurons. However, the cell culture was performed in conditions of hyperglycemia (glucose concentration in Neurobasal A medium, Invitrogen is 25 mM), i.e., we used a high glucose concentration. An additional increase in the glucose concentration (by addition of azido modified glucose, Figure 6) can cause stress and be the reason for the increased expression of Glut1, as reported for adult neural stem cells [41]. Figure 6. Fucose transporter and transferases gene expression after 7 and 14 days in culture (7D and 14D). 3. Materials and Methods Figure 6. Fucose transporter and transferases gene expression after 7 and 14 days in culture (7D and 14D). Apart from a statistically signiﬁcant (* p < 0.05) increase in Glut1 expression after the additio azido modiﬁed glucose at day 7 in culture (Figure 5), no signiﬁcant diﬀerences were observed fo µm) do not affect significantly the de novo expression of carbohydrate transporters and transferases Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D d 14D) * < 0 05 i di t t ti ti ll i ifi t diff Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D and 14D). * p < 0.05 indicates a statistically signiﬁcant diﬀerence. Molecules 2020, 25, x FOR PEER REVIEW 7 of 12 function. Disruption of the normal glucose homeostasis is the pathophysiological cause for many brain disorders [39]. In euglycemic condition, glucose concentration in the plasma is around 5–8 mM, and this corresponds to brain levels of approximately 1–2.5 mM [40]. different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters Expression of genes coding protein-O-mannosyltransferases (Pomt1 and Pomt2) after 7 and 14 days in culture (7D and 14D). pecificity both for substrate and donor nucleotide carbohydrate. The results described abov howed that neuronal GT tolerate the use of unnatural azido adducts. We have used low oncentration to avoid changes in the machinery used by the cell to transport and modify thes Figure 4. Expression of genes coding protein-O-mannosyltransferases (Pomt1 and Pomt2) after 7 and 14 days in culture (7D and 14D). Figure 4. Expression of genes coding protein-O-mannosyltransferases (Pomt1 and Pomt2) after 7 and 14 days in culture (7D and 14D). 6 of 12 e 0 Molecules 2020, 25, 795 addition of azido mo used conditions (24 h sed conditions (24 h period of exposition to the carbohydrate analogs at a low concentration of 50 m) do not affect significantly the de novo expression of carbohydrate transporters and transferases. Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D d 14D) * 0 05 i di t t ti ti ll i ifi t diff Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D and 14D). * p < 0.05 indicates a statistically signiﬁcant diﬀerence. Molecules 2020, 25, x FOR PEER REVIEW 7 of 12 function. Disruption of the normal glucose homeostasis is the pathophysiological cause for many brain disorders [39]. In euglycemic condition, glucose concentration in the plasma is around 5–8 mM, and this corresponds to brain levels of approximately 1–2.5 mM [40]. The concentration used in this study (50 µM) is far below this range and therefore it is not expected to deleteriously affect neurons. However, the cell culture was performed in conditions of hyperglycemia (glucose concentration in Neurobasal A medium, Invitrogen is 25 mM), i.e., we used a high glucose concentration. An additional increase in the glucose concentration (by addition of azido modified glucose, Figure 6) can cause stress and be the reason for the increased expression of Glut1, as reported for adult neural stem cells [41]. d conditions (24 h period of exposition to the carbohydrate analogs at a low concentration of 50 do not affect significantly the de novo expression of carbohydrate transporters and transferases. Figure 5. Glucose transporters and transferases gene expression after 7 and 14 days in culture (7D and 14D). * p < 0.05 indicates a statistically significant difference. 3.1. Synthesis and Characterization of Biorthogonal Reporters N-azidoacetylmannosamine (ManNAz) and N-azidoacetylglucosamine (GlcNAz) were prepared according to a method described from Bertozzi et al. (SI, Scheme S1) [10,36]. Brieﬂy, hydrochloride of D-aminocarbohydrate (1.0 mmol) was added to azidoacetic acid (1.37 mmol) in methanol (10 mL). After dissolution, triethylamine (0.34 mL, 2.43 mmol) was added and the reaction mixture was stirred for 5 min at room temperature (RT). The solution was cooled to 0 ◦C and hydroxybenzotriazole (HOBt, 0.135 g, 1.0 mmol) was added ﬁrst, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, 0.383 g, 2.0 mmol). The mixture was warmed to RT and the reaction proceeded overnight. Next, the solution was concentrated, and the residue was eluted with water over AG 50WX8 resin and AG 1-X2 resin. After concentration, the residue was further puriﬁed by silica gel chromatography, eluting with CHCl3–MeOH. Of note, the puriﬁcation of azidoderivatives from the ammonium salt is a critical step. We have carried out the reaction of d-aminocarbohydrate with chloroacetic anhydride and used NaOH as a base (SI, Scheme S2). This approach was successfully applied for GlcNAz. In this case, a hydrochloride of D-glucosamine (1.0 mmol) was added to a suspension of NaOH (1.0 mmol) in MeOH (3 mL). The mixture was stirred at RT for 5 min and ﬁltered. Triethylamine (0.93 mmol) and chloroacetic anhydride (4.6 mmol) were added to the ﬁltrate. The reaction mixture was stirred for 24 h at RT. The solvent was removed and column chromatography was applied for partial puriﬁcation of the compound, eluting with a gradient of CHCl3:MeOH (20:1 to 7:1). The resulting oil was dissolved in DMF (3 mL). NaN3 (3.0 mmol) was added to the solution and the reaction mixture was heated at 80 ◦C for 2 h. The solvent was removed and second column chromatography puriﬁcation was applied, eluting with a gradient of CHCl3:MeOH. g g The obtained azides were further peracetylated to obtain Ac4ManNAz and Ac4GlcNAz. Acetic anhydride (2.0 mL) was added to a solution of corresponding N-azidocarbohydrate in pyridine (2 mL) and the reaction mixture was stirred overnight at RT. The solution was concentrated, resuspended in CH2Cl2, and washed consecutive with 1 M HCl, saturated NaHCO3, and saturated NaCl. The organic phase was dried over Na2SO4, ﬁltered, and concentrated. The crude material was puriﬁed by silica gel chromatography, eluting with hexanes–ethyl acetate (2:1, v/v). different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters However, the cell culture was performed in conditions of hyperglycemia (glucose concentration in 7 of 12 Molecules 2020, 25, 795 Neurobasal A medium, Invitrogen is 25 mM), i.e., we used a high glucose concentration. An additional increase in the glucose concentration (by addition of azido modiﬁed glucose, Figure 6) can cause stress and be the reason for the increased expression of Glut1, as reported for adult neural stem cells [41]. Neurobasal A medium, Invitrogen is 25 mM), i.e., we used a high glucose concentration. An additional increase in the glucose concentration (by addition of azido modiﬁed glucose, Figure 6) can cause stress the reason for the increased expression of Glut1, as reported for adult neural stem cells [41]. different conditions. 2.2. Gene Expression Levels of Carbohydrate Transporters Synthesis and Characterization of Biorthogonal Reporters N-azidoacetylmannosamine (ManNAz) and N-azidoacetylglucosamine (GlcNAz) were prepared according to a method described from Bertozzi et al (SI, Scheme S1) [10,36]. Briefly, hydrochloride of D-aminocarbohydrate (1.0 mmol) was added to azidoacetic acid (1.37 mmol) in methanol (10 mL) After dissolution triethylamine (0 34 mL 2 43 mmol) was added and the reaction Apart from a statistically signiﬁcant (* p < 0.05) increase in Glut1 expression after the addition of azido modiﬁed glucose at day 7 in culture (Figure 5), no signiﬁcant diﬀerences were observed for the gene expression of mannose (Figure 4) and fucose (Figure 6) transporters and transferases upon addition of azido modiﬁed carbohydrates to neuronal cell cultures. These results indicate that the used conditions (24 h period of exposition to the carbohydrate analogs at a low concentration of 50 µm) do not aﬀect signiﬁcantly the de novo expression of carbohydrate transporters and transferases. methanol (10 mL). After dissolution, triethylamine (0.34 mL, 2.43 mmol) was added and the reaction mixture was stirred for 5 min at room temperature (RT). The solution was cooled to 0 °C and hydroxybenzotriazole (HOBt, 0.135 g, 1.0 mmol) was added first, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, 0.383 g, 2.0 mmol). The mixture was warmed to RT and the reaction proceeded overnight. Next, the solution was concentrated, and the residue was eluted with water over AG 50WX8 resin and AG 1-X2 resin. After concentration, the residue was further purified by silica gel chromatography, eluting with CHCl3–MeOH. Of note, the purification of azidoderivatives from the ammonium salt is a critical step. We have carried out the reaction of D-aminocarbohydrate with chloroacetic anhydride and used NaOH as a base (SI, Scheme The signiﬁcant change in Glut1 expression indicates that brain cells are highly responsive and sensitive to glucose ﬂuctuations. Mammalian brain cells use glucose as a main source of energy; therefore, they depend on the tight regulation of glucose metabolism for proper physiological brain function. Disruption of the normal glucose homeostasis is the pathophysiological cause for many brain disorders [39]. In euglycemic condition, glucose concentration in the plasma is around 5–8 mM, and this corresponds to brain levels of approximately 1–2.5 mM [40]. The concentration used in this study (50 µM) is far below this range and therefore it is not expected to deleteriously aﬀect neurons. 3.1. Synthesis and Characterization of Biorthogonal Reporters Further puriﬁcation by reversed-phase HPLC (KANUER, Berlin, Germany) was also performed using column Atlantis T3 5 µm (Waters, Manchester, UK), 30 × 150 mm and eluting with a gradient of CH3CN and H2O. p y p g 5 µm (Waters, Manchester, UK), 30 × 150 mm and eluting with a gradient of CH3CN and H2O. µ ( ) g g 3 2 The peracetylated N-azidofucose was obtained following a diﬀerent procedure (SI, Scheme S3) [35]. In the ﬁrst step, we obtained 1,2:3,4-Di-O-isopropylidene-L-galactopyranose. Concentrated sulfuric acid (0.01 mL) was added to anhydrous zinc chloride (2.24 mmol) in dry acetone (6.5 mL). Then powdered anhydrous L-galactose (0.250 g) was added quickly, and the reaction mixture was stirred for 10 min. A suspension of Na2CO3 (0.5 g in 0.9 mL of water) was then added slowly. The suspension was ﬁltered, and the precipitate was washed several times with acetone. Combined ﬁltrates were evaporated under reduced pressure. The 1,2:3,4-Di-O-isopropylidene-l-galactopyranose was obtained from this residue after extraction with ether and puriﬁed by column chromatography (silica gel, eluent CH2Cl2:CH3OH 9:1). The 1,2:3,4-Di-O-isopropylidene-6-azido-l-galactopyranose was obtained in the next step. The 1,2:3,4-Di-O-isopropylidene-l-galactopyranose was dissolved in dry CH2Cl2 (2 mL). We then added dry pyridine (1 mL) and triﬂuoromethanesulfonic anhydride (0.372 mL). This reaction mixture was stirred for 30 min at 0 ◦C. After this time, the mixture was diluted with CH2Cl2 (10 mL), washed with ice-cold water (2 × 10 mL), dried over Na2SO4, ﬁltered, and evaporated. The obtained residue was dissolved in dry DMF (1 mL) and sodium azide (0.36 g) was added. The reaction was carried at RT under stirring for 15 h. At the end of the reaction time, CH2Cl2 (10 mL) was added and the 8 of 12 8 of 12 Molecules 2020, 25, 795 mixture was washed with water several times. After removal of the solvent we obtained a syrup-like product, which was puriﬁed by column chromatography (silica gel, eluent CH2Cl2/CH3OH 9:1). The puriﬁed 1,2:3,4-Di-O-isopropylidene-6-azido-l-galactopyranose was dissolved in CF3COOH/water (9:1, 5 mL), stirred for 15 min at RT and then neutralized with (CH3CH2)3N. After concentration, C6H5CH3 was added to the residue and re-evaporated. The residual syrup was acetylated with (CH3CO)2O (2 mL) in pyridine (2 mL) for 15 h at RT. The acetylated mixture was diluted with CH2Cl2 (10 mL) and washed with cold 1 N HCl, saturated NaHCO3, and ﬁnally with water. 3.2. Hippocampal Neuron Isolation, Characterization and In Vitro Culture Brains from postnatal day 1 (PND1) mice were used to obtain neurons as previously described [41]. Brieﬂy, hippocampi were dissected, under a conventional light microscope (SZX7, Olympus, Hamburg, Germany), into smaller fragments, trypsinized for 30 min at 37 ◦C and mechanically dissociated through a 2 mL pipette and a Pasteur pipette. After that, the hippocampal cells were washed 5 times with Hanks’ balanced salt solution (HBSS) supplemented with 0.5% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 10 mM HEPES solution and 1% sodium pyruvate (Invitrogen, Carlsbad, CA, USA) and re-suspended in minimum essential medium (MEM, Invitrogen) supplemented with 10% FBS, 0.5% glucose (Sigma-Aldrich), 0.5% penicillin-streptomycin, 2 mM l-glutamine and 1% MEM vitamins (Invitrogen). Hippocampal cells were plated on culture wells coated with poly-l-ornithine (Sigma-Aldrich), at a density of 50000 cells/cm2 and left at 37 ◦C in a humid atmosphere (5% CO2) for 5 h. After this, the medium was changed into Neurobasal A (Invitrogen) supplemented with 0.5 mM l-glutamine and 2% B27 (Invitrogen). The culture medium was changed 24 h after for Neurobasal A with 2% B27, 1% newborn calf serum (Invitrogen), 0.5 mM l-glutamine, 0.03 µM uridine (Sigma-Aldrich), 0.07 µM FDU (Sigma-Aldrich) and 1 µM kynurenic acid (Sigma-Aldrich), to prevent the proliferation of cells undergoing mitotic division and to reduce enhanced synaptic transmission. The hippocampal neurons were maintained in culture for at least 14 days, in a humid atmosphere (5% CO2) at 37 ◦C. After 1, 3 and 14 days in culture, cells were probed for β actin, βIII Tubulin (neuron marker) and GFAP (astrocyte marker) and nuclei were counterstained with DAPI. For that, cells were ﬁxed in 4% paraformaldehyde at RT for 20 min. Cells were incubated for 1 h at RT with the primary antibodies for GFAP (1:500, Dako, Golstrup, Denmark) and βIII-TUB (1:500, Millipore Iberica, Madrid, Spain) diluted in PBS. Cells were washed and incubated with speciﬁc Alexa 488-conjugated or Alexa 594-conjugated secondary antibodies (Invitrogen) diluted in PBS (1:500) for 1 h at RT, according to the source and isotype of the primary antibodies. Cells were washed with PBS and incubated with 4,6-diamidino-2-phenylindole (DAPI, 1:1000, Invitrogen) in PBS for 5 min at RT. Finally, cells were washed with PBS, and the glass coverslips were mounted in PermaFluor mounting medium (Thermo Fisher Scientiﬁc, Fremont, CA, USA). Fluorescence analysis and image capture were performed using a conventional (BX61; Olympus) or a confocal (FV1000; Olympus) microscope. 3.1. Synthesis and Characterization of Biorthogonal Reporters Solvent was evaporated and the 6-azido-1,2,3,4-tetra-O-acetyl-6-deoxy-l-galactopyranose was puriﬁed by HPLC chromatography. All ﬁnal products were characterized by 1H-NMR (Bruker, Karlsruhe, Germany) and HPLC (KNAUER, Berlin, Germany). 3.3. Bioorthogonal Labeling of Neuroprogenitor Cells with Peracetylated Azido Carbohydrates Peracetylated azido-functionalized mannose (Ac4ManNAz), glucose (Ac4GlcNAz) and fucose (Ac4FucNAz) were dissolved in absolute ethanol (1 mM) and a 50 µL solution was added in each well of the 24 well plate and allowed to evaporate. The azido-functionalized carbohydrates were then resuspended in culture medium (1 mL, 50 µM ﬁnal concentration) and added to the cell cultures at days 7 and 14. Unmodiﬁed mannose was used as a control. After 24 h, cells were washed with PBS supplemented with 2% FBS (Gibco) and incubated with dibenzylcyclooctyne-Fluor 488/561 (50 µM, DBCO-Fluor 488/DBCO-Fluor 561, Jenabioscience, Jena, Germany) for 1 h at 37 ◦C. Finally, cells were washed twice with PBS supplemented with 2% FBS and ﬁxated with 10% buﬀered formalin for 15 min. Molecules 2020, 25, 795 9 of 12 Nuclei were counterstained with DAPI for 15 min. Imaging of the cells with labelled glycans was performed by confocal microscopy (Olympus). Nuclei were counterstained with DAPI for 15 min. Imaging of the cells with labelled glycans was performed by confocal microscopy (Olympus). 3.4. Gene Expression of Carbohydrate Transporters by qRT-PCR Mouse hippocampal neuronal cultures (P1) were supplemented with azido modiﬁed carbohydrates at day 7 and 14. Cells were harvested 24 h after supplementation and mRNA was isolated for quantiﬁcation of gene expression of glucose transporters (Glut1 and Glut3), UDP-glucose glycoprotein glucosyltransferases (Uggt1 and Uggt 2), fucose transporter (solute carrier family 35, member c1, Slc35c1), protein O-fucosyltransferases (pofut1 and pofut2) and protein-O-mannosyltransferases (Pomt1 and Pomt2). Total RNA was extracted from cells using the RNeasy® Plus Micro Kit (Qiagen, Hamburg, Germany), following the manufacturers’ instructions. RNA quality and quantiﬁcation were assessed in the NanoDrop® ND-1000 (Thermoscientiﬁc, Massachusetts, USA) and 500 ng of RNA from each sample was reverse transcribed into cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturers’ instructions. Primers used to measure the expression levels of selected mRNA transcripts of Mus musculus by qRT-PCR were designed using the Primer3 software, on the basis of the respective GenBank sequences (Table S1). The reference gene hypoxanthine guanine phosphoribosyl transferase (Hprt) was used as internal standard for the normalization of the selected transcripts’ expression. qRT-PCR was performed on a CFX 96TM real-time system instrument (Bio-Rad), with the QuantiTect SYBR Green RT-PCR reagent kit (Qiagen) according to the manufacturer’s instructions, using equal amounts of cDNA from each sample. The cycling parameters were 1 cycle at 95 ◦C for 15 min, followed by 40 cycles at 94 ◦C for 15 s, annealing temperature (primer speciﬁc) for 30 s and 72 ◦C for 30 s, ﬁnishing with 1 cycle at 65 ◦C to 95 ◦C for 5 s (melting curve). Product ﬂuorescence was detected at the end of the elongation cycle. All melting curves exhibited a single sharp peak at the expected temperature. 3.5. Statistical Analysis Values are reported as the mean ± standard error. Each condition was tested at least in triplicates in each independent experiment and the experiments were repeated twice. Statistically signiﬁcant diﬀerences between groups were determined using one-way ANOVA, followed by Tukey’s multiple comparison test. Values were considered to be statistically signiﬁcant for p < 0.05 () and p < 0.01 (*). Supplementary Materials: The following are available online. Scheme S1: Synthesis of peracetylated azidomannose (Ac4ManNAz); Scheme S2: Alternative synthesis of GlcNAz using chloroacetic anhydride and NaOH as a base; Scheme S3: Synthesis of peracetylated azidofucose (Ac4FucAz); Figure S1: 1H-NMR spectra of ManNAz (D2O, 300 MHz); Figure S2: 1H-NMR spectra of Ac4ManNAz (CDCl3, 300 MHz), mixture of anomers; Figure S3: HPLC chromatogram of puriﬁed Ac4ManNAz showing the two anomers; Figure S4: 1H-NMR spectra of GlcNAz (D2O, 300MHz); Figure S5: 1H-NMR spectra of Ac4GlcNAz (CDCl3, 300 MHz), mixture of anomers; Figure S6: HPLC chromatogram of puriﬁed Ac4GlcNAz showing the two anomers; Figure S7: 1H-NMR spectra of 6-azido-1,2,3,4-tetra-O-acetyl-6-deoxy-α,β-L-galactopyranose Ac4FucAz (CDCl3, 300 MHz): mixture of anomers; Table S1: Primer sequences used in qRT-PCR. References 1. Lichtenstein, R.G.; Rabinovich, G.A. Glycobiology of cell death: When glycans and lectins govern cell fate. Cell Death Diﬀer. 2013, 20, 976–986. [CrossRef] 2. 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Imaging the glycome. PNAS 2009, 106, 12–17. [CrossRef] 14. Zheng, M.M.; Zheng, L.; Zhang, P.Y.; Li, J.B.; Zhang, Y. Development of Bioorthogonal Reactions and Their Applications in Bioconjugation. Molecules 2015, 20, 3190–3205. [CrossRef] 15. Saxon, E.; Bertozzi, C.R. Cell Surface Engineering by a Modiﬁed Staudinger Reaction. Science 2000, 287, 2007–2010. [CrossRef] 16. Zhang, X.; Zhang, Y. Applications of Azide-Based Bioorthogonal Click Chemistry in Glycobiology. Molecules 2013, 18, 7145–7159. [CrossRef] 17. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. 4. Conclusions All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n◦668983—FoReCaST, by European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572–The Discoveries CTR, and the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions The heterogeneity of glycans and their multiple regulatory roles, together with their importance in brain development, neuroregeneration and synaptic plasticity strongly suggests that glycans are invaluable tools to characterize neurons functions. Therefore, the development of methods to analyze the dynamics of glycan activity in neurons would be advantageous to decode their neural functions. We have proposed a neurocompatible strategy to image temporal distribution of glycoproteins during neuronal development using Cu-free click chemistry. This methodology unlocks new opportunities to study the dynamics of glycan activity in nervous system allowing decoding their eﬀect over neuron functions. Their applicability in hippocampal tissue sections will allow in vivo tracking of glycan changes and understanding the spatial distribution of glycans within nervous tissues. Supplementary Materials: The following are available online. Scheme S1: Synthesis of peracetylated azidomannose (Ac4ManNAz); Scheme S2: Alternative synthesis of GlcNAz using chloroacetic anhydride and NaOH as a base; Scheme S3: Synthesis of peracetylated azidofucose (Ac4FucAz); Figure S1: 1H-NMR spectra of ManNAz (D2O, 300 MHz); Figure S2: 1H-NMR spectra of Ac4ManNAz (CDCl3, 300 MHz), mixture of anomers; Figure S3: HPLC chromatogram of puriﬁed Ac4ManNAz showing the two anomers; Figure S4: 1H-NMR spectra of GlcNAz (D2O, 300MHz); Figure S5: 1H-NMR spectra of Ac4GlcNAz (CDCl3, 300 MHz), mixture of anomers; Figure S6: HPLC chromatogram of puriﬁed Ac4GlcNAz showing the two anomers; Figure S7: 1H-NMR spectra of 6-azido-1,2,3,4-tetra-O-acetyl-6-deoxy-α,β-L-galactopyranose Ac4FucAz (CDCl3, 300 MHz): mixture of anomers; Table S1: Primer sequences used in qRT-PCR. 10 of 12 10 of 12 Molecules 2020, 25, 795 Author Contributions: Conceptualization, I.P.; methodology, D.S.d.C., I.P. and J.C.S.; validation, I.P. and J.C.S.; formal analysis, F.M., N.I.P.-Y.; investigation, D.S.d.C., J.C.S., S.D.M., F.M. and N.I.P.-Y.; resources, J.C.S., R.L.R. and N.S.; data curation, D.S.d.C., J.C.S. and I.P.; writing—original draft preparation, D.S.d.C.; writing—review and editing, I.P., J.C.S. and F.M.; visualization, D.S.d.C., J.C.S. and I.P.; supervision, I.P., J.S.C., N.S. and R.L.R.; project administration, I.P. and R.L.R.; funding acquisition, R.L.R. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, I.P.; methodology, D.S.d.C., I.P. and J.C.S.; validation, I.P. and J.C.S.; formal analysis, F.M., N.I.P.-Y.; investigation, D.S.d.C., J.C.S., S.D.M., F.M. and N.I.P.-Y.; resources, J.C.S., R.L.R. and N.S.; data curation, D.S.d.C., J.C.S. and I.P.; writing—original draft preparation, D.S.d.C.; writing—review and editing, I.P., J.C.S. and F.M.; visualization, D.S.d.C., J.C.S. and I.P.; supervision, I.P., J.S.C., N.S. and R.L.R.; project administration, I.P. and R.L.R.; funding acquisition, R.L.R. References Chem. Int. Ed. 2002, 41, 2596–2599. [CrossRef] 18. Agard, N.J.; Prescher, J.A.; Bertozzi, C.R. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modiﬁcation of biomolecules in living systems. JACS 2004, 126, 15046–15047. [CrossRef] 19. 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https://openalex.org/W4206029261	https://e-journal.usd.ac.id/index.php/IJASST/article/download/3606/2514	English	null	Study of Nickel Extraction Process from Spent Catalysts with Hydrochloric Acid Solution: Effect of Temperature and Kinetics Study	International Journal of Applied Sciences and Smart Technologies	2,021	cc-by	3,773	Kevin Cleary Wanta1,, Ivanna Crecentia Narulita Simanungkalit1, Elsha Pamida Bahri1, Ratna Frida Susanti1, Gelar Panji Gemilar2, Widi Astuti3, Himawan Tri Bayu Murti Petrus4 1Department of Chemical Engineering, Parahyangan Catholic University, Jl. Ciumbuleuit 94, Bandung 40141, Indonesia 2PT Petrokimia Gresik, Jl. Jenderal Ahmad Yani, Gresik 61119, Indonesia 3Research Unit for Mineral Technology, Indonesian Institute of Sciences (LIPI), Jl. Ir. Sutami Km. 15, Tanjung Bintang 35361, Indonesia 4Department of Chemical Engineering, Universitas Gadjah Mada, Jl. Grafika 2, Kampus UGM, Yogyakarta, 55281, Indonesia Corresponding Author: kcwanta@unpar.ac.id International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 Study of Nickel Extraction Process from Spent Catalysts with Hydrochloric Acid Solution: Effect of Temperature and Kinetics Study Kevin Cleary Wanta1,, Ivanna Crecentia Narulita Simanungkalit1, Elsha Pamida Bahri1, Ratna Frida Susanti1, Gelar Panji Gemilar2, Widi Astuti3, Himawan Tri Bayu Murti Petrus4 1Department of Chemical Engineering, Parahyangan Catholic University, Jl. Ciumbuleuit 94, Bandung 40141, Indonesia 2PT Petrokimia Gresik, Jl. Jenderal Ahmad Yani, Gresik 61119, Indonesia 3Research Unit for Mineral Technology, Indonesian Institute of Sciences (LIPI), Jl. Ir. Sutami Km. 15, Tanjung Bintang 35361, Indonesia 4Department of Chemical Engineering, Universitas Gadjah Mada, Jl. Grafika 2, Kampus UGM, Yogyakarta, 55281, Indonesia Corresponding Author: kcwanta@unpar.ac.id (Received 20-08-2021; Revised 19-09-2021; Accepted 20-09-2021) Abstract As one of the hazardous and toxic solid wastes, spent catalysts need to be International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 p-ISSN 2655-8564, e-ISSN 2685-9432 p-ISSN 2655-8564, e-ISSN 2685-9432 can be achieved at a temperature of 85 oC. Kinetic studies were carried out using two kinetic models. The results of both models evaluation on the research data show that the lump model gives better results than the shrinking core model. The average error percentage of the lump model is smaller than the shrinking core model. It indicates that the extraction process was controlled by the diffusion step through the ash layer in the solid and chemical reactions simultaneously. Keywords: Extraction, lump model, nickel, shrinking core model Abstract As one of the hazardous and toxic solid wastes, spent catalysts need to be treated before the waste is discharged into the environment. One of the substances that need to be removed from the spent catalysts is the heavy metal ions and/or compounds contained therein. The method that can be applied is the extraction method using an acid solvent. In this study, the extraction process was carried out on spent catalysts samples from PT. Petrokimia Gresik. The focus of the study is on nickel extraction by varying the temperature in the range of 30–85 oC. A 1 M hydrochloric acid (HCl) solution was used as a solvent while the extraction process was 120 minutes. The experimental results show that the maximum nickel recovery of 14.70% 161 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 1 Introduction Various chemical industries, such as the oil, fertilizer, and petrochemical industries require catalysts to increase the rate of chemical reactions. The catalysts used can be solid catalysts containing different metal contents, such as nickel (Ni), iron (Fe), cobalt (Co), vanadium (V), molybdenum (Mo), and various other heavy metals [1]. The use of catalysts in the long term will make the catalyst saturated and no longer adequate for use. Thus, these catalysts will be replaced and disposed of as a used catalyst or what is usually called spent catalysts. Spent catalysts can not be disposed of directly into the environment because these catalysts are classified as hazardous solid waste. Therefore, this waste needs to be treated first with the aim of taking hazardous compounds, such as heavy metals contained in it. One of the spent catalysts solid waste treatment that can be done is extracting ions or compounds contained in the catalysts. This method is usually referred to as the leaching method. This method is a commonly used method and has been done by several researchers before. This extraction process requires a solvent to react and dissolve the metal ions and/or compounds. The solvents usually used are acidic solvents, both strong acids and weak acids [1], [2], [3], [4]. In this study, the extraction process was carried out using a hydrochloric acid solution. Furthermore, this research also studies parameters that have a significant impact on the extraction process, such as temperature. Temperature is an important parameter in the extraction process because temperature affects the rate of molecular 162 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 diffusion and the rate of chemical reactions. Furthermore, by studying the effect of temperature in the extraction process, the kinetic study of this process can be investigated by utilizing the existing kinetic models, such as the shrinking core model and the lump model. By studying the kinetics of the extraction process, a proper extractor can be designed. A proper extractor design must follow the applicable mechanism of the extraction process. Thus, the results of this study are expected to provide the appropriate information for the extractor design process. 2 Research Methodology Materials. The main raw material of this research is the spent catalyst from PT. Petrokimia Gresik. These catalysts have a nickel content of 16.7% wt, where the largest nickel phase in these catalysts is nickel elements (metals) and nickel oxide compounds (NiO). In addition, another main material used is a 1 M hydrochloric acid (HCl) solution. This HCl solution acts as a solvent in this extraction process. Equipment. The main equipment used for the extraction process is a series of equipment consisting of a three–neck flask (as an extractor), stirrer and motor, condenser, water bath and thermostat (to maintain a constant operating temperature), and a thermometer. As a sample analysis instrument, the instrument used is Atomic Absorption Spectroscopy (AAS). Research procedure. 180 mL of 1 M hydrochloric acid solution was put into a three– neck flask. After the equipments were assembled, the solution was heated to the desired temperature. In this study, the operating temperature was varied at 30, 60, and 85o C. After the temperature was reached, 36 grams of the spent catalyst solids (<74 microns) were put into the extractor. This solids intake will be counted as t = 0. The extraction process lasted for 120 minutes where during the operation time, the sampling was carried out periodically at 5, 10, 15, 30, 60, and 120 minutes. The samples that had been taken would be separated first between the solid and the liquid phase. This separation process was carried out using a centrifuge which was operated at 1,000 rpm for 10 163 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 minutes. The supernatant formed was then analyzed for the nickel content in t International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 minutes. The supernatant formed was then analyzed for the nickel content in the solution using Atomic Absorption Spectroscopy (AAS) instruments. International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 p g p-ISSN 2655-8564, e-ISSN 2685-9432 minutes. The supernatant formed was then analyzed for the nickel content in the solution using Atomic Absorption Spectroscopy (AAS) instruments. minutes. The supernatant formed was then analyzed for the nickel content in the solution using Atomic Absorption Spectroscopy (AAS) instruments. Data analysis. 2 Research Methodology The data obtained from the analysis using the AAS instrument is processed to obtain the result in the form of nickel recovery percentage where the equation used to calculate the value is: (1) (1) where x is the percentage of nickel recovery, CNi is the concentration of nickel extracted during the extraction process in ppm, CNi,tot is the total nickel concentration extracted from raw materials in ppm. where x is the percentage of nickel recovery, CNi is the concentration of nickel extracted during the extraction process in ppm, CNi,tot is the total nickel concentration extracted from raw materials in ppm. Furthermore, the nickel recovery data will be used to study the kinetics of the extraction process. There are two kinetic models applied in this study, namely the shrinking core model and the lump model. For the shrinking core model, the equations used are [5], [6], [7]: Diffusion in liquid film layer controlling : (2) Diffusion in ash layer controlling : – – – (3) Chemical reaction controlling : – – (4) Diffusion in liquid film layer controlling : (2) Diffusion in ash layer controlling : – – – (3) Chemical reaction controlling : – – (4) (2) (4) where x' is the nickel recovery fraction, kf, kd, and kr are the rate constants for the extraction process, and t is the operating time. For the lump model, the equations used are [8]: where x' is the nickel recovery fraction, kf, kd, and kr are the rate constants for the extraction process, and t is the operating time. For the lump model, the equations used are [8]: [ – { – ⁄ – } – ⁄ ] (5) (5) where is a constant related to the rate at the chemical reaction step while is a constant related to the rate at the diffusion step in the ash layer. where is a constant related to the rate at the chemical reaction step while is a constant related to the rate at the diffusion step in the ash layer. Determination of the suitable mathematical model is done by calculating the percentage error of research data and simulation data. 2 Research Methodology The equation used is as follows: \| – \| (6) (6) 164 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 where %E is the percentage of error, is the percentage of nickel recovery from experimental data, and is the percentage of nickel recovery from the simulation results of mathematical models. 3 Results and Discussion Simulation results of the shrinking core model when (a) diffusion in the liquid film layer controlling; (b) diffusion in the ash layer controlling; (c) chemical reactions controlling 166 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 Figure 2 shows the simulation results of the experimental data. The simulation results show that the shrinking core model in which the diffusion step in the ash layer controlling is the best model to illustrate the overall mechanism during the extraction process. This can be concluded because the R2 value obtained in this model is better than the other two models (diffusion step in the film layer and chemical reactions that control the process). A good R2 value is an R2 value that is close to 1. The evaluation results obtained indicate that the diffusion step through the ash layer in the solid is the step with the slowest rate. In solids, there are pathways used for each molecule (both reactant and product molecules) to diffuse. This pathway has a small size so that the reactant molecules that diffuse from the surface of the liquid to the unreacted surface in the solid will interfere with each other with the product molecules that diffuse from the surface that has reacted in the solid to the liquid body. It causes diffusion through the ash layer in the solid to be the step that controls the extraction process. Thus, the total rate of nickel extraction from the spent catalyst is determined by the rate of diffusion in the solid (the slowest rate). Another parameter commonly used to evaluate the kinetics of a process is the activation energy. Activation energy is the minimum energy required for a reaction to occur. The value of this parameter can be found using the Arrhenius equation as follows [9]: [9]: (– ) (7) – (8) (7) (8) where A is the collision frequency, Ea is the activation energy, R is the gas constant, and T is the absolute temperature. In this extraction process, the value of the collision frequency (A) obtained is 0.1384, while the activation energy value is 22.65 kJ/mol. 3 Results and Discussion Effect of temperature on nickel recovery. Temperature is a very important parameter and has a significant influence on determining the rate of the extraction process. In this study, the temperature used is in the range of 30–85 oC. The experimental results are presented in Figure 1. Figure 1. Effect of temperature on nickel recovery Figure 1. Effect of temperature on nickel recovery Figure 1 shows that the higher the temperature used, the more nickel can be obtained. In this study, the highest nickel recovery was obtained during the process at a temperature of 85 oC for 120 minutes, where the percentage of nickel recovery was 14.70%. In general, the nickel recovery that occurs during the process can reach 1.44 (for a temperature of 60 oC) and 2.25 times (for a temperature of 85 oC) compared to nickel recovery at a temperature of 30 oC. This phenomenon can occur because an 165 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 increase in temperature will enhance the kinetic energy of each molecule in the system. Consequently, each molecule will collide more often so that chemical reactions will also take place more quickly. In addition, an increase in temperature will also enhance the rate of diffusion, both molecular diffusion in the liquid film layer and diffusion in the ash layer in the solid. Kinetics study using the shrinking core model. The first kinetic model to be evaluated against the experimental data above is the shrinking core model. This model is the model most widely used by previous researchers in the hydrometallurgy or metal extraction process. The evaluation of this model is carried out using mathematical equations (2–4) and the evaluation results obtained are presented in Figure 2. Figure 2. Simulation results of the shrinking core model when (a) diffusion in the liquid film layer controlling; (b) diffusion in the ash layer controlling; (c) chemical reactions controlling Figure 2. 3 Results and Discussion According to Havlík, if the activation energy value is in the range of 20–35 kJ/mol, the extraction process is controlled by the diffusion and chemical reactions simultaneously [10]. Therefore, this kinetic study will be continued by using the lump model, which combines the two stages to prepare the mathematical model. 167 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 Kinetics study using the lump model. One of the weaknesses of the shrinking core model is that mathematical problem compiled in the model only assume one step that controls the extraction process; other steps are ignored because those steps are considered to have a very fast rate. In fact, in the solid–liquid extraction process, there are five steps involved in the system. For some cases, the use of assumptions as mentioned above does not represent the actual mechanism that occurs during the extraction process. As a result, the designed extractor will not be suitable for this process. Therefore, in this study, to complete the kinetic study, the lump kinetic model is evaluated against the experimental data where the equation used for the simulation process follows equation (5). The simulation results of the lump model are then compared with the evaluation results of the shrinking core model, where the diffusion step in the ash layer controls the extraction process. The comparison of the two kinetic models is presented in Table 1. Table 1. 4 Conclusion Based on the experimental dan simulation results, temperature significantly affects the nickel extraction process from spent catalysts with hydrochloric acid as solvent. At a temperature of 85 oC, the nickel recovery can reach 14.70% after the process lasts for 120 minutes. From the research data, the mechanism of the extraction process was studied and it was found that the rate of the extraction process was determined by the diffusion step through the ash layer in the solid and the chemical reaction step. Both of these stages occur simultaneously. The use of the lump model proves the conclusions obtained. Based on the average error percentage, the model gives a smaller error value than the shrinking core model. The average error percentage for the lump model is 10.26%. Acknowledgements The authors thank the Institute for Research and Community Service of Parahyangan Catholic University, which has supported this research financially. In addition, the authors also thank PT. Petrokimia Gresik, which has provided the main raw materials for this research. 3 Results and Discussion International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 models, namely 10.26% for the lump model and 18.82% for the shrinking core model. These results further corroborate the previous information that the spent catalyst extraction process using 1 M hydrochloric acid solution is controlled by the diffusion step through the ash layer and chemical reactions simultaneously. Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 models, namely 10.26% for the lump model and 18.82% for the shrinking core model. These results further corroborate the previous information that the spent catalyst extraction process using 1 M hydrochloric acid solution is controlled by the diffusion step through the ash layer and chemical reactions simultaneously. models, namely 10.26% for the lump model and 18.82% for the shrinking core model. These results further corroborate the previous information that the spent catalyst extraction process using 1 M hydrochloric acid solution is controlled by the diffusion step through the ash layer and chemical reactions simultaneously. 3 Results and Discussion Comparison of the simulation results of the shrinking core model (diffusion step in the ash layer) with the lump model Time, minutes Nickel Recovery – Experimental, % Nickel Recovery - SCM, % Nickel Recovery – Lump Model, % Error - SCM, % Error – Lump, % 30⁰C 60⁰C 85⁰C 30⁰C 60⁰C 85⁰C 30⁰C 60⁰C 85⁰C 30⁰C 60⁰C 85⁰C 30⁰C 60⁰C 85⁰C 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 1.92 3.02 3.66 0.74 1.17 2.35 1.41 1.89 3.04 61.35 61.28 35.67 26.56 37.42 16.94 10 2.41 3.16 4.32 1.29 1.96 3.49 1.99 2.67 4.28 46.36 37.90 19.23 17.43 15.51 0.93 15 3.13 3.68 5.46 1.75 2.60 4.51 2.43 3.27 5.23 44.07 29.27 17.44 22.36 11.14 4.21 30 3.61 4.13 6.88 2.84 4.09 6.83 3.43 4.60 7.34 21.32 0.91 0.78 4.99 11.38 6.69 60 4.39 5.65 9.73 4.44 6.24 10.10 4.83 6.46 10.27 1.08 10.36 3.80 10.02 14.34 5.55 120 6.51 9.34 14.70 6.73 9.27 14.67 6.78 9.05 14.30 3.33 0.76 0.23 4.15 3.10 2.72 The average error percentage per temperature: 25.36 20.07 11.02 12.22 13.27 5.29 The average error percentage per kinetics model: 18.82 10.26 *SCM: the shrinking core model with a diffusion step in the ash layer controls the process. able 1. Comparison of the simulation results of the shrinking core model (diffusion step in the ash layer) with the lump model Based on Table 1, the lump kinetics model provides better evaluation results than the shrinking core model. This can be concluded from the average error percentage for both 168 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 models, namely 10.26% for the lump model and 18.82% for the shrinking core model. These results further corroborate the previous information that the spent catalyst extraction process using 1 M hydrochloric acid solution is controlled by the diffusion step through the ash layer and chemical reactions simultaneously. International Journal of Applied Sciences and Smart Technologies Volume 3, Issue 2, pages 161–170 p-ISSN 2655-8564, e-ISSN 2685-9432 References [1] M. Marafi dan A. Stanislaus, “Waste Catalyst Utilization: Extraction of Valuable Metals from Spent Hydroprocessing Catalysts by Ultrasonic–Assisted Leaching with Acids.” Industrial & Engineering Chemistry Research, 50, 9495–9501, 2011. [1] M. Marafi dan A. Stanislaus, “Waste Catalyst Utilization: Extraction of Valuable Metals from Spent Hydroprocessing Catalysts by Ultrasonic–Assisted Leaching with Acids.” Industrial & Engineering Chemistry Research, 50, 9495–9501, 2011. [2] P.K. Parhi, K.H. Park, G. Senanayake, “A kinetic study on hydrochloric acid leaching of nickel from Ni–Al2O3 spent catalyst.” Journal of Industrial and Engineering Chemistry, 19, 589–594, 2013. 169 p-ISSN 2655-8564, e-ISSN 2685-9432 [3] J. Ramos–Cano, G. González–Zamarripa, F.E. Carrillo–Pedroza, M. de J.Soria– Aguilar, A. Hurtado–Marcías, A. Cano–Vielma, “Kinetics and statistical analysis of nickel leaching from spent catalyst in nitric acid solution.” International Journal of Mineral Processing, 148, 41–47, 2016. [4] W. Mulak, B. Miazga, A. Szymczycha, “Kinetics of nickel leaching from spent catalyst in sulphuric acid solution.” Int. J. Miner. Process, 77, 231–235 2005. [5] O. Levenspiel, Chemical Reaction Engineering. 3rd Edition, New York: John Wiley & Sons, Inc., 1999. [6] O.S. Ayanda, F.A. Adekola, A.A. Baba, O.S. Fatoki, B.J. Ximba, “Comparative Study of the Kinetics of Dissolution of Laterite in some Acidic Media.” Journal of Minerals & Materials Characterization & Engineering, 10 (15), 1457–1472, 2011. [7] K.C. Wanta, F.H. Tanujaya, R.F. Susanti, H.T.B.M. Petrus, I. Perdana, W. Astuti, “Studi Kinetika Proses Atmospheric Pressure Acid Leaching Bijih Laterit Limonit Menggunakan Larutan Asam Nitrat Konsentrasi Rendah.” Jurnal Rekayasa Proses 12 (2), 77–84, 2018. [8] K.C. Wanta., W. Astuti, I. Perdana, H.T.B.M. Petrus, “Kinetic Study in Atmospheric Pressure Organic Acid Leaching: Shrinking Core Model versus Lump Model.” Minerals, 10, 1–10, 2020. [9] H.S. Fogler, Element of Chemical Reaction Engineering. 4th Edition, New Jersey: Prentice Education, Inc., 2006. [10] T. Havlík, Hydrometallurgy. Cambridge: Woodhead Publishing Limited, 2006. 170
W889601139.txt	https://figshare.com/articles/journal_contribution/Increasing_the_productivity_of_the_wire_cut_electrical_discharge_machine_associated_with_sustainable_production/2074639/1/files/3706273.pdf	en		Increasing the productivity of the wire-cut electrical discharge machine associated with sustainable production	Journal of cleaner production	2,015	cc-by	6,023	Accepted Manuscript Increasing the productivity of the wire-cut electrical discharge machine associated with sustainable production Ibrahem Maher, Ahmed A.D. Sarhan, Mohsen Marani Barzani, M. Hamdi PII: S0959-6526(15)00776-3 DOI: 10.1016/j.jclepro.2015.06.047 Reference: JCLP 5702 To appear in: Journal of Cleaner Production Received Date: 16 October 2014 Revised Date: 10 June 2015 Accepted Date: 10 June 2015 Please cite this article as: Maher I, Sarhan AAD, Barzani MM, Hamdi M, Increasing the productivity of the wire-cut electrical discharge machine associated with sustainable production, Journal of Cleaner Production (2015), doi: 10.1016/j.jclepro.2015.06.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Increasing the productivity of the wire-cut electrical discharge machine associated with sustainable production a,b,c,d RI PT Ibrahem Maher a, Ahmed A. D. Sarhan b,, Mohsen Marani Barzani c, M. Hamdi d Centre of Advanced Manufacturing and Material Processing, Department of Mechanical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia a b Department of Mechanical Engineering, Faculty of Engineering, Kafrelsheikh University, Kafrelsheikh 33516, Egypt Department of Mechanical Engineering, Faculty of Engineering, Assiut University, Assiut 71516, Egypt SC Corresponding Author / E-mail: ah_sarhan@yahoo.com, TEL: +603-7967-4593, FAX: +603-7967-5330 Abstract: Wire-cut electric discharge machining is a nontraditional technique by which the required profile is acquired using M AN U sparks energy. Concerning Wire-cut electric discharge machining, high cutting rates and precision machining is necessary to improve productivity and achieve high quality of machined workpieces. In this research work, an experimental investigation was introduced to achieve higher productivity of the wire electrode associated with sustainable production in terms of product quality and less heat-affected zone. For this purpose, the effects of machining parameters including peak current, pulse on time and wire preloading were investigated using adaptive neuro-fuzzy inference system along with the Taguchi method. From this study, the optimal setting of machining parameters to achieve higher productivity and sustainability was identified. Moreover, Neuro-fuzzy modelling was TE D successfully used to build an empirical model for the selection of machining parameters to achieve higher productivity at highest possible surface quality and minimum cost for sustainable production. Keywords: Sustainable production, Energy consumption, Wire-cut EDM, ANFIS, Taguchi EP 1. Introduction Wire-cut Electric Discharge Machining (Wire-cut EDM) is an advanced machining process for machining AC C complicated shapes of hard conductive materials (Sommer and Sommer, 2013). Wire-cut EDM is usually used when low residual stresses are required, because it does not entail high cutting forces for material removal. Wire-cut EDM can machine any electrically conductive materials regardless of the hardness, from common materials such as copper, aluminum, tool steel, and graphite, to unusual modern alloys including wafer silicon, Inconel, titanium, carbide, polycrystalline diamond compacts. Besides, Wire-cut EDM is also used to machine modern composite materials such as conductive ceramics (Ho et al., 2004; Maher et al., 2015c). In Wire-cut EDM, the workpiece is machined with a series of electrical sparks that are produced between the workpiece and the wire electrode. The wire electrode discharges high frequency pulses of alternating or direct current to the workpiece through a very small spark gap with a nonconductive dielectric fluid. Many sparks can be easily seen instantaneously at the cutting zone because sparks happen more than one hundred thousand times per second (El-Hofy, 2005). Cutting rate and surface 1 ACCEPTED MANUSCRIPT quality are usually used to optimize the material removal rate (productivity) during the period of spark discharge. The productivity in Wire-cut EDM associated with sustainable production depend on a large number of machining parameters, as shown in Fig. 1, like electrical spark (discharge current, pulse width, etc.), wire electrode (shape, tension, material and speed), workpiece (high and material), and nonelectrical (dielectric fluid and flow rate) parameters. Productivity and quality are directly related. Practically, the productivity increases with increasing the RI PT energy consumption (voltage, current, and pulse on time) and decreases with decreasing the energy consumption. On the other hand, surface finish would decrease with increasing the discharge voltage, current, and pulse width (Yeh et al., 2013; Yu et al., 2011). In this research work, the authors would like to achieve higher productivity of the wire electrode in Wire-cut EDM associated with sustainable production in terms of work piece good surface quality and less heat-affected SC zone. Several efforts have been made to find the ideal machining conditions to enhance the productivity and improve the sustainability achieving high product quality by increasing the cutting rate and decreasing the surface roughness (Barzani et al., 2015; Kant and Sangwan, 2014). Nourbakhsh et al. (Nourbakhsh et al., 2013) studied the M AN U effect of injection pressure, servo reference voltage, time between two pulses and pulse width on surface integrity, wire rupture and cutting speed. It was revealed that cutting speed increases as pulse width increases; surface roughness decreases as the time between the two pulses decreases; and wire rupture is sensitive to injection pressure, wire mechanical tension, time between two pulses and pulse width. Besides, (Gökler and Ozanözgü, 2000) studied the effects of cutting parameters on the surface roughness of 1040, 2379 and 2738 steel and practical Wire cut-EDM results have been acquired. El-Hofy (El-Hofy, 2005) revealed that during Wire-cut EDM, the discharge temperature can reach up to 12,000°C, and metallurgical changes happen in the workpiece surface layer. Moreover, a thin heat TE D affected zone layer of 1 µm at 5 µJ energy to 25 µm at high energy is formed. (Levy and Maggi, 1990) introduce that the heat-affected zone together with the solidified layer reaches 25 µm. The zone under the machined surface can be annealed. In addition, some of molten material is not ejected into the dielectric fluid and chills quickly, mainly by heat conduction into most of the workpiece, resulting in a hard surface. The annealed layer depth is proportional to EP the energy used in the cutting process. It is around 50 µm for finish cutting to approximately 200 µm for high cutting rates, as shown in Fig. 2 (El-Hofy, 2005; McGeough, 1988). The recast layer appears at different spark erosion conditions and it contains many pockmarks, globules, cracks, and micro cracks. There are three types of recast AC C layers. Type 1 is a featureless, single layer less than 10µm thick. Type 2 is an etchable, single layer 10 to 20µm thick. Type 3 is a multilayer, 20µm thick or greater, and consists of overlapping solidified layers (Tomlinson and Adkin, 1992). Researchers have carried out several investigations and have noted that this layer is obvious under all machining conditions, including when water is used as dielectric material (Jangra et al., 2011; Ramasawmy et al., 2005). To acquire low surface roughness and small heat-affected zone, low discharge energy parameters (low energy consumption) with high dielectric flushing rate are required. However, such parameters decrease the material removal rate. This implies that a high cutting rate with minimum surface defects is difficult to attain from a single parameter setting. To achieve the productivity of the wire electrode and good quality associated with sustainable production and low energy consumption; mathematical modeling between Wire-cut EDM parameters and 2 ACCEPTED MANUSCRIPT performance measures should be obtainable to manufacturers. Theoretical and empirical approaches are commonly used for Wire-cut EDM modeling (Patil and Brahmankar, 2010). Owing to the simplified and mandatory assumptions, the theoretical models yield big errors between the predicted and experimental results. Nevertheless, empirical models are limited to specific experimental conditions. The Taguchi method and response surface methodology (RSM) are the most often employed statistical techniques for determining the relationship between RI PT different controllable parameters and output performance (Davoodi and Tazehkandi, 2014; Hewidy et al., 2005). Moreover, Fuzzy logic and feed forward neural network has been used to model the process and correlate the parameters with the performance measures (Marani Barzani et al., 2015; Ooi et al., 2015). ANFIS has also been applied to model the process of predicting machining performance (Maher et al., 2015a; Maher et al., 2014). Although the optimization of process parameters have been considered in these techniques, there are limited studies SC about energy consumption of the wire-cut EDM, which are important for factor economics. Because Wire-cut EDM involves multi-performance characteristics, the main objective of this study is to find a combination of Wire-cut EDM parameters to achieve rapid cutting speed as well as low surface roughness and small M AN U heat-affected zone to meet the demands for increasing the wire electrode productivity and product quality associated with sustainable production and low energy consumption. For this reason, ANFIS modelling is introduced as it is one of the soft computing techniques that play an important role in input-output parameter relationship modeling. Using ANFIS model, we can predict the required level of performance to increase the productivity at highest possible level of product quality for sustainable production. TE D 2. Experimental work In this research work, the experiments are carried out in wire-cut electric discharge machine to improve the productivity associated with better surface quality and minimum energy consumption for sustainable production. characterization. AC C 2.1 Experimental setup EP This section describe the experimental setup and the machined samples preparation for morphological The experiments were performed using a computer numerical control (CNC) Sodick A500W Wire-cut EDM machine tool. Hard brass wire with 0.2 mm diameter, tensile strength of 1000 N/mm2, elongation of 1.5% and electrical conductivity of 22 % IACS was used for the machining blocks of AISI 1050 carbon steel under specific machining conditions. The cutting speed was recorded directly from the Wire-cut EDM machine tool monitor. The surface roughness was measured with a stylus-based profilometer (Mitutoyo SJ-201, 99.6% accuracy). Scanning electron microscope (SEM) was used to examine the surface characteristics of the machined part and to measure the heat-affected zone thickness. The average cutting speed was calculated from the three, recorded data points under the same conditions. The average surface roughness was calculated for three different measurements under the same conditions with a 3 ACCEPTED MANUSCRIPT sampling length of Lc=2.5 mm at a specific workpiece area. The average heat-affected zone thickness was calculated from three measurements using image analysis software. 2.2 Machined samples preparation for morphological characterization RI PT The raw material with 100x60x25 mm dimensions was machined into 5x5x25 mm for each specimen. The chemical compositions of AISI 1050 carbon steel was achieved by EDX machine as shown in Table 1. The electrical resistivity and thermal conductivity of AISI 1050 carbon steel were 1.63x10-5 Ω·cm and 49.8 W/(m·K) respectively. Microscopic surface examinations after each grinding and finishing step were carried out using Olumpus BX 61 light optical microscopy (OM). A scanning electron microscope (SEM) equipped with Energy-dispersive X-ray SC spectroscopy (EDS) (Hitachi tabletop microscope TM3030) was used to examine the surface microstructural and topographical characteristics and heat affected zone of the machined part. M AN U 3. Taguchi approach Taguchi’s parameter design is a powerful tool for determining near optimum design parameters for performance, quality, and cost. Orthogonal arrays, one of the major tools used in Taguchi design which study a large number of design variables with a small numbers of experiments. The second important tool used in Taguchi design is signal to noise (S/N) ratio, which used as a measurable values instead of standard deviations and mean. Taguchi introduces a two stage optimization technique, which produces a parameter level combination with minimum standard deviation TE D while maintaining the mean on target (Su, 2013). Taguchi’s approach is completely based on design of experiments (DOE), which optimize the process design and economically solving the problem. DOE is effective in studying the effects of many variables on performance as well as studying the influence of individual parameters to determine which variable has more influence on the performance measure (Roy, 2001). EP The Taguchi full factorial design (L18) was selected with two machining variables with three levels and one machining variable with two levels, as shown in Table 2. The machining parameters, including peak current (IP), pulse width (Ton) and wire tension (WT) were selected for this study to investigate the effect on machining AC C performance, i.e., cutting speed (CS), surface roughness (Ra) and heat-affected zone (HAZ). The machining parameters and levels were selected based on previous experiments using one-factor-at-a-time approach. The other machining parameters were kept constant (Table 3) as recommended by the machine maker. These parameter levels were selected within the limit range of a machine working with no wire breakage based on the initial investigation. The goal of the experiment is to optimize the Wire-cut EDM variables to obtain high cutting speed values using the larger-is-better characteristic criteria (Equation 1), and low surface roughness and heat-affected zone values using the smaller-is-better characteristic criteria (Equation 2). S/N = -10 log(1/n(∑(1/y2))) 2 S/N = -10 log(1/n(∑y )) (1) (2) 4 ACCEPTED MANUSCRIPT where n is the number of experiments, and y is the observed data. Table 4 introduce the real data for cutting speed, surface roughness, and heat-affected zone. Table 5 shows signal to noise ratios of cutting speed, surface roughness, and heat affected zone. Tables 6 to 8 present the mean S/N ratios for each level of cutting speed, surface roughness, and heat-affected zone, respectively. These data were plotted as shown in Figs. 3 to 5, respectively. Figures 3 to 5 indicate that peak current and pulse width are more significant to RI PT the mean S/N ratios for cutting speed, surface roughness, and heat-affected zone. Moreover, Figs. 3 to 5 depict that wire tension does not affect cutting speed or the heat-affected zone. In addition, wire tension is significant on the average S/N response for surface roughness. SC 4. ANFIS modeling ANFIS is a neuro-fuzzy approach, in which a fuzzy inference system employed in the framework of an adaptive neural network. ANFIS can be used to build an input-output mapping based on fuzzy if-then rules as well as preset M AN U input-output data pairs for neural network. The membership function parameters was computed by the ANFIS modelling to track the known experimental input-output data (Jang et al., 1997; Zalnezhad et al., 2013). ANFIS uses five network layers to perform the following fuzzy inference steps as shown in Fig. 6: (1) input parameters, (2) fuzzy set database layer, (2) fuzzy rule base construction, (4) decision-making, and (5) output defuzzification. Layer 1: the output of this layer is the degree to which the given input satisfies the linguistic label associated to this node. Gaussian membership functions are usually used to denote the linguistic terms because the relationship TE D between the cutting parameters and machining performance in Wire-cut EDM is not linear, as shown in Fig. 7(a). First parameter membership functions Ai(IP) = exp[-0.5((IP-ai1)/bi1 )2] (3) Second parameter membership functions 2 Bi(Ton) = exp[-0.5((Ton-ai2)/bi2 ) ] (4) Third parameter membership functions Ci(WT) = exp[-0.5((WT-ai3)/bi3 )2] (5) EP where ai1, ai2, ai3, bi1, bi2, bi3 are the parameter set and i = 1-2 is for IP and i=1-3 is for Ton and WT. When the values of the parameter set change, the membership functions shape differ consequently (Fig. 7(b)), thus displaying several forms of membership functions on linguistic labels Ai, Bi, and Ci. The parameters in this layer are AC C referred to as principal parameters. Layer 2: each neuron in this layer computes the firing strength of the associated rule. The output of each node is as follows: αj = Ai(IP) × Bi(Ton) × Ci(WT) (6) Where j=1-n, n is the number of rules Layer 3: each neuron in this layer compute the normalization of the firing levels. The output of each neuron is as follows: βj=αj/(α1+α2+…+ αn) (7) Layer 4: each neuron in this layer compute the product of the normalized firing levels with the outputs of individual associated rule. 5 ACCEPTED MANUSCRIPT βjFj = βj (ai1 + bi2 + ci3) (8) Layer 5: the neuron in this layer computes the total system output as the sum of all incoming values. CS, Ra,or HAZ = ∑βjFj (9) If a crisp training set ((IPk, Tonk, WTk)k = 1, . . . ,K) is given, then the parameters of the neural net can be learned by Ek = (zk − ok)2 (10) k k RI PT descent type methods. The error function for pattern k is given by: where z is the wanted output and o is the calculated output by the neural network. ANFIS was constructed in MATLAB, with 18 readings comprising the training data set listed in Table 4. Different membership functions were used to train ANFIS. Two membership functions of peak current and three membership functions of the other two parameters (pulse width and wire tension) were chosen to create the ANFIS SC model. The Gaussian membership function (gaussmf) gives the lowest training error of all performance measures, so it was adopted for the ANFIS training process in this study. The fuzzy rule architecture of ANFIS when gaussmf is adopted consists of 18 fuzzy rules generated from the input-output data set based on the Sugeno fuzzy model. M AN U During training, the 18 performance measure values (training data set) were used to conduct 50 cycles of learning with an average error of 8.37×10-7, 2.6×10-6, and 1.5×10-5 for cutting speed, surface roughness, and heat-affected zone, respectively. Figures 8 to 10 introduce the effects of the cutting parameters on the output performance based on the ANFIS models. Figures 8 to 10 also show that the cutting speed, surface roughness, and heat-affected zone increase with increasing peak current and pulse width. Moreover, surface roughness decreases with increasing wire 5. Discussion TE D tension. Nevertheless, wire tension does affect the cutting speed and heat-affected zone. Figure 3 suggests that peak current and pulse width are more significant for cutting speed. Wire tension is not significant on the average S/N ratio for cutting speed. The highest peak current and pulse width appear to be the best particular. EP choice to obtain a high cutting speed value, thus making the process robust to peak current and pulse width in The same conclusions were drawn from Figs. 8(a) and (b) where pulse width and peak current had considerable AC C effect on cutting speed, while an increase in both pulse width and/or peak current led to an increase in cutting speed, but wire tension had a minor effect on cutting speed, same end obtained by (Maher et al., 2015b). Moreover, cutting speed (productivity) has major effect on energy consumption where the increase of cutting speed increases the energy consumption as shown in Fig 8(b). Increasing pulse width and peak current values is recommended for higher productivity. The ANFIS model shows that maximum cutting speed is achieved at the highest levels of peak current and pulse width (high level of energy consumption and process cost). That is because the combination of pulse width and peak current determines the spark energy as shown in equation (11) and hence the quantity of heat required to remove a definite volume of material. By increasing the pulse width and peak current, a large crater has to be cut per spark, as shown in Figs. 11(a) and (b) thus, the energy consumption is high. Consequently, this would increase the heat energy, leading to increased cutting speed as shown in equation (12) (El-Hofy, 2005). 6 ACCEPTED MANUSCRIPT Es = IP × V × Ton (11) CS α Es/(Ton+Toff) (12) Wher V is the spark gab set voltage Figure 4 indicates that pulse width and wire tension are more significant, followed by peak current on average S/N ratio for surface roughness. The lowest values of pulse width and peak current and the highest value of wire RI PT tension are the best choices to attain a low surface roughness value. In addition, Figs. 9(a) and (b) show that surface roughness increases as peak current, pulse width, and spark energy increase, but there are minor changes as wire tension increases. The SEM micrographs of the machined surface at 2000x magnification with the lowest and highest levels of peak current and pulse width are shown in Figs. 12(a) and (b) respectively. The SEM micrographs show that the cavity and redeposited particle with the highest SC levels of peak current and pulse width is higher than the lowest levels of peak current and pulse width. This is because the discharge energy increases with peak current and pulse width. Hence, larger craters are produced that lead to greater workpiece surface roughness (Kumar and Agarwal, 2012). This can be proved by the theory shown in M AN U Figs. 11(a) and (b) and equation (13) (El-Hofy, 2005). hm α (Es)1/3 (13) A study of Fig. 5 suggests that pulse width and peak current are more significant, and wire tension is insignificant on the average S/N response of the heat-affected zone. The smallest values of peak current and pulse width appear to be the best choices to obtain a less thick heat-affected zone. Based on the ANFIS model (Figs. 10(a), (b)), pulse width, and peak current has a great effect on the heataffected zone but wire tension has a minor effect. The heat-affected zone width increases with increasing pulse TE D width and/or peak current as shown in Figs. 10(a) and (b). Figures 13(a) and (b) introduce SEM micrograph of the heat-affected zone. This micrograph verifies that the maximum width of the heat-affected zone is obtained at the highest levels of peak current and pulse width and the smallest heat-affected zone width is at the lowest values of pulse width and peak current. This is because the heat energy increases with peak current and pulse width. Hence, al., 2008). AC C 6. Conclusions EP more heat is produced on the machined surface that leads to a larger heat-affected zone on the workpiece (Saha et In the present case study, two data analysis techniques were used to achieve high productivity and best surface quality for sustainable production and low process cost in terms of product quality and less heat affected zone, from which similar conclusions were drawn. The peak current and pulse width (spark energy parameters) are the most significant parameters affecting cutting speed, surface roughness, and heat-affected zone. Wire tension has minor effect on cutting speed and heat-affected zone but it has pronounced effect on surface roughness. ANFIS was successfully used to develop an empirical model for modeling the relation between the predictor variables (Ton, IP, and WT) and the performance parameters (CS, Ra, and HAZ) to reduce the energy consumption and cost. The ANFIS models results are also compared with the Taguchi model results. As anticipated, the ANFIS models provide 7 ACCEPTED MANUSCRIPT efficient prediction because they generally offer the ability to model more complex nonlinearities and interactions. In addition, by analyzing the results in Wire-cut EDM using the conceptual ANFIS approach, the following can be concluded: 1. ANFIS was used to introduce technological knowledge base for the selection of machining parameters to achieve high productivity at highest possible surface quality for sustainable production and lowering process cost. definite test range. RI PT 2. High pulse width (0.25 µs) and high peak current (17 A) are recommended to obtain high productivity for the 3. Low pulse width (0.15 µs), low peak current (16 A), and high wire tension (400 g) lead to smaller surface roughness values and heat affected zone for the specific test range. SC Acknowledgements This research was funded by the high impact research (HIR) grant number: HIR-MOHE-16001-00-D000027 from the Ministry of Higher Education, Malaysia, and the University of Malaya Postgraduate Research Grant (PPP) M AN U Program No. PG020-2013B. References AC C EP TE D Barzani, M.M., Sarhan, A.A.D., Farahany, S., Ramesh, S., Maher, I., 2015. Investigating the Machinability of Al– Si–Cu cast alloy containing bismuth and antimony using coated carbide insert. Measurement 62, 170-178, http://dx.doi.org/10.1016/j.measurement.2014.10.030. Davoodi, B., Tazehkandi, A.H., 2014. Experimental investigation and optimization of cutting parameters in dry and wet machining of aluminum alloy 5083 in order to remove cutting fluid. Journal of Cleaner Production 68, 234-242, http://dx.doi.org/10.1016/j.jclepro.2013.12.056. El-Hofy, H., 2005. Advanced machining processes. McGraw-Hill Professional, http://dx.doi.org/10.1036/0071453342. Gökler, M.İ., Ozanözgü, A.M., 2000. Experimental investigation of effects of cutting parameters on surface roughness in the WEDM process. International Journal of Machine Tools and Manufacture 40, 1831-1848, http://dx.doi.org/10.1016/S0890-6955(00)00035-3. Hewidy, M., El-Taweel, T., El-Safty, M., 2005. Modelling the machining parameters of wire electrical discharge machining of Inconel 601 using RSM. Journal of Materials Processing Technology 169, 328-336, Ho, K.H., Newman, S.T., Rahimifard, S., Allen, R.D., 2004. State of the art in wire electrical discharge machining (WEDM). International Journal of Machine Tools and Manufacture 44, 1247-1259, http://dx.doi.org/10.1016/j.ijmachtools.2004.04.017. Jang, J.-S.R., Sun, C.-T., Mizutani, E., 1997. Neuro-fuzzy and soft computing : a computational approach to learning and machine intelligence. Prentice Hall, Inc, U.S.A., Jangra, K., Grover, S., Aggarwal, A., 2011. Simultaneous optimization of material removal rate and surface roughness for WEDM of WC-Co composite using grey relational analysis along with Taguchi method. International Journal of Industrial Engineering Computations 2, 479-490, http://dx.doi.org/10.5267/j.ijiec.2011.04.005. Kant, G., Sangwan, K.S., 2014. Prediction and optimization of machining parameters for minimizing power consumption and surface roughness in machining. Journal of Cleaner Production, http://dx.doi.org/10.1016/j.jclepro.2014.07.073. Kumar, K., Agarwal, S., 2012. Multi-objective parametric optimization on machining with wire electric discharge machining. Int J Adv Manuf Technol 62, 617-633, http://dx.doi.org/10.1007/s00170-011-3833-1. Levy, G.N., Maggi, F., 1990. WED Machinability Comparison of Different Steel Grades. CIRP Annals Manufacturing Technology 39, 183-185, http://dx.doi.org/10.1016/S0007-8506(07)61031-2. Maher, I., Eltaib, M.E.H., Sarhan, A.A.D., El-Zahry, R.M., 2015a. Cutting force-based adaptive neuro-fuzzy approach for accurate surface roughness prediction in end milling operation for intelligent machining. Int J Adv Manuf Technol 76, 1459-1467, http://dx.doi.org/10.1007/s00170-014-6379-1. 8 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Maher, I., Eltaib, M.E.H., Sarhan, A.D., El-Zahry, R.M., 2014. Investigation of the effect of machining parameters on the surface quality of machined brass (60/40) in CNC end milling-ANFIS modeling. Int J Adv Manuf Technol 74, 531-537, http://dx.doi.org/10.1007/s00170-014-6016-z. Maher, I., Ling, L.H., Sarhan, A.A.D., Hamdi, M., 2015b. Improve wire EDM performance at different machining parameters – ANFIS modeling, 8th Vienna International Conference on Mathematical Modelling (MATHMOOD 2015). IFAC, Vienna University of technology, Vienna, Austria, pp. 105-110, http://dx.doi.org/10.3182/201502183-AU-30250//978-3-902823-71-70082. Maher, I., Sarhan, A.A.D., Hamdi, M., 2015c. Review of improvements in wire electrode properties for longer working time and utilization in wire EDM machining. Int J Adv Manuf Technol 76, 329-351, http://dx.doi.org/10.1007/s00170-014-6243-3. Marani Barzani, M., Zalnezhad, E., Sarhan, A.A.D., Farahany, S., Ramesh, S., 2015. Fuzzy logic based model for predicting surface roughness of machined Al–Si–Cu–Fe die casting alloy using different additives-turning. Measurement 61, 150-161, http://dx.doi.org/10.1016/j.measurement.2014.10.003. McGeough, J.A., 1988. Electrodischarge machining, Advanced methods of machining. Springer, Nourbakhsh, F., Rajurkar, K.P., Malshe, A.P., Cao, J., 2013. Wire electro-discharge machining of Titanium alloy. Procedia CIRP 5, 13-18, http://dx.doi.org/10.1016/j.procir.2013.01.003. Ooi, M.E., Sayuti, M., Sarhan, A.A.D., 2015. Fuzzy logic-based approach to investigate the novel uses of nano suspended lubrication in precise machining of aerospace AL tempered grade 6061. Journal of Cleaner Production 89, 286-295, http://dx.doi.org/10.1016/j.jclepro.2014.11.006. Patil, N., Brahmankar, P.K., 2010. Determination of material removal rate in wire electro-discharge machining of metal matrix composites using dimensional analysis. Int J Adv Manuf Technol 51, 599-610, http://dx.doi.org/10.1007/s00170-010-2633-3. Ramasawmy, H., Blunt, L., Rajurkar, K.P., 2005. Investigation of the relationship between the white layer thickness and 3D surface texture parameters in the die sinking EDM process. Precision Engineering 29, 479-490, http://dx.doi.org/10.1016/j.precisioneng.2005.02.001. Roy, R.K., 2001. Design of experments using Taguchi approach. John Wiley & Sons, Inc., Saha, P., Singha, A., Pal, S., Saha, P., 2008. Soft computing models based prediction of cutting speed and surface roughness in wire electro-discharge machining of tungsten carbide cobalt composite. Int J Adv Manuf Technol 39, 74-84, http://dx.doi.org/10.1007/s00170-007-1200-z. Sommer, C., Sommer, S., 2013. Complete EDM Handbook. Reliable EDM, Texas, USA, Su, C.-T., 2013. Quality Engineering: Off-Line Methods and Applications. CRC Press, Tomlinson, W.J., Adkin, J.R., 1992. Microstructure and properties of electrodischarge machined surfaces. Surface Engineering 8, 283-288, http://dx.doi.org/10.1179/sur.1992.8.4.283. Yeh, C.-C., Wu, K.-L., Lee, J.-W., Yan, B.-H., 2013. Study on surface characteristics using phosphorous dielectric on wire electrical discharge machining of polycrystalline silicon. Int J Adv Manuf Technol 69, 71-80, http://dx.doi.org/10.1007/s00170-013-4995-9. Yu, P.-H., Lin, Y.-X., Lee, H.-K., Mai, C.-C., Yan, B.-H., 2011. Improvement of wire electrical discharge machining efficiency in machining polycrystalline silicon with auxiliary-pulse voltage supply. Int J Adv Manuf Technol 57, 991-1001, http://dx.doi.org/10.1007/s00170-011-3350-2. Zalnezhad, E., Sarhan, A.D., Hamdi, M., 2013. Investigating the effects of hard anodizing parameters on surface hardness of hard anodized aerospace AL7075-T6 alloy using fuzzy logic approach for fretting fatigue application. Int J Adv Manuf Technol 68, 453-464, http://dx.doi.org/10.1007/s00170-013-4743-1. 9 ACCEPTED MANUSCRIPT Table 1. Chemical compositions of AISI 1050 carbon steel Element C Mn P S Fe Weight, 0.47-0.55 0.60-0.90 ≤0.04 ≤0.05 98.46-98.92 Wt (%) SC Table 3. Constant machining parameters Machining parameter Value Pulse off time 2 µs 21 Volt Flush Pressure 14 kgf/cm² Water Resistivity Meter 6 x 10⁴ Ω.cm Wire speed 3 m/min Cut Length 21.6 mm Main Power Supply Voltage 265 V M AN U Spark gab set voltage EP TE D Table 4 Experimental design using L18 orthogonal array and results Input Es CS (mm/min) No. IP Ton WT (µj) (A) (µs) (g) 1 2 3 Avg 1 1 300 50.4 0.64 0.59 0.55 0.59 2.50 0.15 350 50.4 2 0.60 0.57 0.58 0.58 2.41 3 400 50.4 0.64 0.62 0.62 0.63 2.40 4 300 67.2 0.67 0.69 0.64 0.67 2.59 5 16 0.20 350 67.2 0.72 0.69 0.66 0.69 2.50 6 400 67.2 0.71 0.66 0.70 0.69 2.42 7 300 84 0.85 0.85 0.81 0.84 2.88 8 0.25 350 84 0.83 0.87 0.79 0.83 2.77 9 400 84 0.85 0.87 0.84 0.85 2.72 10 300 53.55 0.83 0.85 0.79 0.82 2.49 11 0.15 350 53.55 0.81 0.76 0.79 0.79 2.45 12 400 53.55 0.77 0.80 0.81 0.79 2.47 13 300 71.4 0.95 0.91 0.92 0.93 2.64 14 17 0.20 350 71.7 0.98 0.96 0.93 0.96 2.56 15 400 71.4 1.01 0.95 0.97 0.98 2.59 16 300 89.25 1.11 1.07 1.12 1.10 2.94 17 0.25 350 89.25 1.11 1.15 1.09 1.12 2.88 18 400 89.25 1.14 1.14 1.12 1.13 2.73 AC C RI PT Table 2. Levels of machining parameters Levels Machining Symbol Units parameter Level 1 Level 2 Level 3 Pulse on time Ton µs 0.15 0.20 0.25 Wire tension WT g 300 350 400 Peak current IP A 16 17 - Output Ra (µm) 2 3 2.44 2.45 2.37 2.43 2.38 2.30 2.61 2.58 2.56 2.47 2.48 2.40 2.79 2.87 2.80 2.79 2.72 2.72 2.52 2.54 2.55 2.44 2.43 2.45 2.61 2.72 2.62 2.60 2.49 2.53 2.91 2.86 2.79 2.87 2.79 2.75 Avg 2.46 2.40 2.36 2.59 2.51 2.43 2.85 2.79 2.72 2.52 2.48 2.45 2.66 2.59 2.54 2.90 2.85 2.76 1 10.39 9.42 9.78 17.08 17.22 17.32 19.44 19.44 19.23 14.68 12.43 12.68 18.91 19.44 19.11 22.75 22.04 21.01 HAZ (µm) 2 3 10.18 10.12 9.53 9.12 10.01 9.87 16.98 16.11 16.22 16.92 17.63 17.71 19.44 19.44 18.77 19.44 19.44 19.65 12.02 13.35 12.79 12.76 12.05 13.35 18.11 19.18 18.77 19.26 18.11 20.08 20.32 19.44 24.04 18.82 21.43 22.84 Avg 10.23 9.36 9.89 16.72 16.79 17.55 19.44 19.22 19.44 13.35 12.66 12.69 18.73 19.16 19.10 20.84 21.63 21.76 15 ACCEPTED MANUSCRIPT Table 5 S/N ratios of machining performance measures S/N ratios No. CS Ra HAZ -20.20 -19.43 -19.90 -24.46 -24.50 -24.89 -25.77 -25.68 -25.77 -22.51 -22.05 -22.07 -25.45 -25.65 -25.62 -26.38 -26.70 -26.75 RI PT -7.82 -7.60 -7.46 -8.27 -7.99 -7.71 -9.10 -8.91 -8.69 -8.03 -7.89 -7.78 -8.50 -8.27 -8.10 -9.25 -9.10 -8.82 SC -4.61 -4.70 -4.01 -3.53 -3.19 -3.19 -1.49 -1.67 -1.42 -1.78 -2.05 -2.07 -0.59 -0.33 -0.16 0.84 0.98 1.08 M AN U 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 EP TE D Table 6 Response Table for S/N Ratios of cutting speed (Larger is better) Level IP Ton WT 1 -3.0905 -3.2035 -1.8605 2 -0.4519 -1.8299 -1.8247 3 -0.2803 -1.6284 Delta 2.6386 2.9232 0.2321 Rank 2 1 3 AC C Table 7 Response table for S/N ratios of surface roughness (Smaller is better) Level IP Ton WT 1 -8.173 -7.764 -8.493 2 -8.414 -8.139 -8.294 3 -8.977 -8.093 Delta 0.241 1.214 0.399 Rank 3 1 2 Table 8 Response table for S/N ratios of heat affected zone (Smaller is better) Level IP Ton WT 1 -23.40 -21.03 -24.13 2 -24.80 -25.10 -24.00 3 -26.18 -24.17 Delta 1.40 5.15 0.17 Rank 2 1 3 16 ACCEPTED MANUSCRIPT WORKPIECE PARAMETERS ELECTRICAL PARAMETERS Servo feed (SF) Pulse on time (Ton) Gap voltage (VP) Material Pulse off time (Toff) Spark gap set voltage (SV) Dielectric flow rate RI PT Height Peak current (IP) Wire-cut EDM Performance Measures Cutting rate (CR)-Surface roughness (SR) Gap current (IG)-Wire rupture (WR) Dimensional deviation (V) Heat affected zone (HAZ) Wire size Wire tension (WT) Wire speed (WS) SC Dielectric conductivity Wire material NON ELECTRICAL PARAMETERS M AN U WIRE ELECTRODE PARAMETERS EP TE D Fig. 1 W-EDM process parameters AC C Fig. 2 EDM heat affected zone (El-Hofy, 2005) 10 SC RI PT ACCEPTED MANUSCRIPT TE D M AN U Fig. 3 Effect of process parameters on cutting speed AC C EP Fig. 4 Effect of process parameters on surface roughness Fig. 5 Effect of process parameters on heat affected zone 11 ACCEPTED MANUSCRIPT Layer 1 IP A1 Layer 2 A1(IP) ∏ A2 Layer 3 α1 Layer 4 IP Ton WT Layer 5 β1 N β1F1 B1 B2 ∑ B3 C1 WT C2 C3 ∏ βnFn βn αn N C3(WT) IP Ton WT Adaptive node Fixed node CS, Ra, or HAZ RI PT Ton Membership grades M AN U Membership grades SC Fig. 6 ANFIS architecture for a three-input Sugeno fuzzy model Pulse on time (µs) Pulse on time (µs) AC C EP TE D (a) (b) Fig. 7 Initial and final membership function of pulse on time (a) Initial membership function (b) Final membership function (a) (b) Fig. 8 The modeled cutting speed by ANFIS in relation to parameters change (a) Cutting speed in relation to change of Pulse on time and peak current (b) Cutting speed in relation to change of wire tension and discharge energy 12 RI PT ACCEPTED MANUSCRIPT TE D M AN U SC (a) (b) Fig. 9 The modeled Surface roughness by ANFIS in relation to parameters change (a) Surface roughness in relation to change of Pulse on time and peak current (b) Surface roughness in relation to change of wire tension and discharge energy AC C EP (a) (b) Fig. 10 The modeled Heat affected zone by ANFIS in relation to parameters change (a) Heat affected zone in relation to change of Pulse on time and peak current ( b) Heat affected zone in relation to change of wire tension and discharge energy (a) (b) Fig. 11 Effect of spark energy on removal rate and surface roughness (a) Effect of pulse current on removal rate and surface roughness (b) Effect of pulse on time on removal rate and surface roughness 13 ACCEPTED MANUSCRIPT High cavity RI PT Low cavity High redeposited particles SC Low redeposited particles TE D M AN U (a) (b) Fig. 12 SEM micrograph at different levels of spark energy (a) SEM micrograph at the lowest levels of peak current (IP=16A) and pulse on time (Ton=0.15µs) (b) SEM micrograph at the highest levels of peak current (IP=17A) and pulse on time (Ton=0.25µs) 21.76 µm AC C EP 9.36 µm (a) (b) Fig. 13 Light optical microscope micrograph at different levels of spark energy (a) Light optical microscope micrograph for the lowest levels of peak current (IP=16A) and pulse on time (Ton=0.15µs) (b) Light optical microscope micrograph for the highest levels of peak current (IP=17A) and pulse on time (Ton=0.25µs) 14 ACCEPTED MANUSCRIPT Highlights An experimental investigation is introduced to achieve higher productivity in WEDM • The effects of the peak current, pulse width and wire preloading are investigated • Adaptive neuro-fuzzy inference system (ANFIS) and Taguchi analysis methods are used • Sustainable production in terms of product quality and heat zone is investigated AC C EP TE D M AN U SC RI PT •
https://openalex.org/W3207838497	https://hal-insu.archives-ouvertes.fr/insu-03713736/file/Martinovi%C4%87_2021_ApJ_923_116.pdf	English	null	Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends	Astrophysical journal/The Astrophysical journal	2,021	cc-by	8,332	To cite this version: Mihailo M. Martinović, Kristopher G. Klein, Tereza Ďurovcová, Benjamin L. Alterman. Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends. The Astrophysical Journal, 2021, 923, 10.3847/1538-4357/ac3081. insu-03713736 Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends Mihailo M. Martinović, Kristopher G. Klein, Tereza Ďurovcová, Benjamin L. Alterman To cite this version: Mihailo M. Martinović, Kristopher G. Klein, Tereza Ďurovcová, Benjamin L. Alterman. Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends. The Astrophysical Journal, 2021, 923, 10.3847/1538-4357/ac3081. insu-03713736 Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends Mihailo M. Martinović, Kristopher G. Klein, Tereza Ďurovcová, Benjamin L. Alterman Distributed under a Creative Commons Attribution 4.0 International License 1. Introduction unstable were very sensitive to the model used to describe the VDF, and that inclusion of multiple ion populations could both enhance or diminish the predicted growth rates. Instabilities, driven by departures from local thermodynamic equilibrium (LTE), are frequently credited with affecting the behavior of rapidly evolving plasma systems, e.g., the expanding solar wind (Matthaeus et al. 2012). To quantify these departures, the underlying charged particle velocity distribution functions (VDFs) are typically modeled as bi-Maxwellians, having anisotropic temperatures T⊥,j and T∥,j with respect to the local magnetic ﬁeld B; relative ﬁeld-aligned drifts between each pair of constituent VDF components i and j being Δvi,j= (Vi −Vj) · B/\|B\|; and temperature disequilibrium between species Ti ≠Tj (Marsch et al. 1982). These anisotropies, drifts, and disequilibrium serve as distinct sources of free energy capable of driving the growth of a number of distinct unstable solutions (see, e.g., Section 5 of Verscharen et al. 2019). The presence of multiple free-energy sources makes it difﬁcult to determine which subset of sources drives a given instability; parametric models accounting for a single source of free energy—e.g., the temperature anisotropy of a single population (Gary et al. 1997; Yoon 2017)—do not account for the diminishment or enhance- ment of predicted linear growth rates associated with the introduction of other departures from LTE, e.g., relative drifts of proton beams (Daughton & Gary 1998; Woodham et al. 2019; Liu et al. 2021), helium (Podesta & Gary 2011; Bourouaine et al. 2013; Verscharen et al. 2013), or their combined effects (Chen et al. 2016). To account for these effects, previous studies implemented Nyquist’s instability criterion (Nyquist 1932; Klein et al. 2017) on limited sets of in situ measurements from the Wind (Klein et al. 2018), Parker Solar Probe (Verniero et al. 2020; Klein et al. 2021), and Helios (Klein et al. 2019) missions, ﬁnding that a majority of intervals were unstable, that the kinds of waves driven In this work, we apply the Nyquist criterion to a recent reprocessing of the VDF measurements that provides ﬁts for a proton core, proton beam, and helium (α) component for nearly the entirety of both Helios I and II missions (Ďurovcová et al. 2019). Details of the method are given in Section 2. Due to Helios I1a and I1b instruments (Schwenn et al. 1975) limitations in resolution, range, and ﬁeld of view (Rosenbauer et al. 1981; Marsch et al. Abstract Instabilities described by linear theory characterize an important form of wave–particle interaction in the solar wind. We diagnose unstable behavior of solar wind plasma between 0.3 and 1 au via the Nyquist criterion, applying it to ﬁts of ∼1.5M proton and α particle Velocity Distribution Functions (VDFs) observed by Helios I and II. The variation of the fraction of unstable intervals with radial distance from the Sun is linear, signaling a gradual decline in the activity of unstable modes. When calculated as functions of the solar wind velocity and Coulomb number, we obtain more extreme, exponential trends in the regions where collisions appear to have a notable inﬂuence on the VDF. Instability growth rates demonstrate similar behavior, and signiﬁcantly decrease with Coulomb number. We ﬁnd that for a nonnegligible fraction of observations, the proton beam or secondary component might not be detected, due to instrument resolution limitations, and demonstrate that the impact of this issue does not affect the main conclusions of this work. Uniﬁed Astronomy Thesaurus concepts: Solar wind (1534); Space plasmas (1544); Solar corona (1483); Heliosphere (711) HAL Id: insu-03713736 https://insu.hal.science/insu-03713736v1 Submitted on 5 Jul 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License https://doi.org/10.3847/1538-4357/ac3081 The Astrophysical Journal, 923:116 (9pp), 2021 December 10 © 2021. The Author(s). Published by the American Astronomical Society. 57/ac3081 Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends Mihailo M. Martinović1,2 , Kristopher G. Klein1 , Tereza Ďurovcová3 , and Benjamin L. Alterman4 1 University of Arizona, Tucson, AZ, USA; mmartinovic@arizona.edu 2 LESIA, Observatoire de Paris, Meudon, France 3 Charles University, Faculty of Mathematics and Physics, V Holešovičkách 2, 180 00 Prague 8, Czech Republic 4 Space Science and Engineering, Southwest Research Institute, San Antonio, TX, USA Received 2021 August 10; revised 2021 October 13; accepted 2021 October 14; published 2021 December 15 Ion-driven Instabilities in the Inner Heliosphere. I. Statistical Trends Mihailo M. Martinović1,2 , Kristopher G. Klein1 , Tereza Ďurovcová3 , and Benjamin L. Alterman4 1 University of Arizona, Tucson, AZ, USA; mmartinovic@arizona.edu 2 LESIA, Observatoire de Paris, Meudon, France 3 Charles University, Faculty of Mathematics and Physics, V Holešovičkách 2, 180 00 Prague 8, Czech Republic 4 Space Science and Engineering, Southwest Research Institute, San Antonio, TX, USA Received 2021 August 10; revised 2021 October 13; accepted 2021 October 14; published 2021 December 15 5 The I1a instrument could take 144 records over its entire angular domain (nine elevation and 16 azimuth channels) and its energy-to-charge range was covered by 32 channels. However, this exceeded the telemetry limits and data reduction had to be applied. In the normal data mode, the channel with the maximum count rate was found. In the next measurement cycle, only a limited number of channels around this maximum are recorded (nine energy, ﬁve azimuth, and ﬁve elevation channels). These nine energy channels are designed to also cover the helium core part of the VDF, making the resolution sparse. This complicates the ability to identify the proton beam. The α beam, which occurs at higher energy-to-charge ratios and has very low abundance, is almost never detected. 6 2.2. Instability Analysis The Nyquist instability criterion (Nyquist 1932), using the hot plasma dispersion relation for an arbitrary number of relatively drifting bi-Maxwellian components as calculated by the Plasma in a Linear Uniform Magnetized Environment (PLUME) numerical dispersion solver (Klein & Howes 2015), has been described in detail in previous publications (Klein et al. 2017). This method determines the stability of a linear system of equations through a conformal mapping of the contour integral of the dispersion relation ( )   k , , w over the upper-half of the complex frequency plane, with the real (ωr) and imaginary (γ) components of the frequency ω = ωr + iγ representing the oscillation and growth or damping rates respectively. This integral counts the number of normal mode solutions that are unstable, having γ > 0, for a speciﬁc wavevector k and set of dimensionless parameters . Iterating this process for multiple contours with increasing values of γ enables the determination of the maximum growth rate and associated characteristics of the fastest growing mode supported by a particular k. We have set γ = 10−4Ωp as the minimum growth rate for a wavevector to be considered unstable, where proton gyrofrequency is given as Ωp = ecB/mp, ec being the elementary charge. We choose this particular minimum value of γ, as for lower values the growth time of the unstable mode becomes long enough for other physical effects—such as propagation, reconnection, or turbulence—to start notably changing the VDF parameters (Livi & Marsch 1987; Kasper et al. 2017). We repeat this process over a log-spaced grid in wavevector space k⊥ρc ä [10−3, 3] and k∥ρc ä [10−2, 3], enabling the determi- nation of the fastest growing mode for ion-scale wavevectors given a particular observed set of parameters . This set is The number of intervals with identiﬁed proton core, proton beam, and α components are listed in Table 1. Proton beams and α populations were distinguished in 843,224 (59.7%) and 744,609 (52.7%) of the ﬁts. It is important to note that both populations could be completely or partially undetected in some of the ﬁts5, but due to different reasons. The beam population can be misinterpreted as part of the core if it has low density6 nb/nc = 1 and/or the relative drift velocity between the two populations is small Δvb,c/vAc = 1. 1. Introduction 1982), the beam population is not always detected. In general, mischaracterization of the beam as a part of the core can lead to signiﬁcant variations in prediction of modes excited by the VDF (Klein et al. 2021). For example, a stable VDF consisted of an almost isotropic core and mildly shifted isotropic beam could potentially be ﬁtted as a single population with T∥,c ? T⊥,c, highly susceptible to ﬁrehose (FH) instability. To ensure this issue does not have a misleading effect on our analysis, we detail the procedure of diagnosing insufﬁciently well-resolved observations in Section 3.1, and remove them from consideration of the results. We ﬁnd that the traditional way of organizing Helios observations over radial distance (Matteini et al. 2007; Hellinger et al. 2011, 2013) does not provide a complete description of the evolution of linear instabilities, neither in terms of their occurrence rate nor the growth rate. The obtained linear trend for both parameters turns out to be a result of a mixture of varying solar wind parameters at each distance. On the contrary, organization of the results with respect to the solar wind bulk velocity vsw and, even more signiﬁcantly, the Coulomb number NC, reveals a speciﬁc region in which either speed or collisionallity seem to be main candidates for the solar wind parameter that governs the behavior of instabilities. The Coulomb number is a dimensionless measure that characterizes the VDF relaxation due to Coulomb collisions. It is deﬁned as NC(cc) = νccr/vsw,c, where νcc is the Coulomb self-collision Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. 1 The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. core density and mass, respectively. This type of under- detection is central technical issue of our data analysis and is discussed in detail in Section 3.1. On the other hand, the helium population is shifted in the instrument frame due to doubled mass-to-charge ratio compared with protons. This effect can cause the helium beams, and rarely even parts of theα bulk population to be out of the instrument ﬁeld of view, or to be impossible to resolve due to lower instrument resolution at high energies. Therefore, the ﬁts performed by Ďurovcová et al. 6 Following notation in Ďurovcová et al. 2019, the VDF components are labeled in such a way that, by deﬁnition, nb/nc 1, while the drift velocity of the beam can be negative. 2.1. Models for Ion Distributions from Helios I and II The previous application of the Nyquist criterion to Helios observations focused entirely on selected fast-wind streams with good ﬁts to the proton (Stansby et al. 2018) and helium (Stansby et al. 2019) components, approximately 45,000 intervals in total (Klein et al. 2019), while entirely neglecting proton beams. p In this work, we apply the same analysis method to 1,480,214 intervals, from the Helios I and II missions, which also include the reprocessed version of intervals used by Klein et al. (2019). From this database, 49,316 (3.3%) entries are excluded to remove observations of coronal mass ejections (Wang et al. 2005). Further on, we remove 3500 (0.2%) cases from further consideration, due to the dispersion solver (Section 2.2) encountering numerical precision issues, and also 14,620 (0.98%) cases when the predicted growth rates could not be accurately described with linear physics (see Section 2.2). Fits assume bi-Maxwellian VDFs for the proton core, proton beam, and α component, and are described in Ďurovcová et al. (2019). By not restricting our study to fast solar wind, we aim to remove selection bias, focusing mostly on faster, and therefore hotter and less collisionally processed solar wind, is naturally biased toward more unstable plasma, and has produced discrepancy in fractions of unstable intervals found by Klein et al. (2019) for Helios and Klein et al. (2018) for Wind at 1 au. In this paper, we aim to provide a more comprehensive picture of the role instabilities play in all kinds of solar wind as it expands in the inner heliosphere. 1. Introduction (2019), as well as in the ﬁts by Stansby et al. (2019) used for linear stability analysis by Klein et al. (2019), represent α particles with a single shifted bi-Maxwellian. A direct consequence of this approach is that majority of the measure- ments yield T⊥,α/T∥,α 1. Similar behavior is reported for protons if the core and beam populations were not separated (Marsch et al. 1981, 1982), where core and moment proton anisotropy are signiﬁcantly different when beams are present. While recent analysis of Parker Solar Probe (PSP) observations (Klein et al. 2021) demonstrated that treating protons as a single population could fail to correctly identify the beam- induced wave modes, the presence of which can be conﬁrmed by electromagnetic ﬁeld measurements (Vech et al. 2020), there is no such study dealing with α particles. New observations of the secondary helium population by PSP (McManus et al. 2020) have the potential to provide precise measurements necessary for detailed analysis of the effect of these structures on plasma linear stability. frequency among core protons (see Hernandez et al. 1987; Kasper et al. 2017 for details). We do note that these two quantities are connected due to νcc scaling with the proton temperature, which is in turn correlated with vsw (Elliott et al. 2012; Perrone et al. 2019; Maksimović et al. 2020). We discuss the validity of these statistics and their implications to the general description of the instability behavior in the solar wind in Sections 3.2 and 4. 3.1. Observational Issues of I1 Instrument where we deﬁne the thermal-to-magnetic pressure ratio β∥,j = 2μ0njkbT∥,j/B2, the speed of light c, the Boltzmann constant kb, the thermal velocity  w k T m 2 j b j j , , = ^ , and gyroradius ρj = mpw∥,j/ecB. We also consider six parameters for each component j: The trends on the panels (b) and (c) of Figure 1 are matching with intuitive expectations—collisionally young solar wind is strongly unstable, before both occurrence rate and intensity of instabilities start declining. However, unexpected behavior at moderate and large NC(cc) can be noticed for two regions in the phase space. First, in rare occasions when the beam is detected in the VDF at large NC(cc), the interval seems to commonly be very unstable, producing signiﬁcant increase in fraction of unstable intervals and maximum growth rate (red rectangle). Second, intervals at moderate NC(cc) where only core and α populations were detected (green rectangle), show a notable increase in the number of unstable VDFs and p max g W . ( ) ⎛ ⎝ ⎜ ⎞ ⎠ ⎟     n n T T T T v v m m q q , , , , , . 2 j j c j j j c j c j p j p , , , , , Ac = D ^ ( ) 2 For this study, we treat the electrons as a single isotropic distribution with density and drift velocity necessary to enforce quasi-neutrality and zero net current. The number of elements in thus varies depending on the number of characterized ion components. p The ﬁrst issue, related to beams at large NC(cc), is rooted in the difﬁculties of beam detection. Three properties characterize the signiﬁcance of proton beams; nb/nc, Δvb,c/vA,c,and T⊥,b/T∥,b. A comparison of these quantities at large NC(cc) and r demonstrated that a beam was resolved only if at least one of the three was large. Otherwise, the beam population was indistinguishable from the core. As a large value of any of the listed beam parameters can cause the VDF to be unstable, the fraction of intervals where the beams are the major emitting component is signiﬁcantly increased, as shown in panels (d1) and (d2). 3.1. Observational Issues of I1 Instrument As the beam-detection instrumental effects become the dominant driver for the fraction of unstable measurements with a detected proton beam at NC(cc) > 0.4, all observations from this region will be excluded from further consideration. p For a given , we calculated γ/Ωp using the Nyquist method as well as p max g W over the entire wavevector range and the associated r p max w W , k c max r , kB max q , and electromagnetic eigen- functions of the most unstable mode. The number of intervals with growth rates in excess of 10−4, 10−3, and 10 p 2 1 W - - are listed in Table 1, both for the entire database and segregated as a function of the identiﬁed ion components. Slightly less than half of the intervals in the total database support at least a weakly growing mode, with nearly a quarter supporting a relatively rapidly growing mode, with 10 p max 2 1 g > W - - . There is signiﬁcant variation between the intervals with and without proton beams, as well as if an α component has been identiﬁed. Nearly a third of intervals with an identiﬁed proton beam component have 10 p max 2 1 g > W - - , while less than 10 % of intervals without proton beams have such strongly growing modes. As mentioned in Section 2.1, for 0.3 max r max g w > (0.98% of all measurements) we do not consider the wave to be a small linear perturbation and exclude these intervals. g The second issue, related to potentially undetected beams playing a role in the instability analysis at NC(cc) ∼0.1 is more complicated. Namely, even though Δvb,c/vA,c decreases with both NC(cc) and r (Alterman et al. 2018), beam parameters could still have nonnegligible values in this region, and nondetection of beams could signiﬁcantly after the predicted plasma response. The manifestation of undetected beams inﬂuence is illustrated in panels (d2) and (d4) of Figure 1. If the proton beam is detected, then we observe a constant trend transferring from the core being the dominant free-energy source in the young wind toward it is irrelevance as a source of free energy as it isotropizes (Kasper et al. 2017). On the other hand, some of the cases where only core and α components are identiﬁed will have an unresolved beam embedded into the core. populations; and, more importantly, their effects on our linear stability analysis. ( ) ⎛ ⎝ ⎞ ⎠    w c , , 1 c c 0 , , b = ( ) 1 2.2. Instability Analysis Here, the core-proton Alfvén velocity deﬁned as v B n m c p Ac 0 m = , where μ0 is magnetic permeability of vacuum, and nc and mp are proton 2 Table 1 The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. Table 1 Number of Intervals with Characterized Proton Core, Proton Beam, and α Components and Their Percentage of the T Classiﬁed as Supporting an Unstable Mode with Growth Rates Greater than 10−4, 10−3, and 10 2 W - Number of Intervals with Characterized Proton Core, Proton Beam, and α Components and Their Percentage of the Total Data Set, as well as the Number of Intervals Classiﬁed as Supporting an Unstable Mode with Growth Rates Greater than 10−4, 10−3, and 10 p 2 1 W - - , with Cumulative Percentages Number of Intervals with Characterized Proton Core, Proton Beam, and α Components and Their Percentage of the Total Data Set, as well as the Number of Intervals Classiﬁed as Supporting an Unstable Mode with Growth Rates Greater than 10−4, 10−3, and 10 p 2 1 W - - , with Cumulative Percentages Total Core Core + Beam Core + α Core + Beam + α # 1,412,778 315,755 (22.3%) 352,414 (24.9%) 253,779 (18.0%) 490,830 (34.7%) 10 p max 4 g W > - 630,540 (44.6%) 60,513 (19.2%) 215,223 (61.1%) 74,452 (29.3%) 280,352 (57.1%) 10 p max 3 g W > - 510,916 (36.2%) 47,115 (14.9%) 182,875 (51.9%) 47,911 (18.9%) 233,015 (47.5%) 10 p max 2 g W > - 316,825 (22.4%) 26,655 (8.4%) 122,617 (34.8%) 16,350 (6.4%) 151,203 (30.8%) composed of two global reference parameters: composed of two global reference parameters: 3. Inferred Stability as a Function of Speed, Distance, and Coulomb Number The summary of our ﬁndings is given on Figure 1. The results are presented in term of the Coulomb number, which we pick as an initial referent parameter as it measures traits of both solar wind speed and radial distance, and only through their combined effects we were able to determine the parts of phase space that potentially have instrumental effects affecting VDF ﬁts. Therefore, before we address the behavior of instabilities with respect to plasma parameters, it is ﬁrst necessary to understand the consequences of instrument limitations regard- ing the characterization of the core; beam and helium The most important effect of this missed detection is the change, either increase or decrease, of the ﬁtted parallel temperature component of the core. Here, we deﬁne the effective beam parallel temperature in terms of the particle 3 Figure 1. Overview of unstable mode behavior and VDF components that emit the most power as a function of NC(cc). The means and medians of p max g W on pan b) are calculated considering only unstable cases, while 10% and 90% percentiles are given by stars and dots, respectively. Panels (d1)–(d4) show percentages of th omponent that emits the largest amount of power for unstable cases. Panel (e) illustrates the occurrence of speciﬁc intervals where one component emits power, east 50% of which is then absorbed by another population. On the contrary, panel (f) shows the cases where two, or rarely even all three ion components have powe mitting within 50% of each other. Red, green, and black shaded areas are used for beam-detection tests described in Section 3.1. The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et a The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. Figure 1. Overview of unstable mode behavior and VDF components that emit the most power as a function of NC(cc). The means and medians of p max g W on panel (b) are calculated considering only unstable cases, while 10% and 90% percentiles are given by stars and dots, respectively. Panels (d1)–(d4) show percentages of the component that emits the largest amount of power for unstable cases. Panel (e) illustrates the occurrence of speciﬁc intervals where one component emits power, at least 50% of which is then absorbed by another population. 3. Inferred Stability as a Function of Speed, Distance, and Coulomb Number For each interval, the two scenarios are compared—having the core and beam as observed (further on, referred to as “C+B case”), and having core and beam replaced with a single proton component with Teff∥(“effective case”). The contribution of the beam to the perpendicular pressure is not enhanced by the drift, and we approximate Teff⊥≈T⊥,c. As nb = nc, a correction to this value that would arise from a more sophisticated approach that takes into account the difference between T⊥,c and T⊥,b would be very small. Interactions of unstable modes with α particles can be very complicated (see Section 3.2 and also Klein et al. 2019), but even the cases that include identiﬁed helium (right panel) show an intuitive general trend. The effective case is generally more stable than the C + B case, as the sensitive beam structure and related velocity space gradients are is removed. This effect is clearly seen at the left panel, where C + B cases with strongly drifted beams have p max g W increased by one to three orders of magnitude compared with their effective counterparts. However, some of the intervals, usually the ones with low beam drifts, tend to be more unstable in the effective case. This unusual behavior can have two causes, as either (1) the beam drift is very low, so it does not trigger any unstable modes, but it contains enough phase space density to potentially “push” the core population over the marginal stability threshold in the effective case; or (2) the beam is signiﬁcantly drifted but has very low density, and is C(cc) y To further illustrate this point, we show the difference between C + B (core) and effective VDFs on β-anisotropy diagram shown on Figure 3. Only cases where p max g W is increased by at least 20% between C + B and effective case are shown for clarity, and are colored with respect to · n n v v b c b c, 2 Ac 2 D , which is used as a proxy for a contribution to the parallel pressure. This quantity is almost completely determined by v v b c, 2 Ac 2 D (not shown). 3. Inferred Stability as a Function of Speed, Distance, and Coulomb Number Stable intervals are shown with an imposed value of · 7 10 p max 5 g W = - . The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. Martinović et al. Figure 2. Fastest growth rates with effective core parallel temperatures compared with observed temperature with resolved core and beam, for cases where α population was detected (right), or was either undetected or removed from the analysis (left). Stable intervals are shown with an imposed value of · 7 10 p max 5 g W = - . evaluate what the effects on the linear stability would be if that beam was not detected, but rather treated as a part of the core VDF. We focus on the observations with 0.3 < NC(cc) < 0.4 (black shaded area on panel (d4)). In this region, there is a total of 77,148 ﬁtted VDFs, and a subset of 42,033 that contain beam components. Within the intervals of interest, 14,628 VDFs also contain α components. First, we calculate Teff∥for each VDF and reevaluate ( ) 0 eff and eff based on that value. Then, we reanalyze these intervals using the procedure described in Section 2.2. We perform the instability analysis for intervals with α components two times, both including a and with the helium artiﬁcially removed to isolate the undetected beam effects. not as strongly unstable as if it is contribution to the parallel pressure would be added to the core. For the beam-detection issues, only the ﬁrst scenario is of interest, as only beams of Δvb,c/vAc 0.5 have realistic possibility to remain undetected. Only 119 (0.28%) effective VDFs from this subset tend to either be unstable when its equivalent C + B distribution is not, or have higher p max g W if both are unstable, so there will not be a signiﬁcant number of cases where unphysical highly unstable modes are inferred. On the opposite end, we ﬁnd that possible nondetection of the beam could “pacify” unstable VDFs, as 13, 942 (33.15%) of VDFs are unstable in C + B case, but are stable in the effective case. However, only 1177 (2.8%) are cases with Δvb,c/vAc 0.5. Therefore, we proceed to keep the intermedi- ate NC(cc) intervals in our analysis. The results are shown on Figure 2. 3. Inferred Stability as a Function of Speed, Distance, and Coulomb Number On the contrary, panel (f) shows the cases where two, or rarely even all three ion components have power emitting within 50% of each other. Red, green, and black shaded areas are used for beam-detection tests described in Section 3.1. kinetic energy in the reference frame of the core cyclotron (IC) and mirror instability. Also, the core stops being a completely dominant emitting component in this region, but there is a notable fraction of unstable intervals that are driven by very hot α particles (Stansby et al. 2019). On the contrary, older wind features T⊥,c/T∥,c ∼1, and sufﬁcient increase in the parallel temperature can make the core susceptible to FH instability due to an increase in the total parallel pressure. This causes the core to maintain its contribution to the unstable behavior of plasma when beams are undetected, which is diminished when the beams are detected. The impact of the effective decrease in Teff∥are opposite of the ones described. ( ) ( )   T T m v k 2 3 b b p b c b eff , , , 2 = + D ( ) 3 and the joint effective parallel temperature of core and beam as ( )    T n T n T n n , 4 c c b b c b eff , eff , = + + ( ) 4 being the parallel temperature of the proton VDF when the beam is not detected. ∥ As failure to detect beams is more likely to happen for lower densities and drifts, we address the case of intermediate instead of low Coulomb numbers in more detail. It is worth noting that, as we have no knowledge of a potential beam when it is not resolved, we will use the intervals that have a beam, and then The impact of T∥,c approaching Teff∥depends on where in the phase space it occurs. In the young wind, core anisotropy is high, and an artiﬁcial increase in the parallel component pushes the T⊥,c/T∥,c ratio toward unity, making the modeled VDF less prone to instabilities triggered by anisotropy, such as ion- 4 Figure 2. Fastest growth rates with effective core parallel temperatures compared with observed temperature with resolved core and beam, for cases where α population was detected (right), or was either undetected or removed from the analysis (left). 3. Inferred Stability as a Function of Speed, Distance, and Coulomb Number Here, we again emphasize that a hypothetical undetected beam can work two ways: total parallel pressure can either increase or decrease, depending on the value of Teff∥,b (Equation (4)), which can be lower than T∥,c if the drift is low. For Teff∥,b < T∥,c, the core population is shifted to the left on the plot, with effective increase in anisotropy, and if the VDF is close to the IC instability threshold, the value of p max g W can be over- estimated. A similar scenario can happen if the VDF is close to FH threshold and the effective parallel pressure is increased due to Teff∥,b > T∥,c. From Figure 3 is visible that, in general, the shift in the parameter space is not large if the drift is not large. As strongly drifted beams have a signiﬁcantly lower chance to be undetected in this part of phase space, an eventual mistreatment of slightly drifted beams will cause only small changes in the estimated maximum growth rates, while intervals that are incorrectly estimated to be unstable due to 5 The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. Figure 3. Shift of the core population in the ( ) ∣∣ ∣∣ ∣∣  T T T T , , c c c c , , , ,eff , eff b b  ^ ^ space due to possible nondetection of beams, with dots and X marks representing C + B and effective case, respectively. The instability thresholds are calculated by Verscharen et al. (2016). Only intervals where the maximum growth rate is increased in the effective case by at least 20% are shown. i 3 hif f h l i i h b b d ibl d i f b i h d d k Figure 3. Shift of the core population in the ( ) ∣∣ ∣∣ ∣∣  T T T T , , c c c c , , , ,eff , eff b b  ^ ^ space due to possible nondetection of beams, with dots and X marks representing C + B and effective case, respectively. The instability thresholds are calculated by Verscharen et al. (2016). Only intervals where the maximum growth rate is increased in the effective case by at least 20% are shown. this mistreatment are only those that already are very close to marginal stability thresholds. 3. Inferred Stability as a Function of Speed, Distance, and Coulomb Number This adds another level of conﬁdence to our approach of treating the intermediate NC(cc) observations as sufﬁciently reliable. similarities, which are intuitively expected as NC(cc) roughly scales as vsw 1 - (panel (d1)) of Figure 1 is shown again for the sake of completeness). For both parameters, the instability growth and occurrence rates show an exponential rise or decay between maximum and minimum values, both of which seem to be separated by a marginally larger amount for the case of NC(cc). For both underlying variables, the difference between mean and median values of p max g W demonstrates similar behavior, with less than 20% discrepancy close to the maximum. Then, during the phase of exponential decay, the mean-to-median difference jumps to a factor of ∼5, which is quantiﬁed by the zeroth order terms in the ﬁtted exponential functions. The described behavior is matched with the decay of the core population as the main source of free energy, and beams and helium gradually taking over that role. At low speeds the core is not as dominant as at low Coulomb numbers, and is also not as insigniﬁcant at high speeds as at high NC(cc). It also follows the pattern shown on Figure 1, panel (e), where the power emitted by the core is mostly absorbed by α particles at the collisionally young wind. As the core becomes isotropic, beams are more likely to be the main driver of instability, and occasionally some of the emitted power is absorbed by the core. A similar graph is produced if the data is organized by vsw,c (not shown), but the trends are slightly less pronounced, similar as on the panels (b5) and (c5) of Figure 4. 3.2. Dependencies on Solar Wind Parameters We summarize the dependencies of the unstable behavior on r, vsw and NC(cc) in Figure 4. The intervals excluded based on the analysis described in Section 3.1 are shaded in black. We restrict our data analysis as a function of radial distance and solar wind speed by identifying the unexpected rise in the mean and median values of the maximum growth rate and, for the case of vsw,c, also a sudden increase in participation of proton beam as the emitting component. The linear trends in Figure 4, panels (a2)–(a4), are showing that, despite the variation of plasma parameters measured at each radial distance, we still have a steady decline in both instability occurrence and growth rates. The fraction of unstable intervals is steadily growing as we approach the Sun, with linear extrapolation aiming at approximately two thirds of all intervals being unstable in the corona. It is unclear from this data set how close to the Sun this trend is maintained. The differences between line slopes for mean and median values of p max g W are of the order of 20%, which is not a large value for a quantity with spread of three and a half orders of magnitude. and is also not as insigniﬁcant at high speeds as at high NC(cc). It also follows the pattern shown on Figure 1, panel (e), where the power emitted by the core is mostly absorbed by α particles at the collisionally young wind. As the core becomes isotropic, beams are more likely to be the main driver of instability, and occasionally some of the emitted power is absorbed by the core. A similar graph is produced if the data is organized by vsw,c (not shown), but the trends are slightly less pronounced, similar as on the panels (b5) and (c5) of Figure 4. Aside from the Coulomb number, which depends only on in situ characteristics of the plasma, a parameter frequently used in the literature is the collisional age Ac, the integral of all the Coulomb collisions experienced by a parcel of plasma as it propagates outward from the Sun’s surface (see, e.g., Maruca The inferred dependencies become signiﬁcantly more extreme if the results are organized with respect to vsw,c or NC(cc). From proﬁles of the emitting components (panels (5)) is clear that these two ways of organizing the data have many 6 igure 4. 3.2. Dependencies on Solar Wind Parameters Fitted linear and exponential dependencies of the fraction of unstable intervals (b), maximum growth rate mean (c), and median (d), from radial distance ( olar wind speed (2), and Coulomb number (3). Panels (1) and (5) show the number of sampled intervals and the fractions of the emitting VDF componen espectively. Black shaded regions are removed from consideration based on instrumental limitations explained in Section 3.1. he Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et al. Figure 4. Fitted linear and exponential dependencies of the fraction of unstable intervals (b), maximum growth rate mean (c), and median (d), from radial distance (1 solar wind speed (2), and Coulomb number (3). Panels (1) and (5) show the number of sampled intervals and the fractions of the emitting VDF component espectively. Black shaded regions are removed from consideration based on instrumental limitations explained in Section 3.1. The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Martinović et a Figure 4. Fitted linear and exponential dependencies of the fraction of unstable intervals (b), maximum growth rate mean (c), and median (d), from radial distance (1), solar wind speed (2), and Coulomb number (3). Panels (1) and (5) show the number of sampled intervals and the fractions of the emitting VDF components, respectively. Black shaded regions are removed from consideration based on instrumental limitations explained in Section 3.1. et al. 2013; Kasper et al. 2017). We reprocessed the results on Figures 1 and 4 in terms of Ac, as well as different variations of Coulomb number that estimate collisions of core population with beams NC(cb) and helium NC(cα). Results obtained are not visually distinguishable form the ones shown here, but subtle differences between these various collision “clocks” still could exist, and their effect on properties of unstable modes will be examined in detail in the follow-up article. robust statistics of the inferred behavior of linear instabilities between 0.3 and 1 au. Linear trends shown on left panels of Figure 4 are different from the results shown by Klein et al. (2019), who found a constant fraction of unstable intervals to be over 80% at all radial distances. As that work used only single core and helium populations overwhelmingly sampled in the fast solar wind with vsw,p > 600 km s−1, the reasons for this discrepancy can be seen in middle panels. 3.2. Dependencies on Solar Wind Parameters For very high speeds, both fraction of unstable modes and p max g W means and medians are reaching maximum values marginally lower than the ones presented by Klein et al. (2019), probably due to the selection bias of that work in using only clearly distinguished components, which naturally prefers intervals with more extreme parameters and References 2019, SoPh, 294, 97 Ďurovcová, T., Šafránková, J., & Němeček, Z, Z. 2019, SoPh, Elliott, H. A., Henney, C. J., McComas, D. J., Smith, C. W., & Vasquez, B. J. 2012, JGRA, 117, A09102 Gary, S. P. 1993, Theory of Space Plasma Microinstabilities (Cambridge: Cambridge Univ. Press) Cambridge Univ. Press) Gary, S. P., Wang, J., Winske, D., & Fuselier, S. A. 1997, JGR, 102, 27159 Goldstein, B. E., Neugebauer, M., & Smith, E. J. 1995, GeoRL, 22, 3389 Hellinger, P., Matteini, L., Štverák, Š., Trávnček, P. M., & Marsch, E. 2011, Gary, S. P., Wang, J., Winske, D., & Fuselier, S. A. 1997, JGR, 102, 27159 Goldstein, B. E., Neugebauer, M., & Smith, E. J. 1995, GeoRL, 22, 3389 Š Š Goldstein, B. E., Neugebauer, M., & Smith, E. J. 1995, GeoRL, 22, 3389 Hellinger, P., Matteini, L., Štverák, Š., Trávnček, P. M., & Marsch, E. 2011, , , g , , , , , , Hellinger, P., Matteini, L., Štverák, Š., Trávnček, P. M., & Marsch, E. 2011, JGR 116 A09105 Hellinger, P., Matteini, L., Štverák, Š., Trávnček, P. M., & Marsch, E. 2011, JGR, 116, A09105 JGR, 116, A09105 Hellinger, P., Trávníček, P. M., Štverák, Š., Matteini, L., & Velli, M. 2013, JGRA, 118, 1351 , , Hernandez, R., Livi, S., & Marsch, E. 1987, JGR, 92, 7723 Kasper, J. C., Klein, K. G., Weber, T., et al. 2017, ApJ, 849 Kasper, J. C., Maruca, B. A., Stevens, M. L., & Zaslavsky, A. 2013, PhRvL, 110, 091102 Klein, K. G., Alterman, B. L., Stevens, M. L., Vech, D., & Kasper, J. C. 2018, PhRvL, 120, 205102 Finally, although instrument limitations (Section 3.1) limit the conﬁdence in our data close to 1 au, we still can conclude that all the components are moving toward stability in approximately linear rate with radial distance. As there are no signiﬁcant energizing mechanisms that increase in effectiveness around 1 au, we hypothesize that the linear instabilities are least abundant in the solar wind at 3–5 au, as collisional processing moves the VDF away from the FH instability threshold (Bale et al. 2009; Matteini et al. 2013). Further away, energy sources that originate outside of the Solar system, such as pick up ions, could start playing a role in the energy balance (Goldstein et al. 1995). Klein, K. G., & Howes, G. G. 2015, PhPl, 22, 032903 Klein, K. G., Kasper, J. ORCID iDs Mihailo M. Martinović https://orcid.org/0000-0002- 7365-0472 Kristopher G. Klein https://orcid.org/0000-0001-6038-1923 Tereza Ďurovcová https://orcid.org/0000-0003-4247-4864 Benjamin L. Alterman https://orcid.org/0000-0001- 6673-3432 Kristopher G. Klein https://orcid.org/0000-0001-6038-1923 Tereza Ďurovcová https://orcid.org/0000-0003-4247-4864 4. Discussion and Conclusions The database from 15 yr of Helios observations of ion VDFs processed by the PLUME dispersion solver provides sufﬁciently 7 The Astrophysical Journal, 923:116 (9pp), 2021 December 10 The Astrophysical Journal, 923:116 (9pp), 2021 Martinović et al. also could occasionally misidentify beam contribution (Section 3.1). supported by the Grant Agency of the Charles University under the project number 264220 and by the Czech Science Foundation under contract 19-18993S. B.L.A. acknowledges NASA contract NNG10EK25C. An allocation of computer time from the UA Research Computing High Performance Computing at the University of Arizona is gratefully acknowledged. The exponential decay in mode activity signals that very slow, and also highly collisionally processed solar wind is still unstable for about a quarter of intervals, but the intensity of the wave ﬂuctuations is signiﬁcantly decreased. This could be the indicator of core population moving away from the IC and mirror instability thresholds and the plasma becoming more isotropic at larger radial distances (Matteini et al. 2007). Once the core is nearly isotropic and beam and helium drifts are sufﬁciently small, and also the value of β∥ increases for all populations due to expansion, the plasma moves toward the FH instability threshold. The marginal stability could then be violated by a variety processes such as VDF elongation due to magnetic moment conservation (Livi & Marsch 1987), long-wavelength compressive ﬂuctuations (Verscharen et al. 2016), or plasma heating due to large amplitude ﬂuctuations (Chandran et al. 2010) and wave resonances (Kasper et al. 2013; Chen et al. 2019). References As a separate topic, it is worth discussing the power transfer between VDF components, shown on Figure 1, panel (e). Intervals where the stable components absorbs signiﬁcant fraction of the emitted free energy are not frequent, not surpassing 6% at any NC(cc), but are a useful indicator of conditions at which each particular unstable mode resonates with any of the components. As mentioned above, the core is very anisotropic in the collisionally young solar wind and acts as the main source of free energy. The main unstable modes triggered are the Alfvén (IC) and entropy (mirror) mode. Neither of these modes has high resonant velocity (Gary 1993), and does not interact with strongly drifted beam particles (Daughton & Gary 1998; Daughton et al. 1999). Helium has notably smaller drifts in the young wind (Kasper et al. 2017; Ďurovcová et al. 2019), and therefore has more particles that can resonate with the growing modes (light blue shade). When the core anisotropy drops, the less populated components take over the role of the primary free-energy source. Also, beam drifts decrease gradually, getting close enough for the excited IC and fast magnetosonic modes to interact ﬁrst with α (pink) and then with the core particles (tomato red). Alterman, B. L., Kasper, J. C., Stevens, M. L., & Koval, A. 2018, ApJ, 864, 112 Bale, S. D., Kasper, J. C., Howes, G. G., et al. 2009, PhRvL, 103, 211101 Bourouaine, S., Verscharen, D., Chandran, B. D. G., Maruca, B. A., & Kasper, J. C. 2013, ApJL, 777, L3 p p Chandran, B. D. G., Li, B., Rogers, B. N., Quataert, E., & Germaschewski, K. 2010, ApJ, 720, 503 , p , , Chen, C. H. K., Klein, K. G., & Howes, G. G. 2019, NatCo, 10, 740 p Chen, C. H. K., Klein, K. G., & Howes, G. G. 2019, NatCo, 10, 740 Chen, C. H. K., Matteini, L., Schekochihin, A. A., et al. 2016, ApJL, 825, L2 Chen, C. H. K., Matteini, L., Schekochihin, A. A., et al. 2016, ApJL, 825, L26 Daughton, W., & Gary, S. P. 1998, JGR, 103, 20613 Daughton, W., & Gary, S. P. 1998, JGR, 103, 20613 Daughton, W., Gary, S. P., & Winske, D. 1999, JGR, 104, 4657 Ď Š g , , y, , , , , , Ďurovcová, T., Šafránková, J., & Němeček, Z, Z. References C., Korreck, K. E., & Stevens, M. L. 2017, JGRA, 122, 9815 Klein, K. G., Martinović, M., Stansby, D., & Horbury, T. S. 2019, ApJ, 887, 234 , Klein, K. G., Verniero, J. L., Alterman, B., et al. 2021, ApJ, 909, 7 Liu, W., Zhao, J., Xie, H., et al. 2021, ApJ, 920, 158 p Livi, S., & Marsch, E. 1987, JGR, 92, 7255 , , , , , , Maksimović, M., Bale, S. D., Berčič, L., et al. 2020, ApJS, 246, 62 Marsch, E., Rosenbauer, H., Schwenn, R., Muehlhaeuser, K. H., & Denskat, K. U. 1981, JGR, 86, 9199 , , , , Marsch, E., Schwenn, R., Rosenbauer, H., et al. 1982, JGR, 87, 52 While we elaborated on the general trends of instability growth rates and consequences of technical limitations of I1a instrument, a detailed examination of the physical aspects is left for future work, in which we will investigate the dynamics of interaction between collisions and instabilities, and offer the general model of the instability behavior in the inner heliosphere. Maruca, B. A., Bale, S. D., Sorriso-Valvo, L., Kasper, J. C., & Stevens, M. L. 2013, PhRvL, 111, 241101 Verniero, J. L., Larson, D. E., Livi, R., et al. 2020, ApJS, 248, 5 Verscharen, D., Bourouaine, S., & Chandran, B. D. G. 2013, ApJL, 773, 163 Verscharen, D., Chandran, B. D. G., Klein, K. G., & Quataert, E. 2016, ApJ, 831, 128 Verscharen, D., Klein, K. G., & Maruca, B. A. 2019, LRSP, 16, 5 Wang, C., Du, D., & Richardson, J. D. 2005, JGRA, 110, A10107 Woodham, L. D., Wicks, R. T., Verscharen, D., et al. 2019, ApJL, 884, L53 Yoon, P. H. 2017, RvMPP, 1, 4 The Astrophysical Journal, 923:116 (9pp), 2021 December 10 Rosenbauer, H., Schwenn, R., Miggenrieder, H., et al. 1981, The Instruments of the Plasma Experiment Onboard the HELIOS Solar Probe, Final Report Max Planck Institute, BMFT-FB-W 81-015 Schwenn, R., Rosenbauer, H., & Miggenrieder, H. 1975, RF, 19, 226 Stansby, D., Perrone, D., Matteini, L., Horbury, T. S., & Salem, C. S. 2019, A&A, 623, L2 Stansby, D., Salem, C., Matteini, L., & Horbury, T. 2018, SoPh, 293, 155 Vech, D., Martinović, M. M., Klein, K. G., et al. 2020, A&A Martinović et al. 2013, PhRvL, 111, 241101 Matteini, L., Hellinger, P., Goldstein, B. E., et al. 2013, JGRA, 118, 277 Matteini, L., Landi, S., Hellinger, P., et al. 2007, GeoRL, 34, L20105 Matthaeus, W. H., Montgomery, D. C., Wan, M., & Servidio, S. 2012, JTurb, 13, 37 McManus, M., Larson, D. E., Bale, S. D., et al. 2020, in AGU Fall Meeting Abstracts, 2020, SH049–0024 (Washington, DC: AGU) ( Nyquist, H. 1932, BSTJ, 11, 126 M.M.M. and K.G.K. were ﬁnancially supported by NASA grants 80NSSC19K1390 and 80NSSC19K0829. K.G.K. is supported by NASA ECIP grant 80NSSC19K0912. T.Ď. was Perrone, D., Stansby, D., Horbury, T., & Matteini, L. 2019, MNRAS, 488, 2380 Podesta, J. J., & Gary, S. P. 2011, ApJ, 742, 41 8 Martinović et al. Martinović et al. Rosenbauer, H., Schwenn, R., Miggenrieder, H., et al. 1981, The Instruments of the Plasma Experiment Onboard the HELIOS Solar Probe, Final Report Max Planck Institute, BMFT-FB-W 81-015 , Schwenn, R., Rosenbauer, H., & Miggenrieder, H. 1975, RF, 19, 226 Stansby, D., Perrone, D., Matteini, L., Horbury, T. S., & Salem, C. S. 2019, A&A, 623, L2 Stansby, D., Salem, C., Matteini, L., & Horbury, T. 2018, SoPh, 293, 155 Vech, D., Martinović, M. M., Klein, K. G., et al. 2020, A&A 9
https://openalex.org/W2586893715	https://europepmc.org/articles/pmc5299435?pdf=render	English	null	Plasmonically enhanced metal–insulator multistacked photodetectors with separate absorption and collection junctions for near-infrared applications	Scientific reports	2,017	cc-by	6,890	Sina Abedini Dereshgi1,2, Zulkarneyn Sisman1,2, Kagan Topalli2,3 & Ali Kemal Okyay1,2,3 A combination of multi-harmonic geometries is also reported in the literature to combine the absorption peaks resulting in broadband MIM absorbers17. While it is plausible to fabricate perfect absorbers using patterning techniques such as electron beam lithogra- phy, prohibitive costs associated with such methods cast doubt on their practical use for large volume manufac- turing. Therefore, dewetting of thin metallic films has emerged as a strong candidate to form nanostructures in a low cost and large-area compatible fashion. Not only does dewetting result in low-cost fabrication of active plas- monic layers, but also the structures formed with this technique exhibit broad absorption spectra. However, there 1Department of Electrical and Electronics Engineering, Bilkent University, Ankara 06800, Turkey. 2UNAM-National Nanotechnology Research Center, Bilkent University, Ankara 06800, Turkey. 3Institute of Material Science and Nanotechnology, Bilkent University, Ankara 06800, Turkey. Correspondence and requests for materials should be addressed to A.K.O. (email: aokyay@ee.bilkent.edu.tr) Plasmonically enhanced metal-insulator-metal (MIM) type structures are popular among perfect absorbers and photodetectors in which the field enhancement (for increased absorption) mechanism is directly coupled with collection (photocurrent) processes. In this work we propose a device structure that decouples absorption and collection parts for independent optimization. Double-stacked MIM (i.e. MIMIM) photodetectors operating in the near-infrared (NIR) spectrum up to 1200 nm wavelength are demonstrated. In the absorbing MIM (at the top side), we have used Silver nanoparticles resulting from dewetting, yielding a very low reflection of 10% for the most part of the 400 to 1000 nm wavelength range. An unconventional plasmonic material, Chromium, exhibits an absorption peak of over 80% at 1000 nm. The complete device has been fabricated and the photo-collection tunneling MIM (at the bottom) suppresses the leakage current by metal workfunction difference. An optimized stack consisting of Silver – Hafnium Oxide – Chromium – Aluminum Oxide – Silver nanoparticles (from bottom to top) yields a dark current of 7 nA and a photoresponsivity peak of 0.962 mA/W at 1000 nm and a full width at half maximum of 300 nm, while applied bias is 50 mV and device areas are 300 μm × 600 μm. With the advent of plasmonics in the recent decades there has been a lot of work ranging from exotic geometries as absorbers to photovoltaic applications. The concurring trend of scaling down the electronic and photonic devices has led to thriving devices that target this requirement by employing plasmonic nanoscale active layers. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 11 October 2016 accepted: 08 January 2017 Published: 09 February 2017 Plasmonically enhanced metal–insulator multistacked photodetectors with separate absorption and collection junctions for near-infrared applications received: 11 October 2016 accepted: 08 January 2017 Published: 09 February 2017 Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 Sina Abedini Dereshgi1,2, Zulkarneyn Sisman1,2, Kagan Topalli2,3 & Ali Kemal Okyay1,2,3 1 Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 g , y , g p y y Plasmonically enhanced metal-insulator-metal (MIM) type structures are popular among perfect absorbers and photodetectors in which the field enhancement (for increased absorption) mechanism is directly coupled with collection (photocurrent) processes. In this work we propose a device structure that decouples absorption and collection parts for independent optimization. Double-stacked MIM (i.e. MIMIM) photodetectors operating in the near-infrared (NIR) spectrum up to 1200 nm wavelength are demonstrated. In the absorbing MIM (at the top side), we have used Silver nanoparticles resulting from dewetting, yielding a very low reflection of 10% for the most part of the 400 to 1000 nm wavelength range. An unconventional plasmonic material, Chromium, exhibits an absorption peak of over 80% at 1000 nm. The complete device has been fabricated and the photo-collection tunneling MIM (at the bottom) suppresses the leakage current by metal workfunction difference. An optimized stack consisting of Silver – Hafnium Oxide – Chromium – Aluminum Oxide – Silver nanoparticles (from bottom to top) yields a dark current of 7 nA and a photoresponsivity peak of 0.962 mA/W at 1000 nm and a full width at half maximum of 300 nm, while applied bias is 50 mV and device areas are 300 μm × 600 μm. With the advent of plasmonics in the recent decades there has been a lot of work ranging from exotic geometries as absorbers to photovoltaic applications. The concurring trend of scaling down the electronic and photonic devices has led to thriving devices that target this requirement by employing plasmonic nanoscale active layers. While Otto and Kretchmann configurations were the presumptive methods to excite surface plasmons (SP), grat- ings1–3, nanoparticles4–8 and nanopillars9–11 made way to more efficient, omnidirectional and compact structures thanks to advanced patterning methods such as electron beam lithography. MIM structures attract interest due to their ability to efficiently trap the incident light within their spacer insulators1,3. Owing to suitable plasma frequency of Silver metal, grating structures made of Silver have been investigated to enhance absorption in Silicon for solar cell applications12. Fang et al. report using Silver hole-array as corrugated top layer in an MIM configuration in the visible spectrum13. A more exotic trapezoid array was reported that manifests over 50 per- cent absorption within the whole visible spectrum14. There are also reports of using MIM structures for efficient infrared absorbers15,16. Sina Abedini Dereshgi1,2, Zulkarneyn Sisman1,2, Kagan Topalli2,3 & Ali Kemal Okyay1,2,3 (a) Schematic of the MIMIM device, comprised of metal nanoparticles (MNP), spacer insulator (Ispa), absorbing metal (Mabs), tunneling insulator (Itunnel) and bottom contact metal (Mcont), (b) SEM image of the nanoparticles after annealing 10 nm Silver film for 20 min at 500 °C and (c) the size distribution of particles from SEM image of part (b). is a trade-off between the spectral bandwidth and the peak height of the absorption profile. Dewetting approach has also been utilized for solar cells to increase scattered light reaching the active region18,19. Incident light on nanoparticles excites localized surface plasmons (LSPs) or surface plasmons (SPs) and their non-radiative decay results in energetic hot electron-hole pairs that can be harnessed as photocurrent at a Schottky junction20,21. In case of traditional MIM type absorber designs, the burden of both absorption and photocurrent generation lies on the same junction which places stringent restrictions on device engineering to comply with desirable electrical and photonic characteristics simultaneously. Besides, Kretchmann configuration is needed to avoid competing photocurrents resulting from LSP excitation only22. Another configuration for MIM is a design with one metal layer comprised of nanoparticles. In such a structure, we need a transparent conductive oxide (TCO) that encapsulates nanoparticle layer to collect the current. This system would be inefficient due to the fact that we have two counter acting photocurrents, one from LSP excitation in nanoparticle layer and the other from SP excitation in the bottom metal which is therefore not reported in literature to the best of our knowl- edge. In order to remove restrictions of MIM, a new paradigm is proposed to decouple absorbing junction from the photocollection junction allowing the independent optimization of optical and electrical processes. This can be achieved in an MIMIM type multi-stack structure23. yp Traditionally Silver and Gold are metals of choice to construct plasmonic nanostructures in the visible24 and NIR regions25. In this work, we also introduce non-conventional metals to be used in different layers of MIMIM multi-stack, especially the absorber layer in NIR region. The top metal is desired to be a good scatterer with low loss while the absorber metal (in the middle) is desired to have a high loss. Materials with the highest absorp- tion coefficients may not be the most suitable metals for this work since the field penetration into the material is also crucial for significant absorption to take place. Sina Abedini Dereshgi1,2, Zulkarneyn Sisman1,2, Kagan Topalli2,3 & Ali Kemal Okyay1,2,3 While Otto and Kretchmann configurations were the presumptive methods to excite surface plasmons (SP), grat- ings1–3, nanoparticles4–8 and nanopillars9–11 made way to more efficient, omnidirectional and compact structures thanks to advanced patterning methods such as electron beam lithography. MIM structures attract interest due to their ability to efficiently trap the incident light within their spacer insulators1,3. Owing to suitable plasma frequency of Silver metal, grating structures made of Silver have been investigated to enhance absorption in Silicon for solar cell applications12. Fang et al. report using Silver hole-array as corrugated top layer in an MIM configuration in the visible spectrum13. A more exotic trapezoid array was reported that manifests over 50 per- cent absorption within the whole visible spectrum14. There are also reports of using MIM structures for efficient infrared absorbers15,16. A combination of multi-harmonic geometries is also reported in the literature to combine the absorption peaks resulting in broadband MIM absorbers17. p p g While it is plausible to fabricate perfect absorbers using patterning techniques such as electron beam lithogra- phy, prohibitive costs associated with such methods cast doubt on their practical use for large volume manufac- turing. Therefore, dewetting of thin metallic films has emerged as a strong candidate to form nanostructures in a low cost and large-area compatible fashion. Not only does dewetting result in low-cost fabrication of active plas- monic layers, but also the structures formed with this technique exhibit broad absorption spectra. However, there 1Department of Electrical and Electronics Engineering, Bilkent University, Ankara 06800, Turkey. 2UNAM-National Nanotechnology Research Center, Bilkent University, Ankara 06800, Turkey. 3Institute of Material Science and Nanotechnology, Bilkent University, Ankara 06800, Turkey. Correspondence and requests for materials should be addressed to A.K.O. (email: aokyay@ee.bilkent.edu.tr) 1 Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 www.nature.com/scientificreports/ Figure 1. (a) Schematic of the MIMIM device, comprised of metal nanoparticles (MNP), spacer insulator (Ispa), absorbing metal (Mabs), tunneling insulator (Itunnel) and bottom contact metal (Mcont), (b) SEM image of the nanoparticles after annealing 10 nm Silver film for 20 min at 500 °C and (c) the size distribution of particles from SEM image of part (b). Figure 1. Sina Abedini Dereshgi1,2, Zulkarneyn Sisman1,2, Kagan Topalli2,3 & Ali Kemal Okyay1,2,3 Despite their high absorption coefficient (k), widely used plasmonic metals are typically good reflectors due to high optical refractive indices (n′). Electric field does not efficiently penetrate into materials with high optical refractive index. Surface plasmon polariton (SPP) quality factor is a suitable metric for the attenuation of fields in a material, taking into consideration the absorption coef- ficient as well as the permittivity (ε = n2). In this paper, first absorbing top MIM is investigated computationally and experimentally and afterwards, the photocollection MIM is optimized and the overall photoresponsivity performance is examined. Figure 1(a) illustrates the schematic of the MIMIM structure as well as contact metals in detail. The distribution of particle sizes is depicted in Fig. 1(c) which demonstrates an average size of 97.11 nm, average distance of 161.4 nm between particles and an average height of 50 nm for particles. Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 Results Ab Measured and computational 3D simulation results of reflection from a surface of MIMIM (Ag nanoparticles - Al2O3 - Mabs - HfO2 – Ag bottom contact) with Mabs chosen to be (a) Aluminum and Gold, (b) Silver and Chromium, (c) simulated absorption percentage in different absorbing metals. Inset of (c) illustrates the layer being studied (i.e. absorbing metal). Figure 3. Computed H-field distribution at wavelengths of (a) 400 nm and (b) 1000 nm for MIMIM structure with Chromium absorbing (middle) metal, at a cross section of sample which includes and bisects two nanoparticles. The inset between two figures illustrates the cross section plane. Figure 2. Measured and computational 3D simulation results of reflection from a surface of MIMIM (Ag nanoparticles - Al2O3 - Mabs - HfO2 – Ag bottom contact) with Mabs chosen to be (a) Aluminum and Gold, (b) Silver and Chromium, (c) simulated absorption percentage in different absorbing metals. Inset of (c) illustrates the layer being studied (i.e. absorbing metal). Figure 3. Computed H-field distribution at wavelengths of (a) 400 nm and (b) 1000 nm for MIMIM structure with Chromium absorbing (middle) metal, at a cross section of sample which includes and bisects two nanoparticles. The inset between two figures illustrates the cross section plane. Figure 3. Computed H-field distribution at wavelengths of (a) 400 nm and (b) 1000 nm for MIMIM structure with Chromium absorbing (middle) metal, at a cross section of sample which includes and bisects two nanoparticles. The inset between two figures illustrates the cross section plane. and (b). Figure 2(c) shows the amount of absorption in the absorbing metal (Mabs). Both simulation and exper- imental results are normalized to Aluminum coated mirrors. The simulations are carried out in 3D while the nanoparticles are extracted from the SEM image into the simulation environment assuming a 50 nm thickness which is the only simplification in simulation (inset of Fig. 3). The 50 nm average thickness is calculated from the comparison between volume of deposited film and area of resulting particles after dewetting. The n, k data for Aluminum Oxide is extracted using J.A. Woollam Co. Inc. VASE. The mesh size is 3 nm in z-direction (perpen- dicular to the sample surface) and 15 nm in x and y directions and the simulation area is 500 nm × 500 nm.lh y Spectral reflection results calculated computationally agree well with experimental ones. Results Ab Absorption (MIM). Simulations the MIMIM structure using FDTD Solutions (by Lumerical) show that the absorption peak lies in the NIR range of interest for insulator (Al2O3) thickness values between 30–50 nm. The thickness of the insulator is chosen and fixed at 40 nm for all the simulations and experiments. We use atomic layer deposition system (ALD) to deposit insulators and control their thicknesses precisely. The main reason for choosing Aluminum Oxide as the spacer is the fact that among dielectrics, Aluminum Oxide is the least defective one and possesses the lowest k value (imaginary part of refractive index) compared to others like Zinc Oxide, Titanium Oxide and Hafnium Oxide.i Ultra-thin, metal films like gold (<10 nm) are not stable due to surface forces at metal-air interface. As the metal film thickness is a few nanometers, not only the deposited film is unstable but also it can barely be regarded as a continuous film. When the thicknesses are in the order of 10 nm, the film is in a thermodynamically metasta- ble state meaning that under some excitation, the film will evolve by agglomerating into islands to minimize the surface area with air26. The breaking of the continuous film into islands by such a process is called dewetting. The size and morphology of the resulting nanoclusters (islands) on the surface highly depend on the layer beneath metal, the metal type and excitation parameters. 10 nm thick Silver was deposited on Aluminum Oxide film via thermal evaporation, and a consecutive rapid thermal annealing (RTA) at 500 °C for 20 min was carried out. The scanning electron microscope (SEM) image of the resulting nanoparticles is given in Fig. 1(b), which shows nan- oparticles are approximately on the order of 100 nm laterally27.hhl p pp y y The simulated MIMIM structures are shown in Fig. 1(a). The resulting normal reflection measurements simulations for four different absorbing metals (Aluminum, Gold, Silver and Chromium) are depicted in Fig. 2 www.nature.com/scientificreports/ Figure 2. Measured and computational 3D simulation results of reflection from a surface of MIMIM (Ag nanoparticles - Al2O3 - Mabs - HfO2 – Ag bottom contact) with Mabs chosen to be (a) Aluminum and Gold, (b) Silver and Chromium, (c) simulated absorption percentage in different absorbing metals. Inset of (c) illustrates the layer being studied (i.e. absorbing metal). Figure 2. Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 Results Ab SPP quality factor QSPP is defined as ε ε = ′ ″ Q ( ) / SPP 2 where ε′ and ε″ are the real and imaginary part of permittivity respectively31. While there is little attention dragged to choosing the absorbing or scattering metals from the existing ones, this concept fits best for such a purpose. A metal is considered a good absorber in a wavelength of interest, when it has low real permittivity (to boost field penetration) and high complex permittivity (high absorption). Therefore, for this purpose Aluminum and specifically Chromium show minima of SPP quality factor and are the most appropriate choices for the target wavelength range. There is a relative minimum for Aluminum which accounts for increased absorption. The situation is pronounced for Chromium because its plasma frequency occurs at 850 nm and for NIR absorption, it proves to be most suitable. It is worth pointing out that using Silver for nanoparticles is also the best choice due to its high SPP quality factor within 400 nm to 1000 nm wavelengths which justifies high scattering after a preceding excitation of LSP27. where ε′ and ε″ are the real and imaginary part of permittivity respectively31. While there is little attention dragged to choosing the absorbing or scattering metals from the existing ones, this concept fits best for such a purpose. A metal is considered a good absorber in a wavelength of interest, when it has low real permittivity (to boost field penetration) and high complex permittivity (high absorption). Therefore, for this purpose Aluminum and specifically Chromium show minima of SPP quality factor and are the most appropriate choices for the target wavelength range. There is a relative minimum for Aluminum which accounts for increased absorption. The situation is pronounced for Chromium because its plasma frequency occurs at 850 nm and for NIR absorption, it proves to be most suitable. It is worth pointing out that using Silver for nanoparticles is also the best choice due to its high SPP quality factor within 400 nm to 1000 nm wavelengths which justifies high scattering after a preceding excitation of LSP27. Photodetector Design (MIMIM). In order to collect the resulting hot electrons or holes, a tunneling design is proposed. There is another possible choice which is using a semiconductor directly in Schottky contact with absorbing metal. Results Ab The little discrepancy between simulation and experiment stems from the fact that we have approximated the thickness of particles to be 50 nm for all of them. Moreover, there is generally a blue-shift in experimental reflection results compared to the simulation results which is attributed to the tarnishing of silver. This is justified once more in the photorespon- sivity results in the following sections. It can be deduced from Fig. 2(c) that Aluminum and Chromium show loss and absorption peaks of 42 percent and 81 percent around 800 nm and 1000 nm, respectively while Silver and Gold are not desirable since they yield very low absorption beyond 600 nm. The observed broad absorption peaks are innate properties of random nanoparticle enhanced absorbers. The absorption of Chromium based structure is highly broadband and extends further into NIR spectrum. Investigating the field profile (Fig. 3) can be insightful in understanding field penetration. The field profiles in the H-field simulation of all of the absorbing metals are analogous with small differences; thus, the results for Chromium is presented here. It is deduced that field penetration from Chromium to bottom layers is negligible.ii i p y g g At 400 nm (Fig. 3(a)) we observe higher order plasmonic modes with confined field mostly within Silver nan- oparticle – air interface which results in excitation of LSPs and absorption mainly occurs in Silver nanoparticles. At 1000 nm (Fig. 3(b)), however, the field is confined within the Aluminum Oxide – absorbing metal interface giving rise to absorption probability through excitation of SPs in the metal. For Silver and Gold absorbing metals, however, the light is reflected and confined in nanoparticle region mainly. The absorption in Aluminum Oxide is negligible27. One can assume that the same H-field profile may lead same absorption in all absorbing metals. Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 3 www.nature.com/scientificreports/ Figure 4. SPP quality factor for (a) Aluminum, Gold, Silver and (b) Chromium. Figure 4. SPP quality factor for (a) Aluminum, Gold, Silver and (b) Chromium. To confront this, we propose scrutinizing electric SPP quality factor of metals. Figure 4 illustrates SPP quality factor for four different metals using their respective permittivity data, taking Palik model for Aluminum and Chromium28, Johnson and Christy for Gold29 and CRC for Silver30. Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 Results Ab The latter is rather inefficient since the dark current typically is rather high in Schottky con- tacts compared to the tunneling counterparts. Out of the four fabricated samples that are investigated, two are Aluminum – Hafnium Oxide – Aluminum or Chromium absorbing metal – Aluminum Oxide – Silver nanopar- ticles (from bottom to top), and the other two are the same structures without Silver nanoparticle layer (MIMI) as references to the first two, respectively. Studying these samples emphasizes plasmonically enhanced absorption as well as the effect of the choice of absorbing metal type. One important design consideration for the junction is the substantial role of absorbing metal thickness in photocurrent. If the absorbing metal is chosen to be too thick, ohmic loss would degrade photoresponsivity. Therefore, the thickness of absorbing metals is chosen to be 30 nm. The reason for choosing 30 nm thick top metals is that the skin depths of metals in the wavelengths of interest are approximately 20 nm32 and slightly thicker metal layers are chosen in order to block any possible penetration of light through the stack to bottom metal which would result in counteracting reverse photocurrent. This counteracting photocurrent would be as a result of hot-hole generation in bottom metal where a negative bias already exists.fi y In order to design an efficient tunneling junction, the main parameters are the choice of bottom metal and tunneling insulator. The available and suitable insulators for tunneling purpose in our ALD system are Aluminum Oxide and Hafnium Oxide. Aluminum Oxide is chemically etched during lithography developing steps in the process (see methods). Given the fact that the tunneling insulator is thin, we chose Hafnium Oxide which is also a high-k dielectric. The thickness of the insulator is critical in performance. For thicknesses above 4 nm the photo- current is highly suppressed due to the exponential dependence of tunneling current on insulator thickness. For thicknesses lower than 4 nm on the other hand, we observe poor uniformity of insulator film. On the grounds of this discussion the tunneling insulator was selected to be 4 nm.h Another design parameter which is the bottom contact metal was chosen to be Silver. The main reason for choice was to create an offset in the metal work functions at the two sides of the tunneling insulators. This o 4 www.nature.com/scientificreports/ Figure 5. Results Ab Energy band diagram of the photocollecting tunneling junction with Chromium and Silver contacts, after non-radiative decay of SPs. Green arrows illustrate the transport of energetic electrons and the red one shows blocking the flow of the electrons in conduction band of the absorbing metal. Figure 5. Energy band diagram of the photocollecting tunneling junction with Chromium and Silver contacts, after non-radiative decay of SPs. Green arrows illustrate the transport of energetic electrons and the red one shows blocking the flow of the electrons in conduction band of the absorbing metal. Figure 6. I–V characteristics of MIMIM (Ag nanoparticles - Al2O3 - absorbing metal - HfO2 - Ag bottom contact) devices with (a) Aluminunm absorbing metal and (b) Chromium absorbing metal, photoresponsivity at applied negative 50 mV bias for MIMI (without Ag nanoparticles) and MIMIM devices respectively with (c) Aluminum absorbing metal and (d) Chromium absorbing metal. Figure 6. I–V characteristics of MIMIM (Ag nanoparticles - Al2O3 - absorbing metal - HfO2 - Ag bottom contact) devices with (a) Aluminunm absorbing metal and (b) Chromium absorbing metal, photoresponsivity at applied negative 50 mV bias for MIMI (without Ag nanoparticles) and MIMIM devices respectively with (c) Aluminum absorbing metal and (d) Chromium absorbing metal. facilitates the suppression of dark (leakage) current which is highly preferable in photodetector performance. Of the possible low resistivity choices, Silver revealed the best performance for both devices with Aluminum and Chromium absorbing metals, due to the fact that it has lower work function than the two absorbing metals. We could use a bottom metal with higher work function than that of Aluminum and Chromium for hot-hole collec- tion but the scarcity of such low resistive metals lead us to Silver to collect hot electrons. The energy band diagram schematic of the tunneling junction is illustrated in Fig. 5. facilitates the suppression of dark (leakage) current which is highly preferable in photodetector performance. Of the possible low resistivity choices, Silver revealed the best performance for both devices with Aluminum and Chromium absorbing metals, due to the fact that it has lower work function than the two absorbing metals. We could use a bottom metal with higher work function than that of Aluminum and Chromium for hot-hole collec- tion but the scarcity of such low resistive metals lead us to Silver to collect hot electrons. The energy band diagram schematic of the tunneling junction is illustrated in Fig. 5. www.nature.com/scientificreports/ www.nature.com/scientificreports/ important and favorable issue is that the I-V curves go through negligible change after dewetting which would not be the case if we used Schottky junctions. In order to discover the most optimum bias, we increase negative bias as long as we observe increase in photocurrent. After an optimum point, which happens to be a negative 50 mV bias, the photocurrent does not increase anymore and dark current increases solely. This phenomenon also solidifies the fact that our measured current is photocurrent. Fig. 6(c) and (d) represent photoresponsivity of MIMIM devices with and without Silver nanoparticles for Aluminum and Chromium respectively, measured at a negative bias of 50 mV. g As it can be inferred from Fig. 6, the observed dark currents are 5 nA and 7 nA for devices with Aluminum and Chromium absorbing metals respectively. The photocurrent results are highly in accordance with the simu- lated absorptions of Fig. 2(c). Photoresponsivity of MIMIM device with Aluminum has a peak value of 36.84 μ A/W at 850 nm which is 29 times the peak value of same device without nanoparticles (the reference device for Aluminum case). The outstanding performance occurs for the MIMIM device with Chromium absorbing metal which exhibits a peak photoresponsivity of 96.24 μA/W at 980 nm which represents 3 times increase compared to the peak value of same device without nanoparticles (the reference device for Chromium case). This is also a 2.6 times increase compared to MIMIM device with Aluminum. The full width at half maximum is more than 300 nm due to the use of random sized nanoparticles. Thus, our best result justifies almost an order of magnitude enhancement compared to MIMI device with Aluminum, which itself has a little absorption peak around 850 nm already. Besides, our Chromium result exhibits more than an order of magnitude enhancement in photorespon- sivity over the reported spin-coated MIMIM structures23. y p p It should be pointed out that the photoresponsivity curves follow the simulated absorption curves quite acceptably. There is a red-shift in photoresponsivity results compared to absorption results of Fig. 2(c), which is attributed to the oxidation of Silver nanoparticles. To vindicate this statement, Fig. 2(a) and (b) results can be taken into account. Methods Fabrication. A Silicon wafer with unintentional doping was used as the substrate. The substrate was cleaned in Acetone, Isopropanol and de-ionized water for 10 minutes each using ultrasonic agitation. Afterwards, 70 nm silver was deposited with thermal evaporation at a base pressure of 2–4 × 10−6 Torr. Afterwards the sample was masked with a small margin (to get the negative contact) and a deposition of hafnium oxide at 200 °C was carried out with Cambridge Nanotech Inc., Savannah S100 ALD system using Trimethylhafnium and milli-Q water as Hafnium and Oxygen precursors respectively with a Nitrogen flow of 20 sccm. For a lift-off process, HMDS and AZ5214 photoresist was spin coated and exposed to UV light using mask aligner and was developed in AZ400K solution, a follow up deposition of 30 nm absorbing metal (either Aluminum or Chromium) was deposited in the same conditions as the negative contact metal and acetone was used for the lift off process. Afterwards, 40 nm Aluminum Oxide was deposited in the same conditions in ALD using Timethylaluminum and milli-Q water as Aluminum and Oxygen precursors respectively. Then, using an optimized inductively coupled plasma (ICP) dry etching recipe33, Aluminum Oxide was patterned to form the positive contact. At last, 10 nm thick Silver was deposited on Aluminum Oxide film via thermal evaporation at 2–4 × 10−6 Torr, and a consecutive rapid thermal annealing (ATV Technologie GmbH, SRO-704 RTA) step was carried out at 120 sccm N2 flow rate. Temperature was ramped up to 500 °C in 5 min, followed by 20 min annealing at constant temperature, 500 °C. At the end of the annealing step, the temperature is passively reduced to 250 °C followed by manually removing the sample to facilitate faster cooling which resulted in some oxidation27. Characterization. We used a custom reflection measurement setup comprised of a Halogen illuminator con- nected to a microscope and directed perpendicular to sample and the reflected light from the microscope was fed to a Newport OSM2 spectrometer and the data was collected by interfacing the spectrometer with PC (Fig. 7 (a)). All measurements were normalized to an Al coated reference sample.h The dark I-V measurements of the devices were conducted using KEITHLEY 2401 Sourcemeter. In order to measure photoresponsivity for the wavelengths between 400 to 800 nm, we used a Xenon-lamp illuminator. The output of the lamp goes through an Oriel 74004 monochromator (1/8 meter, 1200 lines/mm grating). www.nature.com/scientificreports/ In those results there is a blue-shift in experimental reflection results compared to those of simulations (reflection trend is the opposite of absorption trend) due to oxidation of Silver nanoparticles that is inevitable. Moreover, the smoother photoresponsivity curve of Chromium is because of higher photocurrent for this sample that is far larger than our measurement noise level. Conclusionh The demonstration of absorbers and photodetectors were investigated in this work with focus on NIR region. We investigated the effect of four different bottom metals in plasmonically enhanced MIM absorbers with silver nanoparticles. After identifying MIMs with highest absorption in bottom metals of them, which happen to be Aluminum and Chromium, the complete MIMIM photodetector was fabricated in which the energy band engi- neering is done such that the dark current is suppressed and the photocurrent mechanism is based on tunneling of hot electrons. In an MIMIM structure, light falling on the particles excites SPs in the middle absorbing metal. The non-radiative decay of SPs result in hot EHPs and hot electrons are directed to the bottom metal by tunne- ling. We point out Chromium as an efficient absorbing metal due to high loss and suitable plasma frequency that happens in NIR region rather than UV for other metals of choice here. The resulting absorption and photocurrent curves are highly in agreement. Our photodetector design manages to decouple absorption and photocollection parts which facilitates design of efficient photodetectors by engineering each layer independently. Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 Discussionh The applied bias is negative as with the probes illustrated at Fig. 1(a). With such a bias we collect hot electrons and suppress the dark current flow. Since electrons are energetic they can overcome the barrier and work function offset with appropriate applied bias. The dark I-V curves of devices with aluminum and chromium are depicted in Fig. 6(a) and (b) respectively, which justify the work function offset. The device areas are 300 μm × 600 μm. One Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 5 References 1. Min, C. et al. Enhancement of optical absorption in thin-film organic solar cells through the excitation of plasmonic modes in metallic gratings. Appl. Phys. Lett. 96, 133302 (2010). 1. Min, C. et al. Enhancement of optical absorption in thin-film organic solar cells through the excitation of plasmonic modes in metallic gratings. Appl. Phys. Lett. 96, 133302 (2010). g g pp y 2. Sefunc, M. 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Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 7 www.nature.com/scientificreports/ Acknowledgementsh g This work was partially supported by the Scientific and Technological Research Council of Turkey (TUBITAK), grant nos 112E052, and 113M815. A.K.O. acknowledges partial support from the Turkish Academy of Sciences Distinguished Young Scientist Award (TUBA GEBIP) and BAGEP and FABED awards. Author Contributions S.A.D., Z.S., K.T. and A.K.O. contributed equally to all parts of the research. Author Contributions S.A.D., Z.S., K.T. and A.K.O. contributed equally to all parts of the research. Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Dereshgi, S. A. et al. Plasmonically enhanced metal–insulator multistacked photodetectors with separate absorption and collection junctions for near-infrared applications. Sci. Rep. 7, 42349; doi: 10.1038/srep42349 (2017). ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2017 Scientific Reports \| 7:42349 \| DOI: 10.1038/srep42349 8
https://openalex.org/W2040932482	https://hal.science/hal-01510829/file/LNVMP.pdf	English	null	Periodic orbits, damped transitions and targeted energy transfers in oscillators with vibro-impact attachments	Physica. D, Nonlinear phenomena	2,009	cc-by	19,176	Periodic orbits, damped transitions and targeted energy transfers in oscillators with vibro-impact attachments Young Lee, Francesco Nucera, Alexander Vakakis, Michael Mcfarland, Lawrence A Bergman Lawrence A Bergman To cite this version: Young Lee, Francesco Nucera, Alexander Vakakis, Michael Mcfarland, Lawrence A Bergman. Periodic orbits, damped transitions and targeted energy transfers in oscillators with vibro-impact attachments. Physica D: Nonlinear Phenomena, 2009, 238, pp.1868 - 1896. 10.1016/j.physd.2009.06.013. hal- 01510829 Distributed under a Creative Commons Attribution 4.0 International License Young S. Lee a, Francesco Nucera b, Alexander F. Vakakis c, D. Michael McFarland d, Lawrence A. Bergman d Keywords: Targeted energy transfer (TET) Vibro-impact nonlinear energy sink (VINES) Passive and broadband vibration absorber Keywords: Targeted energy transfer (TET) Vibro-impact nonlinear energy sink (VINES) Passive and broadband vibration absorber generally requires two elements: an essentially nonlinear (i.e., non- linearizable) stiffness and a (usually, linear viscous) damper. The former, which is smooth in many cases, enables the NES to resonate with any of the linearized modes of the primary system to which it is attached, whereas the latter acts as dissipator of the vibrational energy transferred through resonant modal interactions (see [3,4] for an overview of the dynamics governing TET and its various applications). In most previous works smooth essential (purely cubic) stiffness nonlinearities were considered, although in some recent works nonsmooth stiffness nonlinearities were studied as well [5–10]. generally requires two elements: an essentially nonlinear (i.e., non- linearizable) stiffness and a (usually, linear viscous) damper. The former, which is smooth in many cases, enables the NES to resonate with any of the linearized modes of the primary system to which it is attached, whereas the latter acts as dissipator of the vibrational energy transferred through resonant modal interactions (see [3,4] for an overview of the dynamics governing TET and its various applications). In most previous works smooth essential (purely cubic) stiffness nonlinearities were considered, although in some recent works nonsmooth stiffness nonlinearities were studied as well [5–10]. HAL Id: hal-01510829 https://hal.science/hal-01510829v1 Submitted on 20 Apr 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Young S. Lee a, Francesco Nucera b, Alexander F. Vakakis c, D. Michael McFarland d, Lawrence A. Bergman d Francesco Nucera b, Alexander F. Vakakis c, D. Michael McFarland d, d a Department of Mechanical and Aerospace Engineering, New Mexico State University, Las Cruces, NM 88003, USA b Department of Mechanics and Materials, Mediterranean University of Reggio Calabria, 89122 Reggio Calabria, Italy c Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Department of Aerospace Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA a Department of Mechanical and Aerospace Engineering, New Mexico State University, Las Cruces, NM 88003, USA b Department of Mechanics and Materials, Mediterranean University of Reggio Calabria, 89122 Reggio Calabria, Italy c Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Department of Aerospace Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA We study complex damped and undamped dynamics and targeted energy transfers (TETs) in systems of coupled oscillators, consisting of single-degree-of-freedom primary linear oscillators (LOs) with vibro- impact attachments, acting, in essence, as vibro-impact nonlinear energy sinks (VI NESs). First, the complicated dynamics of such VI systems is demonstrated by computing the VI periodic orbits of underlying Hamiltonian systems and depicting them in appropriate frequency–energy plots (FEPs). Then, VI damped transitions and distinct ways of passive TETs from the linear oscillators to the VI attachments for various parameter ranges and initial conditions are investigated. As in the case of smooth stiffness nonlinearity [Y. Lee, G. Kerschen, A. Vakakis, P. Panagopoulos, L. Bergman, D.M. McFarland, Complicated dynamics of a linear oscillator with a light, essentially nonlinear attachment, Physica D 204 (1–2) (2005) 41–69], both fundamental and subharmonic TET can be realized in the VI systems under consideration. It is found that the most efficient mechanism for VI TET is through the excitation of highly energetic VI impulsive orbits (IOs), i.e., of periodic or quasiperiodic orbits corresponding to zero initial conditions except for the initial velocities of the linear oscillators. In contrast to NESs with smooth essential nonlinearities considered in previous works, VI NESs are capable of passively absorbing and locally dissipating significant portions of the energies of the primary systems to which they are attached, at fast time scale. This renders such devices suitable for applications, like seismic mitigation, where dissipation of vibration energy in the early, highly energetic regime of the motion is a critical requirement. Periodic orbits, damped transitions and targeted energy transfers in oscillators with vibro-impact attachments Young S. Lee a, Francesco Nucera b, Alexander F. Vakakis c, D. Michael McFarlan Lawrence A. Bergman d ng S. Lee a, Francesco Nucera b, Alexander F. Vakakis c, D. Michael McFarland d, rence A Bergman d 1.1. Background Indeed, NESs with clearances and vibro-impacts have been considered in applications where vibration reduction at a fast time scale is required. The consequence of the capacity of this class of nonsmooth NESs for rapid energy absorption and dissipation makes them applicable to applications where the energy transfer from the directly forced primary structure to the NES(s) must be accomplished at the early stage of the motion, if possible immediately after the application of an external shock. Examples Passive, broadband targeted energy transfer (TET) refers to the one-way directed transfer of energy from a primary subsystem to a nonlinear attachment; this phenomenon is realized in damped, coupled, essentially nonlinear oscillators through resonance cap- tures and escapes along intrinsic periodic or quasiperiodic orbits of the underlying Hamiltonian systems [1,2]. Indeed, TET is real- ized from primary linear or nonlinear systems to essentially non- linear attachments, acting as nonlinear energy sinks (NESs). An NES 1 Fig. 1. Experimental setup of a 3-story building with a VI NES installed at its top floor [5]. include structures under seismic excitation [5–7] and cars during collision. The regular and chaotic dynamics and bifurcations of vibro- impact (VI) oscillators have been studied extensively in the li- terature [11–16]. In a series of papers [17–24], VI dampers were considered for reducing the vibration levels of structures under periodic or stochastic excitation. Shaw and Holmes [25], Shaw and Shaw [26] and Shaw [27] applied methods from the geomet- rical theory of nonlinear dynamics to analyze the dynamics of free and forced dynamics of systems with piecewise nonlineari- ties, including systems with vibro-impact nonlinearities. Recently, Gorelyshev and Neishtadt [28] discussed the extension of adiabatic perturbation theory to VI systems; Mikhlin et al. [29] and others [30–37] studied periodic orbits, bifurcations and chaos in discrete and continuous oscillators with clearance nonlinearities or vibro- impacts; Zhuravlev [38,39] investigated vibro-impact oscillations using nonsmooth coordinate transformations (for an additional ap- plication of this method see also [40]), and Pilipchuk [41] extended this approach by considering nonsmooth transformations of the dependent (temporal) variable of the problem. Fig. 1. Experimental setup of a 3-story building with a VI NES installed at its top floor [5]. p ( p ) p Pinnington [42] analyzed energy exchange and dissipation due to collisions in a line of coupled oscillators, and Salapaka et al. [43] studied the dynamics of a linear oscillator impacting with a vibrat- ing platform. 1.2. Motivations for the present study In previous works, application of VI NESs for passive seismic mitigation was studied theoretically and experimentally in [5–7] – e.g., Fig. 1, where an experimental model of a 3-story building with an attached VI NES under base excitation is depicted. In particular, Nucera et al. [5] considered a single-degree-of-freedom linear oscillator (LO) with no viscous damping coupled to a VI NES as a simplified model for the fixture of Fig. 1. Fig. 2(a) depicts a VI damped transition for this system, from which we deduce that there exist three distinct regimes for this VI damped transition. Moreover, the instantaneous energy plot and wavelet transform spectra of the relative displacement between the linear oscillator and the VI NES depicted in Fig. 2(b) indicate that there occur multi-frequency resonance captures [57,58] in the dynamics as the energy diminishes due to energy dissipation at the impact instant. Clearly, there is the need for developing techniques capable of analyzing and identifying such complex VI transitions, which will enable us to categorize and classify different types of multi- frequency resonance captures that occur in VI orbits. p In the works by Georgiades et al. [8,9] TETs in coupled me- chanical oscillators with NESs possessing nonsmooth stiffnesses (referred to from now on as nonsmooth NESs or NS NESs) were analyzed by studying the shock isolation properties of systems of coupled nonconservative linear oscillators with NESs possessing clearance nonlinearities (i.e., piecewise linear oscillators). Apart from the fact that such nonsmooth stiffness elements introduce strong nonlinearities to the systems to which they are attached, they are rather easy to implement in practical settings as combi- nations of linear stiffnesses. Numerical evidence of the capacity for shock isolation of NS NESs was provided in these works; that is, significant energy transfers from the primary systems to such NESs can occur right from the beginning of the motion (i.e., during the energetically high regime of the dynamics). The capacity of NS NESs to rapidly absorb shock energy in the initial highly energetic regime is critical to their role as shock isolators. In an attempt to address these issues, in this work we will intro- duce some new tools that will help us understand, classify and sys- tematically study complex transitions in the simplest possible VI system, namely, a linear oscillator with a VI attachment. 1.1. Background Quinn [44] investigated the oscillations of two para- metrically excited pendula undergoing vibro-impacts; and Li and Darby [45] reported experimental work on the effect of an impact damper on an MDOF system. Murphy and Morrison [46] studied, computationally and experimentally, instabilities and bifurcations of a vibro-impacting string; and Hu and Schiehlen [47] discussed multi-scale simulation of impact responses with applications from wave propagation to rigid body dynamics. Sampaio and Soize [48] formulated measures that quantify nonlinear effects for uncertain systems, whereas Azeez and Vakakis [49] approached the issue of nonlinear effects quantification using proper orthogonal decom- position. Vedenova et al. [50] and others [51,52] examined model- ing inelastic impacts with smooth, essentially nonlinear functions of high degree; and Nayeri et al. [53] investigated the action of multi-unit impact dampers in systems under stochastic excitation. Namachchivaya and Park [54] developed an analytical approach based on averaging for studying the dynamics of VI systems un- der stochastic excitation; Wagg [55] used energy balance analysis to examine multi-modal systems undergoing vibro-impacts, and studied effective restitution coefficients; and Shaw and Pierre [56] applied tuned impact dampers in rotating structures and assessed their performance. the two limiting linear systems corresponding to zero and large clearances, respectively), small coupling frequencies, and large mass ratios. Moreover, high shock absorption efficiencies was attained for even a small total number of vibro-impacts, provided that conditions for sufficient momentum and energy exchanges between the primary system and the NES during vibro-impacts were realized. It was also shown that VI NESs can be designed as effective shock isolators, providing significant reduction of maximum responses of the primary systems over broad frequency ranges. Hence, appropriately designed VI NESs can act as broadband, passive shock isolators. The results indicated that, in designing VI NESs as shock isolators, important design parameters are the clearances, the coupling stiffnesses and the NES masses. Moreover, better vibro-impact shock absorption is anticipated for weak coupling stiffness between the primary systems and the attached NESs, and relatively large values of NES masses compared to the masses of the corresponding primary systems. These results are in agreement with findings of Georgiades et al. [8]. 1.2. Motivations for the present study Although these tools will be applied exclusively to this two-DOF system, they are rather general so they can be applied to higher-dimensional VI systems as well. First, we will focus on the detailed computation of Karayannis et al. [10] then extended this study to the case where a primary system experiences an impulsive excitation, with the nonlinear attachment undergoing two-sided inelastic impacts. Superior shock absorption by the VI NES compared to the linear absorber is attained for intermediate clearances (i.e., away from 2 Stage A Stage B Stage C Fig. 2. Damped VI transition for a linear oscillator with a VI attachment (cf. Fig. 3): (a) Responses normalized by the clearance; (b) instantaneous total energy, energy dissipation due to vibro-impacts, and wavelet transform (WT) spectra of the relative displacement between the linear oscillator and the VI NES. Stage A Stage B Stage C Fig. 2. Damped VI transition for a linear oscillator with a VI attachment (cf. Fig. 3): (a) Responses normalized by the clearance; (b) instantaneous Stage A Stage B Stage C Fig. 2. Damped VI transition for a linear oscillator with a VI attachment (cf. Fig. 3): (a) Responses normalized by the clearance; (b) instantaneous total energy, energy dissipation due to vibro-impacts, and wavelet transform (WT) spectra of the relative displacement between the linear oscillator and the VI NES. Fig. 2. Damped VI transition for a linear oscillator with a VI attachment (cf. Fig. 3): (a) Responses normalized by the clearanc dissipation due to vibro-impacts, and wavelet transform (WT) spectra of the relative displacement between the linear oscillator a Fig. 3. An SDOF linear oscillator coupled to a VI NES. Fig. 3. An SDOF linear oscillator coupled to a VI NES. the VI periodic solutions of the underlying Hamiltonian system and on their depiction on a frequency–energy plot (FEP). The rich struc- ture of periodic orbits (and impulsive orbits) of the VI system then will become immediately apparent. In the sequence we will study damped transitions of the VI system undergoing inelastic vibro- impacts, by superimposing the WT spectra of transient VI damped responses on the Hamiltonian FEP. 1.2. Motivations for the present study This will allow us to detect and understand complex nonlinear modal interactions occurring dur- ing the VI transitions; in the process we will study the different dy- namical mechanisms that govern VI targeted energy transfer (TET) in the two-DOF system; by doing so we will generalize previous results on TET in dynamical systems with strong but smooth stiff- ness nonlinearities. Our study will demonstrate schematically the complex resonance captures and multi-modal interactions occur- ring during these VI transitions. In addition, we will provide a nu- merical study of VI TET efficiency by considering the excitation of the two-DOF system by external impulses, and end with some con- cluding remarks. Fig. 3. An SDOF linear oscillator coupled to a VI NES. of damped VI transitions that govern TET in the shock-excited system can be gained by depicting the WT spectra of these motions on an FEP of the underlying VI Hamiltonian system (corresponding to the system with purely elastic impacts and no viscous damping elements). Also, we will demonstrate that, for sufficiently weak dissipation, weakly damped VI transitions take place near branches of periodic or quasiperiodic motions of the corresponding undamped system. Hence, by studying the structure of periodic orbits of the Hamiltonian system, we should be able to understand the governing dynamics of the weakly damped dynamics as well, and to clearly identify complex multi-frequency transitions and resonance captures leading to energy exchanges and TET in the weakly damped VI system. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) The two nonsmooth basis functions, τ(x) and e(x) = dτ(x)/dx. (3) In the numerical algorithm used to compute the VI responses, the linear equations (2) are integrated for as long as the no-impact condition \|u2 −u1\| < 1 is satisfied. When \|u2 −u1\| = 1, a (purely elastic) impact occurs, and discontinuities in the velocities take place, whereas the displacements remain continuous throughout the impact. The velocities immediately after the impact are com- puted by relations (3), and the numerical integration of the lin- ear system (2) resumes with the new initial conditions until the next impact occurs, where this procedure is iterated. As discussed in earlier works [5,10], precise computation of the time instants where vibro-impacts occur is essential for the accuracy of the nu- merical simulations. This accuracy was checked by recording the total energy of the VI motion and ensuring its conservation through the entire interval of the simulation. The total (conserved) normal- ized energy H(u1, ˙u1, u2, ˙u2) is computed in terms of the initial conditions of the normalized system (2) as y′ 1 = y3 y′ 2 = y4 y′ 3 = −σα2(y1 −y2)/µ y′ 4 = −α2y2 −σα2(y2 −y1) y1(±1) = y2(±1) = 0 (7) (7) where primes denote differentiations with respect to the nons- mooth variable τ, and the periodic orbits are computed subject to zero initial velocities. Vibro-impacts occur when \|y2 −y1\| = 1, at which points discontinuities in the velocities through expressions similar to Eq. (3) are imposed. The solution of the NLBVP (7) com- putes the periodic orbit over half of its period T = 4α (i.e., for −1 ≤τ ≤1 ⇒−α ≤ξ ≤α); to extend the periodic orbit over the entire period we take into account the form of the nonsmooth transformations (5), such that the conserved energy of the periodic orbit is expressed as H(u1, ˙u1, u2, ˙u2) = 1 2 µ˙u1(0)2 + ˙u2(0)2 + 1 2 u2(0)2 + σ(u1(0) −u2(0))2 = h. (4) h = 1 2α2 µy′ 1(−1)2 + y′ 2(−1)2 . (8) (8) The NLBVP (7) is solved by a shooting method (see, for example, [61–63]) in the bounded domain −1 ≤τ ≤1. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) However, unlike the shooting method employed for the analogous case of the NES with smooth nonlinearity in [57], matching at τ = 0 of the two solutions initiated from the left and right boundaries (τ = ±1) is not helpful in the current VI problem. This is due to the fact that symmetric VI periodic orbits are expected to exhibit vibro- impacts at τ = 0, so matching solutions at that point becomes meaningless. Therefore, in the VI problem we adopt the following alternative matching condition: For fixed quarter-period α, the set of equations of the NLBVP is solved as an initial value problem with initial conditions at the left boundary, yi(−1) = 0 and y′ i(−1) ̸= 0, i = 1, 2. Then, perform matching at the right boundary through the inequalities, \|yi(+1−)\| < ε ≪1, i = 1, 2, where the tolerance ε is taken as O(10−5)–O(10−6). This procedure ensures that the NLBVP (7) is approximately solved (that is, within the prescribed numerical tolerance). Assuming zero initial velocities, the critical threshold for vibro- impacts to occur is computed as hmin = σ/2. Throughout this paper, the normalized mass and stiffness parameters are taken as µ = 0.1 and σ = 0.1, respectively. The periodic solutions of the Hamiltonian vibro-impacting system can be computed by employing the method of nonsmooth temporal transformations (NSTTs) first introduced by Pilipchuk [41] and Pilipchuk et al. [59]. NSTTs were applied in [57] for computing the periodic orbits (or nonlinear normal modes; NNMs [60]) of the corresponding system with smooth essential nonlinearity (i.e., with a ‘smooth’ NES). To this end, we introduce the coordinate transformation ui(ξ) = e(ξ/α) yi (τ(ξ/α)) , i = 1, 2 (5) ui(ξ) = e(ξ/α) yi (τ(ξ/α)) , i = 1, 2 (5) where α = T/4 represents the quarter-period of the VI periodic motion, and the nonsmooth functions τ(·) and e(·) are defined as (see also Fig. 4) ui(ξ) = e(ξ/α) yi (τ(ξ/α)) , i = 1, 2 (5) where α = T/4 represents the quarter-period of the VI periodic (5) where α = T/4 represents the quarter-period of the VI periodic motion, and the nonsmooth functions τ(·) and e(·) are defined as (see also Fig. 4) where α = T/4 represents the quarter-period of the VI periodic motion, and the nonsmooth functions τ(·) and e(·) are defined as (see also Fig. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) We initiate our study by computing the periodic orbits of the Hamiltonian system with no viscous damping dissipation and purely elastic vibro-impacts and depicting them in a frequency– energy plot (FEP). Considering the two-degrees-of-freedom system of Fig. 3, we note that the equations of motion are linear in time intervals between vibro-impacts (i.e., \|x2 −x1\| < δ), and can be written as To study the underlying dynamical mechanisms and associated transient resonance captures (TRCs) that govern passive TET in systems with VI NESs, and also to demonstrate the complexity that a single VI NES can induce in the dynamics, we consider the simplest primary system — VI NES configuration, namely a single-degree-of-freedom (SDOF) LO coupled to a VI NES (Fig. 3). As in the case of the NES with smooth essential nonlinearities studied in previous works, we will show that a clear interpretation m¨x1 + k1(x1 −x2) = 0 M¨x2 + c˙x2 + kx2 + k1(x2 −x1) = 0 (1) (1) 3 Fig. 4. The two nonsmooth basis functions, τ(x) and e(x) = dτ(x)/dx. or in non-dimensional form, or in non-dimensional form, µ¨u1 + σ(u1 −u2) = 0 ¨u2 + λ˙u2 + u2 + σ(u2 −u1) = 0. (2) (2) Dots in (2) denote differentiation with respect to the transformed temporal variable ξ = √ k/Mt; moreover, µ = m/M, denotes the mass ratio; σ = k1/k, the stiffness ratio; and λ = c/ √ Mk, the damping factor. In addition, in Eq. (2) we consider normalized dis- placements through the rescalings ui = xi/δ, i = 1, 2, δ ̸= 0, so in terms of these normalized displacements vibro-impacts occur when \|u2 −u1\| = 1. Note that the mass of the NES is not neces- sarily small compared to that of the LO, so the ratio µ in Eq. (2) is assumed to be an O(1) quantity [10]. Assuming zero viscous damping (i.e., λ = 0) and considering purely elastic impacts (i.e., with restitution coefficient ρ = 1), the VI system of Fig. 3 becomes Hamiltonian. The velocities of the LO and the NES after an impact (denoted by superscripts ‘+’) are computed in terms of the corresponding velocities before impact (denoted by superscripts ‘−’) by the following relations: ˙u+ 1 = (µ −1)˙u− 1 + 2˙u− 2 1 + µ , ˙u+ 2 = 2µ˙u− 1 + (1 −µ)˙u− 2 1 + µ . (3) Fig. 4. fn(u) = knu2n+1, n = 0, 1, . . . , (9) 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) 4) It is anticipated that the seemingly simple VI system of Fig. 3 will possess a very complicated structure of periodic orbits in the FEP. This expectation is justified by the fact that vibro- impact nonlinearity represents a very strong (and degenerate) form of nonlinearity. Indeed, considering the family of essentially nonlinear stiffnesses τ(x) = 2 π sin−1 sin π 2 x , e(x) = d dxτ(x). (6) (6) Then, we obtain the two-point nonlinear boundary value prob- lem (NLBVP) in terms of the variables y1, y2, y3 = y′ 1 and y4 = y′ 2 Then, we obtain the two-point nonlinear boundary value prob- lem (NLBVP) in terms of the variables y1, y2, y3 = y′ 1 and y4 = y′ 2 (9) fn(u) = knu2n+1, n = 0, 1, . . . , 4 Fig. 5. Vibro-impact nonlinearity as limiting case of a family of smooth, essentially nonlinear stiffnesses. where T is the period of the motion. Similar to the ‘smooth’ NES [57], symmetric VI orbits correspond to synchronous oscilla- tions of the LO and the VI NES, and typically are represented by curves in the configuration plane (u1, u2) (cf. Fig. 29 in Appendix). Unsymmetric VI periodic orbits, labeled as Umnpq±, do not satisfy the conditions of the symmetric ones. These orbits correspond to asynchronous motions of the two oscillators and are represented by open or closed (Lissajous) curves in the configuration plane (u1, u2) (cf. Fig. 30 in Appendix). The integer index m denotes the number of half-waves in the VI NES response within a half-period, whereas the integer index n denotes the corresponding number of half-waves in the LO response; clearly, the ratio m:n indicates the order of nonlinear resonance that occurs between the VI NES and the LO for a given VI periodic orbit. Moreover, the index E or O de- notes the symmetry pattern of the vibro-impacts, and has meaning only for symmetric VI orbits: E(O) denotes an even (odd) symmetry of occurring vibro-impacts within a half-period; it follows that the notation E(O) implies that a vibro-impact occurs (does not occur) at quarter-period ξ = α = T/4. The integer indices p and q de- note the number of vibro-impacts that occur in the first and second quarter-period, respectively, of a given VI periodic orbit; it follows that for symmetric orbits p = q. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Finally, the ‘+’ sign corresponds to in-phase VI periodic motions, where, for zero initial displacements, the initial velocities of the LO and the NES possess identical signs at the beginning of both the first and second half-periods of the periodic motion. Otherwise, the VI periodic motion is deemed to be out-of-phase and the ‘−’ sign is used. Finally, the two in-phase and out-of-phase linear modes of the system with zero clearance (δ = 0) are denoted by Lmm± and are, in fact, equivalent to L11±. The (incomplete) FEP of the Hamiltonian VI system for µ = σ = 0.1 is depicted in Fig. 6, with some representative VI periodic orbits presented in Fig. 7. The complexity of the bifurcations that generate the VI periodic orbits can be inferred from the bifurcation diagrams of Fig. 8 where the initial velocities of the LO and the VI NES (for zero initial displacements) are depicted as functions of the total energy h. As mentioned previously, this complexity was anticipated in view of the degenerate vibro-impact nonlinearity of this system. Fig. 5. Vibro-impact nonlinearity as limiting case of a family of smooth, essentially nonlinear stiffnesses. the coefficient kn depends on the exponent n, and is selected so that the normalization condition fn(±1) = ±1, ∀n is satisfied. Then, the vibro-impact nonlinearity corresponding to purely elas- tic impacts is obtained as the (degenerate) discontinuous limit f∞(u) = limn→∞fn(u) (Fig. 5; [50,64]). Viewed in this context, vibro-impact nonlinearity can be considered as the ‘strongest pos- sible’ stiffness nonlinearity of the family (9). Following this reason- ing, Pilipchuk [41,65,66] developed an asymptotic methodology based on nonsmooth transformations and nonsmooth generating functions that is applicable to strongly nonlinear regimes [59,67]. the coefficient kn depends on the exponent n, and is selected so that the normalization condition fn(±1) = ±1, ∀n is satisfied. Then, the vibro-impact nonlinearity corresponding to purely elas- tic impacts is obtained as the (degenerate) discontinuous limit f∞(u) = limn→∞fn(u) (Fig. 5; [50,64]). Viewed in this context, vibro-impact nonlinearity can be considered as the ‘strongest pos- sible’ stiffness nonlinearity of the family (9). Following this reason- ing, Pilipchuk [41,65,66] developed an asymptotic methodology based on nonsmooth transformations and nonsmooth generating functions that is applicable to strongly nonlinear regimes [59,67]. The (incomplete) FEP of the Hamiltonian VI system for µ = σ = 0.1 is depicted in Fig. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Finally, the ‘+’ sign corresponds to in-phase VI periodic motions, where, for zero initial displacements, the initial velocities of the LO and the NES possess identical signs at the beginning of both the first and second half-periods of the periodic motion. Otherwise, the VI periodic motion is deemed to be out-of-phase and the ‘−’ sign is used. Finally, the two in-phase and out-of-phase linear modes of the system with zero clearance (δ = 0) are denoted by Lmm± and are, in fact, equivalent to L11±. The (incomplete) FEP of the Hamiltonian VI system for µ = σ = 0.1 is depicted in Fig. 6, with some representative VI periodic orbits presented in Fig. 7. The complexity of the bifurcations that generate the VI periodic orbits can be inferred from the bifurcation where T is the period of the motion. Similar to the ‘smooth’ NES [57], symmetric VI orbits correspond to synchronous oscilla- tions of the LO and the VI NES, and typically are represented by curves in the configuration plane (u1, u2) (cf. Fig. 29 in Appendix). Unsymmetric VI periodic orbits, labeled as Umnpq±, do not satisfy the conditions of the symmetric ones. These orbits correspond to asynchronous motions of the two oscillators and are represented by open or closed (Lissajous) curves in the configuration plane (u1, u2) (cf. Fig. 30 in Appendix). The integer index m denotes the number of half-waves in the VI NES response within a half-period, whereas the integer index n denotes the corresponding number of half-waves in the LO response; clearly, the ratio m:n indicates the order of nonlinear resonance that occurs between the VI NES and the LO for a given VI periodic orbit. Moreover, the index E or O de- notes the symmetry pattern of the vibro-impacts, and has meaning only for symmetric VI orbits: E(O) denotes an even (odd) symmetry of occurring vibro-impacts within a half-period; it follows that the notation E(O) implies that a vibro-impact occurs (does not occur) at quarter-period ξ = α = T/4. The integer indices p and q de- note the number of vibro-impacts that occur in the first and second quarter-period, respectively, of a given VI periodic orbit; it follows that for symmetric orbits p = q. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) 6, with some representative VI periodic orbits presented in Fig. 7. The complexity of the bifurcations that generate the VI periodic orbits can be inferred from the bifurcation diagrams of Fig. 8 where the initial velocities of the LO and the VI NES (for zero initial displacements) are depicted as functions of the total energy h. As mentioned previously, this complexity was anticipated in view of the degenerate vibro-impact nonlinearity of this system. The anticipated high complexity of the structure of VI periodic orbits dictates the use of careful notation for their representation in the FEP. In principle, the basic notation introduced in [57] for the FEP of the dynamics of the Hamiltonian system with ‘smooth’ NES is followed, with an additional index introduced characterizing the pattern of the occurring vibro-impacts. To this end, we employ the following notation for depicting the various types of VI periodic orbits in the FEP: Note that the FEP in Fig. 6 is obtained for system (2) with all displacements being normalized with respect to the clearance δ, Symmetric VI periodic orbits are denoted as SmnE(O)pp±, and satisfy the conditions, ui(ξ) = ±ui(ξ + T/2), ∀ξ ∈R, i = 1, 2, Fig. 6. The frequency–energy plot (incomplete) of periodic orbits of the Hamiltonian VI system for µ = σ = 0.1; the manifold of VI IOs is indicated by small dots, whereas the thresholds for vibro-impacts for the in-phase and out-of-phase modes are denoted by bullets; unstable branches are denoted by crosses. Fig. 6. The frequency–energy plot (incomplete) of periodic orbits of the Hamiltonian VI system for µ = σ = 0.1; the manifold of VI IOs is indicated by small dots, whereas the thresholds for vibro-impacts for the in-phase and out-of-phase modes are denoted by bullets; unstable branches are denoted by crosses. 5 a b c 7. Solutions of the NLBVP (7) for µ = σ = 0.1: (a) Symmetric VI periodic orbit on the backbone S13O00+ ≡S13+ (α = 1.7, h = 0.58875); (b) Symmetric VI period it S73O33−(α = 1.6459, h = 7.3348); (c) Unsymmetric VI periodic orbit U8853−(α = 4.9065, h = 1.9458) a a b b c Fig. 7. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) 28 in Appendix); and the branch of symmetric in-phase VI NNMs S(2m)(6m)Emm+ (m ≥1) or S(2n + 1)(6n + 3)Onn+ (n ≥0) which bifurcates out of the in-phase linear mode L11+, after which this linear mode also becomes unstable (cf. Fig. 27 in Appendix). For convenience, from hereon the shortened notations, S11−and S13+, will be adopted for these two main backbone branches, which will be referred to as backbone (global) branches of the FEP. Both backbone branches exist over broad frequency and energy ranges and, except for the neighborhoods of the bifurcation points with L11±, they correspond to oscillations that are mainly localized to the VI NES. A basic bifurcation in the VI FEP is the saddle-node (SN) bifurcation of the backbone branch S11−(at h ≈0.06), which signifies the elimination of the unstable branch S11−bifurcating out of L11−; the stable branch S11−generated after this SN bifurcation maintains its stability for increasing energies. As shown below, this SN bifurcation of the backbone branch S11−significantly affects the capacity of the VI NES for TET. The additional in-phase backbone branch S13+ system is purely linear. Clearly, only the in-phase and out-of-phase linear normal modes L11± exist below the energy thresholds. As we increase the energy of the motion above these energy thresholds, vibro-impacts start occurring, giving rise to two main branches of symmetric periodic VI NNMs: the branch of out-of- phase symmetric VI NNMs S(2m)(2m)Emm−(m ≥1, integer) or S(2n+1)(2n+1)Onn−(n ≥0, integer), which bifurcates from the out-of-phase linear mode L11−, after which this mode becomes unstable (cf. Fig. 28 in Appendix); and the branch of symmetric in-phase VI NNMs S(2m)(6m)Emm+ (m ≥1) or S(2n + 1)(6n + 3)Onn+ (n ≥0) which bifurcates out of the in-phase linear mode L11+, after which this linear mode also becomes unstable (cf. Fig. 27 in Appendix). For convenience, from hereon the shortened notations, S11−and S13+, will be adopted for these two main backbone branches, which will be referred to as backbone (global) branches of the FEP. Both backbone branches exist over broad frequency and energy ranges and, except for the neighborhoods of the bifurcation points with L11±, they correspond to oscillations that are mainly localized to the VI NES. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Solutions of the NLBVP (7) for µ = σ = 0.1: (a) Symmetric VI periodic orbit on the backbone S13O00+ ≡S13+ (α = 1.7, h = 0.58875); (b) Symmetric VI periodic orbit S73O33−(α = 1.6459, h = 7.3348); (c) Unsymmetric VI periodic orbit U8853−(α = 4.9065, h = 1.9458) Fig. 7. Solutions of the NLBVP (7) for µ = σ = 0.1: (a) Symmetric VI periodic orbit on the backbone S13O00+ ≡S13+ (α = 1.7, orbit S73O33−(α = 1.6459, h = 7.3348); (c) Unsymmetric VI periodic orbit U8853−(α = 4.9065, h = 1.9458) so that vibro-impacts occur whenever the absolute value of the relative displacement between the two particles becomes equal to unity in magnitude. Considering the original system (1) with clearance δ, its Hamiltonian is expressed as, ˆH = δ2H where H is the normalized Hamiltonian defined by (4). This implies that, for fixed system parameters, µ = 0.1 and σ = 0.1, the Hamiltonian structure of the original (non-normalized) system will be identical to that of Fig. 6; it follows that for larger (smaller) clearances, the entire structure of VI periodic orbits will be preserved but just shifted towards higher (lower) energy regimes. So, the introduced normalization allows us to study all possible VI responses of the original system by considering a single ‘normalized’ FEP for fixed mass and coupling stiffness ratios. It is interesting to note that this normalization does not hold for the system with smooth stiffness nonlinearity (i.e., the ‘smooth’ NES); this can be easily deduced when noting that the introduced normalization changes the form of the system with smooth nonlinearities. Indeed, the topological structure of the FEP of the system with ‘smooth’ NES with pure cubic stiffness nonlinearity (and the corresponding bifurcation structure of the Hamiltonian periodic orbits) is affected by both the mass ratio and the essential stiffness nonlinearity of the NES [57]. We now make some comments and remarks regarding the system is purely linear. Clearly, only the in-phase and out-of-phase linear normal modes L11± exist below the energy thresholds. As we increase the energy of the motion above these energy thresholds, vibro-impacts start occurring, giving rise to two main branches of symmetric periodic VI NNMs: the branch of out-of- phase symmetric VI NNMs S(2m)(2m)Emm−(m ≥1, integer) or S(2n+1)(2n+1)Onn−(n ≥0, integer), which bifurcates from the out-of-phase linear mode L11−, after which this mode becomes unstable (cf. Fig. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Bifurcation diagrams (incomplete) depicting the initial velocities as a function of the total energy h for µ = σ = 0.1: (a) VI NES; (b) LO (unstable branches a k d b ) Total energy Linear oscillator b Linear oscillator Fig. 8. Bifurcation diagrams (incomplete) depicting the initial velocities as a function of the total energy h for µ = σ = 0.1: (a) VI NES; (b) LO (unstable branches are marked by crosses). Fig. 8. Bifurcation diagrams (incomplete) depicting the initial velocities as a function of the total energy h for µ = σ = 0.1: marked by crosses). bifurcating out of the in-phase linear mode L11+ is stable until high energies where zones of instability appear. periodic motions, possessing frequencies that are rational multi- ples of one of the linearized eigenfrequencies of the system. Similar to the FEP of the system with ‘smooth’ NES [57], each subharmonic tongue is defined over a finite energy range, and is composed of a pair of branches of in- and out-of-phase subharmonic oscillations. Depending on the behavior of these VI subharmonic tongues with varying energy, the FEP is portioned into four main regimes, which are labeled as (I)–(IV) in Fig. 6. In Fig. 7(a) we depict a typical stable VI orbit lying on the in- phase backbone branch S13+. We note that, in the corresponding FEP of the system with ‘smooth’ NES studied in [57], there exist two backbone branches S11±. As indicated by the time series of Fig. 7(a), however, on the in-phase VI backbone branch three sign changes for the LO velocity within half a period are realized, com- pared to only one for the NES velocity; this high-frequency compo- nent becomes more prominent at higher energies (in addition, as will be shown in Section 3.1, this explains the 3:1 resonant modal interactions observed in wavelet transform spectra when the dy- namics is initiated on branch S13+ for weak damping). In the highest energy regime (IV), out-of-phase VI subharmonic orbits (both symmetric and unsymmetric) bifurcate out of the backbone branch S11−. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) A basic bifurcation in the VI FEP is the saddle-node (SN) bifurcation of the backbone branch S11−(at h ≈0.06), which signifies the elimination of the unstable branch S11−bifurcating out of L11−; the stable branch S11−generated after this SN bifurcation maintains its stability for increasing energies. As shown below, this SN bifurcation of the backbone branch S11−significantly affects the capacity of the VI NES for TET. The additional in-phase backbone branch S13+ so that vibro-impacts occur whenever the absolute value of the relative displacement between the two particles becomes equal to unity in magnitude. Considering the original system (1) with clearance δ, its Hamiltonian is expressed as, ˆH = δ2H where H is the normalized Hamiltonian defined by (4). This implies that, for fixed system parameters, µ = 0.1 and σ = 0.1, the Hamiltonian structure of the original (non-normalized) system will be identical to that of Fig. 6; it follows that for larger (smaller) clearances, the entire structure of VI periodic orbits will be preserved but just shifted towards higher (lower) energy regimes. So, the introduced normalization allows us to study all possible VI responses of the original system by considering a single ‘normalized’ FEP for fixed mass and coupling stiffness ratios. It is interesting to note that this normalization does not hold for the system with smooth stiffness nonlinearity (i.e., the ‘smooth’ NES); this can be easily deduced when noting that the introduced normalization changes the form of the system with smooth nonlinearities. Indeed, the topological structure of the FEP of the system with ‘smooth’ NES with pure cubic stiffness nonlinearity (and the corresponding bifurcation structure of the Hamiltonian periodic orbits) is affected by both the mass ratio and the essential stiffness nonlinearity of the NES [57]. We now make some comments and remarks regarding the ‘normalized’ FEP of Fig. 6. First, we note that the two bullets indicate the critical energy thresholds below which oscillations without vibro-impacts occur, and the dynamics of the two-DOF 6 Total energy Total energy VINES Linear oscillator a b grams (incomplete) depicting the initial velocities as a function of the total energy h for µ = σ = 0.1: (a) VI NES; (b) LO the in-phase linear mode L11+ is stable until periodic motions, possessing frequencies that Total energy VINES a VINES gy Total energy Linear oscillator b Fig. 8. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Finally, the lowest energy regime (I) of the FEP is defined for energies below the bifurcation point of the linear mode L11−. The manifold of VI IOs meets the stable out-of-phase linear mode L11− at a bifurcation point that coincides with the critical energy level hmin = σ/2; we recall that for h < hmin no vibro-impacts are possible, and the dynamics of the system is completely linear. It is interesting to note that the complexity of the FEP is solely due to the existence of the clearance δ that gives rise to vibro- impacts. Indeed, in the limit of zero clearance, δ →0, the entire structure of VI orbits depicted in the FEP of Fig. 6 collapses to two horizontal lines corresponding to the linear modes L11±. We conclude that, due to the degeneracy of the VI dynamics, even a small clearance can generate significant complexity, including chaotic orbits, as discussed below. As in the case of the system with ‘smooth’ NES [57], there exists a countable infinity of subharmonic tongues, corresponding to symmetric or unsymmetric VI subharmonic motions with different patterns of vibro-impacts realized during a cycle of the oscillation. Unsymmetric VI periodic orbits are represented by closed (Lissajous) curves in the configuration plane of the system. In Fig. 7(b) and(c) we depict two representative symmetric and unsymmetric VI orbits on two subharmonic branches of the FEP. The global dynamics of the Hamiltonian VI system (2) can be studied by constructing Poincaré maps resulting from the projection of the isoenergetic dynamics (i.e., of the dynamical flow corresponding to fixed value of h) on the two-dimensional ‘cut section’ There exists a third class of VI motions in the FEP, which are denoted as VI impulsive orbits (VI IOs). These are VI periodic solutions corresponding to zero initial conditions of the system, except for the initial velocity of the LO. A VI IO represents, in essence, the response of the system being initially at rest and forced by a single impulse applied to the LO at time ξ = 0+. Apart from the clear similarity of a VI IO to the Green’s function defined for the corresponding linear system, the importance of studying this class of orbits stems from their essential role regarding passive targeted energy transfer (TET) from the linear oscillator to the NES [57,58,68,69]. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) With increasing energy they form sub- harmonic tongues of out-of-phase motions with almost constant frequencies, until they reach the manifold of VI IOs (see discus- sion below) after which they change to in-phase motions until they coalesce with the in-phase backbone branch at specific energy p g) A different class of VI periodic solutions of the FEP lies on subharmonic tongues (local branches); these are multi-frequency 7 The numerical results indicate that VI periodic and quasiperiodic IOs form a manifold in the FEP, containing a countable infinity of periodic IOs and an uncountable infinity of quasiperiodic IOs. The numerical results indicate that VI periodic and quasiperiodic IOs form a manifold in the FEP, containing a countable infinity of periodic IOs and an uncountable infinity of quasiperiodic IOs. levels; this signifies the end of these tongues and the elimination of the corresponding subharmonic motions for higher energy val- ues. This is similar to what was observed in the FEP of the system with ‘smooth’ NES [57]. However, the unsymmetric subharmonic tongues do not fold on themselves to reach back to the out-of- phase backbone branch S11−, unlike the case of smooth essential nonlinearity of the NES [57]. For the system under consideration, the approximation to the manifold of VI IOs was computed numerically, and is depicted in Fig. 6; in general, the manifold appears as a smooth curve, with the exception of a number of outliers (which is due to the adopted convention for the frequency index (m:n) in the FEP). In each VI subharmonic tongue, a VI IO is realized whenever the relative motion between that LO and the VI NES changes from in-phase to out-of-phase. Representative VI IOs are depicted in Fig. 9. In general, the IOs become increasingly more localized to the VI NES as their energy decreases, a result which is in agreement with previous results for NESs with smooth essential nonlinearities [69]. As energy increases, the VI IOs tend towards the in-phase mode; that is, their representation in the configuration plane (u1, u2) tends to a straight line of slope 0.084π because the eigenvector for L11+ on the (u1, u2) plane is equal to (1, 3.702). 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Moreover, we note there is no critical energy threshold for the appearance of VI IOs since there are no low energy VI motions (the system is linear for low energy levels); the dominant frequency of a VI IO depends on the clearance δ in the physical model (i.e., on the energy regime where the VI periodic orbits exist). In regime (III) of the FEP the bifurcation behavior of subhar- monic tongues is similar to regime (IV). The apparent difference is that the manifold of VI IOs undergoes a discontinuous transi- tion on branch S31O11−, caused by the two bifurcations of that branch with the unsymmetric subharmonic branches U(15)578− and U8353−in that region (see Detail I in Fig. 6). The subharmonic orbits in regime (II) exhibit different bifur- cation behavior than in regimes (IV) and (III). In fact, it appears that there are no subharmonic tongues bifurcating from S11−; in- stead, small subharmonic tongues appear to lie along the manifold of VI IOs, and eventually merge with the in-phase backbone branch S13+ with decreasing energy. For example, the in-phase unsym- metric branch U21+ bifurcates from S13+ and transforms itself to the out-of-phase unsymmetric branch U21−after it crosses the manifold of VI IOs. In addition, for frequencies in between the two linearized frequencies ω1 and ω2 there exist multiple subharmonic branches bifurcating in a degenerate (higher co-dimensional) bi- furcation from the in-phase linear mode L11+ (see Detail II in the FEP of Fig. 6). These subharmonic branches coexist with the in-phase backbone branch S13+, which is unstable in most of regime (II). Examples of this type of subharmonic branches are U2201+, U5511+, U4421+, . . . in the FEP of Fig. 6. Due to the degenerate VI nonlinearity of the system under con- sideration, it is expected that higher co-dimensional bifurcations will occur in its dynamics. One case of such degenerate bifurca- tions in presented in Detail II of the FEP of Fig. 6, where mul- tiple branches of symmetric and unsymmetric VI periodic orbits (branches S95E22−, U5421−, U4520−, S55O11−, U3201−, . . .) are noted to bifurcate from the in-phase linear mode L11+ at the point of generation of the in-phase backbone branch S13+. In ad- dition, VI periodic orbits below the branch S73E33−appear to lie along the VI IO manifold; for example, tongues U21±, S95E22−, U5421−, U4520−, S55O11−, U3401−and U44−in the FEP of Fig. 6. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Two representative VI IOs for µ = σ = 0.1 (left U5231−; right U4122−): (a) Displacements u1 (VI NES) and u2 (LO); (b) relative responses u1−u2; (c) representations in the configuration plane (u1, u2). µ = σ = 0.1 (left U5231−; right U4122−): (a) Displacements u1 (VI NES) and u2 (LO); (b) relative responses u1−u2; (c) representatio ‘stochastic seas’ (i.e., the regions of chaotic motions) in the Poincaré maps diminish, and the domains of regular motion expand. homoclinic orbits of S11−provides conditions for optimal VI TET, since large-amplitude relative displacements between the LO and the VI NES are realized in that region, and the time scale of the re- sulting TET is affected as well. An additional use of the Poincaré map is that it can help us iden- tify or infer the existence of global features of the dynamics, such as homoclinic and heteroclinic loops. For example, at the energy level h = 0.06 (cf. Fig. 10(c)) we identify stable and unstable VI pe- riodic orbits U44−in the neighborhood of the linear mode L11− and the unstable NNM S11−(note also the location of this branch in the FEP of Fig. 6). This infers the existence of a homoclinic loop that connects the unstable periodic orbit S11−. The topologies of VI IOs on branches such as U44−(which lie in the neighborhood of the SN bifurcation of S11−) are greatly influenced by the family of homoclinic orbits of the unstable branch S11−and significantly affect the efficiency of TET from the LO to the NES. This is similar to what occurs for the case of smooth nonlinearity [69], where it was found that, close to this family of homoclinic orbits, conditions for optimal TET are realized. Indeed, as shown in the next section, excitation of stable VI IOs in the neighborhood of the family of Apart from the compact representation of VI periodic motions, the FEP is a valuable tool for understanding the nonlinear reso- nant interactions (transient resonance captures or TRCs [57,58]) that govern energy exchanges and TET during damped transitions in the weakly dissipative VI system. This is due to the fact that, for sufficiently weak dissipation (caused by inelastic vibro-impacts, viscous damping or both), the damped VI dynamics is expected to be realized in neighborhoods of branches in the FEP of the un- derlying VI Hamiltonian system. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Indeed, in the case of the NES with smooth nonlinearities, IOs (which, under some conditions are in the form of nonlinear beats) play the role of bridging orbits occurring in the initial phase of TET and ‘channeling’ a significant portion of the applied impulsive energy from the linear primary system (in this case the LO) to the NES at a relatively fast time scale; this represents the most efficient scenario for passive TET (i.e., TET through nonlinear beats [58,68,69]). Although the aforementioned results refer to damped impulsive orbits, the dynamics of the underlying Hamiltonian system determines, in essence, the dynamics of the damped system as well, provided that damping is sufficiently small. It follows that the IOs of the VI Hamiltonian system govern, in essence, the initial (critical) phase of TET from the LO to the NES. Σ = (u1, u2, ˙u1, ˙u2) ∈R4/H(u1, u2, ˙u1, ˙u2) : u2 ≡0, ˙u2 > 0 (10) (10) which is transverse to the flow except at points where ˙u2 = 0. Similar Poincaré map construction for vibro-impact dynamics were considered in Mikhlin et al. [29]. The resulting Poincaré maps are depicted in Fig. 10. Below the energy level h = hmin = σ/2 = 0.05 no vibro-impacts occur (cf. Fig. 10(a)), and the only possible periodic solutions are the linear stable modes L11±. At energy levels above this critical threshold (cf. Fig. 10(b)–(h)), vibro-impacts occur (at \|u2 −u1\| = 1), and generate a countable infinity of subharmonic VI orbits that replace the two linear modes L11±; this complex structure of orbits is a direct consequence of the non-integrability of the Hamiltonian VI dynamics. When vibro- impacts occur, the sections of the Poincaré maps corresponding to \|u1\| > 1 are cut-off from the Poincaré maps, and the last boun- ding points that are included in the map are those for which the conditions {u2 = 0 and \|u1\| = 1} hold. For increasing energies, the 8 a b c Fig. 9. Two representative VI IOs for µ = σ = 0.1 (left U5231−; right U4122−): (a) Displacements u1 (VI NES) and u2 (LO); (b) relative responses u1−u2; (c) representati in the configuration plane (u1, u2). a a b b c c Fig. 9. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) This aspect was demonstrated in earlier work [5], where the wavelet transform (WT) spectra of the damped response of the (unnormalized) system were super- imposed on the Hamiltonian FEP. The motion was initiated on a subharmonic tongue, and three distinct stages were distinguished in the resulting damped VI transition: In an initial stage, the motion 9 Fig. 10. Poincaré maps of VI dynamics for µ = σ = 0.1 varying energy. Fig. 10. Poincaré maps of VI dynamics for µ = σ = 0.1 varying energy. 10 for the case of combined inelastic impacts and viscous dissipation (cf. Fig. 13(b), (d)), where the damped dynamics traces, primarily the backbone branch (i.e., there occurs an immediate 1:1 TRC of the dynamics of the NES and the in-phase mode L11± right from the beginning of the motion), and, secondarily, higher-frequency subharmonic tongues. Hence, the addition of weak viscous dissipa- tion in the LO does not appear to affect significantly the VI damped transitions. remains in the neighborhood of the subharmonic tongue where it is generated, yielding an initial persistent subharmonic TRC. As a result, subharmonic VI TET takes place from the LO to the NES, and efficient energy dissipation occurs. In the second stage of the damped motion, the dynamics makes a transition to a differ- ent lower energy subharmonic tongue, which signifies the occur- rence of a different subharmonic TRC (and subharmonic TET) in the damped dynamics. Escape from this second TRC regimes leads to a transition of the dynamics to the manifold of VI IOs during the third stage of the motion, before the dynamics becomes linear, and un- dergoes a final transition to the linear mode L11+ (the final stage of the response). We focus now on the study of the mechanisms that govern VI TET by fixing ρ = 0.7 and λ = 0.005λcr, in order to compare the dynamical mechanisms for VI TET to the corresponding mechanisms for the case of ‘smooth’ NES (i.e., with purely cubic essential stiffness nonlinearity) discussed in [58]. In the smooth case the following three mechanisms for TET were established: (a) fundamental TET, where the damped in-phase NNM invariant manifold S11+ is excited; (b) subharmonic TET, where a low- frequency subharmonic tongue is excited; and (c) TET through nonlinear beats, where an IO close to the 1:1 resonance manifold of the dynamics is excited. 3. Vibro-impact transitions in the dissipative case: VI TET In Fig. 14 we present the damped response of the system for initial conditions on the in-phase backbone branch S13+ and initial normalized energy h ≈10.0. There are four distinct stages in the damped response, which are denoted as stages A–D in Fig. 14. In the highly energetic initial stage A there occurs a 1:1 TRC in the dynamics, with the response possessing a strong harmonic at the frequency ω1 of the in-phase linear mode and a weaker harmonic at 3ω1. This is evident by examining the detailed plot depicted in Fig. 15(a), where it is clear that the relative transient response u1−u2 in stage A possesses a third harmonic component; moreover, it is noted that the WT spectrum of the relative response possesses two clear components, a main one at frequency ω1, and a secondary one at 3ω1 on the subharmonic branch S31O11−. This leads to fundamental VI TET from the LO to the VI NES, with almost 85% of the initial energy (nearly 40% by the LO and 45% by the VI NES) being dissipated during this initial stage of the motion. The nonlinear modal interactions that lead to fundamental VI TET will be examined in more detail later by the method of empirical mode decomposition (EMD), where the governing 1:1 TRC will be more clearly identified. We consider now the weakly dissipative normalized system (2) with λ ̸= 0 and inelastic impacts. Then, the relations (3) that compute the normalized velocities of the LO and the VI NES immediately after an impact in terms of the corresponding velocities before impact are replaced by the following expressions ˙u+ 1 = (µ −ρ)˙u− 1 + (1 + ρ)˙u− 2 1 + µ , ˙u+ 2 = µ(1 + ρ)˙u− 1 + (1 −ρµ)˙u− 2 1 + µ (11) (11) where 0 < ρ ≤1 is the coefficient of restitution. Through the numerical simulations of this section, and unless otherwise noted, we assume that µ = σ = 0.1, ρ = 0.7 and λ = 0.005λcr, where λcr = 2 is the value of critical viscous damping for the LO (hence, weak viscous damping is assumed). p g ) Before we proceed to analyze damped transitions, we investi- gate the competition between the two energy dissipation sources present in the system, namely, viscous dissipation in the LO and in- elastic impacts in the VI NES. 2. Hamiltonian system with elastic vibro-impacts: Periodic orbits in the frequency–energy plot (FEP) Our study of TET in the VI case will follow similar lines, by considering energy exchanges between the LO and the NES for alternative types of initial excitation of the system. In particular, we will study VI TET when in-phase or out-of-phase periodic orbits lying on backbone and subharmonic tongues are excited, as well as when the damped motion is initiated by exciting VI IOs at various energy levels. In what follows we examine each of these cases separately. It will be shown, in the next section, that the transition of the damped dynamics along the manifold of VI IOs during the third stage of the motion is associated with a complex series of multiple TRCs with subharmonic tongues existing in the vicinity of this manifold. It follows that, by studying VI transitions in the FEP and relating them to rates of energy dissipation by the VI NES, we should be able to identify the most effective damped transitions from a TET point of view. In a more general context, we will perform a systematic study of the dynamics of TET in the two-DOF system of Fig. 3 in the next section by assuming inelastic impacts and viscous dissipation in the LO, and analyzing the resulting transient responses by numerical WTs. Then, we will superimpose the resulting WT spectra on the FEP of Fig. 6, in an effort to interpret the damped transitions in terms of the underlying Hamiltonian dynamics, and to identify the governing dynamical mechanisms for VI TET. 3. Vibro-impact transitions in the dissipative case: VI TET For this purpose, the damped motion was initiated on the stable VI IO lying on the subharmonic tongue U8344−. In Figs. 11–13 we depict the damped responses for the following cases: (a) for no viscous dissipation in the LO and inelas- tic impacts (ρ = 0.7, λ = 0 — Fig. 11); (b) for viscous dissipation and purely elastic impacts (ρ = 1, λ = 0.005λcr — Fig. 12); and (c) for a combination of viscous dissipation and inelastic impacts (ρ = 0.7, λ = 0.005λcr — Fig. 13). Comparing Figs. 11(b), (d) and 12(b), (d), we note distinct patterns of energy exchange and dis- sipation in the damped transient dynamics. For purely elastic im- pacts (i.e., after the last vibro-impact) the response is nearly linear and the WT spectra lie along the two linear modes L11±; when in- elastic impacts occur, there occurs a strongly nonlinear transition of the VI dynamics along strongly nonlinear subharmonic tongues and the in-phase backbone branch S13+, until, at the later stage of the response, the dynamics settles into linearized motion along the modes L11±. A similar, albeit weaker, nonlinear transition is noted Stage B (cf. Fig. 15(b)) corresponds to a regime of pure 1:1 TRC as the third harmonic component is nearly eliminated, and the LO and the VI NES execute in-phase oscillations with frequencies approximately equal to ω1. It is clear that the weakly damped dynamics follows approximately the in-phase backbone branch S13+ until this branch becomes unstable (i.e., at the bifurcation point where the subharmonic branches U11+ and U21± bifurcate out of this branch; cf. Fig. 6). This provides further evidence of the connection between the weakly damped dynamics and the dynamics of the underlying Hamiltonian system. During stage C of the damped motion there occurs a complex series of TRCs along subharmonic tongues on the FEP, whereas in the low energy stage D vibro-impacts cease to occur, the motion is purely linear, and energy dissipation is solely due to viscous damping in the LO. As expected, the linear dynamics consists of a combination of the damped analogs of the linear in-phase and 11 a b c d ee 1. 3. Vibro-impact transitions in the dissipative case: VI TET Damped transition for ρ = 0.7, λ = 0: (a) Time series; (b) instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d um of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. of-phase modes L11±, with mode L11+ being more dominant e response. We conclude that in this numerical simulation there occurs amental VI TET due to 1:1 TRC of the dynamics of the VI NES at The next simulation examines the damped response of system for initial conditions on the out-of-phase backbone bra S11−at h ≈0.8 (cf. Fig. 16). There is insignificant TET from th to the NES in this case, since (as in the case of smooth nonlinea a b a b b a b c d c d d ee 11. Damped transition for ρ = 0.7, λ = 0: (a) Time series; (b) instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT ee Fig. 11. Damped transition for ρ = 0.7, λ = 0: (a) Time series; (b) instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. out-of-phase modes L11±, with mode L11+ being more dominant in the response. The next simulation examines the damped response of the system for initial conditions on the out-of-phase backbone branch S11−at h ≈0.8 (cf. Fig. 16). There is insignificant TET from the LO to the NES in this case, since (as in the case of smooth nonlinearity) the initial energy of the motion localizes predominantly to the VI NES right from the beginning of the motion; then, localization to the VI NES is maintained throughout, as the damped VI motion approximately traces the backbone branch S11−. In fact, in this case vibro-impacts occur only during a short initial stage of the We conclude that in this numerical simulation there occurs fundamental VI TET due to 1:1 TRC of the dynamics of the VI NES at frequency ω1. Recalling that ω1 is the natural frequency of the in- phase linear mode L11+, we conclude that during fundamental VI TET the LO and the VI NES engage in in-phase 1:1 resonance capture. 3. Vibro-impact transitions in the dissipative case: VI TET This VI TET mechanism is analogous to fundamental TET discussed in the case of smooth nonlinearity [58]. 12 Damped transition for ρ = 1, λ = 0.005λcr: (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the e VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. (i.e., for ξ < 10; Fig. 16(a)), where almost 90% of total is dissipated. In the purely linear regime where no vibro- (f ξ 10) th i i l d f We conclude that there is immediate escape of the transient damped dynamics from the initially excited out-of-phase back- bone branch S11 follo ed b settlement of the response in alter ped transition for ρ = 1 λ = 0 005λ : (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the Fig. 12. Damped transition for ρ = 1, λ = 0.005λcr: (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. motion (i.e., for ξ < 10; Fig. 16(a)), where almost 90% of total energy is dissipated. In the purely linear regime where no vibro- impacts occur (for ξ > 10) the response is mainly composed of the damped analogue of the out-of-phase linear mode L11−, with a weaker participation of the in-phase damped mode L11+. The participation of these closely spaced modes in the linear response produces a beat phenomenon, which is evidenced by the strong energy exchanges between oscillators noted in Fig. 16(b). motion (i.e., for ξ < 10; Fig. 16(a)), where almost 90% of total energy is dissipated. In the purely linear regime where no vibro- impacts occur (for ξ > 10) the response is mainly composed of the damped analogue of the out-of-phase linear mode L11−, with a weaker participation of the in-phase damped mode L11+. The participation of these closely spaced modes in the linear response produces a beat phenomenon, which is evidenced by the strong energy exchanges between oscillators noted in Fig. 16(b). 3. Vibro-impact transitions in the dissipative case: VI TET We conclude that there is immediate escape of the transient damped dynamics from the initially excited out-of-phase back- bone branch S11−, followed by settlement of the response in alter- native response regimes. This is a general conclusion drawn from the performed numerical simulations, and holds for motions that are initiated on all branches and tongues of the FEP other than the in-phase backbone branch S13+ (we note that this was also the case for the case of smooth nonlinearity [58]). 13 Damped transition for ρ = 0.7, λ = 0.005λcr: (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by e VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. TET through excitation of VI IOs ng established the mechanism of fundamental TET in the case of NES with smooth nonlinearity IOs play an important rol far as TET is concerned; this holds especially for IOs lying in neighborhood of the family of homoclinic orbits of the unsta Fig. 13. Damped transition for ρ = 0.7, λ = 0.005λcr: (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. 3.2. VI TET through excitation of VI IOs 3.2. VI TET through excitation of VI IOs case of NES with smooth nonlinearity IOs play an important role as far as TET is concerned; this holds especially for IOs lying in the neighborhood of the family of homoclinic orbits of the unstable out-of-phase damped NNMs S11−, close to the 1:1 resonance manifold of the damped dynamics [69]. Having established the mechanism of fundamental TET in the system with VI NES, we now consider the possibility of alternative mechanisms for VI TET based on the excitation of VI IOs. As shown in [58], excitation of IOs on certain energy ranges provides the mechanism for most efficient TET in the system with an NES with smooth nonlinearity. In the following we investigate efficiency of TET from the LO to the NES when VI IOs are excited in the four previously defined regimes (I)–(IV) of the FEP. We note that in the In Fig. 17 we depict the damped response of the system when a VI IO in regime (I) is excited. Since the initial energy of the motion is relatively low, vibro-impacts occur only during the short- duration initial stage of the dynamics, and afterwards the dynamics become completely linear, involving continuous energy exchanges 14 Damped transition initiated on S13+ (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of tota d by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP; Stages A–D of the n are indicated. n the two linear modes of the system (with no vibro- s) at frequencies ω1 and ω2. Due to the closely spaced natural frequencies, a linear beat develops and energy is the in-phase backbone branch S13+ is unstable at the s initial energy level considered in this simulation. As a resu damped dynamics may be divided into four distinct stages, l Fig. 14. Damped transition initiated on S13+ (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP; Stages A–D of the damped transition are indicated. between the two linear modes of the system (with no vibro- impacts) at frequencies ω1 and ω2. 3.2. VI TET through excitation of VI IOs Due to the closely spaced linear natural frequencies, a linear beat develops and energy is predominantly dissipated by viscous dissipation in the LO. In this case insignificant TET from the LO to the VI NES occurs. the in-phase backbone branch S13+ is unstable at the specific initial energy level considered in this simulation. As a result, the damped dynamics may be divided into four distinct stages, labeled by A–D in Fig. 18. Stages A–C are strongly nonlinear, whereas, the low energy stage D is linear with no vibro-impacts occurring there. In stage A the damped dynamics follows approximately the tongue S95E22−(where the motion is initiated) with decreasing energy. Nearly 50% of the total initial energy is dissipated during this Stage The damped responses for initial excitation of a VI IO on the subharmonic tongue S95E22−in regime (II) of the FEP are depicted in Fig. 18. In this case the dynamics cannot exhibit a 1:1 TRC, since 15 Fig. 15. Close-ups of the time series (left column) and WTs on the FEP (right column) depicted in Fig. 14. e response, with 33% of total energy being dissipated due to the initial energy level considered), and five distinct stages Fig. 15. Close-ups of the time series (left column) and WTs on the FEP (right column) depicted in Fig. 14. the initial energy level considered), and five distinct stages of the damped motion (labeled as A–E in Fig. 19) are inferred. In stage A the damped motion follows the subharmonic tongue S31O11− which acts as bridging orbit for the dynamics to make the transition from its initial state to 1:1 TRC (which is realized in stage B). In stage B there occurs a 1:1 TRC as the stable in-phase backbone branch S13+ is excited; as a result, fundamental TET from the LO to the VI NES is realized, so that nearly 50% of the total initial energy is dissipated by the VI NES by the end of this stage of the motion. As energy decreases due to viscous dissipation and inelastic impacts, the in-phase backbone branch S13+ becomes unstable and the damped dynamics makes a transition to stages of the response, with 33% of total energy being dissipated due to TET from the LO to the VI NES. 3.2. VI TET through excitation of VI IOs The damped dynamics in stages B and C is complex, as it undergoes transitions along subharmonic tongues such as U5421−, U4520−, S55O11−and U3201−lying close to the manifold of IO. Finally, when sufficient energy is dissipated and no additional vibro-impacts can occur the dynamics settles into the linear stage D, where predominant contribution of mode L11+ is realized. In Fig. 19 the damped dynamics for excitation of the VI IO on the subharmonic tongue S31O11−in regime (III) of the FEP is presented. For the selected initial energy level for this simulation, 1:1 TRC is possible (since the backbone branch S13+ is stable at 16 Damped transition initiated on S11−(ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of tota d by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. D; these stages are similar to those occurring in regime (II) FEP, and the dynamics follows complex transitions along monic tongues, similar to the ones depicted in Fig. 18. At Finally, when IOs in the high energy regime (IV) of th are excited (cf. Fig. 20) the damped transitions are sim those realized in regime (III), with TET efficiency at the Fig. 16. Damped transition initiated on S11−(ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. C and D; these stages are similar to those occurring in regime (II) of the FEP, and the dynamics follows complex transitions along subharmonic tongues, similar to the ones depicted in Fig. 18. At the later, low energy stage E the dynamics is linear and dominated by mode L11+. We conclude that by exciting VI IOs lying in Regime III of the FEP the ‘bridging orbit’ scenario is realized (as in the case of the system with ‘smooth’ NES), leading eventually to the fundamental VI TET. This scenario yields efficient TET from the LO to the VI NES. Finally, when IOs in the high energy regime (IV) of the FEP are excited (cf. Fig. 3.2. VI TET through excitation of VI IOs 20) the damped transitions are similar to those realized in regime (III), with TET efficiency at the end of fundamental VI TET reaching the level of nearly 55%. In conclusion, we identify two mechanisms for VI TET, namely, fundamental VI TET due to 1:1 TRC and VI TET through excitation of a VI IO leading eventually to fundamental VI TET. These are sim- ilar to the corresponding TET mechanisms for the case of ‘smooth’ NES. No subharmonic VI TET (caused by TRC of the dynamics on an 17 7. Damped transition initiated on a VI IO in Regime (I) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) perce l energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. ted VI subharmonic tongue) could be realized in the numerical lations of the dynamics of the VI system under consideration, e VI dynamics seem to engage in series of TRCs involving mul- mechanism for TET in systems with very weak viscous damping weakly inelastic impacts. A final conclusion drawn from the pr ous simulations is that lack of fundamental TET in regimes (I) Fig. 17. Damped transition initiated on a VI IO in Regime (I) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. isolated VI subharmonic tongue) could be realized in the numerical simulations of the dynamics of the VI system under consideration, as the VI dynamics seem to engage in series of TRCs involving mul- tiple subharmonic tongues (instead of an isolated one) lying close to the manifold of VI IOs. However, as shown in the simulations of [5], subharmonic VI TET is indeed possible in the VI system of Fig. 3. Therefore, we conjecture that subharmonic VI TET can be a isolated VI subharmonic tongue) could be realized in the numerical simulations of the dynamics of the VI system under consideration, as the VI dynamics seem to engage in series of TRCs involving mul- tiple subharmonic tongues (instead of an isolated one) lying close to the manifold of VI IOs. 3.2. VI TET through excitation of VI IOs However, as shown in the simulations of [5], subharmonic VI TET is indeed possible in the VI system of Fig. 3. Therefore, we conjecture that subharmonic VI TET can be a mechanism for TET in systems with very weak viscous damping and weakly inelastic impacts. A final conclusion drawn from the previ- ous simulations is that lack of fundamental TET in regimes (I) and (II) of the FEP can be attributed to the instability of the in-phase backbone branch S13+ in the corresponding energy ranges. This is an additional indication of the strong relation that exists between the Hamiltonian dynamics and the weakly damped transitions. 18 Damped transition initiated on a VI IO in Regime (II) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) perce nergy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. ndamental VI TET mechanism by means of empirical mode position (EMD) Decomposition of nonlinear damped transitions by EMD lea multi-scale nonlinear identification of the governing dyna and provides the means for interpreting nonlinear reson (modal) interactions between coupled oscillators as we Fig. 18. Damped transition initiated on a VI IO in Regime (II) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. Fig. 18. Damped transition initiated on a VI IO in Regime (II) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total in of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u on a VI IO in Regime (II) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percenta nd the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. Fig. 18. 3.2. VI TET through excitation of VI IOs Damped transition initiated on a VI IO in Regime (II) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. 3.3. Fundamental VI TET mechanism by means of empirical mode decomposition (EMD) 3.3. Fundamental VI TET mechanism by means of empirical mode decomposition (EMD) Decomposition of nonlinear damped transitions by EMD leads to multi-scale nonlinear identification of the governing dynamics, and provides the means for interpreting nonlinear resonance (modal) interactions between coupled oscillators, as well as the time (or frequency) scales where these modal interactions occur [71]. The specific nonlinear resonance interactions that lead to TET in the VI system can be analyzed through EMD (for example, see [70]). We demonstrate this by analyzing in detail the mechanism for fundamental VI TET and showing that it is a 1:1 TRC. To this end, we analyze the high energy damped transition of Fig. 14 for the motion initiated on the in-phase backbone branch S13+. [ ] In Fig. 21 we depict the results of EMD analysis utilizing Matlab- based codes [72], from which we conclude that both responses of the LO and VI can be decomposed into two intrinsic mode functions (IMFs) (Fig. 21(a) and (b)), respectively. Thus, the exact 19 amped transition initiated on a VI IO in Regime (III) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) perc ergy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. es can be accurately reconstructed with the second IMFs, apture the dominant frequency components of the original ies (Fig. 21(c)). It is interesting to observe that the first IMFs oscillators detect all occurring impacts, so that the second e smooth functions possessing frequency components one can detect that the motions of both oscillators are ou phase whenever vibro-impacts occur. This observation exp (i) the enhanced energy dissipation due to inelastic vibro-im in the system considered, and (ii) the reason behind the that vibro impacts hardly contribute to the observed nonl Fig. 19. 3.2. VI TET through excitation of VI IOs Damped transition initiated on a VI IO in Regime (III) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. one can detect that the motions of both oscillators are out-of- phase whenever vibro-impacts occur. This observation explains (i) the enhanced energy dissipation due to inelastic vibro-impacts in the system considered, and (ii) the reason behind the fact that vibro-impacts hardly contribute to the observed nonlinear resonance interactions, as these occur in-phase and involve mainly 1:1 resonance captures. This suggests that the seemingly 3:1 resonant interaction (particularly at the initial TET stage) indicated by the WT spectra superimposed to the FEP (Fig. 14(e)) may be, in responses can be accurately reconstructed with the second IMFs, which capture the dominant frequency components of the original time series (Fig. 21(c)). It is interesting to observe that the first IMFs for both oscillators detect all occurring impacts, so that the second IMFs are smooth functions possessing frequency components completely free of nonsmooth effects due to vibro-impacts. Fig. 22 provides a more detailed study of the first IMFs of the LO and the VI NES, from where the patterns of vibro-impacts at different stages of the damped response can be clearly inferred; indeed, responses can be accurately reconstructed with the second IMFs, which capture the dominant frequency components of the original time series (Fig. 21(c)). It is interesting to observe that the first IMFs for both oscillators detect all occurring impacts, so that the second IMFs are smooth functions possessing frequency components completely free of nonsmooth effects due to vibro-impacts. Fig. 22 provides a more detailed study of the first IMFs of the LO and the VI NES, from where the patterns of vibro-impacts at different stages of the damped response can be clearly inferred; indeed, 20 Damped transition initiated on a VI IO in Regime (IV) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) per nergy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. urely 1:1 TRC following the in-phase NNM branch S13+ from rt. 3.2. VI TET through excitation of VI IOs ore demonstrating this latest argument by Hilbert spectral s, we compute the (averaged) frequency of vibro-impact 2π. The period and frequency of vibro-impacts evaluated th are assumed to correspond to the instants at (ξi+1−ξi)/2, ∀ Fig. 23, we observe that the three distinct VI TET regimes are represented by these two quantities. In stage A which d Fig. 20. Damped transition initiated on a VI IO in Regime (IV) (ρ = 0.7, λ = 0.005λcrit): (a) Time series; (b) percentage of total instantaneous modal energy; (c) percentage of total energy dissipated by the LO and the VI NES; (d) WT spectrum of the relative displacement u1 −u2; (e) WT spectrum of u1 −u2 superimposed on the FEP. fact, purely 1:1 TRC following the in-phase NNM branch S13+ from the start. 2π. The period and frequency of vibro-impacts evaluated this way are assumed to correspond to the instants at (ξi+1−ξi)/2, ∀i. From Fig. 23, we observe that the three distinct VI TET regimes are clearly represented by these two quantities. In stage A which dictates the initial VI TET period, fluctuations of impact frequency in the high-frequency regime above 3 are observed; that is, occurrence of vibro-impacts is irregular. On the other hand, vibro-impacts occur almost at a constant rate during stage B (at circular frequencies around 1.5); and in stage C the vibro-impacts exhibit a more 2π. The period and frequency of vibro-impacts evaluated this way are assumed to correspond to the instants at (ξi+1−ξi)/2, ∀i. From Fig. 23, we observe that the three distinct VI TET regimes are clearly represented by these two quantities. In stage A which dictates the initial VI TET period, fluctuations of impact frequency in the high-frequency regime above 3 are observed; that is, occurrence of vibro-impacts is irregular. On the other hand, vibro-impacts occur almost at a constant rate during stage B (at circular frequencies around 1.5); and in stage C the vibro-impacts exhibit a more Before demonstrating this latest argument by Hilbert spectral analysis, we compute the (averaged) frequency of vibro-impact occurrences in Fig. 23. First, the period of vibro-impacts on average can be estimated by Ti = ξi+1 −ξi, i = 1, 2, . . . , N −1 where N is the total number of impacts. 3.2. VI TET through excitation of VI IOs Then, its inverse 1/Ti can be regarded as the averaged impact frequency; and the corresponding circular frequency can be computed by multiplying this result by 21 Fig. 21. EMD analysis of the damped transition depicted in Fig. 14: (a) and (b) IMFs of the VI NES and LO responses, respectively; (c) comparison of the original responses o the corresponding second IMFs; (d) evolution of the phase difference, θ1; (e) phase plane representation of the dynamics of the phase difference θ1; (f) refined damped ransition of the FEP where only the second IMFs of the responses are considered. rregular pattern. In particular, the irregular occurrence of vibro- mpacts at the initial stage of the damped transition can be another evidence that the 3:1 resonant interaction at stage A is, in fact, a numerical artifact exclusively due to impacts, whereas in actuality the LO and the VI NES are engaged in 1:1 in-phase transient instantaneous phase of the second IMF of the VI NES response computed by application of the numerical Hilbert transform similarly, we denote by φ2 the corresponding phase of the second IMF of the LO response; finally, we define θ1 = φ1 −φ2 as the phase difference between the two oscillators Then TRCs occurring Fig. 21. EMD analysis of the damped transition depicted in Fig. 14: (a) and (b) IMFs of the VI NES and LO responses, respectively; (c) comparison of the original responses to the corresponding second IMFs; (d) evolution of the phase difference, θ1; (e) phase plane representation of the dynamics of the phase difference θ1; (f) refined damped transition of the FEP where only the second IMFs of the responses are considered. instantaneous phase of the second IMF of the VI NES response computed by application of the numerical Hilbert transform; similarly, we denote by φ2 the corresponding phase of the second IMF of the LO response; finally, we define θ1 = φ1 −φ2 as the phase difference between the two oscillators. Then, TRCs occurring between the second (dominant) IMFs of the responses of the two oscillators can be studied in detail. Indeed, when a specific phase difference exhibits time-like (i.e., monotonic with time) behavior irregular pattern. 3.2. VI TET through excitation of VI IOs In particular, the irregular occurrence of vibro- impacts at the initial stage of the damped transition can be another evidence that the 3:1 resonant interaction at stage A is, in fact, a numerical artifact exclusively due to impacts, whereas in actuality the LO and the VI NES are engaged in 1:1 in-phase transient resonance capture. Recalling that only the second IMFs represent accurately the exact responses of the LO and the VI NES, we denote by φ1 the 22 a b Fig. 22. Detailed depiction of vibro-impacts in the first IMFs of Fig. 21. a precludes the direct application of the averaging theorem with respect to that angle, and the possibility for resonance interaction between the corresponding IMFs exists. a In Fig. 21(d) we note non-time-like behavior of the phase dif- ference during the entire VI damped motion, which indicates that a 1:1 TRC between the two oscillators occurs. The phase plane (θ1, ˙θ1) in Fig. 21(e) more clearly depicts this non-time-like be- havior, indicated by the spirals in the dynamics during the en- tire regime of the VI transition. Finally, the wavelet transform of the difference between the two second IMFs of the two os- cillators, superimposed on the FEP, clarifies the true nonlinear resonant interactions without being ‘polluted’ by the occurring vibro-impacts (Fig. 21(f)). It is interesting to note that the EMD results can separate effects of vibro-impacts from the occurring nonlinear resonance interactions, so that the residuals represent smooth functions that describe true nonlinear resonant interac- tions between the two oscillators. This demonstrates that the EMD technique, although applied (by construction) in an ad hoc manner, can still lead to physically relevant results. Currently, efforts towards a rigorous physical interpretation of EMD results in terms of the slow-flow dynamics of a system, and application of EMD in the context of nonlinear nonparametric system identification are in progress [71]. b Fig. 22. Detailed depiction of vibro-impacts in the first IMFs of Fig. 21. Fig. 23. Period and frequency of impacts. over a specified time interval it may be regarded as a ‘fast’ angle b We end this section by mentioning that the presented VI TET results are by no means optimized; that is, higher TET efficiencies may be achieved when alternative sets of initial conditions or system parameters are considered. 4. Efficiency of VI TET We aim to study the efficiency of VI TET in the system of Fig. 3 by introducing certain definitions related to the capacity of the VI NES to passively absorb and dissipate vibration energy from the LO, as well as the time required for this VI dissipation to occur. Specifically, we denote by ξLI the normalized time instant when the last vibro-impact in a given simulation occurs (that is, for ξ > ξLI the transient response is purely linear); by ξ95% the time required for 95% of the initial energy of the system to get dissipated by viscous damping and inelastic vibro-impacts; and by EVINES the percentage of initial energy eventually dissipated by the VI NES due to inelastic vibro-impacts (i.e., during the entire duration of the damped motion). In this context, the ratio EVINES/ξLI represents the average measure of the percentage of energy dissipated by the VI NES per unit time, whereas the ratio 95/ξ95% the average percentage of energy dissipated per unit time until 95% of total energy is dissipated. It follows that the ratio EVINES/ξLI provides a measure of VI TET efficiency per unit time as long as vibro-impacts occur (i.e., for ξ ≤ξLI), and is used as a means of judging the rate (time scale) of energy dissipation (efficiency) by the VI NES only. Alternatively, the measure 95/ξ95% is used to study the overall rate of energy dissipation in the system (including the combined effects of inelastic vibro-impacts and viscous damping dissipation). Fig. 22. Detailed depiction of vibro-impacts in the first IMFs of Fig. 21. Fig. 23. Period and frequency of impacts. Clearly, higher values of the energy measure EVINES, and/or lower values of the time measure ξ95%, indicate more efficient VI TET in the system under consideration. Moreover, if there are no other sources of dissipation, higher values of the average rate EVINES/ξLI indicate high VI TET efficiency in the nonlinear regime of the damped response; i.e., in the regime where vibro-impacts are realized. However, in the presence of additional viscous damping dissipation in the LO, the average rate 95/ξ95% provides a better indicator of the overall efficiency of TET to dissipate a significant portion of the total initial energy of the system throughout the damped motion. Fig. 23. Period and frequency of impacts. 3.2. VI TET through excitation of VI IOs This leads us naturally to the discussion of TET efficiency in the system with VI NES carried out in the next section. 4. Efficiency of VI TET over a specified time interval, it may be regarded as a ‘fast’ angle, and, hence, may be averaged out of the dynamics; this eliminates the possibility of resonance interaction between the corresponding IMFs. On the contrary, non-time-like behavior of a phase difference 23 a b iciency of TET in the VI system when VI IOs are excited: (a) measures ξLI, ξ95% and EVI NES as functions of total energy; (b) ave of total energy (ρ = 0.7, λ = 0.005λcrit). e depict the measures ξ ξ E and the Focusing now on the average rates depict a a b b . Study of efficiency of TET in the VI system when VI IOs are excited: (a) measures ξLI, ξ95% and EVI NES as functions of total energy; (b) average rates EVI NES/ξL % as functions of total energy (ρ = 0.7, λ = 0.005λcrit). Fig. 24. Study of efficiency of TET in the VI system when VI IOs are excited: (a) measures ξLI, ξ95% and EVI NES as functions of total energy; (b) average rates EVI NES/ξLI and 95/ξ95% as functions of total energy (ρ = 0.7, λ = 0.005λcrit). In Fig. 24 we depict the measures ξLI, ξ95%, EVINES, and the average rates EVINES/ξLI and 95/ξ95% as functions of initial energy, for damped VI responses initiated on VI IOs over a wide energy range of the FEP. The system parameters used for these simulations are ρ = 0.7, λ = 0.005λcr and µ = σ = 0.1. As expected, with increasing energy more vibro-impacts occur, as indicated by the increase of the normalized time measure ξLI with increasing energy in Fig. 24(a). Judging from the dependence of the energy measure EVINES on energy, we conclude that most efficient VI TET is realized when VI IOs are excited in regimes (III) and (IV) of the FEP (the highest VI TET efficiency is above 65% for this series of simulations). Moreover, VI TET in these regimes occurs at a relatively fast time scale, as indicated by the relatively small values of the normalized time measure ξ95% in the corresponding energy ranges. Focusing now on the average rates depicted in Fig. 4. Efficiency of VI TET This last result is not as obvious as it might seem from a first reading; indeed, al- though it is clear that the average rate EVINES/ξLI increases when the restitution coefficient increases (as this results in increased energy dissipated per vibro-impact), this does not necessarily imply that the overall TET efficiency as measured by the average rate 95/ξ95% corresponding to excitations of VI IOs at varying energies and restitution coefficients ρ, and fixed viscous damping coefficient λ = 0.005λcr = 0.01. This study identified the regimes of ef- ficient VI TET when both the energy of the excited VI IO and the coefficient of restitution of vibro-impacts are varied. The results are presented in Figs. 25 and 26, from which we conclude that the most efficient TET takes place when highly energetic VI IOs are ex- cited in regimes (III) and (IV) of the FEP, and for smaller restitution coefficients, i.e., for highly inelastic vibro-impacts. This last result is not as obvious as it might seem from a first reading; indeed, al- though it is clear that the average rate EVINES/ξLI increases when the restitution coefficient increases (as this results in increased energy dissipated per vibro-impact), this does not necessarily imply that the overall TET efficiency as measured by the average rate 95/ξ95% 4. Efficiency of VI TET 24(b), we deduce again that the most efficient rates of the overall energy dissipation measure 95/ξ95%, are realized in regimes (III) and (IV), although the highest rates of energy dissipated by the VI NES during vibro-impacts (EVINES/ξLI) are realized in the lower energy regimes (I) and (II). We conclude that, although in these regimes there occurs strong TET from the LO to the VI NES as long as vibro-impacts last (i.e., there is more efficient energy dissipation per vibro-impact), the overall duration of vibro-impacts is small (due to the small level of overall energy), as reflected by the rela- tively small values of the corresponding overall TET efficiency rates 95/ξ95%. / In an additional series of numerical simulations we com- puted the previous energy dissipation measures for simulations 24 . Efficiency of TET in the VI system when VI IOs are excited: (left column) measures EVI NES, ξLI, ξ95% as functions of total energy and coefficient of restituti 01; (right column) corresponding projections in the plane of total energy versus coefficient of restitution. sponding to excitations of VI IOs at varying energies and ution coefficients ρ, and fixed viscous damping coefficient 0 005λ 0 01 Thi t d id tifi d th i f f also increases (for example, refer to the average rates depict Fig. 24(b)). Fig. 25. Efficiency of TET in the VI system when VI IOs are excited: (left column) measures EVI NES, ξLI, ξ95% as functions of total energy and coefficient of restitution for λ = 0.01; (right column) corresponding projections in the plane of total energy versus coefficient of restitution. also increases (for example, refer to the average rates depicted in Fig. 24(b)). corresponding to excitations of VI IOs at varying energies and restitution coefficients ρ, and fixed viscous damping coefficient λ = 0.005λcr = 0.01. This study identified the regimes of ef- ficient VI TET when both the energy of the excited VI IO and the coefficient of restitution of vibro-impacts are varied. The results are presented in Figs. 25 and 26, from which we conclude that the most efficient TET takes place when highly energetic VI IOs are ex- cited in regimes (III) and (IV) of the FEP, and for smaller restitution coefficients, i.e., for highly inelastic vibro-impacts. A.1. Main backbones See Figs. 27 and 28. See Figs. 27 and 28. See Figs. 27 and 28. 5. Conclusions S11−branch: (a) h = 4.5636; (b) h = 0.14673; (c) h = 0.069495; (d) h = 0.086339. a b a b c d Fig. 29. Symmetric branches: (a) S51O22−; (b) S21 −5 −O −10 −10−; (c) S31E22+; (d) S55E22+. b a b d c c d c Fig. 29. Symmetric branches: (a) S51O22−; (b) S21 −5 −O −10 −10−; (c) S31E22+; (d) S55E22+. and by the National Science Foundation through Grant Number CMS03–24433. and by the National Science Foundation through Grant Number CMS03–24433. NESs with smooth essential nonlinearities, however, VI NESs are capable of passively absorbing and locally dissipating significant portions of the energy of the primary systems to which there are attached, at sufficiently fast time scales. This renders them suitable for applications like seismic mitigation, where shock elimination in the early, highly energetic regime of the motion is a critical requirement. 5. Conclusions We showed that an SDOF primary linear oscillator (LO) with an attached vibro-impact nonlinear energy sink (VI NES) possesses very complicated dynamics. In the absence of energy dissipation, vibro-impacts produced by even small clearances give rise to a variety of periodic (and quasiperiodic) motions, which produce a complex topology of periodic orbits when represented in a frequency–energy plot (FEP). In the limit of zero clearance the entire FEP degenerates to two linear modes. By superimposing wavelet transform (WT) spectra of weakly damped responses 25 Fig. 26. Efficiency of TET in the VI system when VI IOs are excited: (left column) average rates EVI NES/ξLI and 95/ξ95% as functions of total energy and coefficient of restitution for λ = 0.01; (right column) corresponding projections in the plane of total energy versus coefficient of restitution. Fig. 26. Efficiency of TET in the VI system when VI IOs are excited: (left column) average rates EVI NES/ξLI and 95/ξ95% as functions of total energy and coefficient of restitution for λ = 0.01; (right column) corresponding projections in the plane of total energy versus coefficient of restitution. a b c d Fig. 27. S13 + branch: (a) h = 11.4294; (b) h = 5.1365; (c) h = 0.76989; (d) h = 0.1184. b a b a b d d c c Fig. 27. S13 + branch: (a) h = 11.4294; (b) h = 5.1365; (c) h = 0.76989; (d) h = 0.1184. and subharmonic TET can be realized by the VI NES. The most efficient mechanism for TET, however, is through the excitation of highly energetic VI impulsive orbits (IOs), similar to the case of the NES with smooth (cubic) stiffness nonlinearity. In contrast to the and subharmonic TET can be realized by the VI NES. The most efficient mechanism for TET, however, is through the excitation of highly energetic VI impulsive orbits (IOs), similar to the case of the NES with smooth (cubic) stiffness nonlinearity. In contrast to the on the Hamiltonian FEP we were able to study complicated transitions, and deduce the different mechanisms for passive targeted energy transfer (TET) from the LO to the VI NES. As in the case of smooth stiffness nonlinearity, both fundamental 26 a b c d Fig. 28. S11−branch: (a) h = 4.5636; (b) h = 0.14673; (c) h = 0.069495; (d) h = 0.086339. b d a b a d c c d c Fig. 28. Acknowledgments A.2. Subharmonic tongues See Figs. 29 and 30. A.2. Subharmonic tongues See Figs. 29 and 30. References [21] B. Blazejczyk-Okolewska, Analysis of an impact damper of vibration, Chaos Solitons Fractals 12 (2001) 1983–1988. [21] B. Blazejczyk-Okolewska, Analysis of an impact damper of vibration, Chaos Solitons Fractals 12 (2001) 1983–1988. [22] R. Leine, H. 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https://openalex.org/W4230557849	https://publishings.eaae.be/index.php/annual_conference/article/download/29/29	English	null	Effects of Restorative Environments on Creativity in Case of Architecture Education	EAAE Annual Conference Proceedings	2,019	cc-by	1,411	Effects of Restorative Environments on Creativity in Case of Architecture Education Beste Sabir Istanbul Technical University SESSION II PLAC SESSION II Environments on Creativity in Case of Architecture Education PLACE / ENVIRONMENTS PLACE / ENVIRONMENTS architectural education, architectural creativity, meditative spaces, neuroscience of creativity, restorative environments Creativity is a mental process, as Andreasen (2006) describes, it happens when a thought comes up to surface in the mind, it has a complex nature and it does not happen in a tabula rasa condition, instead interaction of human thoughts with socio-cultural situations creates this phenomenon, as Portillo (1996) defines the creativity as an interconnected and mul- tidimensional construct involving person, process, product and place (environment/ press). One of the main intentions of this paper is to address the relationship between the crea- tivity and its supportive environment in case of architectural education which can be defined as a design study that get its origins from creativity. 157 Effects of Restorative Environments on Creativity in Case of Architecture Education Freeman (1971) categorizes creativity with the stages such as: Preparation; Incubation; Enlightenment (A-ha moment, enlight- enment); Affirmation/Verification. Following that organization, we can consider that first two stages are very complex, intricate and interactive processes and in case of architectural education and its environmental necessities, designs should be answering to this complexity and interactivity as well. It is important to consider creativity as the major source in any kind of thinking, and as inseparable from life itself (DeBono, 1993). Any understanding of education and learning includes the term creativity, and thus, the learning environment should carry the necessary components to support it (Demirbas & Demirkan, 2000; Hasirci, 2000). The learning environment in which learning and creative activities take place, should provide students the ground on which creativity can more readily flourish (Hasirci, 2000). There are also arguments whether creativity is latent potentiality or an improvable characteristic (Potur, Barkul, 2006). For the first time in Utah Research Conference in 1959, researchers argued about creativity and its relationship with education, and if it can be developed. (Andreasen, 2006) As Guil- ford (1950) describes, like many other activities, creativity is a behavior that can be developed and learned. This paper focuses on to the neuroscience of creativity and its supportive environ- ments that can develop this ability in architectural education. PLACE / ENVIRONMENTS Andreasen (2006) supports Freeman’s creativity categori- zation; and deepens the A-ha moment stage: Eventhough crea- tivity is a complex process, while the brain is in the REST mode — which is described as ‘free association’, the mind is relaxed and focused on to only one subject such as breath, it creates new synapses and connections by picking up topics from the unconscious fountain of knowledge that have been collected. The psychologists researched with experiments such as divergent thinking questionnaires and declared that, in the REST mode, while the mind is quiet, meditative and free thinking, the brain generates new creative ideas. Effects of Restorative Environments on Creativity in Case of Architecture Education 158 SAB Thus, the paper aims to ask, what is the effect of spaces to this REST mode and creative process? Can meditative and restorative environments support the REST mode and enhance the creativity during architectural education? Free association REST mode focuses on the principle of free circulating thought, allowing relaxation and free thinking to lead to new connec- tions (creative moments) in the brain. As Andreasen (2006) noted, the source of unconsciousness brings about a creation process as the result of new connections and synapsis during the free-movement thought. Working in this sense; accepting that creativity is a complex process and that it depends on senses, observation, culture, field, stimuli and tests, the paper aims to focus on how spaces affect creative process in case of architectural education, production and creation. If REST mode as relaxation, meditation focus and awareness support the process of creation how does restorative (calming, med- itative) spaces and environments affect this process as well? Kaplan, Kaplan, and Ryan (1998) described a restorative environment as a place to rest and recuperate and they stated that ‘natural settings are particularly effective for R&R’ (p 67). A natural landscape can produce a restorative experience and can renew a person’s cognitive powers and they are described as spaces with the following characteristics: ‘quiet fascination; wandering in small spaces; separation from distraction; wood, stone, and old; and the view from the window (Kaplan, Kaplan, & Ryan, 1998). Thus, paper aims to ask; can restorative spaces enhance and stimulate the creative process -described as A-ha moment and REST mode? PLACE / ENVIRONMENTS As a case study, a questionnaire is prepared and asked to third- and fourth-year students of İTÜ Architecture Faculty, in order to get a data from the results if restorative spaces and meditative moments support their creativity in design process and how does creative process being affected by the environment? As neuroscience declares; our environment has many effects on our behaviours. Thus, for to develop the crea- tive process in architecture education, learning environments should inherit related qualities. The result of the questionnaire is expected to give feedback about architecture education en- vironments and if meditative spaces are affecting the creative process in a positive way. 158 References SABIR Andreasen, N. (2006). The Creative Brain: The Science of Genius, Plume. Bohm, D. (1998). On Creativity, Routledge. Bohm, D. (1998). On Creativity, Routledge. Csikszentmihalyi, M. (2013). Creativity: Flow and The Psychology of Discovery and Invention. Harper Perennial. DEBONO, E. (1993). Serious Creativity: Using the Power of Lateral Thinking to Create New Ideas. London: Harper Collins Publishers. DEMIRBAS, O. & DEMIRKAN, H. (2000). Privacy dimensions: a case study in the interior architecture design studio. Journal of Environmental Psychology, 20, (1), 53–64. Effects of Restorative Environments on Creativity in Case of Architecture Education Freeman, J. (1971). Creativity: A Selective Review of Research, Society for Research into Higher Education Ltd, pp.41. Guilford, J. P. (1950). Creativity. American Psychologist, 5(9), 444–454.f HASIRCI, Deniz. (2000). The Effects of the Design and Organi- zation of Learning Environments on Creativity: The Case of Two Sixth Grade Art-Rooms. MFA Thesis. Bilkent University. Kaplan, R., Kaplan, S. & Ryan, R (1998). With people in mind: Design and management of everyday nature. Washington, D.C.: Island Press. Kaufman, S., Gregoire, C. (2015). Wired to Create: Unravelling the Mysteries of the Creative Mind. TarcherPerigee. Mallgrave, H.F. (2011). The Architect’s Brain: Neuroscience, Creativity, And Architecture. Wiley-Blackwell. Parnes, J. (1963). Education and Creativity, Teachers College Record, vol64, 1963, pp. 331–339. Portillo, M. (1996). Uncovering implicit theories of creativity in beginning design students. Journal of Interior Design, 22(2), 16–24. Potur, A., Barkul, A. (2006). Creative Thinking in Architectural Design Education, 1st International CIB Endorsed METU Postgraduate Conference Built Environment & Information Technologies, Ankara. Runco, M. (2014). Creativity: Theories and Themes: Research, Development, and Practice, Academiz Press. 1: Kew Gardens Hills Library, photo by Michael Moran. (https://www.curbed. PLACE / ENVIRONMENTS com/2018/5/24/17389648/library-architecture-teens-public-space) Effects of Restorative Environments on Creativity in Case of Architecture Education Effects of Restorative Environments on Creativity in Case of Architecture Education 160 SA 1: Kew Gardens Hills Library, photo by Michael Moran. (https://www.curbed. com/2018/5/24/17389648/library-architecture-teens-public-space) ments on Creativity in Case of Architecture Education 2: Photo from ITU, architecture studios. Photo by the author. 2: Photo from ITU, architecture studios. Photo by the author. 3: explains the different phases of creativity and their environmental relations. Prepared by the author from the readings and interpretations of Freeman (1971) and Andreasen (2006). 3: explains the different phases of creativity and their environmental relations. Prepared by the author from the readings and interpretations of Freeman (1971) and Andreasen (2006). 4: Tanner Fountain in Harvard University, photo by Alan Ward. (https://www. asla.org/awards/2008/08winners/312.html)0 SABIR 161 s on Creativity in Case of Architecture Education Effects of Restorative Environments on Creativity in Case of Architecture Education Effects of Restorative Environments on Creativity in Case of Architecture Education 4: Tanner Fountain in Harvard University, photo by Alan Ward. (https://www. asla.org/awards/2008/08winners/312.html)0 5: Metropolitan State University, Library’s Labyrinth Garden. (http://www. bestcounselingschools.org/best-campus-meditation-spaces/) 5: Metropolitan State University, Library’s Labyrinth Garden. (http://www. bestcounselingschools.org/best-campus-meditation-spaces/) Image 1 and 2 explain the complex and interactive quality of creative process, and show the interactive environments (that support first two stages of creativity stated by Freeman); for data sharing, preparation, research, data hunting etc. which are all dynamic and complex processes that need suitable environments such as gathering, exhibiting, sharing, experimenting etc. Image 4 and 5 show restorative environments that can support the REST mode by creating meditative state and supporting the creative process in educational environments.
https://openalex.org/W4213273068	https://seer.ufu.br/index.php/biosciencejournal/article/download/55572/33376	English	null	Seed size and presence of thorns in Caryocar brasiliense on seedling emergence	Bioscience journal	2,022	cc-by	3,155	Abstract Commercial propagation of pequizeiro, which stands out among the main native fruits of the Cerrado region for its high economic potential, has been done by seeds, despite their erratic germination. The objective of this study was to evaluate the effect of seed size and presence of thorns on the endocarp on seedling emergence of pequizeiro in the field. Seeds were collected in a pequizeiro commercial area in Canarana-MT, Brazil. Due to availability, 2,353 thorny seeds (3 sizes) and 106 seeds without thorns (medium sized) were tested. Thorny seeds were classified as large – longer than 50 mm; medium – between 40 and 50 mm; small – less than 40 mm. Seedling emergence started after 50 days in all seed categories, extending up to 100 days in the large seeds and to about 1 year after sowing in the small and medium sized ones. Small seeds showed a lower rate and speed of emergence compared to the others. Large seeds showed higher emergence speed compared to the medium ones. The presence of thorns did not affect seedling emergence. These results indicate the advantages of pequizeiro seed classification for sowing purposes. Keywords: Emergence Speed. Germination. Seed Classification. Seed Dormancy. Ricardo CARMONA1 , Daniel Franklin Nunes FERREIRA2 , Thiago Estácio da COSTA3 , Luís Carlos CARVALHO JÚNIOR3 , Antônio Alves de OLIVEIRA JÚNIOR4 ardo CARMONA1 , Daniel Franklin Nunes FERREIRA2 , Thiago Estácio da COSTA3 , Luís Carlos CARVALHO JÚNIOR3 , Antônio Alves de OLIVEIRA JÚNIOR4 1 Faculty of Agriculture, University of Brasília, Brasília, Distrito Federal, Brazil. 2 Fazenda Água Limpa, University of Brasília, Brasília, Distrito Federal, Brazil. 2 Fazenda Água Limpa, University of Brasília, Brasília, Distrito Federal, Brazil. 3 G d t P i A U i it f B íli B íli Di t it F d l B Corresponding author: Ricardo Carmona Email: rcarmona@unb.br How to cite: CARMONA, R., et al. Seed size and presence of thorns in Caryocar brasiliense on seedling emergence. Bioscience Journal. 2022, 38, e38010. https://doi.org/10.14393/BJ-v38n0a2022-55572 1. Introduction Pequizeiro is a fruit tree species largely distributed in the Brazilian tropical savanna biome (Cerrado), with a diversity of uses. Santos et al. (2013) highlighted its economic and cultural importance, since the pequi fruit pulp, in addition to cooking uses, stands out for its high oil content that can be used for therapeutic and biofuel purposes. It is rich in vitamin A and proteins, and can be used for treatment of bronchitis, asthma, and common colds, besides the antioxidant characteristics of the peel (Rocha et al. 2015). It can also be used in cosmetics industry, such as soap production. The pequi peel can be used as animal fodder, and the flour obtained from this part of the fruit can be included in quail diet (Oliveira et al. 2016). In addition, the peel extract and the mesocarp have a nematicide effect on the root knot nematode (Meloidogyne javanica) in tomato plants (Ribeiro et al. 2012). The increasing demand for pequi fruits has led to the intensification of pequizeiro cultivation, which demands the production of commercial plants. According to Maranho et al. (2013) there is a growing need for the commercial production of native plants to supply the demand for the recovery of degraded areas and conservation areas; however, these authors emphasize the lack of information on the propagation of Bioscience Journal \| 2022 \| vol. 38, e38010 \| ISSN 1981-3163 1 1 1 Seed size and presence of thorns in Caryocar brasiliense on seedling emergence these species. In addition, commercialization of these fruits can yield an extra profit during the harvest season. these species. In addition, commercialization of these fruits can yield an extra profit during the harvest season. Pequizeiro plants are usually produced from seeds, despite their erratic germination rates, due in part to the occurrence of dormancy mechanisms (Rocha 2009) and other causes, such as high lipid contents, which are susceptible to peroxidation (rancification) processes, with negative effects on seed longevity. Lima et al. (2007) observed that lipids are the major component in both seeds (51.5%) and mesocarp (33.4%). Lipid peroxidation process (rancification) contributes to seed deterioration (Mendes 2015), thus reducing seed longevity. These factors contribute to the low success in obtaining seedlings from seeds, which limits the commercial expansion of this species. 1. Introduction Rocha (2009) described two mechanisms of dormancy in pequizeiro seeds – physical dormancy of the endocarp and embryonic dormancy, which can be alleviated with gibberellic acid (GA3). This growth regulator has been the main treatment in use to overcome pequizeiro seed dormancy (Carvalho et al. 1994). Even so, the responses of pequizeiro seeds to GA3 are variable. Leão et al. (2012) observed average germination percentages ranging from 5% to 57% with gibberellic acid (GA3) and from 3% to 56% without it. The average period required for pequizeiro seedling emergence was 52 days in the presence and 59 days in the absence of GA3, confirming its low effect. According to these authors, seedling emergence starts at 30 days after sowing, extending up to 128 days, probably due to seed dormancy mechanisms. Germination depends on several factors, such as seed viability and vigor (physiological quality) besides environmental factors. Seed size can also be correlated with physiological quality, since larger seeds store more reserves, which generally results in more vigorous seedlings (Carvalho and Nakagawa 2012; Geritz and Toivonen 2018). Silva et al. (2010) observed that smaller seeds of jackfruit (Artocarpus heterophyllus Lam.) resulted in lower seedling emergence rates compared to the larger ones. In contrast, Nietsche et al. (2004) did not find significant effects of seed size on emergence rates of cagaita (Eugenia dysenterica) seedlings; however, larger seeds germinated faster than the small ones. There is evidence about the occurrence of germination inhibitors in the endocarp and thorns of pequizeiro. Melo and Gonçalves (2001) observed a reduction in the speed of seed germination on lettuce (Lactiva sativa L.) in the presence of aqueous thorns and endocarp extract of pequi. The removal of these structures, however, can cause mechanical damage to the seeds, due to the rigidity of the endocarp combined with the fragility of the seed. The objective of the present study is to evaluate the effects of pequizeiro seed size and the presence of thorns on seedling emergence in the field. 2. Material and Methods Pequizeiro fruits were collected on November 10, 2018, from planted matrices of about 20 years old, in the region of Canarana/MT. Ripe fruits were manually collected on the ground, separately from matrices containing or not thorns on the endocarp. In order to avoid possible effects of the genotype on seedling emergence, fruits of different matrices were mixed for homogenization purposes and transported in polypropylene bags to Fazenda Água Limpa (Universidade de Brasília), in Brasília/DF. Two days after collection, the fruits were cut and the exocarp (peel) discarded. In order to remove the mesocarp, putamens (endocarp containing seed and mesocarp, hereafter referred to as seeds) were naturally fermented openly in a warehouse for four days, at environmental temperatures (Melo and Gonçalves 2001). The seeds were then shaken in a concrete mixer, along with sand, gravel, and water, for 35 minutes, to separate the fermented mesocarp from the endocarp. Seeds were then washed over a sieve, until the complete removal of sand, gravel and the remainings of mesocarp and dried out for 3 days in a warehouse (Melo and Gonçalves 2001). Damaged seeds (rotten, perforated, dark) were discarded, and the rest were treated with 500 grams of Orthocide 500 (Captan) per 40 kg of seeds, to prevent fungi incidence. Seed average mass, regardless the presence of thorns or classification, evaluated in eight replicates of 100 seeds, was 18.5 grams. Thorny seeds were classified in three sizes: large – more than 50 mm length (56 seeds), medium – between 40- and 50- mm length (1,147 seeds) and small – less than 40 mm (1,150 seeds), using an amount of 2,353 seeds. One hundred and six seeds without thorns (all medium sized, due to the absence of other sizes in this lot) were also tested. Bioscience Journal \| 2022 \| vol. 38, e38010 \| https://doi.org/10.14393/BJ-v38n0a2022-55572 2 2 CARMONA, R., et al. The classified seeds were sown on November 20, 2018, in a 1-meter width seedbed containing 50% sand and 50% subsoil (red latosoil collected at 20-40 cm layer), in 7 cm depth furrows spaced 20 cm apart. Seeds were arranged in an upright position with the epicotyl facing upwards and the radicle downwards, in a density of 20 medium or large seeds and 25 small seeds per linear meter. 3. Results and Discussion Pequizeiro seeds showed slow initial germination, as seedling emergence started about 50 days after sowing in field conditions of the present experiment (Figure 1). This period is longer than the 35 days observed by Silva and Leonel (2017) and the 30 days observed by Leão et al. (2012) for the beginning of germination of pequizeiro seeds, probably due to different environmental conditions or seed dormancy. At least two dormancy mechanisms have been attributed to pequizeiro seeds - mechanical restriction of the endocarp and physiological embryonic dormancy (Rocha 2009). At 100 days after sowing, the relative emergence reached 100% in the large seeds (over 50 mm length), while at this moment the relative emergence was around 60% in the medium and small seeds (up to 50 mm length) (Figure 1). In these seeds, emergence extended until about 1 year after sowing (Figure 1). Similar results were obtained by Leão et al. (2012), who observed the emergence of pequizeiro seedlings up to 128 days after seed sowing. In addition to the long germination period, germination process was also dependent on seed size (Figure 1). The results of the present study show that the size of pequizeiro seeds is inversely proportional to the time necessary for seedling emergence. Wagner Júnior et al. (2011) also observed that larger seeds showed higher germination and seedling emergence rates compared to the smaller ones in Jabuticaba Sabará (Myrciaria jaboticaba (Vell.) O. Berg) and in Jabuticaba de Cabinho (M. peruviana var. trunciflora) seeds. In contrast, Nietsche et al. (2004) did not observe significant effect of seed size on seedling emergence rate of cagaita (Eugenia dysenterica) – a native Cerrado fruit – but also concluded that bigger seeds entail faster seedling emergence. For Pereira et al. (2011), the influence of seed mass on germination and vigor can be relative, varying between species and regions. Seed size also affected the final seedling emergence percentage, once the accumulated emergence in small seeds was about 20% after one year (Figure 1), whereas in medium and large ones (> 40 mm length) the accumulated emergence exceeded 30%. These emergency rates agree with the results obtained by Leão et al. (2012), who observed germination rates varying from 3% to 56% in pequizeiro seeds. The presence of thorns in the endocarp did not affect seedling emergence. 2. Material and Methods The seeds were covered with a 2 to 3 cm sand/subsoil (1:1) layer mixture and the seedbed with dry straw, to reduce direct sunlight, evaporation and weed development. The seedbed was irrigated throughout the experimental period, as needed, by sprinklers. Manual weed control was undertaken every 15 days. Seedling emergence was evaluated on a weekly basis, from January 07, 2019, when emergence begun and transplanted to 20 x 35 cm perforated plastic bags. The percentage of accumulated seedling emergence, in relation to the total sown (observed), and the accumulated seedling emergence in relation to the total of seedlings emerged (relative) were evaluated, using polynomial regression with each respective equation, for all seed categories. Thirty days after the end of the seedling emergence period, 20 large seeds, 100 medium seeds and 100 small seeds were exhumed from the soil in order to observe if their tissues were still fresh or rotten. 3. Results and Discussion The observed and relative accumulated emergence of medium seeds (40 to 50 mm length) with or without thorns were similar (Figure 1). In contrast, Melo and Gonçalves (2001) observed a decrease in germination speed of lettuce seeds (Lactuca sativa L.) in the presence of aqueous thorn and endocarp extract of the pequi fruits, which denotes the presence of germination inhibitors in these structures. All the seeds retrieved from the soil thirty days after the end of the seedling emergence period (20 large, 100 medium and 100 small size) were rotten and smelly, which indicates that non-germinated seeds were non-viable at the end of the experiment. These results demonstrate the importance of seed classification by size on pequizeiro. In order to improve rate and speed of emergence, small seeds (less than 40 mm length, in the studied accesses) should be discarded. Medium and large seeds (more than 40 mm) have similar final emergence, but large seeds (more than 50 mm length) germinate faster (up to 100 days after sowing). Apparently, in this species there Bioscience Journal \| 2022 \| vol. 38, e38010 \| https://doi.org/10.14393/BJ-v38n0a2022-55572 3 Seed size and presence of thorns in Caryocar brasiliense on seedling emergence is a direct correlation between the amount of reserve in the seeds and the rate and speed of emergence and, consequently, in seed vigor. Thus, large seeds are the most suitable for sowing purposes. Figure 1. Observed (♦, Oe) and relative (●, Re) emergence of pequizeiro seedlings over time after sowing for: A – small seeds (smaller than 40 mm); B – medium seeds (40 to 50 mm); C – large seeds (above 50 mm); and D – seeds without thorns (medium size). Figure 1. Observed (♦, Oe) and relative (●, Re) emergence of pequizeiro seedlings over time after sowing for: A – small seeds (smaller than 40 mm); B – medium seeds (40 to 50 mm); C – large seeds (above 50 mm); and D – seeds without thorns (medium size). Ethics Approval: Not applicable. Ethics Approval: Not applicable. Acknowledgments: The authors are grateful to Mr. Edemo Correia (Sítio Água Limpa, Canarana/MT) for providing the seeds for this study. The authors are also grateful to the University of Brasília (UnB) for the infrastructure support. Conflicts of Interest: The authors declare no conﬂicts of interest. Conflicts of Interest: The authors declare no conﬂicts of interest. 4. Conclusions Seedling emergence is a slow process in pequizeiro, taking up to a year for med Seed (putamen) size affects rate and speed of seedling emergence. Small seeds (smaller than 40 mm length, in the studied accesses) should be discarded since they generate smaller rate and speed of seedling emergence. Medium and large seeds (longer than 40 mm) imply higher seedling emergence rates, and large seeds (from 50 mm length) generate also higher emergence speed. The presence of thorns in the pequizeiro seeds does not affect rate and speed of seedling emergence. Authors' Contributions: CARMONA, R.: conception and design, acquisition of data, analysis and interpretation of data, drafting the article; FERREIRA, D.F.N.: acquisition of data, drafting the article; COSTA, T.E.: acquisition of data, drafting the article; CARVALHO JÚNIOR, L.C.: acquisition of data, drafting the article; OLIVEIRA JÚNIOR, A.A.: analysis and interpretation of data, drafting the article. All authors have read and approved the final version of the manuscript. References CARVALHO, C.G.S., et al. Efeito de diferentes tratamentos na germinação do pequi (Caryocar brasiliense Camb.). Acta Botânica Brasilica. 1994, 8(1), 109-120. https://doi.org/10.1590/S0102-33061994000100011 CARVALHO, C.G.S., et al. Efeito de diferentes tratamentos na germinação do pequi (Caryocar brasiliense Camb.). Acta Botânica Brasilica. 1994, 8(1), 109-120. https://doi.org/10.1590/S0102-33061994000100011 CARVALHO, N.M. and NAKAGAWA, J. Sementes: ciência tecnologia e produção. 5th ed. Jaboticabal: Funep, 2012. CARVALHO, N.M. and NAKAGAWA, J. Sementes: ciência tecnologia e produção. 5th ed. Jaboticabal: Funep, 2012. CARVALHO, N.M. and NAKAGAWA, J. Sementes: ciência tecnologia e produção. 5th ed. Jaboticabal: Funep, 2012. GERITZ, S., GYLLENBERG, M. and TOIVONEN, J. Adaptive correlations between seed size and germination time. Jornal of mathematical biology. 2018, 77, 1943-1968. https://doi.org/10.1007/s00285-018-1232-z GERITZ, S., GYLLENBERG, M. and TOIVONEN, J. Adaptive correlations between seed size and germination time. Jornal of mathematical biology. 2018, 77, 1943-1968. https://doi.org/10.1007/s00285-018-1232-z GERITZ, S., GYLLENBERG, M. and TOIVONEN, J. Adaptive correlations between seed size and germination time. Jornal of mathematical biology. 2018, 77, 1943-1968. https://doi.org/10.1007/s00285-018-1232-z Bioscience Journal \| 2022 \| vol. 38, e38010 \| https://doi.org/10.14393/BJ-v38n0a2022-55572 CARMONA, R., et al. LEÃO, E.F., PEIXOTO, N. and MORAIS JÚNIOR, O.P. Emergência de plântulas de pequizeiro em função da planta matriz e uso de ácido giberélico. Pesquisa Agropecuária Tropical. 2012, 42(4), 416-423. https://doi.org/10.1590/S1983-40632012000400009 LEÃO, E.F., PEIXOTO, N. and MORAIS JÚNIOR, O.P. Emergência de plântulas de pequizeiro em função da planta matriz e uso de ácido giberélico. Pesquisa Agropecuária Tropical. 2012, 42(4), 416-423. https://doi.org/10.1590/S1983-40632012000400009 LIMA, A., et al. Composição química e compostos bioativos presentes na polpa e na amêndoa do pequi (Caryocar brasiliense, Camb.). Revista Brasileira de Fruticultura. 2007, 29(3), 155-159. http://dx.doi.org/10.1590/S0100-29452007000300052 MARANHO, A., PAIVA, A.B. and PRADO DE PAULA, S.R. Initial growth of native species with timber potential in Amazon, Brazil. Revista Árvore. 2013, 37(5), 913-921. https://doi.org/10.1590/S0100-67622013000500014 MELO, J.T. and GONÇALVES, A. N. Inibidores de germinação em frutos e sementes de pequi. 1st ed. Brasília: Embrapa Cerrados, 2001. Available from: http://ainfo.cnptia.embrapa.br/digital/bitstream/CPAC-2010/23731/1/bolpd-23.pdf MENDES, D.S.T. Germinação e armazenabilidade de sementes de pequizeiro. Montes Claros: Universidade Federal de Minas Gerais, 2015. Available from: http://hdl.handle.net/1843/NCAP-9Y7G4L NIETSCHE, S., et al. Tamanho da semente e substratos na germinação e crescimento inicial de mudas de cagaiteira. Ciência e Agrotecnologia. 2004, 28(6), 1321-1325. OLIVEIRA, M.C., et al. Pequi peel flour in diets for Japanese quail. Acta Scientiarum. Animal Sciences. 2016, 38(1), 101-106. https://doi.org/10.4025/actascianimsci.v38i1.28381 PEREIRA, S.R., et al. Tamanho de frutos e de sementes e sua influência na germinação de jatobá-do-cerrado (Hymenaea stigonocarpa var.stigonocarpa Mart. ex Hayne, Leguminosae – Caesalpinoideae). Revista Brasileira de Sementes. 2011, 33(1), 141-148. https://doi.org/10.1590/S0101-31222011000100016 RIBEIRO, H.B., et al. Resíduos de frutos de pequi no controle do nematoide das galhas em tomateiro. Horticultura Brasileira. 2012, 30(3), 453- 458. http://dx.doi.org/10.1590/S0102-05362012000300016 ROCHA, J.P. Fatores genéticos e ambientais na emergência de plântulas de pequizeiro (Caryocar brasiliense camb.). Diamantina: Faculdade de Ciências Agrárias, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2009. Tese de doutorado. ROCHA, L.B., et al. Gallic acid as the major antioxidant in pequi (Caryocar brasiliense Camb.) fruit peel. Revista Brasileira de Plantas Medicinais. 2015, 17(4), 592-598. https://doi.org/10.1590/1983-084X/14_062 SANTOS, F.S., et al. A cultura do Pequi (Caryocar brasiliense Camb.). Acta Iguazu. 2013, 2(3), 46-57. SILVA, K.S., et al. Influência do tamanho da semente na germinação e vigor de mudas de jaqueira (Artocarpus heterophyllus Lam.). Revista Verde. 2010, 5(4), 217 – 221. SILVA, E.C. and LEONEL, L.V. Avaliação da germinação de sementes de pequizeiro (Caryocar brasiliense Camb) submetidas a diferentes concentrações de ácido giberélico. Cultura Agronômica. 2017, 26(2), 217-223. WAGNER JÚNIOR, A., et al. Germinação e desenvolvimento inicial de duas espécies de jabuticabeira em função do tamanho de sementes. Acta Scientiarum Agronomy. 2011, 33(1), 105-109. https://doi.org/10.4025/actasciagron.v33i1.4881 Received: 7 May 2020 \| Accepted: 16 July 2021 \| Published: 16 February 2022 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bioscience Journal \| 2022 \| vol. 38, e38010 \| https://doi.org/10.14393/BJ-v38n0a2022-55572 5
https://openalex.org/W4390340697	https://jpionline.org/storage/2023/12/IntJPharmInvestigation-14-1-1.pdf	English	null	Navigating Global Regulatory Requirements for Generic Drugs: A Comparative Study of MIST, BRICS, and ICH Countries	International journal of pharmaceutical investigation	2,023	cc-by	6,186	INTRODUCTION Regulatory affairs play a crucial role in the pharmaceutical industry as they ensure the safety and efficacy of medicines for human use. The regulatory authorities of different countries have different requirements for the development, registration, and post-approval of pharmaceutical products. To ensure a consistent and harmonized approach to the regulatory affairs of pharmaceuticals, global organizations such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) have been established. The ICH brings together regulatory authorities and the pharmaceutical industry from around the world to develop and implement guidelines and standards that support the development of safe and effective medicines.1 Regulatory affairs play a crucial role in the pharmaceutical industry as they ensure the safety and efficacy of medicines for human use. The regulatory authorities of different countries have different requirements for the development, registration, and post-approval of pharmaceutical products. To ensure a consistent and harmonized approach to the regulatory affairs of pharmaceuticals, global organizations such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) have been established. The ICH brings together regulatory authorities and the pharmaceutical industry from around the world to develop and implement guidelines and standards that support the development of safe and effective medicines.1 from those of ICH countries (Australia, Brazil, Canada, China, European Union, India, Japan, Russia, South Africa, South Korea, Singapore, Switzerland, Taiwan, United Kingdom, United States). These countries have their own unique regulations and guidelines, which may vary in terms of stringency, documentation requirements, and the length of the approval process. As a result, it is important for pharmaceutical companies to understand the specific requirements of each country to ensure a smooth and efficient regulatory process.2 In this study regulatory dossier requirements of MIST and BRICS countries with those of ICH countries are reviewed and compared. The comparison will provide an insight into the similarities and differences between the requirements of the different regions and help pharmaceutical companies to better understand the regulatory landscape in each country. The regulatory requirements of the countries under the MIST (Mexico, Indonesia, South Korea, and Turkey) and BRICS (Brazil, Russia, India, China, and South Africa) are different MIST countries have made significant progress in recent years in terms of regulatory reform and harmonization, but there is still a long way to go before they can be considered fully harmonized with ICH guidelines. ABSTRACT Bringing a new healthcare product to market can be challenging, especially when navigating the complex regulatory requirements of different countries. The approval process can be a maze of differing regulations, but this study provides a roadmap to success. This study aims to provide a clear and concise comparison of the regulatory dossier requirements of MIST and BRICS with ICH countries. The results of this study highlight key similarities and differences in their standards for quality, safety, efficacy, preclinical and clinical trials, and more. Whether you're looking to meet stringent requirements or find areas of alignment, this study offers valuable insights to help you bring your product to market with confidence. This comprehensive comparative analysis helps in succeeding in today’s rapidly evolving pharmaceutical market. 1Associate Professor, JSS College of Pharmacy, JSS Academy of Higher Education and Research, S.S. Nagar, Mysuru, Karnataka, INDIA. 2Guest Assistant Professor, Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, MALAYSIA. Email: venkateshmpv@jssuni.edu.in ORCID ID: 0000-0002-6804-1023 Keywords: Generic drugs, Regulatory requirement, Dossier requirement, MIST, BRICS, ICH. Received: 06-07-2023; Revised: 20-08-2023; Accepted: 07-10-2023. Received: 06-07-2023; Revised: 20-08-2023; Accepted: 07-10-2023. Int. J. Pharm. Investigation, 2024; 14(1):1-9. https://www.jpionline.org Int. J. Pharm. Investigation, 2024; 14(1):1-9. https://www.jpionline.org Review Article Jayant Kumar1, Ajmera Ramkishan2, Madhugiri Prakash Venkatesh1,3,* 1Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education and Research, S.S. Nag Karnataka, INDIA. 1Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education and Research, S.S. Nagar, Mysuru, k D 2Deputy Drugs Controller, Central Drugs Standard Control Organisation, Hyderabad, INDIA. 3Guest Assistant Professor, Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, MALAYSIA. Navigating Global Regulatory Requirements for Generic Drugs: A Comparative Study of MIST, BRICS, and ICH Countries Jayant Kumar1, Ajmera Ramkishan2, Madhugiri Prakash Venkatesh1,3,* Jayant Kumar1, Ajmera Ramkishan2, Madhugiri Prakash Venkatesh1,3,* BRICS The BRICS countries are major players in the global economy and are home to some of the fastest-growing pharmaceutical industries in the world. The dossier requirements for BRICS countries are designed to ensure the safety and efficacy of new drugs for human use, and to provide a framework for the development and registration of pharmaceuticals in these countries. The dossier requirements for BRICS countries are designed to reflect the specific needs and regulatory practices of each country. For example, the requirements in Brazil, Russia, and South Africa may be influenced by local laws and regulations, as well as cultural and medical practices. In India and China, the requirements may be influenced by the large and rapidly growing populations, as well as the need to ensure access to essential medicines. The dossier requirements for BRICS countries are developed through a collaborative process involving regulatory authorities and the pharmaceutical industry. This collaboration helps to ensure that the requirements are relevant, practical, and responsive to the needs of both regulators and industry. The dossier requirements for BRICS countries are designed to be adaptable to changing circumstances. The guidelines and standards are regularly reviewed and updated to ensure that they remain relevant and up to date with the latest advances in scientific and medical knowledge. The introduction of dossier requirements for BRICS countries is also intended to help these countries become more competitive in the global pharmaceutical industry. The harmonization of requirements and the development of a robust regulatory framework can help to attract investment and innovation to these countries and support the growth of their pharmaceutical industries.6 Regional harmonization of the MIST countries is in different regions of the world, and the introduction of dossier requirements is intended to help promote regional harmonization in the regulatory approval process for pharmaceuticals. The harmonization of requirements can help to reduce the regulatory burden for the pharmaceutical industry and promote cross-border trade and investment in the pharmaceutical sector.8 The regulatory authorities in the MIST countries may provide technical assistance and support to the pharmaceutical industry in developing and submitting dossiers. This can help to ensure that the requirements are practical and easy to implement, and that the industry is able to take full advantage of the opportunities provided by the new requirements and are responsible for monitoring and enforcing compliance with the dossier requirements. ICH The ICH guidelines and standards are developed through a collaborative process involving regulatory authorities and the pharmaceutical industry and are designed to be adaptable to changing circumstances and to reflect the specific needs and regulatory practices of each country.5 This also intended to help these countries become more competitive in the global pharmaceutical industry. The harmonization of requirements and the development of a robust regulatory framework can help to attract investment and innovation to these countries and support the growth of their pharmaceutical industries. As the MIST countries are important players in the global pharmaceutical industry, and the introduction of dossier requirements is a critical step in ensuring the continued growth and development of their pharmaceutical sectors. The requirements help to ensure that new drugs are safe and effective, and that the regulatory approval process is predictable and transparent.7 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 MIST The MIST countries are rapidly developing economies with growing pharmaceutical industries. The primary purpose of the dossier requirements for MIST countries is to ensure the safety and efficacy of new drugs for human use. The requirements provide a framework for the development and registration of pharmaceuticals and help to ensure that the drugs meet the same high standards for safety and efficacy in different countries. Also, it is designed to reflect the specific needs and regulatory practices of these countries. The requirements may be influenced by local laws and regulations, as well as cultural and medical practices. These are developed through a collaborative process involving regulatory authorities and the pharmaceutical industry. This collaboration helps to ensure that the requirements are relevant, practical, and responsive to the needs of both regulators and industry. The guidelines and standards are regularly reviewed and updated to ensure that they remain relevant and up to date with the latest advances in scientific and medical knowledge.3 INTRODUCTION In many MIST countries, there is still a lack of clarity and consistency in the regulatory requirements, which can result in longer approval times and higher costs for pharmaceutical companies.3 DOI: 10.5530/ijpi.14.1.1 Copyright Information : Copyright Author (s) 2024 Distributed under Creative Commons CC-BY 4.0 Publishing Partner : EManuscript Tech. [www.emanuscript.in] International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 1 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 Kumar, et al.: Regulatory requirements for Generic drugs On the other hand, the BRICS countries have some of the largest pharmaceutical markets in the world and are of strategic importance to the pharmaceutical industry. In recent years, the BRICS countries have made significant efforts to improve their regulatory systems and harmonize their requirements with those of ICH. However, there are still significant differences between the regulations in each country, which can make it challenging for pharmaceutical companies to navigate the regulatory landscape. Understanding the specific requirements and challenges of each country can help companies to develop a successful regulatory strategy, reduce the risk of regulatory delays and costs, and ultimately bring safe and effective medicines to patients more quickly.4 MIST DISCUSSION The Common Technical Document (CTD) is a standardized format for the submission of regulatory applications for pharmaceutical products. The CTD is divided into five modules, each with specific sections that provide detailed information about the product being submitted for approval with respect to ICH guidelines. Module 5 – Clinical Study Reports BRICS countries: Each of the BRICS countries has its own regulatory agency responsible for approving new drugs. The submissions required for regulatory approval may differ from the ICH guidelines and can include additional data or information specific to the country.10 This module provides information on the clinical trials conducted to evaluate the efficacy and safety of the product in human subjects. It includes information on the study protocols, results, and statistical analysis, as well as a summary of the overall clinical benefit-risk assessment.9 ICH countries: In ICH countries, the regulatory submission process typically involves submission of a Marketing Authorization Application (MAA) to the relevant national regulatory agency. The MAA must include data generated from clinical trials and other preclinical studies, as well as information on the manufacturing process and quality control measures.4 Module 3 – Quality This module provides detailed information on the quality aspects of the product, including the active pharmaceutical ingredient, the finished product, and the manufacturing process. It includes information on the controls and specifications used to ensure the quality of the product. Module 1 – Administrative Information and Prescribing Information This module provides an overview of the product, including its name, formulation, and intended use. It also includes information on the manufacturer, the marketing authorization holder, and the relevant regulatory submissions. Module 2 – Quality Overall Summaries This module provides a summary of the quality-related information for the product, including the results of stability studies and an overview of the manufacturing process. Clinical trial requirements BRICS countries: The clinical trial requirements for BRICS countries vary widely. For example, in Brazil, clinical trials may be conducted in two phases (I and II) before regulatory approval is granted. In Russia, the number of clinical trials required for approval may be higher compared to ICH countries. In India, the clinical trial requirements may be less stringent compared to ICH countries, but they have recently implemented stricter regulations for clinical trials.10 Module 4 – Non-Clinical Study Reports ICH countries: The ICH guidelines recommend a three-phase clinical trial process (Phase I, Phase II, and Phase III) for regulatory approval. The trials must be conducted in accordance with Good Clinical Practice (GCP) guidelines. This module provides information on the pre-clinical studies conducted to evaluate the safety of the product. It includes information on the study design, methods, and results, as well as an evaluation of the product's safety profile. BRICS This helps to ensure that the safety and efficacy of new drugs are protected, and that the regulatory approval process is transparent and predictable.7 2 Kumar, et al.: Regulatory requirements for Generic drugs Kumar, et al.: Regulatory requirements for Generic drugs Dossier Requirements ICH Countries BRICS Countries Clinical trial requirements Three-phase clinical trial process (I, II, and III) for regulatory approval. Vary widely regarding clinical trial phases. Regulatory submissions Submission of Marketing Authorization Application to the relevant national regulatory agency. Submission may differ from ICH and approval by respective country regulatory agency. Data requirements Data requirements are based on the guidelines set by the ICH. May be more extensive than those in ICH countries. Language requirements Accepted in English. Some of the countries require that regulatory documents to be submitted in their local language. Approval timeline Several months to several years. Timelines vary from ICH due to difference in regulatory process. Table 1: Comparison of MIST and ICH countries dossier requirement. Table 1: Comparison of MIST and ICH countries dossier requirement. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 Approval timeline MIST countries: Clinical trial requirements for the MIST countries vary, but they generally follow the ICH guidelines. In some cases, additional trials may be required to support regulatory approval.15 BRICS countries: Approval timelines for new drugs in BRICS countries can vary widely and may be longer than in ICH countries due to differences in the regulatory process and requirements. In some cases, the approval process can take several years. ICH countries: The ICH guidelines recommend a three-phase clinical trial process (I, II, and III) for regulatory approval. The trials must be conducted in accordance with Good Clinical Practice (GCP) guidelines.16 ICH countries: Depending on the country, approval timeline in ICH countries varies as it typically takes several months to several years to obtain regulatory approval.4 Language requirements BRICS countries: Some of the BRICS countries require that regulatory documents be submitted in their local language. For example, in Russia, submissions must be made in Russian, while in China, submissions may need to be in both English and Chinese.10 Kumar, et al.: Regulatory requirements for Generic drugs Kumar, et al.: Regulatory requirements for Generic drugs data, as well as data on pharmacokinetics, pharmacodynamics, and toxicity in specific populations.10 data, as well as data on pharmacokinetics, pharmacodynamics, and toxicity in specific populations.10 standards. However, China has more stringent requirements in terms of manufacturing information and stability data compared to ICH. Additionally, China requires a risk management plan and information on labeling and packaging, which are not required by ICH.13 ICH countries: In ICH countries, data requirements are based on the guidelines set by the ICH. The data submitted must demonstrate the safety and efficacy of the drug for its intended use.4 South Africa: The SAHPRA's requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, South Africa has more stringent requirements in terms of manufacturing information and stability data compared to ICH. Additionally, South Africa requires a risk management plan and information on labeling and packaging, which are not required by ICH.14 Regulatory submissions MIST countries: Each of the MIST countries has its own regulatory agency responsible for approving new drugs. The submissions required for regulatory approval may differ from the ICH guidelines and can include additional data or information specific to the country.17 Brazil: In terms of quality, safety, and efficacy requirements, ANVISA's guidelines are in line with ICH standards. However, in terms of environmental and social impact assessment, Brazil has more stringent requirements compared to ICH. Additionally, companies must provide information on the raw materials used and the results of environmental and social impact assessments, which are not required by ICH.11 ICH countries: In ICH countries, the regulatory submission process typically involves submission of a Marketing Authorization Application (MAA) to the relevant national regulatory agency. The MAA must include data generated from clinical trials and other preclinical studies, as well as information on the manufacturing process and quality control measures. Russia: The Roszdravnadzor's requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, Russia has more stringent requirements in terms of risk-benefit assessment compared to ICH. Additionally, Russia requires a risk management plan, which is not required by ICH. Dossier requirement comparison of MIST and ICH countries (Table 2) ICH countries: ICH countries typically accept regulatory submissions in English.11 Data requirements BRICS countries: The data requirements for BRICS countries may be more extensive than those in ICH countries. For example, in China, submissions may require Traditional Chinese medicine International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 3 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 Indonesia Clinical trial requirements: Clinical trials in Indonesia are generally conducted in accordance with ICH guidelines. However, some additional trials may be required to support regulatory approval. Regulatory submissions: The regulatory submission process in Indonesia involves submitting a Marketing Authorization Application (MAA) to the Indonesian regulatory agency, the National Agency of Drug and Food Control (BPOM). The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Language requirements MIST countries: Some of the MIST countries require that regulatory documents be submitted in their local language. For example, in Turkey, submissions must be made in Turkish. Data requirements: Indonesia requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements may be more extensive than those in ICH countries. Language requirements: Regulatory documents must be submitted in Indonesian. Data requirements: Indonesia requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements may be more extensive than those in ICH countries. ICH countries: ICH countries typically accept regulatory submissions in English.11 Language requirements: Regulatory documents must be submitted in Indonesian. Data requirements MIST countries: The data requirements for the MIST countries may be more extensive than those in ICH countries. In some cases, additional data may be required to support regulatory approval. India: The CDSCO's requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, India has more stringent requirements in terms of manufacturing information and stability data compared to ICH. Additionally, India requires a risk management plan, which is not required by ICH.12 ICH countries: The data requirements in ICH countries are based on the guidelines set by the ICH. The data submitted must demonstrate the safety and efficacy of the drug for its intended use.11 China: The NMPA's requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 4 Kumar, et al.: Regulatory requirements for Generic drugs Kumar, et al.: Regulatory requirements for Generic drugs Dossier Requirements ICH Countries MIST Countries Clinical trial requirements Three-phase clinical trial process (I, II, and III) for regulatory approval. Clinical trial requirements vary but follow ICH guidelines. Additional trials may be required. Regulatory submissions Submission of Marketing Authorization Application to the relevant national regulatory agency. Submission may differ from ICH and approval by respective country regulatory agency. Data requirements Data requirements are based on the guidelines set by the ICH. May be more extensive than those in ICH countries that demonstrates safety and efficacy. Additional data may require. Language requirements Accepted in English. Some of the countries require that regulatory documents be submitted in their local language. Ex: Turkish. Approval timeline Several months to several years. Timelines vary widely and may be longer from ICH due to difference in regulatory process. Table 2: Comparison of MIST and ICH countries dossier requirement. Mexico Clinical trial requirements: Clinical trials in Mexico are generally conducted in accordance with ICH guidelines. However, some additional trials may be required to support regulatory approval. Regulatory submissions: The regulatory submission process in Mexico involves submitting a Marketing Authorization Application (MAA) to the Mexican regulatory agency, the Federal Commission for the Protection Against Sanitary Risk (COFEPRIS). The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Data requirements: Mexico requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements may be more extensive than those in ICH countries. Language requirements: Regulatory documents must be submitted in Spanish. Approval timeline: The approval timeline in Mexica for medicines is 180 days which includes API or therapeutic indications already approved and 240 days for not approved.3,19 Approval timeline MIST countries: For new drugs in the MIST countries approval timelines can vary widely and may be longer than in ICH countries due to differences in the regulatory process and requirements. In some cases, the approval process can take several years. Approval timeline: The approval timeline in Indonesia is 100 working days with electronic standardized information and 150 days without electronic standardized information countries that have been marketed.20 ICH countries: The approval timeline in ICH countries varies depending on the country, but it typically takes several months to several years to obtain regulatory approval.18 Dossier requirements comparison of MIST with ICH countries Table 2: Comparison of MIST and ICH countries dossier requirement. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 South Korea Clinical trial requirements: Clinical trials in South Korea are generally conducted in accordance with ICH guidelines. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 5 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 Kumar, et al.: Regulatory requirements for Generic drugs Details Countries USA EUROPE JAPAN CHINA INDIA Regulatory authority Food and Drug Administration European Medicines Agency Ministry of Health Labour and Welfare National Medical Product Administration Central Drug Standard and Control organization Types of Application NDA ANDA BLA Marketing Authorization Application NDA ANDA BLA NDA ANDA Supplemental Import Drug application Renewal Marketing Authorization Application Types of Registration procedure Direct submission to FDA or through approved contact agent CP, DCP, MRP and NP J-NDA (Japan New Drug Application) Standard review and Special review procedure N/A Administrative and prescribing information 356h FDA form Notice to Applicant: Volume 2B Application Approval Form (AAF) Chinese specific application Form Form 44 Drug product labeling Package inserts are provided SmPL (Summary of Product Labeling) Draft package inserts -- Proposed Draft labels and cartoons Provided in Module 1 Information about clinical investigator In Module 5 In Module 1 In Module 2 -- In Module 1 BE study against RLD European reference product Japan reference product -- Innovator marketed product Regulatory approval of CTA/IND application Time 30 days 60 days 30 days 9-10 months 16-18 weeks MAA Time 180 Days 210 Days 150 Days 180 Days 8-12 weeks Table 3: Comparison table for CTD requirements in some ICH countries.26 Table 3: Comparison table for CTD requirements in some ICH countries.26 However, some additional trials may be required to support regulatory approval. Turkey Clinical tr Clinical trial requirements: Clinical trials in Turkey are generally conducted in accordance with ICH guidelines. However, some additional trials may be required to support regulatory approval. Regulatory submissions: The regulatory submission process in South Korea involves submitting a Marketing Authorization Application (MAA) to the Korean regulatory agency, the Ministry of Food and Drug Safety (MFDS). The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Regulatory submissions: The regulatory submission process in Turkey involves submitting a Marketing Authorization Application (MAA) to the Turkish regulatory agency, the Turkish Medicines, and Medical Devices Agency (TMDA). The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 South Korea Data requirements: South Korea requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements may be more extensive than those in ICH countries. Data requirements: Turkey requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements may be more extensive than those in ICH countries. Language requirements: Regulatory documents must be submitted in Turkish. Data requirements: Turkey requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements may be more extensive than those in ICH countries. Language requirements: Regulatory documents can be submitted in English or Korean. Approval timeline: The approval timeline in South Korea can be months or several years.21 Language requirements: Regulatory documents must be submitted in Turkish. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 6 Kumar, et al.: Regulatory requirements for Generic drugs Language requirements: Regulatory documents can be submitted in English. Approval timeline: The approval timeline in Turkey is 210 days having addition of 30 days for examining suitability of application.17 Approval timeline: The approval timeline in the US can be 180 days or less other times it may take several revisions.12 Dossier requirements of some countries under ICH (Table 3) Data requirements: Japan requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements are more extensive than those in other ICH countries. United States (US) United States (US) United States (US) Clinical trial requirements: Clinical trials in the US are conducted in accordance with ICH guidelines and are subject to review by the US Food and Drug Administration (FDA). Language requirements: Regulatory documents can be submitted in English or Japanese. Regulatory submissions: The regulatory submission process in the US involves submitting a New Drug Application (NDA) to the FDA. The NDA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Approval timeline: The approval timeline in Japan is generally 150 days to 12 months.22 European Union (EU) Mexico: The COFEPRIS' requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, Mexico has more stringent requirements in terms of environmental impact assessment compared to ICH. Additionally, Mexico requires a risk management plan and information on labeling and packaging, which are not required by ICH.15 Clinical trial requirements: Clinical trials in the EU are conducted in accordance with ICH guidelines and are subject to review by the European Medicines Agency (EMA). Regulatory submissions: The regulatory submission process in the EU involves submitting a Marketing Authorization Application (MAA) to the EMA. The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Indonesia: The BPOM's requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, Indonesia has more stringent requirements in terms of manufacturing information and stability data compared to ICH. Additionally, Indonesia requires a risk management plan, which is not required by ICH.20 Data requirements: The EU requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements are more extensive than those in other ICH countries. Language requirements: Regulatory documents can be submitted in English. Turkey: The Turkish Ministry of Health's requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, Turkey has more stringent requirements in terms of environmental impact assessment compared to ICH. Additionally, Turkey requires a risk management plan and information on labeling and packaging, which are not required by ICH.17 Approval timeline: The approval timeline in the EU is generally 210 days and can vary based on the procedure and revisions.12 Japan Clinical trial requirements: Clinical trials in Japan are conducted in accordance with ICH guidelines and are subject to review by the Japanese Ministry of Health, Labour, and Welfare (MHLW). South Korea: The MFDS' requirements for quality, safety, and efficacy, as well as preclinical and clinical trials, are in line with ICH standards. However, South Korea has more stringent requirements in terms of manufacturing information and stability data compared to ICH. Additionally, South Korea requires a risk management plan, which is not required by ICH.21 Regulatory submissions: The regulatory submission process in Japan involves submitting a Marketing Authorization Application (MAA) to the MHLW. The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 ACKNOWLEDGEMENT Regulatory submissions: The regulatory submission process in Australia involves application submit for Registration of a Therapeutic Goods to the TGA. The application must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. The author thanks JSS College of Pharmacy and JSS Academy of Higher Education and Research, Mysuru for providing the necessary facilities for carrying out this work. ABBREVIATIONS AAF: Application Approval Form; ANDA: Abbreviated New Drug Application; ANVISA: Agência Nacional de Vigilância Sanitária; API: Active Pharmaceutical Ingredients; BE: Bioequivalence; BLA: Biologics Licence Application; BPOM: Badan Pengawas Obat dan Makanan; BRICS: Brazil, Russia, India, China, and South Africa; CDSCO: Central Drugs Standard Control Organisation; COFEPRIS: Commission for the Protection Against Sanitary Risk; CP: Centralized Procedure; CTA: Clinical Trial Application; CTD: Common Technical Document; DCP: Decentralized Procedure; EMA: European Medicines Agency; EU: European Union; FDA: Food and Drug Administration; GCP: Good Clinical Practices; ICH: International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; IND: Investigational New Drug; MAA: Marketing Authorization Application; MAH: Marketing Authorization Holder; MFDS: Ministry of Food and Drug Safety; MHLW: Ministry of Health, Labour, and Welfare; MIST: Mexico, Indonesia, South Korea, and Turkey; MRP: Mutual Recognition Procedure; NDA: New Drug Application; NDS: New Drug Submission; NMPA: National Medical Products Administration; NP: National Procedure; RLD: Reference Listed Drug; SAHPRA: South African Health Products Regulatory Authority; SmPL: Summary of Product Labeling; TGA: Therapeutic Goods Administration; TMDA: Turkish Medicines, and Medical Devices Agency. Language requirements: Regulatory documents can be submitted in English. Approval timeline: The approval timeline in Australia can vary widely and may take several months.24 Canada Clinical trial requirements: Clinical trials in Canada are conducted in accordance with ICH guidelines and are subject to review by Health Canada. Data requirements: The US requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements are more extensive than those in other ICH countries. Regulatory submissions: The regulatory submission process in Canada involves submitting a New Drug Submission (NDS) International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 7 Kumar, et al.: Regulatory requirements for Generic drugs to Health Canada. The NDS must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Approval timeline: The approval timeline in Switzerland for authorization takes 10 and 12 months or between 4 and 5 months if applicant choose expedited procedure.25 Data requirements: Canada requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements are more extensive than those in some other ICH countries. CONCLUSION The regulatory environment in MIST and BRICS countries continues to evolve, and it is important for pharmaceutical companies to stay informed and adapt their regulatory strategies as needed. While there may be significant differences between the regulatory requirements of each country, the goal of ensuring the safety and efficacy of medicines for human use remains the same. By working closely with regulatory authorities and taking a proactive approach to regulatory affairs, pharmaceutical companies can help to promote greater harmonization and consistency in the regulatory requirements of MIST and BRICS countries. Language requirements: Regulatory documents can be submitted in English or French. Approval timeline: The approval timeline in Canada can vary from 6 months to 2 years.23 The authors declare no conflict of interest. The authors declare no conflict of interest. Australia Clinical trial requirements: Clinical trials in Australia are conducted in accordance with ICH guidelines and are subject to review by the Therapeutic Goods Administration (TGA). International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 CONFLICT OF INTEREST Data requirements: Australia requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements are more extensive than those in some other ICH countries. Switzerland Clinical trial requirements: Clinical trials in Switzerland are conducted in accordance with ICH guidelines and are subject to review by the Swiss Agency for Therapeutic Products (Swissmedic). Regulatory submissions: The regulatory submission process in Switzerland involves submitting a Marketing Authorization Application (MAA) to Swissmedic. The MAA must include data generated from clinical trials, as well as information on the manufacturing process and quality control measures. Data requirements: Switzerland requires data that demonstrate the safety and efficacy of the drug for its intended use. The data requirements are more extensive than those in some other ICH countries. Language requirements: Regulatory documents can be submitted in English, French or German. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 8 REFERENCES 1. Medicines Agency E. 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REFERENCES Annex 11 Good regulatory practices in the regulation of medical products Background WHO Expert Committee on Specifications for Pharmaceutical Preparations Fifty-fifth report. Available from: https://www.wto.org/english/tratop_ e/trips_e/techsymp_290621/gaspar_pres2.pdf. doi: doi. 18. Tannoury M, Attieh Z. The influence of emerging markets on the pharmaceutical industry. Curr Ther Res Clin Exp. 2017;86:19-22. doi: 10.1016/J.CURTHERES.2017.04. 005, PMID 29234483. 8. Introduction – international regulatory harmonization amid globalization of drug development – NCBI bookshelf [cited Feb 14, 2023]. Available from: https://www.nc bi.nlm.nih.gov/books/NBK174222/. 19. PharmaBoardroom – regulatory. Mexico: Pricing and Reimbursement [cited Mar 16, 2023]. Available from: https://pharmaboardroom.com/legal-articles/ regulatory-pricing-and-reimbursement-mexico/. Cite this article: Kumar J, Ramkishan A, Venkatesh MP. Navigating Global Regulatory Requirements for Generic Drugs: A Comparative Study of MIST, BRICS, and ICH Countries. Int. J. Pharm. Investigation. 2024;14(1):1-9. Cite this article: Kumar J, Ramkishan A, Venkatesh MP. Navigating Global Regulatory Requirements for Generic Drugs: A Comparative Study of MIST, BRICS, and ICH Countries. Int. J. Pharm. Investigation. 2024;14(1):1-9. 9
https://openalex.org/W2765172651	https://www.epj-conferences.org/articles/epjconf/pdf/2017/24/epjconf-jdn22_00003.pdf	English	null	Nuclear structures: Twinning and modulation in crystals	EPJ web of conferences	2,017	cc-by	7,270	C ⃝The Authors, published by EDP Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Nuclear structures: Twinning and modulation in crystals Václav Petˇríˇcek and Michal Dušek Institute of Physics, Czech Academy of Sciences v.v.i., Na Slovance 2, 182 21Praha 8, Czech Repu titute of Physics, Czech Academy of Sciences v.v.i., Na Slovance 2, 182 21Praha 8, Czech Republic Institute of Physics, Czech Academy of Sciences v.v.i., Na Slovance 2, 182 21Praha 8, Abstract. Crystal structure analysis is a standard technique routinely applied to single crystals as well as powders. However the process is not so straightforward if the crystal sample is affected by twinning or if the structure is modulated. In such cases the standard procedures are not directly applicable. The main purpose of this contribution is to show how to solve and reﬁne such difﬁcult structures. While for twinned structures the basic property of crystal – translation symmetry in three dimensional space–remains valid, for modulated crystals a special superspace theory must be exploited in order to describe the atomic structure with crystallographic methods generalized for superspace. DOI: 10.1051/epjconf/201715500003 DOI: 10.1051/epjconf/201715500003 EPJ Web of Conferences 155, 00003 (2017) JDN 22 1. Introduction Modern structure analysis of single crystals based on diffraction experiment is nowadays a standard discipline which allows solving and reﬁning structures of most new crystalline materials. Data are usually acquired on laboratory X-ray diffractometers, and structure solution and reﬁnement is made with standard program packages such SHELX [1], Olex2 [2], Crystals [3] or Jana2006 [4] in almost automatic way. Such a work can be made by a non- specialist in crystallography because most of possible problems can be detected and corrected with help of checking program such as Platon [5]. Simple structure of a well diffracting sample can be now measured, solved and reﬁned in less than one hour and the task can be done without a knowledge what is behind these powerful tools. However, not all structures can be solved by using these standard approaches. Two effects – twinning and modulations – often make serious problems in structure solution and reﬁnement if they are present in the crystals. These effects are especially important during phase transitions where electric and/or magnetic properties of the studied crystal may depend substantially on the phase and therefore a full description of the corresponding structural changes is very important. If these changes affect atomic positions as well as ordering of magnetic moments, such crystals must be studied by both X-ray and neutron diffraction technique. In this paper we shall concentrate on problems of solution and reﬁnement of nuclear structure from the neutron diffraction experiment. However, most of explanations are also valid for X-ray diffraction. In spite of the fact that most of standard programs can handle twinning in the crystal, application of these tools needs much deeper understanding of crystallography. Even more complicated are modulated crystals, which miss the basic property of the classical crystal, i.e. translation symmetry. In this paper we will introduce shortly the problem of solution and reﬁnement of difﬁcult structures. For more details about the underlying theory, we recommend the monographs [6] and [7]. EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 Figure 1. Diffraction pattern of monoclinic crystal, with monoclinic angle different for 90◦, twinned by a general rotation 180◦along c axis. Figure 1. Diffraction pattern of monoclinic crystal, with monoclinic angle different for 90◦, twinned by a general rotation 180◦along c axis. Figure 1. 1. Introduction Diffraction pattern of monoclinic crystal, with monoclinic angle different for 90◦, twinned by a general rotation 180◦along c axis. 2.1 Overlaps in twinned crystals (h, k, 0) diffraction plane of a simulated structure with point group 4/m: (a) without twinning; (b) twinned by 180◦rotation along b axis with volume fractions (3:1); (c) twinned by 180◦rotation along b axis with volume fractions (1:1). overlaps of diffraction spots, using the distance in reciprocal space between (h21, k21, l21) and the closest (h1, h1, l1). The reﬂections are supposed to be fully overlapped if their distance is smaller than the resolution limit of the data collection and fully separated if the distance is larger than the selected limit for such separation. Reﬂections having partial overlaps, i.e. with their distance larger than the resolution limit but smaller than the limit for full separation should be discarded from reﬁnement. Such a criterion is very rough and it might lead to relatively large number of deleted reﬂections. Moreover, the resolution and separation limits are not the same for all pairs of reﬂections, because they may be registered in an area detector at different geometry. For this reason, the standard data processing programs like Crysalis, Saint or Eval recognize the overlaps during data processing and encode corresponding information (i.e. which reﬂections are overlapped and separated) in a form of so-called hklf5 format. In such case the partially overlapped reﬂections are considered together in the reﬁnement and no information needs to be deleted. The case presented in the Fig. 1 is simple: the twinning is easily recognized from the diffraction pattern, the structure is solved by standard techniques from one domain or from a detwinned data and, ﬁnally, the structure is reﬁned using the twinning matrix or hklf5 format. Twins with full overlap of all reﬂections are much more difﬁcult case since they cannot be easily recognized. In the Fig. 2 we present a simulated diffraction pattern of a tetragonal structure with point symmetry 4/m, having different portion of twin domains related by 180◦ rotation along b axis. In cases (b) and (c) no indication of twinning is apparent and, moreover, the symmetry of the diffraction pattern (c) is higher (4/mmm) than the Laue symmetry of the structure. This means that presence of twinning in the crystal may obscure the correct symmetry, which can be deduced only during solution and reﬁnement process. From Fig. 2, the necessary condition for such full overlaps is obvious: the point symmetry of the reciprocal lattice must be higher than the point symmetry of the structure. 2.1 Overlaps in twinned crystals Twinned crystals are supposed to be composed from structurally identical domains mutually related by proper or improper rotations. The number of different domains is usually not very large and in most cases there are present just two differently oriented domains. The simplest case leads to existence of two mutually rotated diffraction patterns which can be simply recognized from diffraction pattern, see Fig. 1. In this example, reﬂections (h,k,0) are systematically overlapped with reﬂections (−h, −k, 0) of the second domain. Depending on the monoclinic angle , additional (random) overlapping reﬂections may occur for (h, k, l) with l being close to 2na/c cos . pp g y g The relationship between orientations of two domains is related by so called twinning matrix T2which is deﬁned by the equation: a1 b1 c1 = a2 b2 c2 · T2 (1) (1) where ai, bi and ci are cell parameters of the ith domain. The twinning matrix can be as used to express diffraction indices of the second domain in the reciprocal coordinate system of the ﬁrst (reference) system: h21 k21 l21 = h2 k2 l2 · T2 (2) (2) While the indices (h2, k2, l2) are integers deﬁning some reﬂections from the second domain, their corresponding coordinates (h21, k21, l21) in the reference subsystem of the ﬁrst domain need not to be integers. The twinning matrix can be used for predicting the full and partial While the indices (h2, k2, l2) are integers deﬁning some reﬂections from the second domain, their corresponding coordinates (h21, k21, l21) in the reference subsystem of the ﬁrst domain need not to be integers. The twinning matrix can be used for predicting the full and partial 2 DOI: 10.1051/epjconf/201715500003 EPJ Web of Conferences 155, 00003 (2017) EPJ Web of Conferences 155, 00003 (2017) JDN 22 JDN 22 l=0 h k l=0 h k l=0 h k (a) (b) (c) Figure 2. (h, k, 0) diffraction plane of a simulated structure with point group 4/m: (a) without twinning; (b) twinned by 180◦rotation along b axis with volume fractions (3:1); (c) twinned by 180◦rotation along b axis with volume fractions (1:1). l=0 h k (a) l=0 h k (b) l=0 h k (c) (b) (c) (a) Figure 2. 2.2 Symmetry of the twinned diffraction pattern Overlapping of diffraction spots from n twin domains can be expressed by the following formula for the combined structure factor F(h): F2(h) = v1F 2(hT1) + v2F 2(hT2) + . . . + vnF 2(hTn) (3) F2(h) = v1F 2(hT1) + v2F 2(hT2) + . . . + vnF 2(hTn) (3) (3) where vi is the volume fraction of ith domain and F(h) is the structure factor. The volume fractions are normalized to 1: n i=1 vi = 1 (4) (4) The Eq. (3) is based on the assumption that diffractions from twin domains are independent. The structure factors F(h) are coefﬁcients in the Fourier summation of periodic density of scatterers (r): (r) = r + 3 i=1 niai (5) (5) (r) = h F(h)exp(−2ihr) (6) (6) The combined structure factor is F(h) and it can be calculated from the integrated intensity I(h) of the relevant diffraction spot as for a non-twinned crystal: I(h) = S.Lp(\|h\|).A(h).F2(h) (7) (7) where S stands for the scaling factor, A for absorption correction and Lp for Lorentz- polarization factor. where S stands for the scaling factor, A for absorption correction and Lp for Lorentz- polarization factor. The full symmetry of the crystal is expressed by the space group symmetry which consists from the symmetry operations of the type: r′ = Rjr + sj + ck + n (8) (8) where Ri and si are rotation and translation part of the ith symmetry operation, cj is jth centering vector and n is an arbitrary unit cell translation. Then the generalized equation for symmetry condition is: where Ri and si are rotation and translation part of the ith symmetry operation, cj is jth centering vector and n is an arbitrary unit cell translation. Then the generalized equation for symmetry condition is: (r) = Rjr + 3 i=1 (sji + cki + ni)ai (9) (9) From the Eqs. (9) and (6) we can get: From the Eqs. (9) and (6) we can get: From the Eqs. (9) and (6) we can get: F(hRj) = F(h)exp[2ih(sj + ck)] (10) F(hRj) = F(h)exp[2ih(sj + ck)] (10) and for squared structure factors the following symmetry relationship holds: and for squared structure factors the following symmetry relationship holds: F 2(hRj) = F 2(h) (11) (11) This means that the diffraction pattern follows the point group symmetry. 2.1 Overlaps in twinned crystals Similar effects can be present even for structures of lower symmetry (triclinic, monoclinic, . . . ) if their cell parameters correspond within the experimental accuracy to a higher lattice point symmetry. This may happen as a result phase transitions in which the original symmetry is reduced but cell parameters are almost unchanged. In the following we shall concentrate on problems of data processing, solution and reﬁnement of twins with full overlaps. 3 3 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 JDN 22 2.2 Symmetry of the twinned diffraction pattern Moreover, the fact that the nuclear density is a real function leads to the following relationship for the complex conjugated structure factor: F ∗(h) = F(−h) (12) (12) and therefore the diffraction pattern shows contains always an inversion center even for non- centrosymmetric structures. EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 As mentioned above, complete overlaps of diffraction spots of the twinned crystals are possible only if the point symmetry of the lattice H is higher than the point symmetry of the structure G. Say that the order of the subgroup G with respect to H is n. Then the point group H can be decomposed into left cosets: H = T1G + T2G + . . . + TnG (13) (13) where the set of twinning operations {T1, T2, . . . .Tn} are selected arbitrary as representatives of each coset. Without loss of generality the ﬁrst twinning element can be chosen as an identity operation. Symmetry of the diffraction pattern in the case of equal volume fractions follows the lattice symmetry H. But as volume fractions need not be exactly equal we cannot a priory use during data merging all symmetry operations from H. Question is if all symmetry operations from the point group G are always present in the diffraction pattern. For the reﬂection hGj where Gj is an arbitrary operation from G the Eq. (3) gives: F2(hGj) = v1F 2(hGjT1) + v2F 2(hGjT2) + . . . + vnF 2(hGjTn) mmetry requirement F2(hGj) = F2(h) leads to set of conditions: F2(hGj) = v1F 2(hGjT1) + v2F 2(hGjT2) + . . . + vnF 2(hGjTn) then the symmetry requirement F2(hGj) = F2(h) leads to set of conditions: GjTk = TkGl ⇒Gl = T−1 k GjTk F2(hGj) = v1F 2(hGjT1) + v2F 2(hGjT2) + . . . + vnF 2(hGjTn) GjTk = TkGl ⇒Gl = T−1 k GjTk (14) (14) where Gl is again an operation from G. These conditions are fulﬁlled for a subgroup G only if it is a normal subgroup in H. In case the subgroup G is not a normal subgroup of H only operations fulﬁlling the Eq. (14) can be used in the merging procedure of symmetry equivalent reﬂections. Note that if the order of the subgroup G in H is two, the subgroup is always normal. 2.2 Symmetry of the twinned diffraction pattern The conclusion about symmetry of the diffraction pattern is that the symmetry can mimic the lattice symmetry but it can even mimic a symmetry lower than the point group of the crystal. 2.3 Systematic extinctions of twins Systematic extinctions are used to determine a correct space group of the studied compound. From the Eq. (10) it follows that reﬂections fulﬁlling the condition hRj = h (i.e. h invariant with respect to Rj) can have non-zero structure factor only if: exp 2ih sj + ck = 1 ⇒ h sj + ck = n (15) (15) where n is an arbitrary integer. Table 1. Systematic extinctions for m(001). Table 1. Systematic extinctions for m(001). (s1, s2, s3,) Symmetry operation Extinction condition (0, 0, 0) m – (1/2, 0, 0) a h = 2n + 1 (0, 1/2, 0) b k = 2n + 1 (1/2, 1/2, 0) n h + k = 2n + 1 l=0 h k l=0 h k (a) (b) Figure 3. (h, k, 0) diffraction plane of simulated structure with an a-glide plane: (a) without twinning; (b) twinned by 90◦rotation around c axis with volume fractions 1:1. l=0 h k (a) l=0 h k (b) (b) (a) Figure 3. (h, k, 0) diffraction plane of simulated structure with an a-glide plane: (a) without twinning; (b) twinned by 90◦rotation around c axis with volume fractions 1:1. Overlapping of reﬂections in twinned crystals can partially hide the extinction conditions. In the example shown in the Fig. 3 such an effect is demonstrated for the a-glide plane: As a result of the twinning, the absent reﬂections must fulﬁll the conditions h = 2n + 1 and k = 2n + 1, which cannot occur for a non-twinned crystal. Thus the extinctions are violated in very speciﬁc way which can help to recognize that the crystal in twinned. where n is an arbitrary integer. Example: The mirror plane with its normal parallel to the c axis has the following matrix form with respect to the crystal coordinate system a, b and c: Example: The mirror plane with its normal parallel to the c axis has the following matrix form with respect to the crystal coordinate system a, b and c:     1 0 0 0 1 0 0 0 −1  ,   s1 s2 s3     (16)     1 0 0 0 1 0 0 0 −1  ,   s1 s2 s3     (16) (16) From the space group theory it follows that in a primitive unit cell s1 and s2 are equal either to 0 or 1/2 and that the third component s3 in not restricted and depends on the origin selection. Taking into account all combinations and the Eq. (15), we can get extinctions as summarized in the Table 1. 5 DOI: 10.1051/epjconf/201715500003 EPJ Web of Conferences 155, 00003 (2017) JDN 22 JDN 22 Table 1. Systematic extinctions for m(001). (s1, s2, s3,) Symmetry operation Extinction condition (0, 0, 0) m – (1/2, 0, 0) a h = 2n + 1 (0, 1/2, 0) b k = 2n + 1 (1/2, 1/2, 0) n h + k = 2n + 1 3.1 Superspace approach for modulated structures Translational periodicity is the basic property of the classical crystal, see the Eq. (5). A direct consequence of this periodicity is the existence of a diffraction pattern by X-ray, neutrons or electrons with sharp diffraction spots which are localized at the points of the reciprocal lattice deﬁned by basic vectors a∗ 1, a∗ 2 and a∗ 3: h = h1a∗ 1 + h2a∗ 2 + h3a∗ 3 (19) (19) The reciprocal lattice vectors are related to translational (direct space) lattice vectors by equations: The reciprocal lattice vectors are related to translational (direct space) lattice vectors by equations: a∗ 1 = a2 × a3 a1 (a2 × a3) a∗ 2 = a3 × a1 a1 (a2 × a3) a∗ 3 = a1 × a2 a1 (a2 × a3) (20) (20) However, there are compounds which also give additional sharp diffraction spots, so-called satellites, localized out of the nodes of the reciprocal lattice. These spots can be indexed only if one or more additional (so called modulation) vectors are used: However, there are compounds which also give additional sharp diffraction spots, so-called satellites, localized out of the nodes of the reciprocal lattice. These spots can be indexed only if one or more additional (so called modulation) vectors are used: (21) H = h1a∗ 1 + h2a∗ 2 + h3a∗ 3 + mq . . . (21) In most cases one modulation vector is sufﬁcient for indexing of satellites, and in the following text we shall conﬁne ourselves to the case with one modulation vector. The diffraction pattern of such modulated crystal looks like the one shown in the Fig. 4. In most cases one modulation vector is sufﬁcient for indexing of satellites, and in the following text we shall conﬁne ourselves to the case with one modulation vector. The diffraction pattern of such modulated crystal looks like the one shown in the Fig. 4. The fact that the additional (satellite) reﬂections are sharp and regularly distributed in the diffraction space means that the violation of the 3d translation symmetry must be somehow regular. For handling of modulated structure a special theory of superspace and its symmetry has been developed. The development is closely connected with names Peter de Wolff, Aloysio Janner and Ted Janssen [8]. 2.4 Solution and reﬁnement of twinned structures Solution of crystal structures of twinned samples with completely overlapping diffraction patterns may be a difﬁcult task. In cases where twinning is a result of a phase transition from higher to lower symmetrical phase, the known structural model of the higher symmetrical phase can be used as a starting point for the structural solution of the lower symmetrical phase. However, in many cases several different subgroups are to be tested to ﬁnd correct solution. In the case that domains are not equally occupied and an approximate volume fraction is known, the observed structure factors can be corrected to get “detwinned” structure factors useful for ab-initio solution by standard methods: F 2(h) = [(1 −v)F2(h) −vF2(h · T2)] (1 −2v) (17) (17) wever, this correction cannot be used for equally or almost equally occupied domains. On the other hand, as soon as some starting structural model is known, the reﬁnement and completing the structure is very similar to that of a regular structure. The number of parameters in the reﬁnement is enlarged only by the (n-1) volume fractions. 6 EPJ Web of Conferences 155, 00003 (2017) JDN 22 EPJ Web of Conferences 155, 00003 (2017) DOI: 10.1051/epjconf/201715500003 For completing the structure a Fourier maps can be used in which the observed structure factors are corrected for twinning by one of these methods: F 2 obs,corr (h) = 1 v1 F2 obs (h) −v2F 2 calc (h · T2) −v3F 2 calc (h · T3) −. . . −vnF 2 calc (h · Tn) F 2 obs,corr (h) = 1 v1 F2 obs (h) −v2F 2 calc (h · T2) −v3F 2 calc (h · T3) −. . . −vnF 2 calc (h · Tn) F 2 obs,corr (h) = 1 v1 F2 obs (h) −v2F 2 calc (h · T2) −v3F 2 calc (h · T3) −. . . −vnF 2 calc (h · Tn) (18) (18) Fobs,corr (h) = \|Fobs (h)\| \|Fcalc(h)\| \|Fcalc(h)\| The above described methods handling twinned crystals have been implemented in the program Jana2006 and several typical practical examples were included into Jana cookbook (examples 3.1–3.4). Both can be downloaded the Jana home page: http://jana.fzu.cz/. The above described methods handling twinned crystals have been implemented in the program Jana2006 and several typical practical examples were included into Jana cookbook (examples 3.1–3.4). Both can be downloaded the Jana home page: http://jana.fzu.cz/. 3.1 Superspace approach for modulated structures The superspace approach is based on the construction which moves artiﬁcially all satellite reﬂections into the 4th dimension with a shift proportional to the satellite index – see Fig. 5. The spots are now making a lattice in the four dimensional reciprocal space described by the reciprocal vectors: A∗ i = a∗ i for i = 1, 2, 3 A∗ 4 = q + e (22) (22) The lattice character of the diffraction pattern in (3 + 1)d superspace means that the modulated structure has a translation periodicity in (3 + 1)d superspace. Thus the generalized The lattice character of the diffraction pattern in (3 + 1)d superspace means that the modulated structure has a translation periodicity in (3 + 1)d superspace. Thus the generalized 7 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 Figure 4. (h,0,l) diffraction plane of Na2CO3. The white grid represents the lattice of the main reﬂections. The additional (satellite) diffraction spots are regularly displaced from regular (main) spots by a q-vector (red arrows). Figure 4. (h,0,l) diffraction plane of Na2CO3. The white grid represents the lattice of the main reﬂections. The additional (satellite) diffraction spots are regularly displaced from regular (main) spots by a q-vector (red arrows). Figure 5. Introducing superspace in the reciprocal space: the vector e is perpendicular to the diffraction space R∗ 3. Black spots are main reﬂections located at the nodes of the three-dimensional reciprocal lattice. Gray spots are satellite reﬂections. The new reciprocal lattice vector: A∗ 4 = q + e. White spots are satellites projected to the new lattice vector; the blue area is a (3 + 1)d reciprocal cell describing main reﬂections as well as satellites. Figure 5. Introducing superspace in the reciprocal space: the vector e is perpendicular to the diffraction space R∗ 3. Black spots are main reﬂections located at the nodes of the three-dimensional reciprocal lattice. Gray spots are satellite reﬂections. The new reciprocal lattice vector: A∗ 4 = q + e. White spots are satellites projected to the new lattice vector; the blue area is a (3 + 1)d reciprocal cell describing main reﬂections as well as satellites. 3.1 Superspace approach for modulated structures Due to the translation symmetry, each such point can be transformed to the ﬁrst unit cell (as white points). where the lattice vectors Ai fulﬁl the equations: where the lattice vectors Ai fulﬁl the equations: 3.1 Superspace approach for modulated structures nuclear density function fulﬁlls the equation: nuclear density function fulﬁlls the equation: ˜ (r) = ˜ r + 4 i=1 niAi (23) (23) 8 EPJ Web of Conferences 155, 00003 (2017) JDN 22 EPJ Web of Conferences 155, 00003 (2017) DOI: 10.1051/epjconf/201715500003 JDN 22 Figure 6. The positionally modulated structure in the (3 + 1)d superspace: the atomic positions are described by the modulation function deﬁned in a (3 + 1)d unit cell. This unit cell can be repeated by the (3 + 1)d translational symmetry to ﬁll the whole (3 + 1)d superspace. Large blue points indicate intersections of the modulation function with R3 (the real space). These intersections deﬁne the crystal structure. Due to the translation symmetry, each such point can be transformed to the ﬁrst unit cell (as white points). Figure 6. The positionally modulated structure in the (3 + 1)d superspace: the atomic positions are described by the modulation function deﬁned in a (3 + 1)d unit cell. This unit cell can be repeated by the (3 + 1)d translational symmetry to ﬁll the whole (3 + 1)d superspace. Large blue points indicate intersections of the modulation function with R3 (the real space). These intersections deﬁne the crystal structure. Due to the translation symmetry, each such point can be transformed to the ﬁrst unit cell (as white points). Figure 6. The positionally modulated structure in the (3 + 1)d superspace: the atomic positions are described by the modulation function deﬁned in a (3 + 1)d unit cell. This unit cell can be repeated by the (3 + 1)d translational symmetry to ﬁll the whole (3 + 1)d superspace. Large blue points indicate intersections of the modulation function with R3 (the real space). These intersections deﬁne the crystal structure. Due to the translation symmetry, each such point can be transformed to the ﬁrst unit cell (as white points). Figure 6. The positionally modulated structure in the (3 + 1)d superspace: the atomic positions are described by the modulation function deﬁned in a (3 + 1)d unit cell. This unit cell can be repeated by the (3 + 1)d translational symmetry to ﬁll the whole (3 + 1)d superspace. Large blue points indicate intersections of the modulation function with R3 (the real space). These intersections deﬁne the crystal structure. 3.2 Superspace symmetry mmetry operation ˆS in the superspace must keep the generalized electron density invariant: Symmetry operation ˆS in the superspace must keep the generalized electron density invariant: ˜(ˆSr) = ˜(r) (25) ˜(ˆSr) = ˜(r) (25) (25) ˜(ˆSr) = ˜(r) A set of symmetry operations fulﬁlling this equation constitutes a superspace group. In analogy to 3d structures, the superspace symmetry induces diffraction symmetry, and the whole diffraction pattern including satellite reﬂections follows the corresponding point group. Introducing symmetry in (3 + 1)d superspace does not mean that all possible four- dimensional space groups are available for crystal symmetry. There are some restrictions which considerably reduce the number of four dimensional space groups acceptable for description of modulated structures. Any superspace operation R has the following matrix form with respect to the given crystallographic base: R = E 0 M I , sE sI (26) (26) The left-upper part E is 3 × 3 matrix which represents proper or improper rotation in three dimensional space (known from 3d space groups). The right-upper part is 3× 1 matrix (a column) of zeros. This restriction follows from the fact that superspace is based in a mathematical construction which uses an auxilliary vector e. This vector must stay perpendicular to the real three dimensional space when the symmetry operation is applied. The right-lower block I is 1 × 1 matrix with value equal ±1. Finally, the left-lower block M is 1 × 3 matrix (row) fulﬁlling the equation: qE −Iq = M (27) (27) This means that the rotation part of the symmetry operation is fully determined from the 3d rotation part and modulation vector. This means that the rotation part of the symmetry operation is fully determined from the 3d rotation part and modulation vector. The translation part sE follows from the corresponding 3d space group. The only new (compared with standard crystallography) part is sI which deﬁnes the translation component along the additional 4th direction. Value of this component can induce systematic extinctions of satellite reﬂections. For example the mirror plane with its normal parallel to the c axis and I = 1 causes extinctions as described in the Table 2. where the lattice vectors Ai fulﬁl the equations: where the lattice vectors Ai fulﬁl the equations: Ai = ai −(ai · q) e i = 1, 2, 3 A4 = e (24) (24) as follows from orthogonality conditions between direct Ai and reciprocal A∗ i : as follows from orthogonality conditions between direct Ai and reciprocal A∗ i : Ai · A∗ j = ij Ai · A∗ j = ij In the Fig. 6 the translation symmetry is demonstrated for positionally modulated structure. There is again a unit cell which contains structural information needed to generate whole modulated structure. The superspace approach allows generalization of standard structure tools such as Fourier and Patterson syntheses, structure solution and reﬁnement techniques. As the really observed 3d diffraction pattern is a projection from an auxilliary (3 + 1)d diffraction pattern in superspace along the vector e, corresponding 3d Fourier map which shows a nuclear density, is a section perpendicular to the vector e through a (3 + 1)d periodic superspace map. Moving this section along the vector e gives sections (so-called t-sections) of the same modulated structure which differs only by the origin shift. The (3 + 1)d Fourier maps are calculated from structure factors according to the equation analogical to the Eq. (6). Structure factor amplitudes are based on observed intensities (Eq. (7)) while phases follow from the actual model. The sections, in which modulation of one atomic coordinate x1, x2 or x3 is visualized as a function of x4, are called de Wolff’s sections. They play a crucial role in ﬁnding and completing modulation models for individual atoms in the structure. 9 EPJ Web of Conferences 155, 00003 (2017) EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 Table 2. Systematic extinctions for m(001). Table 2. Systematic extinctions for m(001). 3.3 Basic modulation types Modulations in the crystal can affect all structural parameters i.e. atomic occupancies, positions, ADP, multipole coefﬁcients or magnetic moments. Any structural parameter p(x4) due to the translation symmetry in the superspace can be expressed as a Fourier expansion: p (x4) = p0 + n ps,n sin 2nx4 + pc,n cos 2nx4 (28) (28) where p0, ps,n, pc,n are expansion coefﬁcients which are used to describe modulation wave. The number of used terms in the Eq. (28) depends on complexity of the modulation in crystal which is usually connected with the maximal number of observable satellite reﬂections. In the following a simple modulation models and their inﬂuence to the diffraction pattern is presented. 3.2 Superspace symmetry In this table, the symbol used for symmetry elements consists from two parts, the ﬁrst one deﬁnes the 3d symbol and the second one the additional information about sI, which in our example is 0 and s for sI equal 0 and 1/2, respectively. The superspace symbols are described in the International Tables for Crystallography (IT) vol. C [9]. The symbol consists of three parts – space group symbol, speciﬁcation of the modulation vector and speciﬁcation of the sI components of the symmetry operations. The components of modulation vector are either restricted by the Eq. (27) to speciﬁc rational values or they can have general (irrational) value. Irrational components are speciﬁed by Greek letters , or . Example: Pmna(0 1/2 )s00. Information available in the IT vol. C is limited, without a list of superspace group operations which restrict modulation functions of atoms located at special positions. Fortunately the list of symmetry operations for superspace groups up to (3 + 3)d superspace as well as additional details about their standard settings is accessible from [10]. The symmetrical restrictions of modulated functions expressed as a combination of harmonic waves for (3 + 1) superspace groups have been published [11] and they are automatically applied in the program Jana2006. 10 DOI: 10.1051/epjconf/201715500003 EPJ Web of Conferences 155, 00003 (2017) JDN 22 Table 2. Systematic extinctions for m(001). (s1, s2, s3, s4) Symmetry operation Extinction condition (0, 0, 0, 0) m 0 – (0, 0, 0, 1/2) m s m = 2n + 1 (1/2, 0, 0, 0) a 0 h = 2n + 1 (1/2, 0, 0, 1/2) a s h + m = 2n + 1 (0, 1/2, 0, 0) b 0 k = 2n + 1 (0, 1/2, 0, 1/2) b s k + m = 2n + 1 (1/2, 1/2, 0, 0) n 0 h + k = 2n + 1 (1/2, 1/2, 0, 1/2) n s h + k + m = 2n + 1 Table 2. Systematic extinctions for m(001). 3.2 Superspace symmetry (s1, s2, s3, s4) Symmetry operation Extinction condition (0, 0, 0, 0) m 0 – (0, 0, 0, 1/2) m s m = 2n + 1 (1/2, 0, 0, 0) a 0 h = 2n + 1 (1/2, 0, 0, 1/2) a s h + m = 2n + 1 (0, 1/2, 0, 0) b 0 k = 2n + 1 (0, 1/2, 0, 1/2) b s k + m = 2n + 1 (1/2, 1/2, 0, 0) n 0 h + k = 2n + 1 (1/2, 1/2, 0, 1/2) n s h + k + m = 2n + 1 3.3.1 Occupational modulation e simplest occupational modulation with only one harmonic wave can be expressed like: p (x4) = p0 + ps,1 sin (2x4) + pc,1 cos (2x4) (29) (29) From the kinematical theory of diffraction it can be shown that such modulation will generate only ﬁrst order satellites, as visualized in the Fig. 7(a). The corresponding de Wolff’s section through the (3 + 1)d Fourier map is shown in Fig. 7(b): This simple harmonic model for occupation modulation is not very common in real- life examples. Usually the diffraction pattern also contains higher order satellites and the description of modulations needs more harmonic waves. In the limiting case the modulation takes step like character: p(x4) = 1 x4 ∈⟨x0 4 − 2 , x0 4 + 2 ⟩ p(x4) = 0 x4 /∈⟨x0 4 − 2 , x0 4 + 2 ⟩ (30) (30) where x0 4 and deﬁne the center and length of the x4 interval in which the atom is present. where x0 4 and deﬁne the center and length of the x4 interval in which the atom is present. 11 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 EPJ Web of Conferences 155, 00003 (2017) DOI: 10.1051/epjconf/201715500003 JDN 22 (a) (b) Figure 7. The simple harmonic occupational modulation: (a) diffraction pattern, (b) de Wolff’s section. (a) (b) Figure 8. The step like occupational modulation (so-called crenel modulation): (a) diffraction pattern, (b) de Wolff’s section. (a) (b) Figure 7. The simple harmonic occupational modulation: (a) diffraction pattern, (b) de Wolff’s section. (a) (b) (b) (a) Figure 7. The simple harmonic occupational modulation: (a) diffraction pattern, (b) de Wolff’s secti (a) (a) (b) Figure 8. The step like occupational modulation (so-called crenel modulation): (a) diffraction pattern, (b) de Wolff’s section. (b) (b) (a) Figure 8. The step like occupational modulation (so-called crenel modulation): (a) diffraction pattern, (b) de Wolff’s section. As shown in the Fig. 8, the step like modulation leads to a high number of satellite reﬂections. Such a modulation is called crenel modulation [12]. Description of step-like modulation by harmonic waves would require high number of parameters. Instead of them we can use a crenel function (30) which has only two parameters – the center and the width. 3.3.2 Positional modulation The diffraction patterns for weak and strong modulation amplitude are shown in the Figs. 9 and 10. On contrary to occupational modulation here one harmonic can generate higher satellites. With growing amplitude of the modulation the order and relative intensity of satellites increases. 3.3.3 Composite structures However, the interaction between both subsystems usually induces a mutual modulations as shown in the Fig. 12. 3.3.3 Composite structures These structures consist of two or more modulated subsystems each having its (3 + 1)d reciprocal lattice A∗ν i i = 1, 2, 3, 4 (ν denotes the subsystem) related to a reference lattice 12 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 (a) (b) Figure 9. The weak positional modulation: (a) diffraction pattern, (b) de Wolff’s section. (a) (b) (b) (a) Figure 9. The weak positional modulation: (a) diffraction pattern, (b) de Wolff’s section. (a) (b) Figure 10. The strong positional modulation : (a) diffraction pattern, (b) de Wolff’s section. (b) (a) (b) (a) Figure 10. The strong positional modulation : (a) diffraction pattern, (b) de Wolff’s section. A∗ i i = 1, 2, 3, 4 by the equation: A∗ i i = 1, 2, 3, 4 by the equation: A∗ν i = 4 i=1 Zν ijA∗ j (31) (31) where the Zν ij is an integer matrix. Figure 11 shows a simple example of a composite structure made of two columns of atoms with different periodicity along the a1 1\|\| a2 1 direction, while the either parameters are identical: a1 2 = a2 2 and a1 3 = a2 3. In this example, the subsystems are not modulated and each of them makes a diffraction pattern which can be fully indexed by three indexes. We need four indexes, however, to index these patterns together. p g If we choose the ﬁrst subsystem as a reference subsystem, the Z matrices take form Z1 =   1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1   Z2 =   0 0 0 1 0 1 0 0 0 0 1 0 1 0 0 0   13 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 (a) (b) Figure 11. Composite structure composed of non-modulated subsystems: (a) diffraction pattern, (b) de Wolff’s section. (b) (a) (b) (a) Figure 11. Composite structure composed of non-modulated subsystems: (a) diffraction pattern, (b) de Wolff’s section. (a) (b) (a) (b) Figure 12. Composite structure composed of modulated subsystems: (a) diffraction pattern, (b) de Wolff’s section. (b) (a) Figure 12. Composite structure composed of modulated subsystems: (a) diffraction pattern, (b) de Wolff’s section. Diffraction pattern of non-modulated composite crystal contains only main reﬂections of both subsystems. 3.4 Solution of modulated structures In many cases modulated structures can be solved in two steps. In the ﬁrst step only main reﬂections are used to solve an average structure by standard methods. Such a structural model can show features like split atomic positions or unusually large ADP ellipsoids (see Fig. 13). Such effects can help to predict type of modulations and recognize atoms which are strongly modulated. In the second step, the modulation waves are found by reﬁnement from the small randomly chosen displacements. However in case of strong modulations the second step or even the ﬁrst step can fail because both the average structure and the initial modulation wave may be too far of the 14 EPJ Web of Conferences 155, 00003 (2017) DOI: 10.1051/epjconf/201715500003 JDN 22 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 JDN 22 JDN 22 (a) (b) Figure 13. Anion AsF4(OH)2 [16] (a) average structure calculated from main reﬂections; (b) modulated structure. (a) (a) (b) (b) Figure 13. Anion AsF4(OH)2 [16] (a) average structure calculated from main reﬂections; (b) modulated structure. correct solution. As for the standard crystallography, reﬁnement and Fourier methods only work for “almost ﬁnished” structures. This was the reason why the standard solution methods were generalized for modulated structures with the aim to solve them ab initio. The direct methods based on the Sayre equation were used for the development the program DIMS written by Fan Hai-fu [13, 14]. The heavy atom method for modulated crystals was developed by Steurer [15] and Petricek et al. [16]. However, these methods are not applicable to all cases. The most promising method for ab initio solution of strongly modulated structures is so-called charge ﬂipping [17, 18]. The method has been implemented into the program Superﬂip [19] and it is distributed together with program Jana2006. This method can solve modulated structures just in one step and starting modulation functions can be deduced directly from the output of the charge ﬂipping, which is the nuclear density map in superspace. With help of the modern diffractometer software and charge ﬂipping, the steps like indexing, data collection, data processing and structure solution can be done almost routinely. However, it does not mean that complete solution of modulated structures is a routine job. Difﬁculties start after the solution step, where e.g. interpretation of the charge ﬂipping output requires experience, manual work and analysis of Fourier sections. 3.4 Solution of modulated structures This interpretation is crucial for proper selection of modulation parameters and it can be speciﬁc for different modulated structures. This selection is important not only for reﬁnement but especially for interpretation of results where we aim to understand the reasons why the structure is modulated. Similarly as for twinned structures, the Jana cookbook contains several worked examples (5.1–5.5 and 7.1–7.3) covering typical cases of modulated structures. Moreover, everybody can participate in workshops which are offered regularly in the Jana web page for people interested in practical solution of modulated structures, as well as in bi-annual workshops organized by university in Bayreuth and in many other events focused to aperiodic crystallography. 15 EPJ Web of Conferences 155, 00003 (2017) JDN 22 DOI: 10.1051/epjconf/201715500003 References [1] G. Sheldrick, Acta Cryst. A64, 112 (2008) [2] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, J. Appl. Cryst. 42, 339 (2009) [3] P.W. Betteridge, J.R. Carruthers, R.I. Cooper, K. Prout, D.J. Watkin, J. Appl. Cryst. 36, 1487 (2003) [4] V. Petˇríˇcek, M. Dušek, L. Palatinus, Z. Kristallogr. 229, 345 (2014) [5] A.L. Spek, PLATON, A Multipurpose Crystallographic Tool, Utrecht University, Utrecht, The Netherlands (2008) [6] S. van Smaalen, Incommensurate Crystallography. (Oxford University Press, Oxford, 2007) [7] T. Janssen, G. Chapuis, M. de Boissieu, Aperiodic Crystals. (Oxford University Press, Oxford, 2007) [8] P.M. de Wollf, T. Janssen T., A. Janner, Acta Cryst. A37, 625 (1981) [9] International Tables for Crystallography, Volume C, Ed. E. Prince (Kluwer Acad. Publ., Dordrecht, 2004) [10] H.T. Stokes, B.J. Campbell, S. van Smaalen, Acta Cryst. A67, 45 (2011) [11] V. Petˇríˇcek, P. Coppens, Acta Cryst. A44, 1051 (1988) [12] V. Petˇríˇcek, A. van der Lee, M. Evain, Acta Cryst. A51, 529 (1995) [13] Hao Quan, Liu Yi-wei, Fan Hai-fu, Acta Cryst. A43, 820 (1987) [14] Fan Hai-fu, S. van Smaalen, E.J.W. Lam, P.T. Beurskens, Acta Cryst. A49, 704 (1993) [15] W. Steurer, Acta Cryst. A43, 36 (1987) [16] J. Peterková, M. Dušek V. Petˇríˇcek, J. Loub, Acta Cryst. B54, 809 (1998) [17] G. Oszlányi, A. Süto, Acta Cryst. A60, 134 (2004) [18] L. Palatinus, Acta Cryst. A60, 604 (2004) [19] L. Palatinus and G.Chapuis, J. Appl. Cryst. 40, 786 (2007) 16
https://openalex.org/W3156120138	https://www.researchsquare.com/article/rs-353955/v1.pdf?c=1631876432000	English	null	Multi-material Topology Optimization Based on Multiple SIMP of Variable Density Method	Research Square (Research Square)	2,021	cc-by	8,230	Multi-material Topology Optimization Based on Multiple SIMP of Variable Density Method Hongyu Jiao Changshu Institute of Technology https://orcid.org/0000-0002-3025-4170 Ying Li (  cslgjxly@163.com ) Changshu Institute of Technology Original Article Keywords: Topology optimization, Muti-material, Multiple SIMP, Variable density method Posted Date: April 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-353955/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ·1· Multi-material Topology Optimization Based on Multiple SIMP of Variable Density Method Hong-Yu Jiao1 • Ying Li1 Received June xx, 201x; revised February xx, 201x; accepted March xx, 201x © Chinese Mechanical Engineering Society and Springer-Verlag Berlin Heidelberg 2017 Abstract: In this paper a multiple SIMP (solid isotropic material with penalization model) of variable density method is proposed to solve the problem of muli-material topology optimization. All candidate materials including void material are arranged in descending order of elastic modulus. The material conversion scheme of multiple SIMP is based on the elastic modulus of the candidate material after interpolation. The iterative criterion of muti-material topology optimization is derived from the Kuhn-Tucker condition using the guide-weight method. Three examples show that it is effective and moderate to use the proposed method to solve the problem of muti-material topology optimization. Guo el al. [16] proposed a level set-based approach to solve the stress-related topology optimization problem. The absence of gray areas in the design domain is the most attractive feature of this approach during the optimization process. The approach is extended to stress-related topology optimization for muti-materials [17].In comparison with SIMP-based optimization approaches, the level set-based approach has lower solution efficiency. This means that it needs more iteration to get the optimal solution. Tavakoli et al. [18] introduced a new algorithm to solve muti-materials topology optimization problem. The core idea of the algorithm is to divide the multi-material topology optimization problem into a sequence of binary phase topology optimization sub-problems. These sub-problems are solved in a sequential manner according to the traditional binary phase topology optimization problem. There are a large number of binary phase topology optimization sub-problems to be solved in the proposed algorithm. The computational cost of this algorithm increases dramatically. It usually takes over 1000 iterations to find an optimal solution. Keywords: Topology optimization • Muti-material • Multiple SIMP • Variable density method Multi-material Topology Optimization Based on Multiple SIMP of Variable Density Method Hong-yu Jiao, born in 1981, is currently an associate professor at School of Automotive Engineering, Changshu Institute of Technolo China. He received his PhD degree from Tongji University, China, in 2015. His research interests include structural lightweight design Tel: +86-13914927008; E-mail: cslgjhy@163.com Ying Li, born in 1979, is currently a lecturer at School of Automotive Engineering, Changshu Institute of Technology, China. Her research interests include structural lightweight design. Tel: +86-18852981695; E-mail: cslgjxly@163.com Corresponding author：Ying Li E-mail：cslgjxly@163.com Jia-Fan Zhang et al. ·2· ORIGINAL ARTICLE(or REVIEW) 1 School of Automotive Engineering, Changshu Institute of Technology, Suzhou 215500, China  Ying Li cslgjxly@163.com  Ying Li cslgjxly@163.com 1 School of Automotive Engineering, Changshu Institute of Technology, Suzhou 215500, China 1 Introduction As a kind of structural optimization, topology optimization is a mathematical method for optimizing the distribution of materials in a given design domain according to given load conditions, constraints and performance indicators [1-3]. In the past decades, a variety of topology optimization methods such as level set method [4-5], phase field method [6-7] bi-directional evolutionary structural optimization (BESO) method [8-9], and variable density method [10-11] has been extensively and deeply researched. These topology optimization methods gradually expanded from single-material to multi- materials [12-15]. To solve the muti-material topology optimization problem, Xia et al. [19] integrate the material conversion scheme of the BESO with the level set method. This method inherits the advantages of the BESO method and the level set method, and overcomes the shortcomings of the two methods. Among the various popular interpolation schemes of variable density method, SIMP is a commonly used density-stiffness interpolation model. The interpolation model uses the element relatively density as the design variable, and the material properties are simulated by the exponential function of the element relatively density. The Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation ·3· elastic modulus in SIMP for single material [20] can be formulated as descending order of elastic modulus such that E1>E2>…>Em. The structure is divided into n elements and the initial material of all elements is candidate material μ1. ( ) 0 p E x x E = ， (1) (1) In the multiple SIMP of variable density method, the material properties are still simulated by the exponential function of the element relatively density. The elastic modulus in multiple SIMP of variable density method can be formulated as where E(x) is the elastic modulus after interpolation; x is the element relatively density; p is penalization factor and E0 is the elastic modulus of the material. For the two-phase material problem, the SIMP model can be expressed as ( ) ( ) p j j i i j E x x E = ， (4) (4) ( ) 1 2 (1 ) p p E x x E x E = + − ， (2) (2) where ( ) j i E x is the elastic modulus after interpolation by the jth material μj. j ix (i=1,2,…,n) is the relative density of the ith element whose material is the jth material μj. Ej is the elastic modulus of the jth material μj. 1 Introduction 1 1 j j j p i i j E x x E + + = ， 1 1 j j j p i i j E x x E + + = ， (7) (7) The maximum value of 1 j ix + is 1. When 1 j j p i j E x E +  , the element changes from the jth material μj to the (j+1)th material μj+1.At this time, the relative density of the ith element whose material is the (j+1)th material μj+1 is calculated as 1 1 j j j p i i j E x x E + + = ， (8) Figure 1 Elastic modulus in multiple SIMP of variable density method (8) Either the previous method for muti-material topology optimization has more iteration or additional design variables need to be added. In this paper, under the premise of without increasing design variables, we aim to solve the problem of muti-material topology optimization. 1 Introduction where E1 and E2 are the elastic modulus of the two materials, respectively. The three-phase material usually contains two material and void material. The SIMP model of three-phase material requires two design variables to describe. It can be further expressed as In the multiple SIMP of variable density method, the material of an element can change from one to another, as shown in Fig.1. When an element changes from the jth material μj to the (j+1)th material μj+1, the elastic modulus after interpolation does not change. It is expressed as ( ) 1 2 2 1 2 1 2 1 2 2 , ( (1 ) ) p p p E x x x x E x E = + − ，(3) According to this law, the SIMP model m-phase material requires (m-1) design variables to describe. Blasques [21] applied the SIMP-like interpolation model to muti-phase material topology optimization problem of composite beam cross sections. It also requires (m-1) design variables. g p ( ) ( ) 1 j j i i E x E x + = ， (5) (5) We can get Eq. (6) from Eqs.(4) and (5). We can get Eq. (6) from Eqs.(4) and (5). We can get Eq. (6) from Eqs.(4) and (5). ( ) ( ) 1 1 p p j j i j i j x E x E + + = ， ( ) ( ) 1 1 p p j j i j i j x E x E + + = ， (6) (6) The Eq. (7) is expressed in another form as The Eq. (7) is expressed in another form as Zuo et al. [22] proposed an ordered SIMP interpolation method to solve muti-phase material topology optimization problem. This method interpolates the elastic modulus for multiple materials with respect to the normalized density variables by introducing power functions with scaling and translation coefficients. The biggest feature of this method is that there is no need to introduce any new design variables. Yin et al. [23] proposed a new material interpolation model based on SIMP method, which is named the peak function model. This model can easily include multi-phase materials in the design by using a linear combination of normal distribution functions. The outstanding feature of this model is also that there is no need to add any new design variables. 2 Multiple Variable Density Method The structure is composed of m candidate materials μj, with corresponding densities ρj, and elastic modulus Ej(j=1,2,…,m). The candidate material μm is the void material. Let all the m candidate materials be arranged in Figure 1 Elastic modulus in multiple SIMP of variable density method ·4· Jia-Fan Zhang et al. max min max min min max 0 0 0 0 0 ( ) 0 ( ) 0 0 g( ) 0 ( ) 0 1,2, , 1, 2, 3, , X X X X j i j i j j j i i i j i j i x x L f g x x x x x x x x g g g x x x i n j or or orm        =      = + =          =       =   =    =         = =  , (15) 3.1 The Mathematical Model A matrix X is defined to store the relative density of elements. The number of matrix rows is the number of elements n, and the number of matrix columns is the number of candidate materials m. It is expressed as , (15) 1,1 1,2 , ( , , , ) X n m i j x x x R  =  ， (9) 1,2, , i n = 1,2,3, j m = , (9) An element can only be assigned one of m materials. It means that only one element in each row is (0, 1] and the other elements are equal to zero. When the value of j ix is between the upper and lower limits of the design variable, we can obtain the following equation as A column matrix I is defined, whose elements are equal to 1. It is expressed as 1 j i j j i i j i j j i i g g x x x f f x x x       = =   − −   (16) 1 [1,1, ,1]m I  = ， (10) (10) We are setting a matrix X which is equal to X times I. It is expressed as X XI = ， (11) X XI = ， (11) (11) (16) For each i We mainly discuss the problem of multi-material topology optimization under mass constraint in this paper. So f(X) represent muti-material structural performance and g(X) represent the total volume or mass of muti-material structure. We mainly discuss the problem of multi-material topology optimization under mass constraint in this paper. So f(X) represent muti-material structural performance and g(X) represent the total volume or mass of muti-material structure. 3.1 The Mathematical Model , j i i j x x = ， (12) (12) The mathematical model of muti-material topology optimization under the single working condition can be expressed as Using the guide-weight method [24-28], the four key formulas are defined as follows: 1 2 ( , , , ) 1,2, , 1, 2, 3, , X j j j n i x x x R i n j or or orm =  = = i j i g H x  =  , (17) j i i i W x H = , (18) j i i j i f G x x  = −  , (19) 1 n i i G G = = , (20) (17) (13) ( ) min . ( ) 0 X X f s t g →  (13) ( ) min . ( ) 0 X X f s t g →  (18) min max j i x x x   where vector X is an n-dimensional vector of the design variables; f(X) is the objective function and g(X) is the constraint function. n is the number of finite element meshes and m is the number of candidate materials. xmin and xmax are the lower and upper limits of the design variable, respectively. (19) (20) where Hi is the heap density of j ix . Wi is the equivalent mass of j ix . Gi is the guide-weight of j ix and G is the total guide-weight. 4 The Minimum Compliance Problem of Muti-material Topology Optimization A step factor α is introduced into the iterative criterion. The purpose is to ensure convergence of optimization results. Considering the lower and upper limits of the design variable, the iterative criterion is finally indicated as In this section, we mainly discuss the minimum compliance problem of multi-material topology optimization by virtue of the above proposed method. The element relative density is selected as the design variable .The structural mass is as the constraint function. It can be formulated as Case(1):If( ) ( 1) 1 k j j p i j E x E + +  , then ( ) ( ) ( ) ( ) ( ) ( ) ( 1) 1 max ( 1) , ( 1) max max (1 ) k k k j j j i p i i k i j i j k j i E G x x x H E x x x x    + + + +    + −      =       . (23) Case(1):If( ) ( 1) 1 k j j p i j E x E + +  , then It can be formulated as It can be formulated as ( ) ( ) ( ) ( ) ( ) ( ) ( 1) 1 max ( 1) , ( 1) max max (1 ) k k k j j j i p i i k i j i j k j i E G x x x H E x x x x    + + + +    + −      =       . (23) mulated as ( ) f C = X ， (29) 0 0 ( ) 0 g M f M = −   X ， (30) (29) (29) (30) (23) (30) where C is the compliance of muti-material structure. M0 is the initial mass of muti-material structure. M is the mass of muti-material structure after optimization. f0 is a mass fraction. In this case, the element relative density will change and the material of the elements does not change. 4 The Minimum Compliance Problem of Muti-material Topology Optimization Case(2):If( ) ( 1) 1 k j j p i j E x E + +  , then ( ) ( ) ( ) ( 1) , 1 1 1,or2,or3, or 1 k k j j p i j i j E x x j m E + + + = = − , (24) ( ) ( ) , min k i j x x = (25) How to get the explicit expression between structural compliance and the design variable is the key to solving multi-material topology optimization. The structural compliance C can be expressed as T T T 1 1 F U U KU u k u n n i i i i i i C c = = = = = =   , (31) (25) (31) In this case, the ith element changes from the jth material μj to the (j+1)th material μj+1. The relative density of the ith element whose material is the (j+1)th material , 1 i j x + is computed by Eq.(24). Because an element can only be assigned one of m materials. The relative density of the ith element in the jth ,i j x is assigned a value of xmin as Eq. (25). where U is the displacement vector. F is the load vector. K is the structural stiffness matrix. ui is the element displacement vector. ki is and the element stiffness matrix. According to multiple SIMP of variable density method, we know that 3.2 Guide-weight Method Substituting equations (17) and (19) into equation (16), then we have In order to solve the multi-material topology optimization problem, we first need to construct the Lagrange equation as follows 1,2, , 1, 2, 3, , j i i i G x i n j or or orm H  = = = (21) (21) ( ) ( ) L f g  = + X X ， (14) (14) The above formula is the iterative criterion which can solve the problem of muti-material topology optimization taking advantage of the guide-weight method. When the muti-material topology optimization is performed, the iterative criterion can be written as where λ is a Lagrange multiplier. where λ is a Lagrange multiplier. Based on the Kuhn-Tucker condition, the optimal value X* must be satisfied X* must be satisfied Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation ·5· ( ) ( 1) ( ) ( ) 1,2, , k j k i i i G x i n H  + = = . topology optimization using multiple SIMP of variable density method can be solved. (22) 4 The Minimum Compliance Problem of Muti-material Topology Optimization T ( ) u κ u j p j i i i i i p x pc = = T ( ) u κ u j p j i i i i i p x pc = = The total guide-weight G can be calculated as The total guide-weight G can be calculated as According to equations (44-48), the minimum compliance problem of muti-material topology optimization can be solved by virtue of the above proposed method. 1 1 n n i i i i G G p c pC = = = = =   ， (39) (39) The mass of muti-material structure M is expressed as 1 1 n m j i i j i j M x v  = = = ， (40) 3.3Lagrange Multiplier λ ( ) k k j p j i i i x = , (32) (32) When the design variable X takes the optimal value X*, the Lagrange multiplier λ can be obtained by means of where ki is the stiffness matrix of the ith element after penalization. k j i is the initial stiffness matrix of the ith element assigned to the jth material μj. 1,2, , i j i i G i n x H = = = . (26) The structural compliance C is rewritten based on multiple SIMP of variable density method. It can be formulated as Hence 1 1 2 1 2 1 n i n i n n i i G G G G W W W W  = = = = = = =   ， (27) T 1 ( ) u k u n j p j i i i i i C x = = ， (33) (33) (27) Derivation of structural compliance C with respect to design variables is obtained as Derivation of structural compliance C with respect to design variables is obtained as 1 i= T T U F U j j j i i i C F x x x      = +       ， (34) Wi is the equivalent mass of muti-material structure. When performing a multi-material topology optimization, Lagrange multiplier λ can be calculated as (34) The load vector F has nothing to do with the relative density j ix , so the above equation is simplified as below. 0 W G =  , (28) (28) T U F j j i i C x x   =   ， (35) (35) where W0 is the mass or volume constraint of muti-material structure. where W0 is the mass or volume constraint of muti-material structure. From the equation KU=F, the following equation is obtained as Using equations (22-25), and (28), muti-material ·6· Jia-Fan Zhang et al. 6 Numerical Experiments and Discussions 1,2, , 1 2 j i i i j pc x i n j or or m v  = = = ，(43) 5 Convergence Criteria (40) The relative error τ of the two adjacent optimization results is less than the given convergence accuracy τmax for five times in succession, that is, 5 consecutive times satisfying equation (49), it is considered that the muti-material topology optimization has converged. The convergence criteria can be formulated as Derivation of the mass of muti-material structure M with respect to design variables j ix is obtained as [29] i j j i M v x   =  ， (41) (41) g max    − =   = − = − N k k N k k k C C C 1 ) 1 ( 1 ) 1 ( ) ( ) ( , (49) 6 Numerical Experiments and Discussions max    − =   = − = − N k k N k k k C C C 1 ) 1 ( 1 ) 1 ( ) ( ) ( , (49) Let’s substitute equations (30) and (41) into equation (17), then we have (49) i i j j j i i g M H v x x    = = =   ， (42) (42) Equations (38) and (42) are plugged into equation (22), and then it can be written as 3.3Lagrange Multiplier λ 0 K U F U K j j j i i i x x x    + = =    ， (36) om equations (35) and (36) we have ( ) ( ) ( ) ( ) ( ) ( ) ( 1) 1 ( 1) max , ( 1) max max (1 ) k k k j j j i p k i i i j j i j k j i E pc x x x v E x x x x    + + + +    + −       =      (45) (45) From equations (35) and (36), we have From equations (35) and (36), we have q ( ) ( ), T T 1 1 T - - - ( ) k k U U u u u k u n i i i j j j i i i i j p j i i i i C x x x p x = −    = =    =  ， (37) t’s substitute equations (37) and (31) into equation Case(2):If( ) ( 1) 1 k j j p i j E x E + +  , then ( ) ( ) ( ) ( 1) , 1 1,or2,or3, or 1 k k j j p i j i E x x j m E + + = = − , ，(46) Let’s substitute equations (37) and (31) into equation (19), and then sort to get the following equation. ( ) ( ) , min k i j x x = ， (47) ( ) ( ) , min k i j x x = ， (47) j j i i i j j i i f C G x x x x   = − = −   ， (38) Lagrange multiplier λ can be computed by Lagrange multiplier λ can be computed by (38) 0 0 fM pC W G = =  ， (48) (48) 6.1 Bridge Structure under Mass Constraint 6.1 Bridge Structure under Mass Constraint The design domain of the bridge structure [30] is 200×100 rectangular, as shown in Fig.2. The design domain is divided into 200×100 elements during multi-material topology optimization analysis. The left lower endpoint ot the design domain is fully constrained. In the vertical direction of the design domain, the right lower endpoint is constrained. The vertical load F1 is 20 N, located on the midpoint A of lower boundary. The vertical load F2 is 10 N, located on the ¼ and ¾ of lower boundary, respectively. The other necessary parameters corresponding to these test cases are listed in Table 1. The above formula is the iterative criterion which can solve the muti-material topology optimization problem of minimum compliance taking advantage of the guide-weight method. When the muti-material topology optimization is performed, the iterative criterion can be written as ( ) ( 1) ( ) ( ) 1,2, , 1 2 k j k i i i j pc x i n j or or m v  + = = = (44) Taking into account the convergence of optimization and the limits of design variables, the iterative equation is finally expressed as Case(1):If( ) ( 1) 1 k j j p i j E x E + +  , then Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation ·7· (a) k=1 (b) k=6 (c) k=11 (d) k=16 (e) k=21 (f) k=36(optimal topology) μ1 μ2 μ3 Figure 2 Optimization domain of bridge structure Table 1 Parameters corresponding to bridge structure Test Case Candidate materials m Penalizat ion factor p Elastic Modulus Ej Density ρj Mass Fraction f0 Bridge #1 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.2 Bridge #2 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.25 Bridge #3 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.3 Bridge #4 μ1, μ2, μ3 3.5 [2,1,e-9] [2,1,e-9] 0.25 Bridge #5 μ1, μ2, μ3 4 [2,1,e-9] [2,1,e-9] 0.25 Bridge #6 μ1, μ2, μ3, μ4 3 [4,2,1,e-9] [4,2,1,e-9] 0.1 Bridge #7 μ1, μ2, μ3, μ4 3 [9,3,1,e-9] [9,3,1,e-9] 0.05 6.1.1Muti-material topology optimization of bridge structure Figure 3 shows the evolution of muti-material topology optimization for bridge structure #1 with three kinds of materials including void material. It can be seen that most of the elements are converted from material μ1 to material μ2 in the first iteration. 6.1 Bridge Structure under Mass Constraint As the number of optimization increases, the amount of material μ1 is getting less and less and the amount of material μ2 is getting more and more. In the 11th iteration, some elements are changed from material μ2 to material μ3 (void material). After the 16th iteration, the amount of material μ3 is getting more and more and the main body of optimal topology is forming. Until muti-material topology optimization is completed, an optimal topology with 3 materials is obtained, as shown in Figure 3(f). The main body of optimal topology is composed of material μ2 and material μ3. Material μ1 only appears in areas with high stress. (a) k=1 (b) k=6 (c) k=11 (d) k=16 (e) k=21 (a) k=1 (b) k=6 (c) k=11 Figure 2 Optimization domain of bridge structure Table 1 Parameters corresponding to bridge structure Test Case Candidate materials m Penalizat ion factor p Elastic Modulus Ej Density ρj Mass Fraction f0 Bridge #1 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.2 Bridge #2 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.25 Bridge #3 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.3 Bridge #4 μ1, μ2, μ3 3.5 [2,1,e-9] [2,1,e-9] 0.25 Bridge #5 μ1, μ2, μ3 4 [2,1,e-9] [2,1,e-9] 0.25 Bridge #6 μ1, μ2, μ3, μ4 3 [4,2,1,e-9] [4,2,1,e-9] 0.1 Bridge #7 μ1, μ2, μ3, μ4 3 [9,3,1,e-9] [9,3,1,e-9] 0.05 6.1.1Muti-material topology optimization of bridge t t Table 1 Parameters corresponding to bridge structure Table 1 Parameters corresponding to bridge structure Figure 3 shows the evolution of muti-material topology optimization for bridge structure #1 with three kinds of materials including void material. It can be seen that most of the elements are converted from material μ1 to material μ2 in the first iteration. As the number of optimization increases, the amount of material μ1 is getting less and less and the amount of material μ2 is getting more and more. In the 11th iteration, some elements are changed from material μ2 to material μ3 (void material). After the 16th iteration, the amount of material μ3 is getting more and more and the main body of optimal topology is forming. Until muti-material topology optimization is completed, an optimal topology with 3 materials is obtained, as shown in Figure 3(f). The main body of optimal topology is composed of material μ2 and material μ3. Material μ1 only appears in areas with high stress. 6.1 Bridge Structure under Mass Constraint (f) k=36(optimal topology) μ1 μ2 μ3 (f) k=36(optimal topology) Jia-Fan Zhang et al. ·8· Figure 3 Evolution of topology optimization for bridge structure #1 Figure 5 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 6.1.4The effect of the number of candidate materials m on optimization results #1 6.1.2The effect of mass fraction f on optimization results Figure 4 shows the optimization curves of structural compliance C for bridge structure #1, #2 and #3 when mass fraction f is 0.2, 0.25, and 0.3, respectively. As the number of optimization increases, three curves of structural compliance increase rapidly to a maximum value and then fall smooth. The optimal topologies are obtained after 36, 33, and 19 iterations, respectively, as shown in Fig.4. We find a law that the structural compliance and number of iterations when the optimal topology is obtained gradually becomes larger, as the mass fraction gets smaller. But the optimal topologies are similar, which can prove that the proposed method is effective and robust. Figure 5 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 Figure 5 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 Figure 5 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 6.1.4The effect of the number of candidate materials m on optimization results 6.1.4The effect of the number of candidate materials m on optimization results Figure 4 Optimization curves of structural compliance when mass fraction f is 0.2, 0.25, and 0.3 Figure 6 shows the evolution of topology optimization for bridge structure #6 and #7 with four kinds of materials including void material. The main body of optimal topology with four kinds of materials is similar with the main body of optimal topology with three kinds of materials. The optimal topology for bridge structure #6 is composed of material μ3 and material μ4. Materials μ1 and μ2 appears in areas with high stress. The optimal topology for bridge structure #7 also is composed of material μ3 and material μ4. But material μ2 only appears in areas with high stress and material μ1 disappears. Because material μ1 has a higher density, material μ1 has been fully converted to material μ2 under the condition of smaller mass fraction f. 6.2 Cantilever beam structure under the condition of volume constraint or mass constraint The short cantilever beam structure is a rectangle with a length of 200 and a width of 100, as shown in Fig.7. The left boundary of design domain is fixed. A vertical load of F3 = 10 N is applied to the centre point B on the right boundary of the design domain. The short cantilever beam structure is divided into 200×100 finite element meshes. The other necessary parameters corresponding to these test cases are listed in Table 2. 6.1 Bridge Structure under Mass Constraint Figure 4 Optimization curves of structural compliance when mass fraction f is 0.2, 0.25, and 0.3 1 =6 11 6.1.3 The effect of penalization factor p on optimization results 6.1.3 The effect of penalization factor p on optimization results k k k= Figure 5 shows the optimization curves of structural compliance C for bridge structure #2, #4 and #5 when penalization factor p is 3, 3.5, and 4, respectively. The optimal topologies are obtained after 33, 18, and 15 iterations, respectively, as shown in Fig.5. We find the second law that the number of iterations gradually decrease as the penalty factor p increases. This means that increasing the penalty factor can get a lower number of iterations. Similar optimal topologies can be obtained when the penalty factors are different, which again proves that the method proposed in this paper is effective and moderate. Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation ·9· k=16 k=21 Optimal opology (a) Bridge #6 (b) Bridge #7 μ1 μ2 μ3 μ4 Figure 6 Evolution of topology optimization for bridge structure #6 and #7 6 2 C til b t t d th diti f k=16 k=21 Optimal topology (a) Bridge #6 (b) Bridge #7 μ1 μ2 μ3 μ4 Figure 6 Evolution of topology optimization for bridge structure #6 and #7 6 2 C til b t t d th diti f Figure 8 Process of topology optimization for cantilever beam Figure 8 Process of topology optimization for cantilever beam Optimal Figure 8 Process of topology optimization for cantilever beam Figure 8 is the process of topology optimization for cantilever beam #8 and #9 with three kinds of materials including void material under mass or volume constraint. The optimal topologies are obtained after 43 and 48 iterations, respectively. We find that the main body of optimal topology under volume constraint is similar with optimal topology under mass constraint. Because the effect of density is not considered under volume constraint, the structural compliance under the condition of volume constraint is much greater than those under mass constraint. Figure 6 Evolution of topology optimization for bridge structure #6 and #7 6.2 Cantilever beam structure under the condition of volume constraint or mass constraint 6.2 Cantilever beam structure under the condition of volume constraint or mass constraint 6.3 Short MBB beam with real material The short MBB beam structure is a rectangle with 200mm×100mm, as shown in Fig.9. The left lower endpoint of the design domain is full constrained. In the vertical direction of the design domain, the right lower endpoint is constrained. The vertical load F4 is 1000 N, located on the centre point D of lower boundary. The short MBB beam is divided into 200×100 finite element meshes. Figure 7 Optimization domain of cantilever beam structure Table 2 Parameters corresponding to cantilever beam structure Test Case Candidate materials m Penalizat ion factor p Elastic Modulus Ej Density ρj Mass Fraction f0 Cantilever #8 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.3 Cantilever #9 μ1, μ2, μ3 3 [2,1,e-9] [1,1,1] 0.3 The short MBB beam structure is a rectangle with 200mm×100mm, as shown in Fig.9. The left lower endpoint of the design domain is full constrained. In the vertical direction of the design domain, the right lower endpoint is constrained. The vertical load F4 is 1000 N located on the centre point D of lower boundary. The shor MBB beam is divided into 200×100 finite element meshes. Figure 9 Optimization domain of short MBB beam structure The topology optimization problem of short MBB beam Figure 7 Optimization domain of cantilever beam structure Table 2 Parameters corresponding to cantilever beam structure Test Case Candidate materials m Penalizat ion factor p Elastic Modulus Ej Density ρj Mass Fraction f0 Cantilever #8 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.3 Cantilever #9 μ1, μ2, μ3 3 [2,1,e-9] [1,1,1] 0.3 The short M 200mm×100m endpoint of th vertical direct endpoint is co located on the MBB beam is Figure 9 Optimi The topolog Figure 9 Optimization domain of short MBB beam structure Figure 7 Optimization domain of cantilever beam structure Table 2 Parameters corresponding to cantilever beam structure Test Case Candidate materials m Penalizat ion factor p Elastic Modulus Ej Density ρj Mass Fraction f0 Cantilever #8 μ1, μ2, μ3 3 [2,1,e-9] [2,1,e-9] 0.3 Cantilever #9 μ1, μ2, μ3 3 [2,1,e-9] [1,1,1] 0.3 Figure 9 Optimization domain of short MBB beam structure The topology optimization problem of short MBB beam The topology optimization problem of short MBB beam ·10· Jia-Fan Zhang et al. with mass constraint f=0.3 is illustrated here. We use four real materials including steel, aluminium, magnesium and void material. The elastic modulus of steel, aluminium, magnesium and void material are ES=210GPa, EA=70GPa, EM=40GPa and EV=10-6ES, respectively. (50) The dimension of the performance index PI is the same as the dimension of the objective function. The performance index PI can be called the objective function including constraint conditions. In this article it is referred to herein as structural compliance including mass fraction. The smaller the performance index PI, the smaller the objective function including constraint conditions. Therefore, it is of practical significance to use the performance index PI to evaluate the optimal topology of the three material combinations. Consent for publication Not applicable The innovation of this paper is to transform the muti-material topology optimization problem into multiple single-material topology optimization problems. The material conversion scheme is based on the elastic modulus of the candidate material after interpolation. Compared with the single material SIMP method, the proposed method also has the advantages of fast convergence and less iteration. Acknowledgements The authors sincerely thanks to Professor Qicai Zhou of Tongji University for his critical discussion and reading during manuscript preparation. Authors’ contributions The author’ contributions are as follows: Hong-yu Jiao wrote the manuscript; Ying Li wrote the program using ANSYS APDL language and was responsible for proofreading. Table 3 Parameters when the optimal topology is obtained Material combinations k C(N.mm) f(%) PI(N.mm) Combination A 33 132.93 22.15 29.45 Combination B 28 139.01 20.78 28.88 Combination C 39 137.80 19.69 27.13 Competing interests The authors declare no competing financial interests. Availability of data and materials The datasets supporting the conclusions of this article are included within the article. Funding Supported by National Natural Science Foundation of China (Grant No. 51605046) and Jiangsu provincial government scholarship program (JS-2017-188), which are gratefully appreciated. When the optimal topology is obtained, the parameters of the three material combinations are shown in the Table 3. After 39 iterations, the combination C obtained the optimal topology. At this time, the structural compliance is 137.80mm and the mass fraction f is 19.69%. The performance index PI is 27.13N.mm, which is the smallest of the three material combinations. So it can be considered that the optimal topology of combination C is the optimal topology of multi-material topology optimization. 6.3 Short MBB beam with real material The density of steel, aluminium, magnesium and void material are ρS=7890kg/m3, ρA=2630kg/m3, ρM=1740kg/m3 and ρV=10-6ρS, respectively. Poisson's ratio v=0.3. and void. Through a certain number of optimization calculations, we obtain the optimal topologies of the three material combinations. The main body of the three optimal topologies are similar. Steel is mainly distributed in high stress areas. Low stress area is occupied by aluminium and magnesium. But the details of the three optimal topologies are different. Comparison with the optimal topology of combination A, steel appears in the upper middle region of the optimal topology of combination B. In the optimal topology of combination C aluminium replaces magnesium in the upper middle region. At the same time, aluminium also appeared around the steel. Figure 10 shows the evolution of muti-material topology optimization for MBB structure with 4 real materials including void material. The three different material combinations are: (A) Steel, aluminium and void; (B) Steel, magnesium and void; (C) Steel, aluminium, magnesium k=1 k=6 k=11 k=16 k=21 Optimal topology Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation ·11· Combination A (b) Combination B (c) Combination C Steel Aluminum Magnesium Void Figure 10 Evolution of muti-material topology optimization for MBB beam When the topology optimization converges, the values of the mass fraction are different. Due to the different mass fractions, the minimum compliance problems of three material combinations cannot be reasonably compared. problem of muti-material topology optimization based on multiple SIMP of variable density method. It can provide a new thought for researchers to study muti-material topology optimization in the future. At present, the proposed method can only achieve one-way conversion of candidate materials in descending order of elastic modulus. 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Engineering Structures, 2018, 163:1-10. [27] Li ZD, Liu XJ. Guide-weight method on solving topology optimization problems under single load case. Journal of Mechanical Engineering,2011; 47:107-114. [10] Zhao F. Topology optimization with meshless density variable approximations and BESO method[J]. Computer-Aided Design,2014,56:1-10. [28] Xu HY, Guan LW, Chen X, Wang LP. Guide-Weight method for topology optimization of continuum structures including body forces. Finite Elements in Analysis and Design,2013; 75:38-49. [11] Chu S, Miao M, Gao L, et al. Topology optimization of multi-material structures with graded interfaces[J].Computer Methods in Applied Mechanics and Engineering,2019,346: 1096-1117. [29] Xingtong Y , Ming L . Discrete multi-material topology optimization under total mass constraint[J]. Computer-Aided Design, 2018, 102:182-192. [12] Kang Z, Wu C, Luo Y, et al. Robust topology optimization of multi-material structures considering uncertain graded interface[J]. Composite Structures, 2019,208:395-406. [30] Wang L, Liu D, Yang Y, et al. Novel methodology of Non-probabilistic Reliability-based Topology Optimization (NRBTO) for multi-material layout design via interval and convex mixed uncertainties[J]. Computer Methods in Applied Mechanics & Engineering,2019,346:550-573. [13] Zhang X M, Hu K, Wang N F, et al. Multi-objective Topology Optimization of Multiple Materials Compliant Mechanisms Based on Parallel Strategy [J]. Journal of Mechanical Engineering, 2016, 52(19):1-8. Biographical notes [14] Sanders E D , Pereira A , Aguiló, Miguel A, et al. PolyMat: an efficient Matlab code for multi-material topology optimization[J]. Structural & Multidisciplinary Optimization, 2018,58:2727-2759. Hong-yu Jiao, born in 1981, is currently an associate professor at School of Automotive Engineering, Changshu Institute of Technology, China. He received his PhD degree from Tongji University, China, in 2015. His research interests include structural lightweight design. Tel: +86-13914927008; E-mail: cslgjhy@163.com [15] Wu C , Fang J , Li Q . Multi-material topology optimization for thermal buckling criteria[J]. Computer Methods in Applied Mechanics & Engineering, 2019, 346:1136-1155. [16] Guo X , Zhang W S , Wang M Y , et al. Stress-related topology optimization via level set approach[J]. Computer Methods in Applied Mechanics and Engineering, 2011, 200:p.3439-3452. Ying Li, born in 1979, is currently a lecturer at School of Automotive Engineering, Changshu Institute of Technology, China. Her research interests include structural lightweight design. Tel: +86-18852981695; E-mail: cslgjxly@163.com [17] Guo X , Zhang W , Zhong W . Stress-related topology optimization of continuum structures involving multi-phase materials[J]. Computer Methods in Applied Mechanics & Engineering, 2014, 268:632-655. Tel: +86-18852981695; E-mail: cslgjxly@163.com Tel: +86-18852981695; E-mail: cslgjxly@163.com [18] Tavakoli R , Mohseni S M . Alternating active-phase algorithm for multimaterial topology optimization problems: A 115-line MATLAB implementation[J]. Structural & Multidisciplinary Optimization, ·13· Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation Novel 6-DOF Wearable Exoskeleton Arm with Pneumatic Force-Feedback for Bilateral Teleoperation Appendix Appendix and supplement both mean material added at the end of a book. An appendix gives useful additional information, but even without it the rest of the book is complete: In the appendix are forty detailed charts. A supplement, bound in the book or published separately, is given for comparison, as an enhancement, to provide corrections, to present later information, and the like: A yearly supplement is issue. Figures Figure 1 Figures Figures Figures Figures Figure 1 Elastic modulus in multiple SIMP of variable density method Figure 2 Optimization domain of bridge structure g Figure 1 Elastic modulus in multiple SIMP of variable density method Figure 2 Optimization domain of bridge structure Elastic modulus in multiple SIMP of variable density method Elastic modulus in multiple SIMP of variable density method Elastic modulus in multiple SIMP of variable density method Figure 2 Optimization domain of bridge structure Optimization domain of bridge structure Figure 3 E l ti f t l ti i ti f Figure 3 Figure 3 Evolution of topology optimization for bridge structure #1 Figure 4 Optimization curves of structural compliance when mass fraction f is 0.2, 0.25, and 0.3 Figure 4 Optimization curves of structural compliance when mass fraction f is 0 2 0 25 and 0 3 Figure 4 Optimization curves of structural compliance when mass fraction f is 0.2, 0.25, and 0.3 Optimization curves of structural compliance when mass fraction f is 0.2, 0.25, and 0.3 Optimization curves of structural compliance when mass fraction f is 0.2, 0.25, and 0.3 Figure 5 O ti i ti f t t l li h li ti f t i 3 3 5 d 4 Figure 5 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 Figure 5 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 Optimization curves of structural compliance when penalization factor p is 3, 3.5, and 4 Figure 6 Evolution of topology optimization for bridge structure #6 and #7 Figure 6 Evolution of topology optimization for bridge structure #6 and #7 Figure 6 Evolution of topology optimization for bridge structure #6 and #7 Figure 7 Optimization domain of cantilever beam structure Figure 8 Process of topology optimization for cantilever beam Figure 7 Figure 7 Optimization domain of cantilever beam structure Optimization domain of cantilever beam structure Figure 8 Process of topology optimization for cantilever beam Figure 9 Optimization domain of short MBB beam structure Figure 9 Optimization domain of short MBB beam structure Optimization domain of short MBB beam structure Figure 10 E l i f i i l l i i i f MBB b Figure 10 Evolution of muti-material topology optimization for MBB beam
https://openalex.org/W4394853407	https://akjournals.com/downloadpdf/view/journals/650/165/15/article-p568.pdf	Hungarian	null	Az oktenidin-dihidroklorid antiszeptikum alkalmazása a fül-orr-gégészetben	Orvosi hetilap	2,024	cc-by	4,297	ÖSSZEFOGLALÓ KÖZLEMÉNY ÖSSZEFOGLALÓ KÖZLEMÉNY Molnár András dr. ■ Krasznai Magdolna dr. ■ Maihoub Stefani dr. Molnár András dr. ■ Krasznai Magdolna dr. ■ Maihoub Stefani dr. melweis Egyetem, Általános Orvostudományi Kar, Fül-Orr-Gégészeti és Fej-Nyaksebészeti Klinika, Budapest Az oktenidin-dihidroklorid antiszeptikum mint bőr- és felületfertőtlenítő elterjedt a mindennapi gyakorlatban, fül- orr-gégészeti használatának lehetőségei azonban kevésbé ismertek. Az oktenidin-dihidroklorid szintetikus piridin származék, amely az antiszeptikus hatás tekintetében széles spektrumot ír le. Hatékonynak bizonyult Gram-pozitív és Gram-negatív baktériumokkal, burokkal rendelkező vírusokkal és egyes gombákkal szemben is. Az orr, a szájüreg, valamint a garat gazdagon tartalmaz mikrobákat, és mint első behatolási kapuk, infekciók gyakori színhelyei. Sajnála tos tény, hogy folyamatosan nő az antibiotikumokkal és antimikotikumokkal szembeni rezisztencia, a felső légúti ví rusos fertőzések esetében pedig az antivirális készítmények nem alkalmazhatók rutinszerűen. Ennek megfelelően az antiszeptikumok mindennapi alkalmazása előnyös lehet, amelyekkel szemben – aspecifikus hatásuknak köszönhe tően – a rezisztenciák kialakulásának lehetősége kisebb. Az oktenidin-dihidroklorid fül-orr-gégészeti alkalmazásáról számos korábbi tanulmány született, amelyek megerősítették a hatékonyságát különböző infekciók esetében – ezek eredményeit foglaljuk össze a jelen közleményben. Antibakteriális spektruma alapján, amely magában foglalja a pha ryngitist leggyakrabban okozó kórokozókat, az oktenidin-dihidroklorid-tartalmú szopogatótabletta előnyösen hasz nálható a garatgyulladások kezelésében, amit megerősít, hogy antibiotikummal együtt alkalmazva szinergista hatású. Az egyes speciális pharyngitisek közül például korábbi kutatási eredmények a Neisseria gonorrhoeae fertőzéssel szem ben is hatékonynak találták, ami lényeges, hiszen az azitromicinnel szembeni rezisztencia folyamatosan nő. A min dennapi fül-orr-gégészeti gyakorlatban, de más szakmákat érintően is lényeges a tracheakanülök potenciális szerepe, a rajtuk keletkező biofilm által nosocomialis infekciók létrejöttében. Korábbi kutatás például oktenidin-dihidroklorid hatóanyaggal bevont műanyag tracheakanül dekontaminációját vizsgálta Pseudomonas aeruginosa és Staphylococcus aureus kórokozók tekintetében, azzal az eredménnyel, hogy a kolonizáció a bevont tracheakanülök esetében szigni fikánsan csökkent. További fontos eredmény, hogy a meticillinrezisztens S. aureusszal szemben is eredményesnek bizonyult, amely az orrbemenetben gyakran kolonizál és okoz fertőzést. Gombák tekintetében biztató eredmények adódtak a Candida albicans esetében, amely viszonylag gyakran okoz gombás pharyngitist, például immunszuppri mált betegekben vagy antibiotikumok túlzott/indokolatlan használata során. Elmondható ezenkívül, hogy az azo lokkal szembeni rezisztencia növekvő tendenciát mutat. Az oktenidin-dihidroklorid számos fül-orr-gégészeti infekció esetében jó választás lehet, az egyes eredményeket azonban további klinikai kutatásokkal szükséges megerősíteni. Orv Hetil. 2024; 165(15): 568–573. Kulcsszavak: oktenidin-dihidroklorid, pharyngitis, tracheakanül, biofilm, lokális antiszeptikum Application of octenidine-dihydrochloride antiseptic in otorhinolaryngology Octenidine-dihydrochloride, a skin antiseptic and surface disinfectant, is widely used in everyday practice; however, its use in otorhinolaryngological practice is not widespread. Octenidine-dihydrochloride is a pyridine derivate with a relatively broad antimicrobial spectrum, including Gram-positive and Gram-negative bacteria, enveloped viruses, and some fungi. Considering the contamination of the nose, oral cavity, and oropharynx, and the fact that the upper airway is the first line of defense against infections, upper-airway infections are common. Unfortunately, the resist ance against antibiotics and antimycotics is increasing. Additionally, routine use of antiviral agents is unrecommend ed in upper airway viral infections. Therefore, using antiseptics can be beneficial, considering their aspecific effects, which are essential in preventing resistance. Regarding the otorhinolaryngological use of octenidine-dihydrochloride, previous studies concluded promising results, summarised in the present article. Since its antibacterial spectrum in cludes the most frequent bacteria for pharyngitis, octenidine-dihydrochloride-containing tablets might be benefi cially used in pharyngitis. As a further advantage, octenidine-dihydrochloride and antibiotics show a synergistic ef fect. Investigating some special pharyngitis types, e.g., caused by Neisseria gonorrhoeae, also showed its effectiveness, in which case the resistance against azithromycin is increasing. In everyday medical practice, the surface of tracheos tomy tubes, due to the biofilm formulation, is crucial in nosocomial infections. A previous study has investigated the 568 2024 ■ 165. évfolyam, 15. szám ■ 568–573. DOI: 10.1556/650.2024.33019 ■ © Szerző(k) Unauthenticated \| Downloaded 10/24/24 05:18 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY effects of octenidine-dihydrochloride-coated tracheotomy tubes in decontaminating Pseudomonas aeruginosa and Staphylococcus aureus. Their results showed significant decontamination using the coated tracheostomy tubes. Another significant result was the reduction of methicillin-resistant S. aureus, which frequently colonises the nares and results in infections. It also showed effectivity against Candida albicans infections, commonly observed in my cotic pharyngitis, e.g., in the case of immunosuppression and overuse of antibiotics. Furthermore, C. albicans shows an increasing resistance against azoles. In conclusion, octenidine-dihydrochloride shows promising results in otorhi nolaryngological infections; however, some results must be strengthened by further clinical studies. Keywords: octenidine-dihydrochloride, pharyngitis, tracheostomy tube, biofilm, local antiseptic Molnár A, Krasznai M, Maihoub S. [Application of octenidine-dihydrochloride antiseptic in otorhinolaryngology]. Orv Hetil. 2024; 165(15): 568–573. (Beérkezett: 2024. január 25.; elfogadva: 2024. február 20.) (Beérkezett: 2024. január 25.; elfogadva: 2024. február 20.) spektrum írható le: antibakteriális (Gram-pozitív és Gram-negatív baktériumok), antimikotikus (Candida spp.) és parciális antivirális (burokkal rendelkező víru sokkal szembeni) hatásai ismertek. Rövidítések GUMBOS = (group of uniform materials based on organic salts); MRSA = meticillinrezisztens Staphylococcus aureus; OCT = oktenidin-dihidroklorid; RSV = légúti óriássejtes vírus; SARS-CoV-2 = (severe acute respiratory syndrome corona virus) súlyos akut légzőszervi tünetegyüttest okozó korona vírus-2; STD = (sexually trasmitted disease) nemi úton terjedő betegség; ZOI = (zones of inhibition) gátlási zóna Az antiszeptikumok egyik fontos fül-orr-gégészeti vo natkozása, hogy a szájüreg és a szájgarat bőségesen tar talmaz baktériumkolóniákat [1]. Idesorolható például a Streptococcus spp., a Staphylococcus aureus és a Staphy lococcus epidermidis, a Haemophilus influenzae vagy a Neisseria meningitidis is. Baktériumok mellett gombák is megtalálhatók, például a mindennapi klinikai gyakorlat ból jól ismert Candida fajok [2]. A szájüregben vagy a garatban kolonizáló baktériumok klinikai jelentősége, hogy a normálflórát képező baktériumok pusztulása (például antibiotikumok túlzott használata miatt) és/ vagy a patogén fajok elszaporodása fertőzések kialakulá sához vezethet. Sajnálatos tény, hogy az infekciók nem kizárólag a szájüregre vagy a garatra lokalizálódnak, a szájüregi flóra például összefüggésbe hozható pneumo niával, felhívva a figyelmet a lokális antiszeptikumok használatának fontosságára a lélegeztetéssel összefüggő tüdőgyulladás megelőzésében is [3]. A felső légúti infek ciók a mindennapi klinikai gyakorlat jelentős hányadát teszik ki; kiemelendő, hogy mind a rhinitis, mind a pha ryngitis tekintetében dominálnak a vírusos fertőzések. Ez a tény, illetve a baktériumok antibiotikumokkal szem beni növekvő rezisztenciája hangsúlyossá teszi az anti szeptikumok használatának fontosságát. Az egyik lehető ség az oktenidin-dihidroklorid (OCT) hatóanyag, amely Európában több országban elterjedt, és használata nem kizárólag a nyálkahártyák fertőtlenítésére terjed ki [4]. i Application of octenidine-dihydrochloride antiseptic in otorhinolaryngology A baktériumok szem pontjából kiemelendő, hogy hatékonynak bizonyult az MRSA (meticillinrezisztens S. aureus) és Pseudomonas aeruginosa kórokozókkal szemben; mindkettőnél jól ismert az antibiotikumokkal szembeni erős rezisztencia. A vírusok tekintetében lényeges, hogy a SARS-CoV-2- vel szemben is írtak le előnyös hatást [5]. További elő nye, hogy kizárólag lokálisan hat, nem szívódik fel sem bőrön, sem nyálkahártyán keresztül, így szisztémás mellékhatást nem okoz. Az OCT a mindennapi fül-orr- gégészeti vagy fogorvosi gyakorlatban széles körben használt klórhexidinhez hasonló hatásspektrummal ren delkezik, ugyanakkor kevesebb mellékhatással (a klórhe xidin például neurotoxikus hatást fejt ki, ízérzészavart, nagyobb koncentrációban allergiás reakciót okozhat, így hosszú távon nem alkalmazható [6, 7]). A magyaror szági piacon az OCT hatóanyag mint felülfertőtlenítő (Octenisept®) és mint szopogatótabletta (Okteangin® 2,6 mg) érhető el. Oropharyngealis Neisseria gonorrhoeae infekció kezelése gonorrhoeae fertőzés kezelésében, amely reaktív ionpárok együttes alkalmazását jelenti (ebben az esetben az OCT és a kar benicillin között létrejött ionpárokat). Tekintettel arra, hogy az OCT és a karbenicillin kémiailag eltérő módon viselkedik, a „GUMBOS” szempontból új ionpár létre hozására alkalmas vegyületek. A vizsgálatban a gátlási zónákat (ZOI – zones of inhibition) a Kirby–Bauer- korong diffúziós teszt alapján határozták meg. Az anti bakteriális hatékonyság vizsgálata során kimutatható volt, hogy az oktenidin/karbenicillin kombináció additív hatást fejtett ki a N. gonorrhoeae infekcióval szemben. A ZOI-értékek nagyobbak vagy legalább azonosak vol tak az azitromicin és a doxiciklin esetében tapasztaltak kal. Ez szintén jelentős, hiszen a N. gonorrhoeae azitro micinnel szembeni rezisztenciája világszerte növekvő gyakoriságot mutat, különböző nemzetközi statisztikák alapján elérheti akár a 60%-ot N. gonorrhoeae esetében [19]. Citotoxicitás az oktenidin/karbenicillin kombiná ció esetében sem igazolódott [20]. 2024 ■ 165. évfolyam, 15. szám 5 1. táblázat Az oktenidin-dihidroklorid eddig vizsgált antimikrobás haté konyságának összefoglalása Baktériumok Gram-pozitív Streptococcus pyogenes, Streptococ cus pneumoniae, Staphylococcus aureus, MRSA, Enterococcus hirae Gram-negatív Pseudomonas aeruginosa, Haemophilus influenzae Vírusok Influenzavírusok, parainfluenza vírusok, rhinovirusok, korona vírusok, adenovírusok, RSV Lipidburokkal rendelkező vírusok Gombák Candida albicans MRSA = meticillinrezisztens Staphylococcus aureus; RSV = légúti óriás sejtes vírus Pharyngitisek kezelése A pharyngitis az egyik leggyakoribb kórkép a mindenna pi orvosi gyakorlatban, amellyel az érintettek a leggyak rabban háziorvost, sürgősségi osztályt, fül-orr-gégészt vagy gyermekorvost keresnek fel. A pharyngitis a leg gyakrabban (kb. 90%) vírusfertőzés hatására alakul ki (például herpeszvírusok, rhinovírusok, adenovírusok vagy koronavírusok) [8, 9]; ennek megfelelően ezekben az esetekben antibiotikumok használatára nincs szükség, ilyenkor csak szupportív terápiát javaslunk. Amennyiben a folyamat bakteriális, a leggyakrabban a Streptococcus pyogenes baktérium okozza, ezenkívül állhat még a hát térben H. influenzae, Chlamydophila pneumoniae, illetve egyes STD-kórokozók is [10]. Antibiotikumok haszná latára – figyelembe véve a rezisztenciák kialakulását vagy a normálflóra károsodását – csak bakteriális folyamat ese tében van szükség, ez differenciálható a fizikális vizsgá Az OCT hatása aspecifikus, ennek előnye, hogy kisebb eséllyel alakulnak ki a baktériumok részéről rezisztenci ák, ugyanakkor citotoxikus hatása nem ismert (szemben más elterjedt antiszeptikumokkal, például klórhexidin- diglükonát). Antimikrobás hatása tekintetében széles 569 2024 ■ 165. évfolyam, 15. szám ORVOSI HETILAP ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 05:18 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Oropharyngealis Neisseria gonorrhoeae infekció kezelése lat, esetleg bakteriológiai tenyésztés alapján [10, 11]. A magyarországi gyakorlatban az egyik leggyakrabban alkalmazott antiszeptikum – mint szupportív terápia – a klórhexidin-diglükonát hatóanyagot tartalmazó torok öblögető, amellyel kapcsolatban viszont növekvő re zisztencia tapasztalható, citotoxikus és neurotoxikus mellékhatásai vannak, ízérzéscsökkenést okozhat, ezért használata hosszú távon nem javasolt. Szubjektíve az ol datnak kellemetlen mellékíze van, így a betegek részéről előfordulhat nem kielégítő együttműködés; allergiás re akciót is okozhat, ez elsősorban bőrön történő alkalma záskor jelentős (kontaktdermatitis) [12, 13]. Előnyös választás lehet az OCT, amely esetében citotoxikus vagy karcinogén hatás nem igazolódott, korábbi vizsgálatok alapján a placentán sem jut át [14]. Másik fontos, elő nyös tulajdonsága, hogy az OCT nem illékony vegyület, ennek megfelelően sem nyálkahártyáról, sem bőrről nem szívódik fel, így hatását lokálisan fejti ki, szisztémás mel lékhatást nem okoz. Tekintettel arra, hogy Gram-pozitív és Gram-negatív baktériumokra (például S. pyogenes, Streptococcus pneumoniae, S. aureus, H. influenzae stb. – 1. táblázat), burkos vírusokra és gombákra is hat, vi szonylag széles hatásspektrumot ír le, amely előnyös a pharyngitisek tekintetében [15]. Aspecifikus hatása a bakteriális rezisztenciák megelőzése szempontjából is je lentős. Fontos további jellemzője, hogy antibiotikummal együtt alkalmazva bakteriális folyamatoknál szinergista hatású [16]. A jelenleg forgalomban lévő Okteangin® 2,6 mg tabletta OCT mellett borsmentát és ánizsolajat is tartalmaz, amely a nyálkahártya hidegreceptorainak sti mulálása által további fájdalomcsillapító hatással bír. A 2,6 mg-os tabletta 12 éves kor felett alkalmazható típusos szájüregi vagy szájgarati infekciók esetében: 2–3 óránként javasolt egy tabletta elszopogatása, max. 6 tab letta/nap dózisban. A N. gonorrhoeae infekció az egyik leggyakoribb, STD-t okozó fertőzés, amely világszerte növekvő gyakoriságot mutat [17]. A N. gonorrhoeae fertőzések egy része a száj garatra is lokalizálódik, így egyes esetekben pharyngitis hátterében is azonosítható. Fül-orr-gégészeti bakteriális fertőzéseknél kiemelt jelentőségű, hogy az oropharynx ban kolonizáló baktériumok esetében az antibiotiku mokkal szemben szerzett rezisztenciák – különböző mechanizmussal – nagy arányban fordulnak elő. Az anti biotikumok csökkent hatékonysága részben magyaráz ható az antibiotikumok oropharynxszöveteken keresz tüli rossz penetrációjával is. A N. gonorrhoeae fertőzés kezelésének jelenlegi elsőként választandó szere az im. ceftriaxon mellett az orálisan adagolt azitromicin [18], amelyre a N. gonorrhoeae rezisztenciája növekvő tenden ciát mutat. Az antiszeptikumok, mint például az OCT egyik előnye, hogy aspecifikus hatásuk révén kisebb az esély a baktérium részéről rezisztencia kialakulására. Egy konkrét korábbi kutatás OCT/karbenicillin „GUMBOS” (group of uniform materials based on organic salts) ha tékonyságát vizsgálta oropharyngealis N. Rhinologiai vonatkozások gyakorisága az elmúlt évtizedekben jelentősen emelke dett. Korábbi vizsgálatok alapján kimutatható volt, hogy az endotrachealis tubus ’cuff’-ja már pár napos gépi léle geztetést követően vastag bakteriális biofilmmel került bevonásra, amely könnyedén lélegeztetéssel összefüggő pneumoniához vezet [25]. A mindennapi gyakorlatban használt tracheakanülök két fő típusa – aszerint, hogy milyen anyagból készülnek – a fém- és a műanyag kanü lök. A műanyag kanülök növelik ugyan a beteg kényel mét, és általában könnyebben érhetők el, mint a fémtí pusok, a műanyag felszín miatt nagyobb az esély a bakteriális biofilm képződésére. Az invazív eszközökhöz társuló infekció kezelése különösen nagy kihívás: ennek oka egyrészt, hogy az antibiotikumok nagy része nem tudja penetrálni a kialakult biofilmet, másrészt az antibio tikumokkal szembeni növekvő rezisztenciával is magya rázható [26]. Ennek megfelelően felmerült olyan kanü lök kialakításának a lehetősége, amelyek felszíne be van vonva valamilyen antiszeptikummal, ilyen lehet például az OCT. Egy korábbi kutatás OCT-vel bevont trachea kanül bakteriális kontaminációját hasonlította össze nem bevont kanülével. Az eredmények alapján az OCT hatá sára a bakteriális kontamináció szignifikánsan csökkent (103 cfu/ml, összevetve a nem bevont kanüllel, amely esetében az érték 105 cfu/ml-nek adódott) [27]. gyakorisága az elmúlt évtizedekben jelentősen emelke dett. Korábbi vizsgálatok alapján kimutatható volt, hogy az endotrachealis tubus ’cuff’-ja már pár napos gépi léle geztetést követően vastag bakteriális biofilmmel került bevonásra, amely könnyedén lélegeztetéssel összefüggő pneumoniához vezet [25]. A mindennapi gyakorlatban használt tracheakanülök két fő típusa – aszerint, hogy milyen anyagból készülnek – a fém- és a műanyag kanü lök. A műanyag kanülök növelik ugyan a beteg kényel mét, és általában könnyebben érhetők el, mint a fémtí pusok, a műanyag felszín miatt nagyobb az esély a bakteriális biofilm képződésére. Az invazív eszközökhöz társuló infekció kezelése különösen nagy kihívás: ennek oka egyrészt, hogy az antibiotikumok nagy része nem tudja penetrálni a kialakult biofilmet, másrészt az antibio tikumokkal szembeni növekvő rezisztenciával is magya rázható [26]. Ennek megfelelően felmerült olyan kanü lök kialakításának a lehetősége, amelyek felszíne be van vonva valamilyen antiszeptikummal, ilyen lehet például az OCT. Egy korábbi kutatás OCT-vel bevont trachea kanül bakteriális kontaminációját hasonlította össze nem bevont kanülével. Az eredmények alapján az OCT hatá sára a bakteriális kontamináció szignifikánsan csökkent (103 cfu/ml, összevetve a nem bevont kanüllel, amely esetében az érték 105 cfu/ml-nek adódott) [27]. A S. aureus baktérium gyakran kolonizál az orrbemenet ben; ezen belül különös jelentőséggel bír az MRSA, az ehhez kapcsolódó kórházi fertőzések világszerte növek vő gyakoriságot mutatnak [28]. Az oktenidin-dihidroklorid hatása a tracheakanül-biofilmre Tracheotomia elvégzésére, illetve tracheakanül behelye zésére különböző indikációkkal kerülhet sor [21], így tracheakanült viselő beteggel különböző osztályokon ta lálkozhatunk, úgymint fül-orr-gégészet, intenzív osztály, neurológia vagy pulmonológia stb. [22]. Korábbi kuta tási eredmények alapján a gépi lélegeztetéssel összefüggő pneumonia hátterében nagy arányban azonosíthatók a P. aeruginosa és S. aureus kórokozók [23], ezenkívül a Klebsiella pneumoniae szerepe (1. ábra) is kiemelendő [24]. Az ezek által okozott nosocomialis pneumonia 2024 ■ 165. évfolyam, 15. szám 570 ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 05:18 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY ÖSSZEFOGLALÓ KÖZLEMÉNY 1. ábra Klebsiella pneumoniae által képzett biofilm tracheakanülön [27]. a), b), c), a) c) 1. ábra Klebsiella pneumoniae által képzett biofilm tracheakanülön [27]. a), b), c), d) a) b) c) d) a) b) b) a) c) d) d) d) 1. ábra Klebsiella pneumoniae által képzett biofilm tracheakanülön [27]. a), b), c), d) Irodalom Szájüregi és garatgyulladások tekintetében a C. albicans által okozott mycosis nemritkán látható a rutin klinikai gyakorlatban, a gombafaj az egészséges emberek kb. 60%-ában kolonizál. Fertőzés döntően immunszuppri mált betegeknél vagy antibiotikumok túlzott/indokolat lan használatakor fordul elő. Sajnálatos, hogy az antifun gális készítményekkel (például azolok) szembeni rezisz tencia nő [36, 37]. A candidaemia által okozott mortali tási arány intenzív osztályokon nagynak tekinthető [38]. Ennek megfelelően felmerült az antiszeptikumok hasz nálatának lehetősége nemcsak a prevencióban, hanem a Candida-infekció kezelésében is, főleg a rezisztens fajok esetében. Korábbi in vitro kutatási eredmények rámutat tak, hogy csupán 0,05%-os OCT 30 másodpercnyi inku bációt követően hatékonynak bizonyult C. albicans és Candida glabrata esetében is [39]. Ezt megelőzően buccalis epithelsejteken végzett in vitro kutatás kimutat ta, hogy az OCT szignifikánsan csökkentette a Candida spp. sejtekhez történő tapadását [40]. Az OCT haté konyságáról szájüregi és garati infekciók kezelése tekin tetében eddig korlátozott kutatási eredmény áll rendel kezésre, ugyanakkor sikeresen alkalmazták más régiók ban megjelenő mycosisoknál [41, 42]. Az OCT antifun gális hatékonyságáról egyébként is döntően in vitro kutatási eredmények érhetők el [43, 44], így hatékony ságának vizsgálatához klinikai kutatások elvégzése szük séges. [1] Moldován A, Rózsa N, Herczegh A. The role of probiotics in oral health. [A probiotikumok szerepe az orális egészségben.] Orv Hetil. 2023; 164: 942–947. [Hungarian]l [2] Ylikoski J, Savolainen S, Jousimies-Somer H. Bacterial flora in the nasopharynx and nasal cavity of healthy young men. ORL J Otorhinolaryngol Relat Spec. 1989; 51: 50–55. [3] Rohrer N, Widmer AF, Waltimo T, et al. Antimicrobial efficacy of 3 oral antiseptics containing octenidine, polyhexamethylene biguanide, or Citroxx: can chlorhexidine be replaced? Infect Control Hosp Epidemiol. 2010; 31: 733–739. [4] Hübner NO, Siebert J, Kramer A. Octenidine dihydrochloride, a modern antiseptic for skin, mucous membranes and wounds. Skin Pharmacol Physiol. 2010; 23: 244–258. [5] Steinhauer K, Meister TL, Todt D, et al. Comparison of the in- vitro efficacy of different mouthwash solutions targeting SARS- CoV-2 based on the European Standard EN 14476. J Hosp Infect. 2021; 111: 180–183. [6] Henschen A, Olson L. Chlorhexidine-induced degeneration of adrenergic nerves. Acta Neuropathol. 1984; 63: 18–23. [7] Stingeni L, Lapomarda V, Lisi P. Occupational hand dermatitis in hospital environments. Contact Dermatitis 1995; 33: 172–176. [8] Denison MR. Viral pharyngitis. Semin Pediatr Infect Dis. 1995; 6: 62–68. [9] Weber R. Pharyngitis. Prim Care 2014; 41: 91–98. [10] Sykes EA, Wu V, Beyea MM, et al. Pharyngitis: approach to diag nosis and treatment. Irodalom Can Fam Physician 2020; 66: 251–257. [11] Takács A, Szűcs D, Terhes G. Exudative tonsillitis in children. How can we reduce the unnecessary antibiotic consumption? [Lepedékes tonsilla gyermekkorban. Hogyan csökkenthető az indokolatlan antibiotikumfelhasználás?] Orv Hetil. 2020; 161: 50–55. [Hungarian] [12] Silvestri DL, McEnery-Stonelake M. Chlorhexidine: uses and ad verse reactions. Dermatitis 2013; 24: 112–118. [13] Gürgan CA, Zaim E, Bakirsoy I, et al. Short-term side effects of 0.2% alcohol-free chlorhexidine mouthrinse used as an adjunct to non-surgical periodontal treatment: a double-blind clinical study. J Periodontol. 2006; 77: 370–384. Rhinologiai vonatkozások Az MRSA antibiotiku mokkal szembeni rezisztenciája nő [29], beleértve a lo kálisan használható antibiotikumokat (mupirocin) [30]. Az MRSA-hordozókban nagyobb eséllyel alakul ki infek ció [31], valamint hangsúlyozandó a kórházi személyzet mint potenciális hordozó szerepe. Ezek miatt lényeges olyan alternatív antiszeptikumok alkalmazása, amelyek hatékonyak lehetnek az MRSA eradikációjában. A nem zetközi irányelv a hordozókra vonatkozóan lokális intra nasalis antibiotikumkezelést (mupirocinkenőcs) és a teljes test bőrfelszínének antiszeptikummal történő fer tőtlenítését javasolja [32]. Az intranasalis eradikáció te kintetében alternatívaként OCT is alkalmazható, bár az OCT-tartalmú kenőcsök használata korlátozottan terjedt el [33]. Egy korábbi kutatásban 4%-os klórhexidin-dig lükonát és intranasalis OCT-gél hatékonyságát vizsgálták MRSA eradikálásában, három különböző kórházban. A vizsgálat alapján az MRSA-hordozás gyakorisága ha sonló volt a három különböző kórházban, ennek kap csán orrbemenetből és több helyről bőrfelszínről vett 571 2024 ■ 165. évfolyam, 15. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 05:18 AM UTC Unauthenticated \| Downloaded 10/24/24 05:18 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Szerzői munkamegosztás: M. A.: Anyaggyűjtés, a kézirat megírása. K. M.: Kritikai megjegyzések. M. S.: A kézirat megírása, kritikai megjegyzések. A közlemény végleges változatát valamennyi szerző elolvasta és jóváhagyta. bakteriológiai mintákat elemeztek. Az alkalmazott be avatkozás hatására az MRSA prevalenciája 43–58%-kal csökkent [34]. A hatékonyság tekintetében ez hasonló nak mondható ahhoz, mint amit korábban klórhexidin és intranasalis jód használata kapcsán írtak le [35]. Szerzői munkamegosztás: M. A.: Anyaggyűjtés, a kézirat megírása. K. M.: Kritikai megjegyzések. M. S.: A kézirat megírása, kritikai megjegyzések. A közlemény végleges változatát valamennyi szerző elolvasta és jóváhagyta. Szerzői munkamegosztás: M. A.: Anyaggyűjtés, a kézirat megírása. K. M.: Kritikai megjegyzések. M. S.: A kézirat megírása, kritikai megjegyzések. A közlemény végleges változatát valamennyi szerző elolvasta és jóváhagyta. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Következtetés Respir Care 2014; 59: 895–915. [35] Miller L, Mckinnell JA, Raveena Singh R, et al. Reduction of MDRO colonization in nursing home residents with routine use of chlorhexidine bathing and nasal iodophor (Project PROTECT). Open Forum Infect Dis. 2016; 3(Suppl 1): 1386. [22] Bódis F, Orosz G, Szabó M, et al. Role of anthropometric pa rameters in percutaneous tracheostomies performed on cadavers. [Az antropometriai paraméterek szerepe a cadaveren végzett per cutan tracheostomás módszerekben.] Orv Hetil. 2023; 164: 630–635. [Hungarian] [36] Pankhurst CL. Candidiasis (oropharyngeal). BMJ Clin Evid. 2013; 2013: 1304. [37] Krasznai M, Molnár A, Maihoub S. The differential diagnostic role of the tongue. [A nyelv differenciáldiagnosztikai szerepe.] Orvostovábbk Szle. 2023; 5: 67–78. [Hungarian] [23] González C, Rubio M, Romero-Vivas J, et al. Bacteremic pneu monia due to Staphylococcus aureus: a comparison of disease caused by methicillin-resistant and methicillin-susceptible organ isms. Clin Infect Dis. 1999; 29: 1171–1177. [38] Bassetti M, Righi E, Montravers P, et al. What has changed in the treatment of invasive candidiasis? A look at the past 10 years and ahead. J Antimicrob Chemother. 2018; 73(Suppl 1): i14–i25. [24] Ochońska D, Ścibik Ł, Brzychczy-Włoch M. Biofilm formation of clinical Klebsiella pneumoniae strains isolated from tracheos tomy tubes and their association with antimicrobial resistance, virulence and genetic diversity. Pathogens 2021; 10: 1345. [39] Spettel K, Bumberger D, Camp I, et al. Efficacy of octenidine against emerging echinocandin-, azole- and multidrug-resistant Candida albicans and Candida glabrata. J Glob Antimicrob Re sist. 2022; 29: 23–28. [25] Adair CG, Gorman SP, Feron BM, et al. Implications of endotra cheal tube biofilm for ventilator-associated pneumonia. Intensive Care Med. 1999; 25: 1072–1076. [40] Ghannoum MA, Abu Elteen K, Stretton RJ, et al. Effects of octe nidine and pirtenidine on adhesion of Candida species to human buccal epithelial cells in vitro. Arch Oral Biol. 1990; 35: 249– 253. [26] Adair CG, Gorman SP, O’Neill FB, et al. Selective decontamina tion of the digestive tract (SDD) does not prevent the formation of microbial biofilms on endotracheal tubes. J Antimicrob Chemother. 1993; 31: 689–697. [41] Friese K, Neumann G, Siebert J. Topical antiseptics as an alterna tive in the treatment of acute vulvovaginal candidosis. Arch Gynecol Obstet. 2003; 268: 194–197. [27] Zumtobel M, Assadian O, Leonhard M, et al. The antimicrobial effect of octenidine-dihydrochloride coated polymer tracheoto my tubes on Staphylococcus aureus and Pseudomonas aeruginosa colonisation. BMC Microbiol. 2009; 9: 150. Következtetés Az OCT antiszeptikum fül-orr-gégészeti alkalmazását illetően számos biztató korábbi kutatási eredmény áll rendelkezésre. A pharyngitis kezelésében vírusos, gom bás, valamint bakteriális folyamatban is javasolható, az utóbbinál antibiotikummal kombinálva szinergista hatá sú. Olyan ritkább szájgarati infekcióknál is sikeresen al kalmazták, mint egyes STD-k. Kiemelt jelentőségű, hogy tracheakanülön létrejövő biofilm ellen is hatékonynak bizonyult. Olyan, antibiotikummal szemben jelentős rezisztenciát mutató kórokozók, mint az MRSA vagy a P. aeruginosa esetében is igazolódtak előnyös hatásai. Az in vitro kutatási eredmények mellett további klinikai kutatások elvégzése szükséges az egyes vonatkozásokban felvetett hatékonyságáról. [14] Zöllner H, Kramer A, Youssef P, et al. 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Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep. 2015; 64: 1–137. Erratum: MMWR Recomm Rep. 2015; 64: 924. Anyagi támogatás: A közlemény megírása, illetve a kap csolódó munka anyagi támogatásban nem részesült. 572 2024 ■ 165. évfolyam, 15. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 05:18 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY [19] Unemo M, Lahra MM, Escher M, et al. WHO global antimicro bial resistance surveillance for Neisseria gonorrhoeae 2017–18: a retrospective observational study. Lancet Microbe 2021; 2: e627–e636. [33] Hübner NO, Wander K, Ryll S, et al. Antibiotic-free decolonisa tion of MRSA-positive staff. [Antibiotikafreie Sanierung von MRSA-positivem Personal.] GMS Krankenhhyg Interdiszip. 2009; 4: Doc04. [German] [20] Lopez KM, Hobden JA, Warner IM. Octenidine/carbenicillin GUMBOS as potential treatment for oropharyngeal gonor rhoea. J Antimicrob Chemother. 2020; 75: 3576–3581. [34] Chow A, Hon PY, Tin G, et al. Intranasal octenidine and univer sal antiseptic bathing reduce methicillin-resistant Staphylococcus aureus (MRSA) prevalence in extended care facilities. Epidemiol Infect. 2018; 146: 2036–2041. [21] Cheung NH, Napolitano LM. Tracheostomy: epidemiology, in dications, timing, technique, and outcomes. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Következtetés [42] Koburger T, Hübner NO, Braun M, et al. Standardized com parison of antiseptic efficacy of triclosan, PVP-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob Chemother. 2010; 65: 1712–1719. 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https://openalex.org/W2755894917	https://opus.bibliothek.uni-augsburg.de/opus4/files/102758/ijms-18-02003.pdf	English	null	To Wnt or Lose: The Missing Non-Coding Linc in Colorectal Cancer	International journal of molecular sciences	2,017	cc-by	10,959	To Wnt or Lose: The Missing Non-Coding Linc in Colorectal Cancer Peng Shen 1,2, Martin Pichler 1,3, Meng Chen 1, George A. Calin 1,4,* and Hui Ling 1,* 1 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; shenbo20110311@163.com (P.S.); martin.pichler@medunigraz.at (M.P.); MChen11@mdanderson.org (M.C.) 1 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; shenbo20110311@163.com (P.S.); martin.pichler@medunigraz.at (M.P.); MChen11@mdanderson.org (M.C.) Nanfang Hospital, Southern Medical University/The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, Guangdong, China 3 Research Unit of Non-Coding RNA and Genome Editing in Cancer, Division of Oncology, Medical University of Graz, Graz 8010, Austria 4 The Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA * Correspondence: gcalin@mdanderson.org (G.A.C.); linghui1234@gmail.com (H.L.); Tel.: +1-713-792-5461 (G.A.C.); +1-713-563-5635 (H.L.) y * Correspondence: gcalin@mdanderson.org (G.A.C.); linghui1234@gmail.com (H.L.); Tel.: +1-713-792-5461 (G.A.C.); +1-713-563-5635 (H.L.) Received: 9 August 2017; Accepted: 13 September 2017; Published: 20 September 2017 Abstract: Colorectal cancer (CRC) is the third most frequent cancer and one of the leading causes for cancer-related mortality. Aberrant activation of the Wnt signaling is an essential initiating factor in colon carcinogenesis, and a driving force of CRC progression. Recently, long non-coding RNAs (lncRNAs) have emerged as signiﬁcant players in CRC pathogenesis through diversiﬁed mechanisms. Although both Wnt signaling and lncRNAs represent interesting research areas for CRC, an effort of directly connecting these two areas is lacking. To ﬁll in the knowledge gap, we focus on the reported ﬁndings of lncRNAs that regulate Wnt signaling or essential Wnt signaling targets. These include several newly discovered lncRNAs originated from the ampliﬁed cancer-associated chromosome 8q24 region that surrounds the essential Wnt target MYC gene, lncRNAs reported to be involved in CRC stem cells, and several individual lncRNAs connected to Wnt signaling through other mechanisms. This review will provide essential information that assists in understanding the missing link of lncRNAs to the classical Wnt signaling in CRC. Keywords: Wnt; long non-coding RNA; CCAT1; CCAT2; PVT1; H19 International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Int. J. Mol. Sci. 2017, 18, 2003; doi:10.3390/ijms18092003 1. Introduction Colorectal cancer (CRC), accounting for 8% of new cancer cases, is the third most frequent cancer, and one of the leading cause of cancer-related mortalities in the United States [1]. Despite the fact that the CRC incidence rates declined in people aged 50 years or older, CRC incidence rates increased by 22% from 2000 to 2013 in adults younger than 50 years in the United States [2]. Similarly, CRC mortality rates increased by 13% in those less than 50 years old during the same period [2]. The rise of CRC with early age at diagnosis underlines the need for exploration of new avenues for disease understanding, and the development of innovative detection and intervention strategies. Aberrant Wnt signaling is the initiating and driving event underlying the vast majority of CRC carcinogenesis [3–5]. Although the essential molecules of Wnt signaling have been well characterized, how this pathway is ﬁne-tuned at other layers remains largely unexplored. Non-coding RNA transcripts such as microRNAs have been revealed to restrain or activate Wnt signaling, by controlling the expression of Wnt signaling proteins [6–8]. More recently, long non-coding RNAs (lncRNAs), deﬁned as being at least 200 nucleotides in length, show signiﬁcant association with CRC incidence, Int. J. Mol. Sci. 2017, 18, 2003; doi:10.3390/ijms18092003 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 2003 2 of 17 the extent of malignancy, and patient prognosis [9,10]. However, the mechanisms underlying lncRNA involvement in CRC lag far behind its discovery pace and remain largely uncharacterized, partially because of the lack of a uniﬁed molecular mechanism. underlying lncRNA involvement in CRC lag far behind its discovery pace and remain largely uncharacterized, partially because of the lack of a unified molecular mechanism. We reason that because Wnt signaling is an initiating force in CRC carcinogenesis lncRNAs that We reason that because Wnt signaling is an initiating force in CRC carcinogenesis, lncRNAs that participate in this pathway might represent the novel research avenue for mechanisms of understanding the other regulatory layers of CRC. We acknowledge that many excellent reviews have covered each topic of Wnt signaling and lncRNA independently, and refer the readers to this literature for more in-depth information [9,11,12]. In this review, we focus on the knowledge connecting these two topics, and aim to review recent findings linking lncRNAs with essential Wnt signaling. 1. Introduction Herein, we briefly introduce the individual topic, summarize the typical examples that connect these two topics, and discuss the clinical application potential of lncRNAs in CRC. We further separated these lncRNAs into several categories: those transcribed from the 8q24 cancer risk region, associated with CRC stem cells, and others. To keep this review concise while informative, we list only the essential findings limited by our own knowledge, and apologize to those whose work was not referenced due to space restrictions. We reason that because Wnt signaling is an initiating force in CRC carcinogenesis, lncRNAs that participate in this pathway might represent the novel research avenue for mechanisms of understanding the other regulatory layers of CRC. We acknowledge that many excellent reviews have covered each topic of Wnt signaling and lncRNA independently, and refer the readers to this literature for more in-depth information [9,11,12]. In this review, we focus on the knowledge connecting these two topics, and aim to review recent findings linking lncRNAs with essential Wnt signaling. Herein, we briefly introduce the individual topic, summarize the typical examples that connect these two topics, and discuss the clinical application potential of lncRNAs in CRC. We further separated these lncRNAs into several categories: those transcribed from the 8q24 cancer risk region, associated with CRC stem cells, and others. To keep this review concise while informative, we list only the essential findings limited by our own knowledge, and apologize to those whose work was not referenced due to space restrictions. 2. Wnt: The Initiating and Driving Force of Colorectal Cancer (CRC) 2. Wnt: The Initiating and Driving Force of Colorectal Cancer (CRC) In 1982, the first Wnt family member Int1 (now known as Wnt1) was identified as a mouse proto-oncogene that is activated by the integration of mouse mammary tumor virus [13]. Subsequent research demonstrated that Int1 is a vertebrate homologue of the previously identified fly Wingless (Wg) gene, from which the term Wnt originates [14]. As the most well-known and best characterized Wnt signaling pathway, the canonical Wnt signaling that involves β-catenin and members of the lymphocyte-enhancer-binding factor (LEF)/T-cell factor (TCF) family is the fundamental driving force of CRC [15]. In the presence of a Wnt ligand binding to its receptor complex, β-catenin is rescued from ubiquitination and proteasomal breakdown by the destruction complex comprising adenomatous polyposis coli (APC), AXIN1, and GSK3β [16,17]. The stabilized β-catenin translocates to nucleus and forms a complex with LEF/TCF transcription factors to activate the transcription of a wide range of Wnt target genes [18] (Figure 1). In 1982, the first Wnt family member Int1 (now known as Wnt1) was identified as a mouse proto- oncogene that is activated by the integration of mouse mammary tumor virus [13]. Subsequent research demonstrated that Int1 is a vertebrate homologue of the previously identified fly Wingless (Wg) gene, from which the term Wnt originates [14]. As the most well-known and best characterized Wnt signaling pathway, the canonical Wnt signaling that involves β-catenin and members of the lymphocyte-enhancer-binding factor (LEF)/T-cell factor (TCF) family is the fundamental driving force of CRC [15]. In the presence of a Wnt ligand binding to its receptor complex, β-catenin is rescued from ubiquitination and proteasomal breakdown by the destruction complex comprising adenomatous polyposis coli (APC), AXIN1, and GSK3β [16,17]. The stabilized β-catenin translocates to nucleus and forms a complex with LEF/TCF transcription factors to activate the transcription of a wide range of Wnt target genes [18] (Figure 1). Figure 1. Long non-coding RNA (LncRNA) involvement in canonical WNT signaling. Arrow in black: activate; T in Red: inhibit. Figure 1. Long non-coding RNA (LncRNA) involvement in canonical WNT signaling. Arrow in black: activate; T in Red: inhibit. Figure 1. Long non-coding RNA (LncRNA) involvement in canonical WNT signaling. Arrow in black: activate; T in Red: inhibit. Figure 1. Long non-coding RNA (LncRNA) involvement in canonical WNT signaling. Arrow in black: activate; T in Red: inhibit. Int. J. Mol. Sci. 3. LncRNAs: The Emerging Dark Matters That Matter The vast majority of the human genome is transcribed into RNA transcripts, but only a small proportion of these RNA molecules are translated into proteins [28]. The genes that do not code for proteins produce non-coding RNAs (ncRNAs) as the final output. As one type of ncRNA, microRNA (miRNA) received much attention in the last 15 years since the discovery of their cancer involvement [29]. More recently, long ncRNAs (lncRNAs), containing no less than 200 nucleotides, have emerged as important new players in cancer [12,30]. According to their genomic features, these lncRNAs can be further classified into long intergenic ncRNAs (lincRNAs), transcribed ultraconserved regions (T-UCRs), circular RNAs, promoter-associated RNAs, enhancer-associated RNAs, and others [12,31]. In this review, we use the term lncRNA for consistency, although most of the examples in the text are lincRNAs. A previous study summarized the diverse molecular mechanisms of lncRNAs into four archetypes [32]: (i) lncRNA can serve as a molecular sensor to deliver the signal from the hint of cellular context in a temporal and spatial manner; (ii) lncRNA can serve as decoy to interfere the function of proteins, or that of miRNAs by sponging the miRNAs via sequence complementarity; (iii) lncRNA can guide chromatin-modifying proteins onto target genes, either locally in cis or distantly in trans, respectively; (iv) lncRNAs can bridge multiple proteins together to modify chromatin or stabilize subcellular structures. It should be noted that these archetypes are not exclusive, and a single lncRNA may have multiple mechanisms. 2. Wnt: The Initiating and Driving Force of Colorectal Cancer (CRC) 2. Wnt: The Initiating and Driving Force of Colorectal Cancer (CRC) 2017, 18, 2003 3 of 17 The Cancer Genome Atlas (TCGA) consortium revealed that the Wnt signaling pathway was altered in more than 90% of CRC tumors, with mutational inactivation of the APC tumor suppressor gene, located at chromosome 5q21-q22, in ~80% of cases [19]. As an initiating event in both familial adenomatous polyposis and sporadic CRCs [20,21], the mutational inactivation of APC leads to the accumulation of β-catenin in the nucleus, a hallmark of the canonical Wnt signaling, and the transcriptional activation of Wnt target genes by β-catenin/TCF complex [22,23]. Wnt signaling is essential in maintaining the stem cell niche, and high Wnt activity was reported to accurately deﬁne the CRC stem cell population [24]. Experimentally, restoration of APC reverted CRC tumorigenic lesions by re-establishing the normal crypt homeostasis, even in mice harboring oncogenic Kras and mutated p53 [25]. This experimental ﬁnding not only reinforces the essential suppressor function of APC in CRC initiation, but also revealed the critical importance of APC-regulated Wnt signaling in CRC progression. As classical downstream targets that respond to Wnt signaling, CCND1 and MYC are established drivers in CRC formation by regulating cell growth, apoptosis, migration, invasion and stem cell maintenance [26,27]. 4. LncRNAs in Wnt Signaling and CRC Since Wnt signaling is an essential pathway in CRC carcinogenesis and progression, it is not surprising that many CRC-associated lncRNAs exert their function via this pathway. The dynamic molecular mechanisms of lncRNAs also render them large diversity in regulating Wnt activity or essential Wnt downstream targets. The 8q24 region represents one of the most frequently ampliﬁed cancer-associated regions in CRC, and contains the MYC oncogene [33]. Reports in the last several years revealed that the 8q24 region is an oasis for long non-coding RNAs [34–38]. As such, we discuss this unique group of lncRNAs separately. We summarize three lncRNAs in the subsection “CRC stem cells—related lncRNAs” and several lncRNAs that do not share common features in the subsection “Others” (Table 1). 4 of 17 Int. J. Mol. Sci. 2017, 18, 2003 Table 1. LncRNAs involved in Wnt signaling and CRC. LncRNA Loci Length Indentiﬁcation Method Mechanism Related to Wnt Clinical Relevance 8q24 region CCAT1-S (CARLo-5) 8q24.21 2628 nt RACE qRT-PCR Transcription of CCAT1-S is inﬂuenced by the allele status of the single nucleotide polymorphisms (SNP) rs6983267 via a long-range interaction of CCAT1-S promoter with rs6938267-containing region. Promotes CRC growth and invasion; Increased expression correlates with poor prognosis. CCAT1-L 8q24.21 5200 nt RNA-seq qRT-PCR Northern blot RACE Interacts with CTCF to faciliate chromatin looping connecting MYC enhancer and promoter, resulting in MYC transcription. CCAT2 8q24.21 340 nt qRT-PCR Northern blot RACE Interacts with TCF7L2 to promote MYC and other Wnt target gene transcription. Spans the SNP rs6983267 alleles that responds differentially to Wnt signaling. Promotes CRC growth and metastasis; Increased expression correlates with poor prognosis. CASC11 (CARLo-7) 8q24.21 872 nt qRT-PCR Interacts with heterogeneous ribonucleoprotein K (hnRNP-K) to protects β-catenin from degradation, and consequently activates Wnt signaling. MYC binds to the promoter of CASC11 to activate its transcription. Promotes CRC growth and invasion; Increased expression correlates with CRC size, invasion, and lymph metastasis. PVT1 8q24.21 1957 nt RACE Northern blot qRT-PCR Interacts with MYC protein to prevent MYC phosphorylation and degradation. Promotes CRC growth. Increased expression correlates with poor prognosis. PCAT1 8q24.21 1992 nt qRT-PCR, RNA-seq Increases MYC expression. Promotes CRC growth. CRC stem cell Lnc34a 1p36.22? 693 nt qRT-PCR RACE Interacts with Dnmt3a, HDAC1, and PHB2E to epigenetically silences miR-34a expression, resulting in CRC stem cell asymmetric division. Enriched in CRC stem cells, and upregulated in late-stage CRCs. 4.1. LncRNAs from 8q24 Region 4.1. LncRNAs from 8q24 Region The chromosome 8q24 is The chromosome 8q24 is frequently ampliﬁed in human cancer. Particularly, the 8q24.21 genomic region that spans almost 2 Mb but represents a desert for protein coding genes and attracts much attention for several reasons. (i) This region contains the MYC gene, which is a classical Wnt signaling target and an essential oncogene [27,39]. (ii) Genome-wide association studies consistently suggested that multiple single nucleotide polymorphisms (SNPs) in such region are associated with CRC risk [40–42]. (iii) DNA elements in this region have various enhancer activities that are affected by SNPs [43,44], and more recently this region was proposed as a typical example of a super-enhancer [45]. These observations, originating from a different angle but pointing to the same genomic locus, indicate that important unidentiﬁed molecular culprits reside in such region. Indeed, the last decade witnessed an explosion of discoveries of lncRNAs in the 8q24.21 region, most of which show relevance with multiple types of cancers including CRC. Not surprisingly, a large proportion of these lncRNAs were found to regulate MYC, a protein-coding gene in the region, in one way or another (Figure 2). We can envision that strong enhancer activity in this region promotes transcription of lncRNAs because of the presence of abundant transcriptional factors and mediator proteins. These lncRNAs in turn increase the enhancer activity by forming the chromatin loop or bridging the protein partners, thus creating a positive feedback mechanism in controlling MYC expression. Because of their sensitivity in responding to oncogenic signals, the lncRNAs themselves are often found to be prognosis factors in predicting the outcome of CRC patients. The chromosome 8q24 is frequently amplified in human cancer. Particularly, the 8q24.21 genomic region that spans almost 2 Mb but represents a desert for protein coding genes and attracts much attention for several reasons. (i) This region contains the MYC gene, which is a classical Wnt signaling target and an essential oncogene [27,39]. (ii) Genome-wide association studies consistently suggested that multiple single nucleotide polymorphisms (SNPs) in such region are associated with CRC risk [40–42]. (iii) DNA elements in this region have various enhancer activities that are affected by SNPs [43,44], and more recently this region was proposed as a typical example of a super-enhancer [45]. These observations, originating from a different angle but pointing to the same genomic locus, indicate that important unidentified molecular culprits reside in such region. 4. LncRNAs in Wnt Signaling and CRC RBM5-AS1 3p21.31 1386 nt lncRNA array RNA-Seq qRT-PCR Interacts with β-catenin, and promotes the transcriptional activity of β-catenin/TCF7L2 complex. WiNTRLINC1 11p15.5 4117 nt qRT-PCR Northern blot Interacts with TCF7L2/β-catenin to form chromatin loop and activate ASCL2 transcription. Increased expression correlates with metastatic potential and poor prognosis. Mechanism Related to Wnt 5 of 17 Int. J. Mol. Sci. 2017, 18, 2003 Table 1. Cont. LncRNA Loci Length Indentiﬁcation Method Mechanism Related to Wnt Clinical Relevance Others H19 11q15.5 6295 nt RACE cloning Northern blot qRT-PCR Interacts with macroH2A to derepress transcription of CDK8, which positively regulates β-catenin activity. Interacts with hnRNP U to repress Wnt gene transcription. Interacts with EZH2 to repress NKD1 transcription, resulting in Wnt activation. Antagonizes the inhibition of let7 on MYC, which regulates H19 transcription. Increased expression correlates with poor prognosis independent of other factors. CCAL Chr3 1933 nt Microarray RACE qRT-PCR Interacts with and degrades AP-2α, a negative regulator of Wnt activity, resulting in increased MDR1 transcription. Increased expression correlates with poor prognosis and poor response to adjuvant chemotherapy CTD903 14q11.2 903 nt Microarray qRT-PCR Inhibits Wnt signaling and EMT by unknown mechanisms. Increased expression correlates with favorable prognosis. ASBEL 21q21.1 2000 nt qRT-PCR Northern blot Interacts with TCF3 to repress ATF3 transcription. MYU 16q24.3 6310 nt RNA-seq Upregulated by MYC. Interacts with hnRNP-K to stabilize CDK6 mRNA. * RACE: Rapid ampliﬁcation of cDNA ends; CTCF: CCCTC-binding factor. Mechanism Related to Wnt Int. J. Mol. Sci. 2017, 18, 2003 Int. J. Mol. Sci. 201 , 8, 2003 6 of 17 4.1. LncRNAs from 8q24 Region 4.1. LncRNAs from 8q24 Region The chromosome 8q24 is Indeed, the last decade witnessed an explosion of discoveries of lncRNAs in the 8q24.21 region, most of which show relevance with multiple types of cancers including CRC. Not surprisingly, a large proportion of these lncRNAs were found to regulate MYC, a protein-coding gene in the region, in one way or another (Figure 2). We can envision that strong enhancer activity in this region promotes transcription of lncRNAs because of the presence of abundant transcriptional factors and mediator proteins. These lncRNAs in turn increase the enhancer activity by forming the chromatin loop or bridging the protein partners, thus creating a positive feedback mechanism in controlling MYC expression. Because of their sensitivity in responding to oncogenic signals, the lncRNAs themselves are often found to be prognosis factors in predicting the outcome of CRC patients. Figure 2. 8q24 lncRNAs in MYC regulation. Figure 2. 8q24 lncRNAs in MYC regulation. Figure 2. 8q24 lncRNAs in MYC regulation. Figure 2. 8q24 lncRNAs in MYC regulation. 4.1.2. CCAT2 The CCAT2 gene was identiﬁed by the Calin laboratory based on several previous observations [38]. First, this high degree of conservation of this genomic region among mammals suggests the functional importance of this locus and associated transcripts [48]. Second, the rs6983267 SNP in this region is one of the most consistently reported, predisposing SNPs in prostate cancer and CRC [41,49,50]. Third, this region, 335 kb centromeric from the MYC oncogene possesses strong enhancer activity that is inﬂuenced by the rs6983267 SNP variants [43,44]. After cloning and characterization of the gene, CCAT2 was found to express at higher levels in microsatellite-stable CRC tumors that exhibit chromosomal instability (CIN), than in microsatellite-instable tumors or normal mucosae that lack the CIN feature [38]. This led to the discovery of CCAT2 initiation of CIN via cell model systems. Experimental data suggest that CCAT2 not only exerts cis regulatory effects on the nearby gene MYC, but also interacts with TCF7L2 protein to exert trans regulatory effects on Wnt signaling [38]. Together with the DNA element with enhancer activity inﬂuenced by the SNP, the CCAT2 RNA contributes to a unique Wnt signaling regulatory network. This DNA-RNA regulatory network may be essential in CRC, as deletion of this genomic region, which results in loss of both DNA elements and RNA transcripts, reduces the number of the intestine polyps in ApcMin/+ mice [51]. Recently, this non-coding RNA was reported to exert allele-speciﬁc effects on cancer metabolism by interaction with the splicing protein CFIm and ensuring alternative splicing of glutaminase [52]. In addition, multiple meta-analysis studies proved the prognostic value of CCAT2 in predicting cancer patient survival [53–56]. 4.1.1. CCAT1 4.1.1. CCAT1 The CCAT1 gene is located 515 kb upstream of the MYC oncogene, encodes a short isoform CCAT1-S and a long isoform CCAT1-L. CCAT1-S, also known as CARLo-5, is upregulated in all disease stages, including pre-malignant conditions, during CRC transformation [37]. A meta-analysis suggests significant association of increased CCAT1 expression in tumor samples with poor survival of cancer patients [46]. The expression of CCAT1-S is significantly correlated with the allele status of the SNP rs6983267, located telomeric of CCAT1-S [37]. In addition, the genomic region containing rs6983267 forms a chromatin loop with the promoter of CCAT1-S gene, suggesting a long-range interaction of rs6983267-containing region with CCAT1-S promoter in regulating its expression [37]. Experimentally, knockdown of CCAT1-S decreased CRC cell growth in vitro and in vivo [37]. Similar to its shorter isoform, CCAT1-L is highly expressed in CRC tumors, and promotes CRC growth in The CCAT1 gene is located 515 kb upstream of the MYC oncogene, encodes a short isoform CCAT1-S and a long isoform CCAT1-L. CCAT1-S, also known as CARLo-5, is upregulated in all disease stages, including pre-malignant conditions, during CRC transformation [37]. A meta-analysis suggests significant association of increased CCAT1 expression in tumor samples with poor survival of cancer patients [46]. The expression of CCAT1-S is significantly correlated with the allele status of the SNP rs6983267, located telomeric of CCAT1-S [37]. In addition, the genomic region containing rs6983267 forms a chromatin loop with the promoter of CCAT1-S gene, suggesting a long-range interaction of rs6983267-containing region with CCAT1-S promoter in regulating its expression [37]. Experimentally, knockdown of CCAT1-S decreased CRC cell growth in vitro and in vivo [37]. Similar to its shorter isoform, CCAT1-L is highly expressed in CRC tumors, and promotes CRC growth in xenograft mouse 7 of 17 Int. J. Mol. Sci. 2017, 18, 2003 models [47]. CCAT1-L enhances MYC transcription, as elegantly demonstrated by genome engineered cell lines that overexpressed CCAT1-L at its own gene loci [47]. Chromosome conformation capture assay suggests that CCAT1-L facilitates the formation of a long-range physical interaction loop between the MYC enhancer and its promoter [47]. Molecular mechanism study revealed an interaction of CCAT1-L with CTCF, an essential protein regulating 3D structure of the chromatin [47]. The CCAT1 gene is also transcriptionally regulated by MYC [34]. These results reveal a complex molecular interaction connecting SNP, enhancer, lncRNA, and protein in controlling MYC expression and CRC growth. 4.1.3. CASC11 CASC11, also known as CARLo-7, is located ~2.1 kb upstream of the MYC gene. The CASC11 gene encompasses the lymphoma predisposition SNP rs16902359. Similar to the above CCAT transcripts, CASC11 is overexpressed in CRC tumors, and high CASC11 correlates with large primary tumors and metastasis to lymph nodes [35]. Ectopic expression of CASC11 promotes CRC growth and metastasis in vitro and in vivo [35]. Mechanism study showed that CASC11 interacts with and increase the stability of heterogeneous ribonucleoprotein K (hnRNP-K), which protects β-catenin from degradation by the destruction complex, and consequently activates WNT/β-catenin signaling [35]. Forming a feedback mechanism, MYC protein binds to the promoter of the CASC11 gene to activate its transcription [35]. 4.2.1. Lnc34a Loss of miR-34a is commonly seen in many types of cancer [68,69]. Several reports showed that miR-34a directly targets genes involved in Wnt signaling, including Wnt ligands and the essential β-catenin/TCF7L2 components, resulting in suppression of Wnt activity [7,70,71]. Recently, miR-34a was revealed to be directly involved in controlling CRC stem cell asymmetric division by forming a feedforwarded loop targeting Numb and Notch [72]. In an effort to trace the mechanism of reduced miR-34a expression in CRC cells, a new lncRNA termed lnc34a, transcribed in the opposite orientation from the miR-34a, was identified as a key regulator of miR-34a [73]. Lnc34a interacts with several epigenetic regulators, namely, Dnmt3a, HDAC1, and PHB2, to silence the transcription of miR-34a independent of the p53 protein [73]. The functional importance of lnc34a was demonstrated by its enrichment in CRC stem cells, and its ability to initiate asymmetric division by suppressing miR-34a [73]. Interestingly, lnc34a is distributed unevenly during cell division, and represses the transcription of miR-34a in only one daughter cell [73]. Mouse studies proved the function of Lnc34a in regulating self-renewal of cancer stem cell and CRC growth [73]. Concordantly, lnc34a expression was found to be upregulated in clinical samples of late-stage CRCs [73]. This provides an example of lncRNA-miRNA interaction in maintenance of cancer stem cell feature by regulating Wnt and Notch signaling. 4.1.4. PVT1 Different from most of the non-coding genes upstream of MYC, PVT1 is located 100–500 kb downstream of the MYC gene. In addition, different from CCAT1 and CCAT2 that regulate MYC transcription, PVT1 controls MYC protein levels by protecting the MYC protein from degradation [36]. Speciﬁcally, the physical interaction between PVT1 RNA and MYC protein interferes with its phosphorylation at threonine 58, which is essential in leading to MYC protein degradation [36]. As revealed by in vivo chromosome engineering, PVT1 is indispensable for MYC-induced cancer promoting effect [36]. The copy number of PVT1 and MYC gene was co-increased in nearly all CRC cases with MYC gene ampliﬁcation [36]. Depletion of PVT1 reduced the tumorigenic capacity of HCT116, a MYC-driven CRC cell line [36]. In addition, multiple studies reported signiﬁcant association 8 of 17 Int. J. Mol. Sci. 2017, 18, 2003 between PVT1 expression and CRC malignancies, and pointed to PVT1 as a potential diagnostic and prognostic marker in CRC [36,57]. Interestingly, the PVT1 gene locus also harbors a cluster of six annotated microRNA genes (namely, miR-1204, miR-1205, miR-1206, miR-1207-5p, miR-1207-3p, and miR-1208), of which the function remains to be clariﬁed [58]. 4.2.2. RBM5-AS1 The lncRNA RBM5-AS1 was found to be enriched during sphere formation of colon cancer initialing cells [74]. Silencing of RBM5-AS1 impaired Wnt signaling, while overexpression enhances Wnt signaling in CRC cells [74]. The RBM5-AS1 activity on Wnt signaling is critical for enabling the function of CRC stem cells, as loss of RBM5-AS1 impaired the spheroid formation in multiple CRC cell lines [74]. Mechanism study revealed that RBM5-AS1 physically interacts with β-catenin, and promotes the interaction of β-catenin with the TCF7L2 complex [74]. As a result, Wnt target genes such as SGK1, YAP1 and MYC are transcriptionally activated by RBM5-AS1 [74]. 4.2. CRC Stem Cell—Associated LncRNAs Wnt signaling is a determining factor in CRC stem cell maintenance [24,63]. It controls not only essential stem cell genes such as LGR5, but also regulates asymmetric division of CRC stem cells [64,65]. The control of CRC stem cells by Wnt signaling offers growth and selection advantages, which may underlie the resistance of CRC tumors to chemotherapeutic drugs [66,67]. Several lncRNAs have been reported to control the fate of the CRC stem cells. 4.1.5. PCAT1 The PCAT1 gene is located ~725 kb upstream of the MYC oncogene. Besides the involvement of PCAT1 in prostate cancer [59,60], PCAT1 was also found to be overexpressed in CRC tumors [61]. Experimental data showed that downregulation of PCAT1 inhibits CRC growth in vitro and in vivo, partially via its regulatory effect on MYC [62]. High levels of PCAT1 expression in primary CRC tumors were significantly associated with distal metastasis of CRC. Moreover, multivariable analysis revealed that increased PCAT1 expression was an independent factor for poor prognosis in CRC patients [61]. 4.3.2. CCAL LncRNA expression proﬁling of normal, adenoma, and carcinoma tissues identiﬁed CCAL as a crucial regulator of CRC carcinogenesis [66]. In addition, high CCAL levels in the CRC tumor correspond to short overall survival and poor response to adjuvant chemotherapy [66]. The interaction of CCAL with AP-2α protein promotes the degradation of AP-2α, a negative regulator of β-catenin/TCF7L2 interaction in CRC, and thus indirectly activates Wnt signaling [66]. As a consequence, the multidrug resistance (MDR1) gene, which is a Wnt target that encodes the P-glycoprotein 1, is activated [66]. The effect on MDR1 offers a possible explanation on the observed association between CCAL expression and therapeutic outcome in patients with CRC. 4.3.1. H19 H19 is one of the first imprinted non-coding genes discovered in 1990s [76]. H19 is exclusively transcribed from the maternally inherited allele and participates as a key factor in embryonic development [77,78]. Various reports have suggested the involvement of H19 in human cancer [77,79–81]. H19 exerts its function by interaction with EZH2 [82], sponging microRNAs such as let7 and miR-106a [83,84], or production of miR-675 as a primary transcript [85]. Studies from our own work using TCGA CRC data identified H19 as a top candidate in association with worse CRC survival [86]. Knockdown of H19 caused a dramatic reduction of CRC cell proliferation and migration [86]. An unbiased approach with microarray analysis revealed not only the known mechanisms of H19 regulation on let7 and MYC, but also a novel mechanism where H19 regulates β-catenin activity via modulating CDK8 expression [87], which is probably a consequence of H19 interaction with the repressive histone variant macroH2A [86,88]. This study, together with other findings that H19 regulates Wnt signaling by interaction with hnRNP resulting in suppressed expression of Wnt genes in liver cells [89], and by interaction with EZH2, leading to Wnt activation through NKD1 repression in bladder cancer [82], provides a vivid example of diverse mechanisms by a single lncRNA. H19 itself was transcriptionally controlled by the MYC protein, thus forming a connecting loop of Wnt-MYC-H19-Wnt [90]. Multiple studies, including our own work, suggest that H19 is an independent prognostic marker for CRC survival [86]. Combined analysis of H19 with its molecular targets significantly improved the prediction power to a level comparable to stage, validating the clinical significance of CDK8-β-catenin regulation by H19 in CRC [86]. 4.2.3. WiNTRLINC1 ASCL2 is an essential transcription factor in controlling the stemness of intestinal cells in response to Wnt signaling [65]. Using ChIP-seq with antibodies against RNA polymerase II, WiNTRLINC1 9 of 17 Int. J. Mol. Sci. 2017, 18, 2003 (WNT-regulated lincRNA 1), located ~60 kb away from the ASCL2 gene, was identified as one of the direct β-catenin/TCF7L2 targets in CRC [75]. WiNTRLINC1 physically interacts with β-catenin/TCF7L2 to facilitate the looping of regulatory elements, and consequently activate the transcription of the ASCL2 gene [75]. The regulatory network of Wnt-WiNTRLINC1-ASCL2-stemness is further enhanced with the transcriptional activation of WiNTRLINC1 by ASCL2 [75]. The expression levels of WiNTRLINC1 and ASCL2 were both increased in clinical CRC tumors, and high levels of WiNTRLINC1 were correlated with increased metastatic potential and worse prognosis of CRC patients [75]. 4.3. Others 4.3. Others 4.3.5. MYU MYU (c-Myc-upregulated lncRNA), originating from opposite strand of the VPS9D1 gene, is transcriptionally activated by the Wnt target MYC [93]. Downregulation of MYU inhibits the proliferation of MYC-overexpressing cells in vitro and retards xenograft CRC tumor growth in vivo [93]. MYU interacts with the protein hnRNP-K, which binds to the 3′ untranslated region (UTR) of the CDK6 mRNA to prevent the degradation of CDK6 by miR-16 [93]. Consequently, MYU stabilizes CDK6 and controls the cell transition from G1 to S phase [93]. The mechanism of MYC-MYU-hnRNP-K-CDK6 provides another layer of complexity connecting Wnt signaling and CRC growth. 5. Potential Clinical Application Once dark genome matter, lncRNA emerged as an important layer of regulation of the essential signaling in CRC [94]. Many of the Wnt-associated lncRNAs exhibit significant association with clinical parameters of CRC. Being potential candidates, lncRNAs have several advantages as cancer biomarkers. First, tissue-specific patterns of lncRNA expression suggests that lncRNAs may be over-represented in specific types of tumor, as exemplified by the successful development of PCA3 as cancer biomarker to help determine the necessity for repeated prostate biopsies [95]. Second, lncRNAs are sensitive signaling molecules in response to tissue-specific and context-specific stimuli [32]. Genentech researchers identified CCAT1 as a reliable marker for predicting the response to JQ1, a chemical inhibitor of bromodomain containing 4 (BRD4), suggesting the biomarker potential of CCAT1 in stratifying patients for clinical trials [96]. Third, the fact that some lncRNA genes contain predisposition SNPs offers a unique opportunity for using both DNA and RNA information as biomarkers [31]. As an example, the combined detection of rs6983267 allele status and CCAT2 levels might strengthen the disease connection. Indeed, a large-scale study showed that the preventative effect of aspirin in CRC was affected by the rs6983267 status [97]. We hypothesize that the CCAT2 transcript might work together with its DNA counterpart to regulate nearby genes, and influence the effect of aspirin in preventing CRC. Last, a combination of lncRNA profiling with other RNA or protein expression might improve the biomarker sensitivity or specificity. The finding that levels of H19 and its targets together have a prediction power for CRC prognosis similar to tumor stage supports this possibility, and highlights the importance of mechanism understanding in translational applications [86]. Because of the diverse mechanisms of lncRNAs in ﬁne-tuning Wnt signaling, lncRNAs can be potential therapeutic targets. For instance, the multifaceted function of H19 (sponging let7 [84], regulating methylation at a genome wide level [98], targeting CDK8-β-catenin signaling [86], and regulating the cell cycle [86]) in CRC makes it possible to tackle multiple oncogenic mechanisms with a single hit on this lncRNA. The unique mechanism of PVT1 in controlling MYC protein stability can also be explored for therapeutic modulation of MYC protein, which is hard to inhibit by small molecule chemicals [99]. Similarly, ﬁnding out how the transcription of lnc34a is controlled might offer novel insights and strategies to target CRC cancer stem cells. 4.3.3. CTD903 CTD903 is transcribed from the region of chromosome 14q11.2. Ectopic expression of CTD903 inhibits cell proliferation and cell motility of CRC cells [91]. Cell line model showed that downregulation of CTD903 results in the activation of Wnt/β-catenin signaling, and consequently leads to epithelial mesenchymal transition (EMT), as evidenced by the increase of Twist, Snail, and Vimentin, and reduction of the epithelial marker ZO-1 [91]. This effect of CTD903 on Wnt and EMT provides a possible explanation on the observed suppression of CRC cell invasion by CTD903 [91]. Concordantly, CTD903 predicts the favorable prognosis of CRC patients [91]. The exact molecular mechanism by which CTD903 inhibits Wnt signaling remains to be characterized. Int. J. Mol. Sci. 2017, 18, 2003 10 of 17 10 of 17 4.3.4. ASBEL In an effort to identify Wnt-regulated lncRNAs in CRC, ASBEL (a lncRNA also known as BTG3-AS1) was revealed to be a direct target of β-catenin by RNA-seq and ChIP-seq analysis [92]. Knockdown of ASBEL retarded tumor growth in a xenograft mouse model of CRC [92]. Mechanism studies showed that ASBEL forms a complex with TCF3, a transcription factor that is transcriptionally activated by β-catenin, to cooperatively suppress ATF3 gene transcription [92]. This β-catenin-ASBEL-TCF3-ATF3 signaling was demonstrated to be required for CRC proliferation [92]. Together, these data suggest an important role of lncRNA in promoting CRC formation by mediating and executing Wnt activity. 5. Potential Clinical Application There are tremendous challenges ahead for the application of the lncRNA in clinical settings: their expression levels are usually low; some intron-derived or nuclear-localized lncRNAs are unstable with a half-life less than 2 h (http://stability.matticklab.com) [100]; their tissue-speciﬁc expression pattern 11 of 17 Int. J. Mol. Sci. 2017, 18, 2003 may render complexity in the interpretation of proﬁling by mixed cell population; targeting lncRNAs that reside in the nucleus is still facing many technically difﬁculties and uncertainties [101]. However, with the technical advances in sequencing and in situ hybridization, it is possible to obtain precise tissue-speciﬁcity information and spatial expression patterns. Similarly, with the technical advance on siRNA, antisense oligonucleotides (ASOs), and delivery systems, it is possible to overcome technical difﬁculties and safety concerns. For instance, most ASOs are taken up by the clearance organs such as liver, and thus, targeting CRC represents unmet challenges [102]. The conjugation of colon speciﬁc ligands, similar as the improved distribution seen in the hepatocytes by GalNAc ligand [103], might be one strategy for enhancing the targeting potency of siRNA or ASOs for CRC. Understanding the mechanism such as the effect of protein interactions on cellular uptake of modiﬁed ASOs is critical in optimizing the delivery of oligonucleotides [104]. 6. Conclusions As an essential signaling in CRC initiation and progression, Wnt signaling is involved in all key aspects of cancer biology related to tumor growth, metastasis, and therapeutic response. As such, any layer of regulation of this signaling has signiﬁcance in determining the cancer cell fate, and the outcome of CRC patients. The emerging concept of lncRNAs as important players in CRC by regulating Wnt singling deserves the attention of both academia and the biotechnological industry. Currently, the mechanism of understanding lncRNAs in CRC is still in its early infancy, and this represents one of the bottlenecks in bringing them to clinical applications. We propose that the mechanism of elucidation should be facilitated by unbiased and high-throughput experimental analysis including gene expression microarray analysis, RNA sequencing, methylation proﬁling, protein interaction identiﬁcation, genomic occupation by lncRNAs, and others. In addition, large-scale database analysis will reveal the clinical signiﬁcance of lncRNA, and offer hints on the molecular mechanisms. Finally, more effort should be put into the study of the secondary and higher-order structure of lncRNAs, which is critical for detailed understanding of lncRNA function at a molecular level. We expect that the advances on mechanism of understanding lncRNAs in Wnt signaling might bring novel candidates as biomarkers and therapeutics for CRC. We predict that microdissection and single-cell sequencing might represent a trend in fully extracting gene expression information, and foresee that breakthroughs in using oligonucleotides as drugs in the near future will also help achieve the full potential of lncRNA targeting as cancer therapy. Acknowledgments: Work in George A. Calin’s laboratory is supported by National Institutes of Health (NIH/NCATS) grant UH3TR00943-01 through the NIH Common Fund, Office of Strategic Coordination (OSC), the NIH/NCI grant 1 R01 CA182905-01, a U54 grant—UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, a Team DOD (CA160445P1) grant, a Ladies Leukemia League grant, a CLL Moonshot Flagship project, a SINF 2017 grant, and the Estate of C. G. Johnson, Jr. Martin Pichler was supported by the funding of an Erwin Schroedinger Scholarship of the Austrian Science Funds (No. J3389-B23). Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Groden, J.; Thliveris, A.; Samowitz, W.; Carlson, M.; Gelbert, L.; Albertsen, H.; Joslyn, G.; Stevens, J.; Spirio, L.; Robertson, M.; et al. Identiﬁcation and characterization of the familial adenomatous polyposis coli gene. Cell 1991, 66, 589–600. [CrossRef] 2. Siegel, R.L.; Miller, K.D.; Fedewa, S.A.; Ahnen, D.J.; Meester, R.G. S.; Barzi, A.; Jemal, A. 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https://openalex.org/W2525077777	https://hal.sorbonne-universite.fr/hal-01373902/document	English	null	Marinobacter Dominates the Bacterial Community of the Ostreococcus tauri Phycosphere in Culture	Frontiers in microbiology	2,016	cc-by	12,042	To cite this version: Josselin Lupette, Raphaël Lami, Marc Krasovec, Nigel Grimsley, Hervé Moreau, et al.. Marinobacter Dominates the Bacterial Community of the Ostreococcus tauri Phycosphere in Culture. Frontiers in Microbiology, 2016, 7, pp.1414. 10.3389/fmicb.2016.01414. hal-01373902 HAL Id: hal-01373902 https://hal.sorbonne-universite.fr/hal-01373902v1 Submitted on 27 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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ORIGINAL RESEARCH published: 07 September 2016 doi: 10.3389/fmicb.2016.01414 Marinobacter Dominates the Bacterial Community of the Ostreococcus tauri Phycosphere in Culture Josselin Lupette1,2,3, Raphaël Lami4,5, Marc Krasovec1,2, Nigel Grimsley1,2, Hervé Moreau1,2, Gwenaël Piganeau1,2 and Sophie Sanchez-Ferandin1,2* 1 Sorbonne Universités, Université Pierre et Marie Curie Paris 06, UMR 7232 Biologie Intégrative des Organismes Marins, Observatoire Océanologique, Banyuls-sur-Mer, France, 2 Centre National de la Recherche Scientiﬁque, UMR 7232 Biologie Intégrative des Organismes Marins, Observatoire Océanologique, Banyuls-sur-Mer, France, 3 CEA/CNRS/INRA/Université Grenoble Alpes, UMR 5168 Laboratoire Physiologie Cellulaire Végétale, Grenoble, France, 4 Sorbonne Universités, Université Pierre et Marie Curie Paris 06, USR 3579 Laboratoire de Biodiversité et Biotechnologies Microbiennes, Observatoire Océanologique, Banyuls-sur-Mer, France, 5 Centre National de la Recherche Scientiﬁque, USR 3579 Laboratoire de Biodiversité et Biotechnologies Microbiennes, Observatoire Océanologique, Banyuls-sur-Mer, France Microalgal–bacterial interactions are commonly found in marine environments and are well known in diatom cultures maintained in laboratory. These interactions also exert strong effects on bacterial and algal diversity in the oceans. Small green eukaryote algae of the class Mamiellophyceae (Chlorophyta) are ubiquitous and some species, such as Ostreococcus spp., are particularly important in Mediterranean coastal lagoons, and are observed as dominant species during phytoplankton blooms in open sea. Despite this, little is known about the diversity of bacteria that might facilitate or hinder O. tauri growth. We show, using rDNA 16S sequences, that the bacterial community found in O. tauri RCC4221 laboratory cultures is dominated by γ-proteobacteria from the Marinobacter genus, regardless of the growth phase of O. tauri RCC4221, the photoperiod used, or the nutrient conditions (limited in nitrogen or phosphorous) tested. Several strains of Marinobacter algicola were detected, all closely related to strains found in association with taxonomically distinct organisms, particularly with dinoﬂagellates and coccolithophorids. These sequences were more distantly related to M. adhaerens, M. aquaeoli and bacteria usually associated to euglenoids. This is the ﬁrst time, to our knowledge, that distinct Marinobacter strains have been found to be associated with a green alga in culture. Edited by: Edited by: Michael Thomas-Poulsen, University of Copenhagen, Denmark Michael Thomas-Poulsen, University of Copenhagen, Denmark Reviewed by: Tony Gutierrez, Heriot-Watt University, Scotland Garret Suen, University of Wisconsin–Madison, USA University of Wisconsin–Madison, USA *Correspondence: Sophie Sanchez-Ferandin sophie.sanchez-ferandin@obs- banyuls.fr Specialty section: This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology Received: 25 May 2016 Accepted: 26 August 2016 Published: 07 September 2016 Keywords: Ostreococcus tauri, Marinobacter sp., picoalgae, bacteria, interactions, phytoplankton Ostreococcus tauri RCC4221 Culture in Different Conditions Ostreococcus tauri strain RCC4221 was isolated in 1994 from the North-West Mediterranean Thau lagoon (Courties et al., 1994) and maintained in the laboratory (cultures and cryopreservation). The O. tauri strain was grown in liquid medium in aerated ﬂasks (Sarstedt), in growth chambers at 20±1◦C and white light at around 100 µmol photons.m−2.sec−1 using three photoperiods (LD 08:16; LD 12:12; and LD 14:10). Two culture media were prepared, one with natural sea water, L1-MOLA (Guillard and Hargraves, 1993) and one with artiﬁcial sea water, F/2-ESAW (Harrison et al., 1980). Both L1-MOLA and F/2-ESAW were produced by adding nitrogen (NaNO3), phosphorus (NaH2PO4) and vitamins (B1, B12, and H) but at diﬀerent concentrations (Supplementary Table S1). Only L1-MOLA was prepared from seawater, collected from 20 m below sea level at the MOLA station and kept several weeks in the dark before use. The seawater was ﬁltered through 0.22 µm and autoclaved. For nutrient limitation experiments, four conditions were tested: L1-MOLA, F/2-ESAW, F/2-ESAW 50%N (half nitrogen concentration), and F/2-ESAW 10%P (one tenth phosphorous concentration) (see Supplementary Table S1 for the detailed composition of each medium used in this study). For each tested condition, cultures were prepared in triplicates. Considering all of the culture experimentations (three photoperiods and four culture media conditions mentioned above), a total of 12 diﬀerent conditions were used in triplicate, thus 36 diﬀerent ﬂasks maintained in incubators. Half a milliliter of each culture was collected daily during 35 days and then ﬁxed 10 min at room temperature in the dark with glutaraldehyde (Sigma #G5882 – ﬁnal concentration: 1%) before frozen in liquid nitrogen and stored at −80◦C. Some studies demonstrated the presence of speciﬁc bacterial communities associated with algal blooms in marine environments (Gonzalez et al., 2000; Buchan et al., 2014). In axenic (i.e., exempt of bacteria) microalgal monocultures, microalgal growth may be unstable and prone to perturbation (Kazamia et al., 2014), highlighting again the importance of microalgalbacterial interactions, not only in natural environments but also in laboratory culture conditions. However, except for some species (Alavi et al., 2001; Green et al., 2004, 2015; Jasti et al., 2005), particularly diatoms (Grossart et al., 2005; Sapp et al., 2007a,b; Eigemann et al., 2013; Amin et al., 2015; Mishamandani et al., 2016), the bacterial diversity associated with microalgae species is still poorly known. Citation: As examples, stimulation of phytoplankton growth by bacteria can occur via the production of vitamins (Haines and Guillard, 1974; Kurata, 1986; Croft et al., 2005; Kuo and Lin, 2013), siderophores (Martinez et al., 2000, 2003; Amin et al., 2009) or phytohormones (de-Bashan et al., 2008) like auxin (Gonzalez and Bashan, 2000). In contrast, bacteria can even kill the algae by the secretion of algicidal compounds (Mayali and Azam, 2004). problems like sensitivity to herbicides (Sanchez-Ferandin et al., 2013) or tolerance to polluants like arsenic (Zhang et al., 2013). Despite the wealth of genomic data available for this species (Derelle et al., 2006; Blanc-Mathieu et al., 2014), the bacterial community associated with the O. tauri phycosphere is poorly understood, and the nature of the interactions between O. tauri and bacteria remains an open question (Abby et al., 2014). Here, we focus on the nature and dynamics of the microbiome of O. tauri RCC4221 across a large range of culture conditions. Surprisingly, we provide evidence that bacteria from one single genus, Marinobacter (γ-proteobacteria, order Alteromonadales) is largely predominant across culture conditions. Citation: Lupette J, Lami R, Krasovec M, Grimsley N, Moreau H, Piganeau G and Sanchez-Ferandin S (2016) Marinobacter Dominates the Bacterial Community of the Ostreococcus tauri Phycosphere in Culture. Front. Microbiol. 7:1414. doi: 10.3389/fmicb.2016.01414 Phytoplankton, together with viruses, bacteria, and micrograzers constitute diﬀerent communities of species which all play fundamental roles in the functioning of microbial food web (Buchan et al., 2014). Phytoplankton and bacterial communities are closely linked in coastal marine environments (Fuhrman et al., 1980; Rooney-Varga et al., 2005; Amin et al., 2015) and bacterial–algal interactions play a major role in determining bacterial and algal diversity in the ocean (Schäfer et al., 2002). Detailed knowledge of these interactions is thus crucial for understanding marine ecosystems. September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org Microalgae–Bacteria Interactions Lupette et al. Phytoplankton exudates can be important substrates for bacteria, especially in early phytoplankton bloom conditions (Fouilland et al., 2013) although other carbon sources might also be important for bacterial growth (Fouilland and Mostajir, 2010). In turn, some bacteria are known to inhibit or promote microalgal growth (Cole, 1982; Fukami et al., 1997; de-Bashan et al., 2004). The volume of water closely surrounding algal cells in which many metabolic exchanges may occur, is called the phycosphere (Bell and Mitchell, 1972). It is analogous to the rhizosphere in soils and it has direct implications for nutrient ﬂuxes to and from algal cells (Amin et al., 2012b). In this niche, bacteria can live freely around microalgae and interact through metabolic ﬂuxes via the environmental medium, or they may be more closely associated with the cells, such as epiphytic or endophytic bacteria. Epiphytic bacteria adhere to the microalgal surface (Bell and Mitchell, 1972; Shapiro et al., 1998) with a tight functional association (Middelboe et al., 1995; Smith et al., 1995; Grossart et al., 2005). Endophytic bacteria are able to develop inside microalgal cells and have been seen in Chlorophyta (Kawafune et al., 2012). Both commensalism and/or competition for micronutrients may occur between phytoplankton and bacteria (Bratbak and Thingstad, 1985; Amin et al., 2012a). Bacteria might positively (stimulation) or negatively (inhibition, alteration of physiology, death) inﬂuence phytoplankton dynamics. Ostreococcus tauri RCC4221 Culture in Different Conditions Small green algae belonging to the order Mamiellales are ubiquitous in the world oceans including the Arctic zone (de Vargas et al., 2015), and are of prime importance in the ecology of Mediterranean coastal lagoons. Among this group, the smallest free-living eukaryotic cell Ostreococcus tauri was discovered in the Mediterranean lagoon Thau 20 years ago (Courties et al., 1994), and can be observed as a dominant species during phytoplanktonic blooms in coastal seas (O’Kelly et al., 2003). In such lagoons or coastal regions where limnic and oceanic waters meet, the environment is more variable than in open sea, strongly inﬂuencing bacterial diversity (Glöckner et al., 1999; Herlemann et al., 2011; Biži´c-Ionescu et al., 2015). O. tauri is also being used as a model organism for studying diverse environmental Frontiers in Microbiology \| www.frontiersin.org Cell Concentrations and Growth Rates Cell Concentrations and Growth Rates Cell concentrations were determined using ﬂow cytometry (FACSCantoIITM, Becton Dickinson, San Jose, CA, USA). O. tauri cells were detected using the red ﬂuorescence emission (FL3) of chlorophyll pigments. For enumeration of bacteria, nucleic acids were labeled with SYBR R⃝Green I (Lonza, # 50512) and were detected by green ﬂuorescence (FL1) (Marie et al., 1997). Two kinds of bacteria were distinguished: HNA (High September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org 2 Microalgae–Bacteria Interactions Lupette et al. RCC4221, ﬁxed in glutaraldehyde 1% (Sigma), frozen in liquid nitrogen, and stored at −80◦C. Then, in order to follow bacterial diversity at speciﬁc times along O. tauri growth, aliquots were pooled following the four diﬀerent O. tauri RCC4221 growth phases: latency (LAT), exponential (EXP), stationary (STA) and decline (DEC) phases. Only samples corresponding to the LD 14:10 condition (for which the whole O. tauri growth from LAT to DEC phases was observed) were extracted for subsequent ribosomal 16S DNA sequencing (Table 1). apparent Nucleic Acid content) and LNA (Low apparent Nucleic Acid content) bacteria (Gasol et al., 1999). Growth rates (µmax) were determined from cell concentrations measured at diﬀerent times with the following equation (Levasseur et al., 1993): µmax = ln(N2/N1)/(t2 −t1) (1) (1) (where N1, N2, are cell abundances in the exponential phase at times t1 and t2). High Throughput Sequencing Data Analysis Sixteen diﬀerent conditions were analyzed, using four samples (corresponding to the four O. tauri diﬀerent growth phases: latent period, exponential growth, stationary phase, decline) for each medium. The samples were named OtL1LAT, OtL1EXP, OtL1STA, and OtL1DEC for L1 culture medium; Otf2LAT, Ot f2EXP, Ot f2STA, and Ot f2DEC for f/2 culture medium; OtPLAT, OtPEXP, OtPSTA, and OtPDEC for F/2 (50% [P]); and OtNLAT, OtNEXP, OtNSTA, and OtNDEC for F/2 (10% [N]). For each condition, DNA extraction was performed by using the modiﬁed CTAB method (Winnepenninckx et al., 1993). Total microbial 16S rDNA diversity of each of these 16 DNA samples was estimated by Illumina, 2 × 300 bp PE sequencing 20,000 PCR-ampliﬁed sequences (MrDNA, Molecular Research Laboratory, 503 Clovis Road, Shallowater, TX 79363 USA). The 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer were used in a 28 cycles PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94◦C for 3 min, followed by 28 cycles of 94◦C for 30 s, 53◦C for 40 s and 72◦C for 1 min, after which a ﬁnal elongation step at 72◦C for 5 min was performed. After ampliﬁcation, PCR products were checked in 2% agarose gel to determine the success of ampliﬁcation and the relative intensity of bands. Sixteen samples were pooled Statistical Analyses and Graphical Representations An O. tauri RCC4221 culture was diluted serially to permit isolation of single microalgal cells in individual culture wells [by “extinction dilution” or “endpoint dilution,” see for example (Taylor, 1962)], reducing also the density of bacteria. Sixteen independent lines obtained in this way from a single cell were maintained similarly by enumeration and dilution through 27 serial single-cell endpoint dilutions (about 500 cell divisions over the 54 weeks of culture). All of the cell lines were grown in L1 medium in 24 well plates with single-cell endpoint dilutions at every 14-day sub-culturing step (Krasovec et al., 2016) (Table 1). Each line was then screened on marine agar plates to isolate bacterial colonies and identify their diversity by PCR. The W18 bacterial forward (5′-GNTACCTTGTTACGACTT-3′) and W02 universal reverse (5′-GAGTTTGATCMTGGCTCAG-3′) primers were used (Godon et al., 1997). PCR were run with the Kapa Extra HS mix (CliniSciences) and standard ampliﬁcation (35 cycles of 15 s at 95◦C, 15 s at 50◦C and 1 min 20 s at 72◦C). Ampliﬁed DNAs were sequenced with Sanger method by Cogenics (Takeley, Essex, UK) and Bio2Mar platform (Banyuls- sur-mer, France). Statistical analyses were performed with R version 3.2.0 software1. t-tests were done on paired samples by permutation (t.paired.perm.R function)2 were performed. Graphical views from R and Excel were saved in pdf. 1http://www.R-project.org 2http://adn.biol.umontreal.ca/∼numericalecology/Rcode/ September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org Identiﬁcation of Cultivable and Total Bacteria in O. tauri RCC4221 Standard and Limiting Nutrient Conditions For each tested culture condition, aliquots of 100 µL of O. tauri cultures were spread onto L1-MOLA solid medium plates every 5 days. After 2 days of incubation in the same conditions as the ones used for the incubation of cells in liquid medium (see the section of Materials and Methods), DNA samples extracted from four morphologically identical colonies per condition were sequenced for a total of 12 conditions (384 sequences; Table 1). For total bacterial diversity identiﬁcation, an aliquot of 100 µL of culture was taken each day during the growth of O. tauri 1http://www.R-project.org 2http://adn.biol.umontreal.ca/∼numericalecology/Rcode/ TABLE 1 \| Summary of the different experimental conditions. Photoperiod Experimental conditions Number of O. tauri cultures Samples for Sanger sequencing Samples for Illumina sequencing 08:16 Serial endpoint dilutions cultures 16 48 None 08:16 12:12 14:10 L1-MOLA Cultures in triplicates 384 for all photoperiods and all experimental conditions 16 photoperiod 14:10 LAT, EXP, STA, DEC for all experimental conditions F/2-ESAW F/2-ESAW 50%N F/2-ESAW 10%P Frontiers in Microbiology \| www.frontiersin.org 3 September 2016 \| Volume 7 \| Article 1414 TABLE 1 \| Summary of the different experimental conditions. Microalgae–Bacteria Interactions Lupette et al. L1-MOLA and F/2-ESAW media at LD 12:12 light cycle condition (Figure 1). Interestingly, maximal growth rate appears more important in F/2-ESAW medium than in F/2-ESAW 10%P at this LD 12:12 photoperiod (p-value < 0.05) (Figure 1). In the other photoperiods, no signiﬁcant diﬀerence was observed between the distinct F/2-ESAW media. We can also observe a decrease in O. tauri maximal growth rate in L1-MOLA compared to the one of cells cultivated in F/2-ESAW at LD 08:16 light cycle condition (certainly because of a longer exponential growth phase in L1-MOLA than in F/2-ESAW with minimal light) (p-value < 0.005) (Figure 1). After 20 days of growth, the highest O. tauri cell abundances were observed in L1 MOLA medium whatever the photoperiodic conditions (Figure 2). Whatever the culture medium or the photoperiodic condition, diﬀerent phases of O. tauri growth can be identiﬁed and are described in Figure 2. However, depending upon the diﬀerent experimental approaches used, these phases can exhibit various durations (Figure 2). Globally, the cell concentration reached in the diﬀerent culture conditions is the lowest in ESAW 10%P whatever the other experimental conditions (Figure 2). The relative abundance of total bacteria in O. tauri RCC4221 cultures was measured over 35 days in the 12 diﬀerent conditions (Figure 3). Identiﬁcation of Cultivable and Total Bacteria in O. tauri RCC4221 Standard and Limiting Nutrient Conditions HNA (High content DNA) and LNA (Low content DNA) bacteria (Gasol et al., 1999) were observed in the diﬀerent culture conditions (Figure 3). They were considered as dominant when log (HNA bacteria and LNA bacteria/O. tauri) ratios >1 (Figure 3). Overall, the lowest bacterial abundances were observed when the cultures were grown in L1 MOLA medium (Figure 3) and the highest proportions of O. tauri cells relatively to bacteria were observed during the exponential growth phase (approximately together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were puriﬁed using calibrated Ampure XP beads. Then the pooled and puriﬁed PCR products were used to prepare the Illumina DNA library. Sequencing was performed at MR DNA on a MiSeq following the manufacturer’s guidelines. Sequence data were processed using MR DNA analysis pipeline3 and analyzed using QIIME software (Quantitative Insights Into Microbial Ecology)4 (Caporaso et al., 2010). In summary, sequences were joined, depleted of barcodes then sequences <150 bp removed, and sequences with ambiguous base calls removed. A 97% cut-oﬀfor sequence identity was used for classiﬁcation into OTUs. The sequences obtained were dominated by O. tauri mitochondrial sequences (>85%) and the analysis was conducted after eliminating these sequences, leaving 97362 bacterial sequences of interest spread among the 16 diﬀerent conditions (roughly 6085 bacterial sequences per condition). Sequences were submitted to GenBank with the project reference (BioProject ID) PRJNA328274. Alignments and Phylogenetic Reconstructions Among the 97362 total sequences obtained, 88179 (90.6%) were assigned to Marinobacter spp., clustered at 97% identity threshold inside six distinct OTUs after eliminating sequencing errors (instead of 799 initially obtained comprising 617 OTUs with one sequence and 176 OTUs with less than 200 sequences). The majority of Marinobacter sequences (95.3%) grouped inside one unique OTU (84817 sequences in OTU_7771). Among the remaining sequences, 6% (5821 sequences) were assigned to the Hyphomonas genus, and clustered in two main OTUs. The majority of Hyphomonas sequences (96.4%) grouped inside one unique OTU (5609 sequences in OTU_6113). The alignment of OTU sequences with annotated NCBI 16S rDNA sequences was performed using Clustal W (Thompson et al., 1994) implemented in MEGA 6.1 software (Tamura et al., 2013). The sequence alignment was then adjusted manually. The best evolutionary model was chosen using MEGA 6.1 software and resulted in Kimura two parameters with a Gamma correction. Two phylogenetic trees were constructed following this model by Neighbor-Joining (NJ) and Maximum Likelihood (ML) approaches using 1000 bootstrap replicates. Gram positive bacteria (Staphylococcus) and Gram negative δ-proteobacteria (Geobacter) were used as outgroups. Given that the two topologies obtained from the two phylogenetical methods were highly similar, only the NJ phylogenetic tree was shown with the bootstraps values resulting from each method. FIGURE 1 \| O. tauri RCC4221 growth rates comparison in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete media), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorus concentration divided per 10). Photoperiods: LD 12:12, LD 08:16, LD 14:10. ∗p < 0.05 and ∗∗p < 0.005. Abundance and Dynamics of Bacteria in the O. tauri Phycosphere FIGURE 1 \| O. tauri RCC4221 growth rates comparison in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete media), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorus concentration divided per 10). Photoperiods: LD 12:12, LD 08:16, LD 14:10. ∗p < 0.05 and ∗∗p < 0.005. FIGURE 1 \| O. tauri RCC4221 growth rates comparison in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete media), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorus concentration divided per 10). Photoperiods: LD 12:12, LD 08:16, LD 14:10. ∗p < 0.05 and ∗∗p < 0.005. Globally, the highest O. tauri RCC4221 maximal growth rates in exponential phase were observed for cells cultivated in 3www.mrdnalab.com 4http://qiime.org/ September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org 4 Microalgae–Bacteria Interactions Lupette et al. FIGURE 2 \| O. tauri RCC4221 growth for 35 days in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete medium), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. Growth Phases: L, latency; E, exponential; S, stationary, and D, decline. FIGURE 3 \| Ostreococcus tauri RCC4221-bacterial growth ratios for 35 days in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. (A) log (HNA bacteria/O. tauri) growth ratio. (B) log (LNA bacteria/O.tauri) growth ratio. FIGURE 2 \| O. tauri RCC4221 growth for 35 days in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete medium), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. Growth Phases: L, latency; E, exponential; S, stationary, and D, decline. FIGURE 2 \| O. tauri RCC4221 growth for 35 days in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete medium), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. Growth Phases: L, latency; E, exponential; S, stationary, and D, decline. FIGURE 2 \| O. tauri RCC4221 growth for 35 days in four different media and three different photoperiods. Abundance and Dynamics of Bacteria in the O. tauri Phycosphere Media: L1-MOLA, F/2-ESAW (complete medium), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. Growth Phases: L, latency; E, exponential; S, stationary, and D, decline. FIGURE 3 \| Ostreococcus tauri RCC4221-bacterial growth ratios for 35 days in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. (A) log (HNA bacteria/O. tauri) growth ratio. (B) log (LNA bacteria/O.tauri) growth ratio. FIGURE 3 \| Ostreococcus tauri RCC4221-bacterial growth ratios for 35 days in four different media and three different photoperiods. Media: L1-MOLA, F/2-ESAW (complete), F/2-ESAW 50%N (nitrogen concentration divided per 2) and F/2-ESAW 10%P (phosphorous concentration divided per 10). Photoperiods: LD 08:16, LD 12:12, LD 14:10. (A) log (HNA bacteria/O. tauri) growth ratio. (B) log (LNA bacteria/O.tauri) growth ratio. 25 days) phases (Figures 3A,B). Since some bacteria may be adhering to algal cells, these ﬁgures probably underestimate the actual number of bacteria present. The latency and decline phases are advantageous for the development of LNA bacteria while after 10 days growth) (Figures 2 and 3). However, whatever the culture conditions, the highest densities of HNA and LNA bacteria were present in O. tauri RCC4221 culture during latency (from 0 to 8 days approximately) and decline (starting from 25 days) phases (Figures 3A,B). Since some bacteria may be adhering to algal cells, these ﬁgures probably underestimate the actual number of bacteria present. The latency and decline phases are advantageous for the development of LNA bacteria while September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org 5 Microalgae–Bacteria Interactions Lupette et al. to a group comprising free-living M. salsuginis, M. algicola, M. MOLA20 and diverse Marinobacter spp. found associated with diﬀerent microalgae (BPNJ = 90, BPML = 85, Figure 4). More precisely, they exhibit a close relationship to the M. algicola clade (BPNJ = 65, BPML = 56, Figure 4). In addition to the OTU 7771 (84817 sequences) found as 99% identical to the previously identiﬁed culturable Marinobacter strain (Marinobacter sp. Abundance and Dynamics of Bacteria in the O. tauri Phycosphere strain 1, Figure 4), several variants were found from the total diversity analysis (the following percentages of identity are calculated from the partial sequences used in the ﬁnal alignment): OTU 242 (704 sequences), that is 96.7% identical to OTU 7771, OTU 5245 (512 sequences), 96% identical to OTU 7771, OTU 1418 (239 sequences), 96.7% identical to OTU 7771, OTU 11824 (211 sequences), 96.7% identical to OTU 7771 (Figure 4). All of the Hyphomonas sequences obtained from Illumina sequencing were closely related to H. johnsonii sequences, with strong bootstrap support (BPNJ = 87) in the Hyphomonas clade (BPNJ = 99, BPML = 98) of α-proteobacteria (Figure 4). the exponential phase is more suitable for the development of HNA bacteria (except in F2-ESAW medium). A majority of LNA bacteria [with log (LNA bacteria/O. tauri) ratio around 2] are simultaneously present with microalgal cells in exponential phase in F2-ESAW medium (Figure 3B). The proportion of LNA bacteria increases when cultivated in F/2-ESAW 50%N and F/2- ESAW 10%P media compared to L1-MOLA medium and are predominant in F/2-ESAW medium whatever the photoperiod condition (Figure 3B). In contrast, the development of HNA bacteria was higher in nitrogen-limited medium (except in LD 08:16 photoperiodic condition) and in phosphorus-limited medium in a period corresponding to the stationary phase (from 15 to 25 days approximately) in all tested photoperiodic conditions (Figure 3A) while LNA bacteria seem predominant starting from 25 days (Figure 3B). Diversity of Culturable versus Total Bacteria during O. tauri RCC4221 Growth Marinobacter was by far the most frequently found genus and Marinobacter sequences were present at all stages and conditions during the growth of O. tauri RCC4221 (Figure 5). In addition to γ-proteobacteria, sequences from α-proteobacteria (mainly Hyphomonas species) were recovered (Figure 5) at a low percentage in the culture-dependent approach (0.3%), but at a higher frequency by Illumina sequencing (6%). Whatever the culture conditions, the Marinobacter genus always predominated (90.6%, Table 2). The highest proportion of Hyphomonas sequences was observed during exponential, stationary and decline phases, particularly in L1, F/2 and N-limited media conditions (Figure 5). g Whatever the culture conditions used in the culture-dependent approach, bacteria from the γ-proteobacteria group were largely dominant. From the 16 O. tauri independent lines that were endpoint diluted 27 times through serial subcultures, all the bacteria isolated on either solid Marine Agar or L1 media exhibited one unique 16S sequence (“Marinobacter sp. strain 1,” Figure 4), closely related to the Marinobacter genus (γ-proteobacteria, order Alteromonadales) (Table 2; Figure 4). The full-length 16S sequence showed 98% identity to the Marinobacter algicola and Marinobacter sp. DS1930-III sequences. Bacteria isolated on L1 solid medium during the 35 days of O. tauri RCC4221 growth in the diﬀerent culture conditions also exhibited the same full-length 16S sequence with 98% identity to the Marinobacter algicola species in 95.3% of the bacteria identiﬁed (Table 2). Other γ-, β-, and α-culturable proteobacteria were also detected in the diﬀerent cultures, albeit found much less frequently. One sequence belonging to Pseudomonas (γ-proteobacteria) was found with O. tauri cultivated in L1-MOLA medium after 15 days-growth under LD 12:12 photoperiod (Figure 4); one sequence belonging to Hyphomonas (α-proteobacteria) was found with O. tauri cultivated in L1-MOLA medium at the beginning of the growth under LD 08:16 photoperiod (Figure 4); one sequence belonging to Massilia genus (β-proteobacteria) was found with O. tauri cultivated in F/2-ESAW medium at the beginning of the growth under LD 14:10 photoperiod (Figure 4); and one Gram-positive bacteria sequence, from the Staphylococcus genus was found, possibly a contaminant from handling of cultures. Frontiers in Microbiology \| www.frontiersin.org Marinobacter (γ-Proteobacteria) Was the Most Prevalent Genus Across a Wide Range of Culture Conditions Marinobacter (γ-Proteobacteria) Was the Most Prevalent Genus Across a Wide Range of Culture Conditions Globally, the analysis of diversity using Illumina sequencing on total community was entirely congruent with diversity inferred with a culture-dependent approach. Strikingly, all 16 O. tauri RCC4221 cultures regularly submitted to serial endpoint dilutions to one single cell per ml contained only sequences from the Marinobacter genus. These observations strongly suggest that these bacteria are required for O. tauri RCC42221 growth in our culture conditions but we cannot exclude the opportunistic presence of Marinobacter, given the enriched cultures media used and the possible use by bacteria of exudates secreted by microalgae, as shown in other bacteria– phytoplankton interactions (Fouilland et al., 2013). Interestingly, in our study, several distinct Marinobacter strains were present simultaneously and systematically throughout the growth of the O. tauri RCC4221 strain, whatever the conditions tested, as shown by high-throughput Illumina sequencing of PCR- ampliﬁed 16S ribosomal gene sequences directly from the algal culture, with no separate bacterial culture step, revealing an unsuspected genetic diversity. This suggests that in general The total bacterial diversity in O. tauri RCC4221 cultures was investigated by Illumina sequencing at distinct growth phases (latency, exponential, stationary, and decline phases) during the 35 days’ growth in 14:10 light-dark cycles. Globally, the total diversity observed was entirely congruent with our results using a culture-dependent approach showing a large majority of Marinobacter spp. (Table 2). More precisely, in spite of the relatively small 16S sequence length available, the total diversity analysis provided evidence for diverse Marinobacter strains (Figure 4). These partial sequences are clearly closely related September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org 6 Microalgae–Bacteria Interactions Lupette et al. r-Joining phylogenetic tree obtained from partial 16S DNA alignment of bacterial sequences obtained from this study and from reported on the nodes of the tree are the bootstrap values (BP) obtained from NJ and ML methods (BPNJ/BPML). Only those superior to 50 are x shows Marinobacter sequences afﬁliated to M. algicola species from our phylogenetic reconstructions. The name “Marinobacter sp. strain 1” equencing of Marinobacter obtained from the culture-based approach. Sequences obtained in this study were submitted to GenBank with the Project ID) PRJNA328274. Marinobacter (γ-Proteobacteria) Was the Most Prevalent Genus Across a Wide Range of Culture Conditions TABLE 2 \| Numbers of 16S ribosomal RNA sequences analyzed. Approach Experimental conditions Number of bacterial sequences 16S rDNA gene Percentage of Marinobacter sequences Percentage of Hyphomonas sequences Culture-dependant (solid medium) Serial endpoint dilutions cultures 48 Complete (1455 bp) 100% – L1-MOLA/F/2- ESAW/F/2-ESAW 50%N/F/2-ESAW 10%P 384 Complete (1455 bp) 95.3% 0.3% Culture-independent (Illumina) L1-MOLA/F/2- ESAW/F/2-ESAW 50%N/F/2-ESAW 10%P 97362 Partial (270 bp) 90.6% 6% Bacterial colonies were obtained after growth in L1 solid medium. Except for the serial dilution extinction experiment, bacteria were isolated from the 12 different experimental conditions (four different media L1-MOLA, F/2-ESAW, F/2-ESAW 50%N, and F/2-ESAW 10%P- and three different photoperiodic conditions). Bacterial sequences were analyzed after either using Sanger sequencing (culture-dependant approaches) or partial (Illumina) length 16S DNA sequencing (independent of bacterial culture). TABLE 2 \| Numbers of 16S ribosomal RNA sequences analyzed. LE 2 \| Numbers of 16S ribosomal RNA sequences analyzed. Bacterial colonies were obtained after growth in L1 solid medium. Except for the serial dilution extinction experiment, bacteria were isolated from the 12 different experimental conditions (four different media L1-MOLA, F/2-ESAW, F/2-ESAW 50%N, and F/2-ESAW 10%P- and three different photoperiodic conditions). Bacterial sequences were analyzed after either using Sanger sequencing (culture-dependant approaches) or partial (Illumina) length 16S DNA sequencing (independent of bacterial culture). FIGURE 5 \| Proportion of Marinobacter and Hyphomonas sequences obtained from total bacterial diversity (Illumina) analysis in the four experimental conditions and different O. tauri growth phases. Bacterial sequences were obtained from O. tauri RCC4221 cultures in the four different media, noted as OtL1, Otf2, OtN (F/2-ESAW 50%N) and OtP (F/2-ESAW 10%P), and in the four main growth phases (LAT, latency; EXP, exponential; STA, stationary; and DEC, decline). In each culture condition, an increase in the proportion of Hyphomonas sequences is observed mainly in the exponential, stationary and decline growth phases. FIGURE 5 \| Proportion of Marinobacter and Hyphomonas sequences obtained from total bacterial diversity (Illumina) analysis in the four experimental conditions and different O. tauri growth phases. Bacterial sequences were obtained from O. tauri RCC4221 cultures in the four different media, noted as OtL1, Otf2, OtN (F/2-ESAW 50%N) and OtP (F/2-ESAW 10%P), and in the four main growth phases (LAT, latency; EXP, exponential; STA, stationary; and DEC, decline). In each culture condition, an increase in the proportion of Hyphomonas sequences is observed mainly in the exponential, stationary and decline growth phases. Marinobacter (γ-Proteobacteria) Was the Most Prevalent Genus Across a Wide Range of Culture Conditions r-Joining phylogenetic tree obtained from partial 16S DNA alignment of bacter reported on the nodes of the tree are the bootstrap values (BP) obtained from NJ and M x shows Marinobacter sequences afﬁliated to M. algicola species from our phylogeneti equencing of Marinobacter obtained from the culture-based approach. Sequences obt Project ID) PRJNA328274. FIGURE 4 \| Neighbor-Joining phylogenetic tree obtained from partial 16S DNA alignment of bacterial sequences obtained from this study and from GenBank. Numbers reported on the nodes of the tree are the bootstrap values (BP) obtained from NJ and ML methods (BPNJ/BPML). Only those superior to 50 are reported. The gray box shows Marinobacter sequences afﬁliated to M. algicola species from our phylogenetic reconstructions. The name “Marinobacter sp. strain 1” refers to the Sanger-sequencing of Marinobacter obtained from the culture-based approach. Sequences obtained in this study were submitted to GenBank with the project reference (BioProject ID) PRJNA328274. of bacterial sequences obtained from this study and from September 2016 \| Volume 7 \| Article 1414 7 Frontiers in Microbiology \| www.frontiersin.org Microalgae–Bacteria Interactions Lupette et al. TABLE 2 \| Numbers of 16S ribosomal RNA sequences analyzed. Approach Experimental conditions Number of bacterial sequences 16S rDNA gene Percentage of Marinobacter sequences Percentage of Hyphomonas sequences Culture-dependant (solid medium) Serial endpoint dilutions cultures 48 Complete (1455 bp) 100% – L1-MOLA/F/2- ESAW/F/2-ESAW 50%N/F/2-ESAW 10%P 384 Complete (1455 bp) 95.3% 0.3% Culture-independent (Illumina) L1-MOLA/F/2- ESAW/F/2-ESAW 50%N/F/2-ESAW 10%P 97362 Partial (270 bp) 90.6% 6% Bacterial colonies were obtained after growth in L1 solid medium. Except for the serial dilution extinction experiment, bacteria were isolated from the 12 different experimental conditions (four different media L1-MOLA, F/2-ESAW, F/2-ESAW 50%N, and F/2-ESAW 10%P- and three different photoperiodic conditions). Bacterial sequences were analyzed after either using Sanger sequencing (culture-dependant approaches) or partial (Illumina) length 16S DNA sequencing (independent of bacterial culture). FIGURE 5 \| Proportion of Marinobacter and Hyphomonas sequences obtained from total bacterial diversity (Illumina) analysis in the four experimental conditions and different O. tauri growth phases. Bacterial sequences were obtained from O. tauri RCC4221 cultures in the four different media, noted as OtL1, Otf2, OtN (F/2-ESAW 50%N) and OtP (F/2-ESAW 10%P), and in the four main growth phases (LAT, latency; EXP, exponential; STA, stationary; and DEC, decline). In each culture condition, an increase in the proportion of Hyphomonas sequences is observed mainly in the exponential, stationary and decline growth phases. Frontiers in Microbiology \| www.frontiersin.org Dynamics of Bacterial Growth Depends upon the O. tauri RCC4221 Growth Medium In the light of results from high throughput Illumina sequencing, the proportion of γ-proteobacteria and particularly of Marinobacter genus sequences is the largest and encompasses almost all of the bacterial diversity. These diﬀerent Marinobacter strains were subsequently found by ﬂow cytometry as HNA (high apparent nucleic acid content) or LNA (low apparent nucleic acid content) during algal growth. Classically, HNA bacteria are considered as the most active members of a given community while LNA are regarded as inactive, dead or dormant cells (Gasol et al., 1999; Lebaron et al., 2001, 2002). In addition, in numerous studies, both HNA and LNA are considered to be members of diﬀerent (Zubkov et al., 2001; Fuchs et al., 2005; Mary et al., 2006) or identical (Flaten et al., 2003; Servais et al., 2003) phylotypes (Andrade et al., 2007). Our dominant bacterial population (i.e., Marinobacter spp.) was found in both HNA and LNA populations but only further experiments using accurate cell sorting and precise identiﬁcation of these sorted bacteria would conﬁrm that HNA and LNA have identical ribotypes (David Pecqueur, personal communication). The highest O. tauri cell abundance relatively to bacteria was observed during exponential phases where maximal O. tauri growth rates were observed. In contrast, the highest bacterial abundance was observed in other phases of the O. tauri growth. One interesting fact which retained our attention is the relatively low abundance of O. tauri all along the duration of experiments when cultivated in depleted media such as in F/2-ESAW 10%P medium, demonstrating the importance of phosphorous. By the way, HNA bacteria seem particularly abundant in this particular condition. Globally, O. tauri cells growth is higher in L1-MOLA medium than in F/2-ESAW one, and appears highly favored compared to both HNA and LNA bacteria and particularly LNA bacteria growths in this medium. One possible explanation is that additional substances present in natural seawater might promote algal growth. Concerning the modalities of O. tauri growth, our results tend to show the importance of phosphorous, while nitrogen limitation doesn’t signiﬁcantly reduce microalgal growth. However, excepted in LD 08:16 photoperiodic condition, there is no clear diﬀerence in O. tauri RCC4221 maximal growth rates in exponential phase for cells cultivated in L1-MOLA and The presence of Marinobacter species in laboratory cultures raises questions about the possibility of a laboratory artifact. Marinobacter (γ-Proteobacteria) Was the Most Prevalent Genus Across a Wide Range of Culture Conditions We also included two sequences of Marinobacter found associated with diﬀerent diatoms (one with Pseudo-nitzschia, the other with Thalassiosira) (Grossart et al., 2004; Amin et al., 2015). Interestingly, the two sequences emerged in diﬀerent clades, the ﬁrst one with M. algicola and our sequences, the second one with M. adhaerens (Figure 4). this study and Marinobacter 16S sequences associated with very taxonomically distinct microalgae in the literature (eight OTUs), 14 out of these 15 OTUs cluster all together within the same clade. Members of the genus Marinobacter have been detected in numerous dinoﬂagellate and coccolithophorid cultures (Alavi et al., 2001; Hold et al., 2001; Amin et al., 2009) which all emerged in M. algicola clade in our study (Figure 4). In addition, a recent analysis of the ectobiotic bacterial diversity associated with the euglenoid Eutreptiella sp. revealed the occurrence of abundant γ-proteobacteria, speciﬁcally Marinobacter (Kuo and Lin, 2013). All of these Marinobacter strains were closely related to M. adhaerens (Kuo and Lin, 2013). We also included two sequences of Marinobacter found associated with diﬀerent diatoms (one with Pseudo-nitzschia, the other with Thalassiosira) (Grossart et al., 2004; Amin et al., 2015). Interestingly, the two sequences emerged in diﬀerent clades, the ﬁrst one with M. algicola and our sequences, the second one with M. adhaerens (Figure 4). Flavobacteria 16S rDNA sequences were not found from the solid culture medium approach and quasi-absent (0.25% of OTUs) from the Illumina sequencing analysis. Together with α- and γ-proteobacteria (Morris et al., 2002; Sunagawa et al., 2015), they are the bacteria the most commonly detected in microalgal cultures and phytoplanktonic blooms (Buchan et al., 2014). Almost all culturable and visually distinct bacteria isolated from Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala microalgal cultures maintained for several years in laboratory belong to the Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae families (Schwenk et al., 2014), here again found at very low frequency in O. tauri RCC 4221 cultures (0.23, 0.34, and 0.04% of OTUs, respectively). Dynamics of Bacterial Growth Depends upon the O. tauri RCC4221 Growth Medium Recent ﬁndings argue against this, because Marinobacter species were also recently found in coccolithophorids and dinoﬂagellates cultures (Green et al., 2015), although at a much lower proportion in diatom cultures cultivated in the same media (Amin et al., 2009). Rather, we have selected the bacterial species that best supports rapid host growth from a diversity of bacteria observed in the O. tauri culture originally isolated (Abby et al., 2014). If Marinobacter tends to show a speciﬁc adaptation to coccolithophores and dinoﬂagellates (Green et al., 2010), it appears that, from our study, it is not limited to these groups and can also be extended to Chlorophyta, another distant lineage. Marinobacter (γ-Proteobacteria) Was the Most Prevalent Genus Across a Wide Range of Culture Conditions there may be no strict association between one OTU and microalgae, but several closely related OTUs and microalgae, together with Marinobacter spp., like those found in association with dinoﬂagellates, coccolithophorids, and one diatom (Green et al., 2004, 2015; Amin et al., 2009, 2015). To our knowledge, this is the ﬁrst report of an association between Marinobacter strains and a green marine microalga from the class Mamiellophyceae. nitrate reduction) (De La Haba et al., 2011). In our study, the Marinobacter sequences obtained from solid culture medium were closely related to sequences from Marinobacter sp. MOLA20 (AM990796), a bacterial strain isolated from the Gulf of Lion, and to Marinobacter sp. DS1930-III, a bacterial strain which seems to occur as a microbial symbiotic community together with Halomonas sp. and Pelagibaca sp., and in monocultures of the chlorophyte Dunaliella salina (C. Baggesen, unpublished). Several studies have recently described Marinobacter strains related to M. algicola species in association with microalgal cultures maintained in laboratory and belonging to a wide range of species diversity across the eukaryotic tree of life (Amin et al., 2009; Kuo and Lin, 2013; Le Chevanton et al., 2013; Green et al., 2015). Indeed, among the seven OTUs identiﬁed from Marinobacter is the most diversiﬁed genus in the Alteromonadaceae (Gauthier et al., 1992; De La Haba et al., 2011) and is commonly found in the oceans worldwide. These bacteria are bacilli with gram-negative walls, halotolerant, with an aerobic metabolism (Gauthier et al., 1992). They use diﬀerent hydrocarbon sources and are able to perform direct reduction from nitrate to ammonium (known as dissimilatory September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org 8 Microalgae–Bacteria Interactions Lupette et al. this study and Marinobacter 16S sequences associated with very taxonomically distinct microalgae in the literature (eight OTUs), 14 out of these 15 OTUs cluster all together within the same clade. Members of the genus Marinobacter have been detected in numerous dinoﬂagellate and coccolithophorid cultures (Alavi et al., 2001; Hold et al., 2001; Amin et al., 2009) which all emerged in M. algicola clade in our study (Figure 4). In addition, a recent analysis of the ectobiotic bacterial diversity associated with the euglenoid Eutreptiella sp. revealed the occurrence of abundant γ-proteobacteria, speciﬁcally Marinobacter (Kuo and Lin, 2013). All of these Marinobacter strains were closely related to M. adhaerens (Kuo and Lin, 2013). Possible Roles of Bacteria in Algal Cultures The bacteria isolated in this study have probably survived over several years in continuous algal cultures, where only those bacteria capable of growing under nutrient-rich conditions will survive after successive transfers. Some bacterial groups are able to grow rapidly, out-competing other bacteria in enrichment cultures, as seen in Marinobacter species (Handley et al., 2010), when there may be a surplus of organic carbon, or under aerobic to anaerobic conditions (Edwards et al., 2003; Handley et al., 2013). The lifestyle types exhibited by Marinobacter strains such as M. aquaeolei have been described as opportunistic, being able to utilize urea and phosphonate as alternative of N and P sources, or generalist like Shewanella, Pseudomonas, Vibrio, and Roseobacter (Singer et al., 2011). Phytoplankton needs iron in large amounts to support the photosynthetic ﬁxation of carbon. Bacteria from Marinobacter genus can produce siderophores (Vraspir and Butler, 2009) and contribute to iron chelation and internalization (Martinez et al., 2000, 2003). Siderophore (like vibrioferrin) production may be a useful chemotaxonomic marker for algal-associated Marinobacter species (Amin et al., 2009). Our phylogenetic reconstruction (Figure 4) clearly shows the close relationship between our Marinobacter strains and the Marinobacter sp. DG1194 (isolated from an Alexandrium culture), DG1594 (isolated from an Emiliana culture) and DG1597 (isolated from a Coccolithus culture), all shown to produce and uptake vibrioferrin (Amin et al., 2009) (BPNJ = 65, BPML = 56, Figure 4). In their study, the authors suggested that bacteria may promote algal assimilation of iron (Amin et al., 2009). Bacteria of the Marinobacter genus also seem to stimulate the growth of the dinoﬂagellate Gymnodinium catenatum (Bolch et al., 2011), and the accumulation of lipids in the commercially important microalgae Nannochloropsis (David Green, personal communication). Although O. tauri can be cultured easily in the laboratory, the complete eradication of bacteria is diﬃcult and seems to prevent microalgal growth (unpublished results). This persistence of bacteria in O. tauri cultures raises questions regarding the nature of the potential interactions between these microbial populations. Further experiments are needed to investigate the physiological impacts of these newly identiﬁed Marinobacter strains in our O. tauri RCC4221 system. g In serial endpoint dilution subcultures, only the bacteria associated with the single algal cells used to establish the culture have the opportunity to be present as the culture is grown and transferred. We showed that Marinobacter is present from the beginning of the culture growth. Other γ-, β-, and α-Proteobacteria Found in O. tauri Cultures Contrary to expectations, we did not detect any Roseobacter species although this is the most frequently observed genus in data from surface communities (Morris et al., 2002). However, from both the culture-dependent and Illumina approaches, sequences from the α-proteobacteria Hyphomonas sp. were obtained. These sequences are closely related to H. johnsonii and to a sequence from a bacterial strain isolated locally (Hyphomonas MOLA116, AM990890). However, we exclude the hypothesis that this bacterial strain was introduced as a contaminant in L1 MOLA medium since this medium was autoclaved and no trace of bacteria was detectable by ﬂow cytometry before use. Interestingly, in a recent study, not only Marinobacter, but also Hyphomonas bacteria were isolated from diﬀerent isolates of the coastal diatom Pseudo-nitzschia multiseries (Amin et al., 2015) and showed the same phylogenetic position that our sequences in our reconstructions. One sequence belonging to Massilia genus (β-proteobacteria), particularly rare in marine environments, was also obtained. Gram-positive bacteria were also frequently found in marine microalgal cultures in laboratory, but were probably the result of contamination from handling rather than from the marine environment (Nicolas et al., 2004). September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org 9 Microalgae–Bacteria Interactions Lupette et al. was limited following serial subcultures in medium lacking B1. To our knowledge, no such experiment has been conducted to explore the eﬀect of vitamin B7 limitation on O. tauri growth. Bacteria secreting vitamins can stimulate microalgal growth as vitamin-dependent axenic microalgae cannot survive in nutrient-limited medium (Haines and Guillard, 1974; Kurata, 1986; Croft et al., 2005; Grant et al., 2014). Eighteen clones exhibiting a Marinobacter 16S sequence closely related to M. adhaerens were obtained from ectobiotic bacteria living with the euglenoid Eutrepsiella sp. (Kuo and Lin, 2013). These bacteria provided vitamin B12 and other growth-enhancing factors for the euglenoid (Kuo and Lin, 2013). However, the growth rate of the diatom Pseudo-nitzschia multiseries was unaﬀected when co-cultured with Marinobacter in speciﬁc experiments where the diatom was previously treated with antibiotics (Amin et al., 2015). F/2-ESAW media. We also observed that the bacterial abundance proﬁles varied with the microalgal culture phases, a higher abundance of bacteria being found in the LAT and DEC phases of O. tauri growth. Some bacteria, in particular γ-proteobacteria very often live epiphytically on detritic organic particles (DeLong et al., 1993), perhaps explaining the abundance of bacteria when microalgal growth declines. Possible Roles of Bacteria in Algal Cultures It is almost certain that these bacteria beneﬁt from their coexistence with microalga. Macronutrient concentrations found in algal cultivation media such as F/2-ESAW are about 100-fold higher than those found in coastal marine environments, such as the Bay of Fundy (Guillard and Ryther, 1962; Martin et al., 2001), also favoring bacterial development. Inversely, O. tauri RCC4221 cells probably beneﬁt from the presence of bacteria. Indeed, completely axenic cultures of O. tauri RCC4221 have not so far been possible to maintain, despite the use of antibiotics treatment protocols. Abby et al. (2014) focused on algal cultures maintained without antibiotics for several years (Abby et al., 2014), and found a total of 1425 Marinobacter sequences in an O. tauri RCC4221 culture among over 1400000 sequences (thus, representing 0.10%). As in the present study, these sequences were all closely related to M. algicola. It is not surprising that Abby et al. (2014) found a greater diversity of diﬀerent bacterial species present, because cultures were then routinely maintained by subculturing using a larger volume (usually 50 µl) of culture, permitting transfer of a population of bacteria, rather than by dilution through one-cell endpoint dilutions. Over half of all microalgal species require an exogenous supply of vitamin B12 (cobalamin). Among the four B12-dependent enzymes, only one, the methionine synthase, is present in O. tauri genome, and O. tauri was shown to be auxotrophic for vitamin B12 (Helliwell et al., 2011). In addition, a recent study conﬁrmed Ostreococcus to be a thiamine (vitamin B1) auxotroph in laboratory experiments using culture media also containing vitamin B12 (cobalamin) and B7 biotin (Paerl et al., 2015). In this latter study, growth of Ostreococcus Frontiers in Microbiology \| www.frontiersin.org FUNDING JL was supported by Sorbonne Universités, SATS-SU, “ANR- 11-IDEX-0004-02.” This work was funded by the PHYTNESS project “ANR-13-JSV6-0005.” JL was supported by Sorbonne Universités, SATS-SU, “ANR- 11-IDEX-0004-02.” This work was funded by the PHYTNESS project “ANR-13-JSV6-0005.” Bacteria Associated with Microalgae in Natural Environments Buchan et al. (2014) monitored the succession of bacterial communities associated with diatoms, dinoﬂagellates, nanoﬂagellates, and picophytoplankton blooms in a large- scale study (Buchan et al., 2014). The main bloom-associated bacterial groups were, in order of abundance, α-proteobacteria, Flavobacteriia, and γ-proteobacteria. In the earliest stages of a bloom, phytoplankton release amino acids, organic acids, carbohydrates, and sugar alcohols which are hypothesized to function as chemoattractants for beneﬁcial bacteria, including bacteria that produce phytoplankton growth-promoting compounds, such as vitamins. At the height of the bloom, September 2016 \| Volume 7 \| Article 1414 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 10 Microalgae–Bacteria Interactions Lupette et al. in response to nutrient-limiting conditions, the release of small molecules by living phytoplankton increases and further stimulates heterotrophic bacterial activity (Buchan et al., 2014). Then, during the waning stage of the bloom, phytoplankton release higher molecular weight macromolecules (polysaccharides, proteins, nucleic acids, lipids, material resulting from cell lysis) (Buchan et al., 2014). In our experiments, we observed a higher proportion of bacteria in the latency (LAT) and decline (DEC) phases of O. tauri growth. Some bacteria, in particular γ-proteobacteria very often live as epiphytes on detritic organic particles (DeLong et al., 1993). This phenomenon could explain the abundance of bacteria particularly when the microalgal growth declines. We also observed a higher bacterial diversity (including mainly Marinobacter, but also Hyphomonas to a lesser extent) during the exponential (EXP), stationary (STA) and decline (DEC) phases of the O. tauri RCC4221 growth, which is congruent with the stimulation of heterotrophic bacterial activity observed by Buchan et al. (2014). Lastly, we have to keep in mind that the type of a given algal-bacterial interaction can also change depending on environmental conditions. In laboratory conditions, the interaction between Scenedesmus obliquus and a non-identiﬁed bacterial community evolves from a mutualistic to a competitive interaction in phosphorus-limited conditions (Danger et al., 2007). A mutualistic phase and a pathogenic phase (where bacteria probably killed dinoﬂagellate cells), were also observed in co-cultures of Dinoroseobacter shibae and Prorocentrum minimum (Wang et al., 2014). These observations highlight the need of further experimentation in co-cultures to assess the complexity of bacterial–algal interactions. co-cultures with vitamins or nutrient-limiting conditions should provide additional insights in these green algal-bacterial interactions. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01414 TABLE S1 \| Composition of L1-MOLA (Guillard and Hargraves, 1993) and F/2-ESAW (Harrison et al., 1980) media. (nd, not determined). TABLE S1 \| Composition of L1-MOLA (Guillard and Hargraves, 1993) and F/2-ESAW (Harrison et al., 1980) media. (nd, not determined). CONCLUSION We show that bacterial communities associated with O. tauri RCC4221 laboratory cultures are almost exclusively limited to γ-proteobacterial strains from the Marinobacter genus, regardless of the growth phases of O. tauri RCC4221 or cultures conditions tested (photoperiod and nutrient depletion). Interestingly, Marinobacter bacteria identiﬁed in O. tauri RCC4221 cultures by both culture-dependant and high-throughput sequencing are all closely related to the M. algicola clade, regrouping strains known to produce siderophores. Further experiments combining AUTHOR CONTRIBUTIONS JL performed experimental approaches, ﬁgures, and participated to the writing of the manuscript. RL analyzed high throughput Illumina raw data and participated to the writing of the manuscript. MK performed endpoint dilution experiments and participated to the writing of the manuscript. NG, HM, and GP helped to design the experiments and actively participated to the writing of the manuscript. SS-F conceived the study, participated to the experiments, performed phylogenetic analyses, and wrote the manuscript. ACKNOWLEDGMENTS We would like to thank David Pecqueur and Christophe Salmeron from the Cytometry platform (Observatoire Océanologique, Banyuls-sur-mer) and the Genomics of Phytoplankton group for stimulating discussions and technical help. REFERENCES Amin, S. A., Hmelo, L. R., van Tol, H. M., Durham, B. P., Carlson, L. T., Heal, K. R., et al. (2015). Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria. Nature 522, 98–101. doi: 10.1038/nature 14488 Abby, S. S., Touchon, M., DeJode, A., Grimsley, N., and Piganeau, G. (2014). Bacteria in Ostreococcus tauri cultures – friends, foes or hitchhikers? Front. 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https://openalex.org/W2276986596	https://www.revistas.ufg.br/VISUAL/article/download/34485/18189	Portuguese	null	O método cooperação dos Amigos do Cinema	Visualidades	2,015	cc-by	7,918	O método cooperação dos Amigos do Cinema Lara Lima Satler Alice Fátima Martins Lara Lima Satler Alice Fátima Martins Palavras-chave: Realizar filmes, coletivos, cooperação VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Resumo Palavras-chave: Realizar filmes, coletivos, cooperação Neste texto, pretendemos discutir como o Sistema CooperAÇÃO - Amigos do Cinema se articula em coletivo e simultaneamente realiza suas produções audiovisuais. O Sistema CooperAÇÃO é formado por um grupo de amigos que há mais de quatorze anos já produziu um total de quinze filmes no interior do Brasil, no Estado de Goiás. Os dados aqui apresentados foram coletados por meio de observação direta e entrevistas. Objetivamos compreender como o grupo se articula em coletivo e como realiza seus filmes. Como resultados, pretendemos contribuir com discussões que problematizem as relações entre o realizar filmes em coletivos. 203 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Keywords: Filmmaking, collectives, cooperation The cooperation method of the Amigos do Cinema Lara Lima Satler Alice Fátima Martins Lara Lima Satler Alice Fátima Martins VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Abstract In this paper, we intend to discuss how the Sistema CooperAÇÃO – Amigos do Cinema is articulated collectively and simultaneously performs its movies productions. The Sistema CooperAÇÃO is formed by a group of friends for over fourteen years has produced a total of fifteen films in the interior of Brazil, in the State of Goiás. The data presented were collected through direct observation and interviews. We aim to understand how the group is articulated collectively and how make their movies. As a result, we intend to contribute with discussions that think about the relationship between making films in collectives. 204 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 1. Apresentação Neste texto, pretendemos discutir como o Sistema CooperA- ÇÃO - Amigos do Cinema se articula em um coletivo artístico e desse modo realiza suas produções audiovisuais. O Sistema CooperAÇÃO se apresenta como “um grupo de amigos que produzem [de modo] independente. Técnicos e atores unidos com um só objetivo, estudar cinema” (encarte dos DVDs). i j O grupo já lançou quinze produções, entre filmes de cur- ta, média e longas metragem1. Acompanhamos as filmagens da produção intitulada O Capitão do Mato (MARTINS MU- NIZ, 2013), entre fevereiro e março de 2013, em locações que incluíram Goiânia, Aparecida de Goiânia e Cedro, um povoa- do próximo a Trindade, todos no estado de Goiás. Assim, in- teressa-nos compreender, nesta reflexão: como a organização do grupo se articula em coletivo? Como o grupo articula seu duplo objetivo de realizar filmes e fazê-los em cooperação? Para tanto, investigaremos as implicações que o fazer filmes em coletivo exercem na produção audiovisual deste grupo. Este texto é fruto de duas pesquisas em andamento, uma intitulada “Outros fazedores de cinema”, e a outra “Eu, a câmera e o outro: aprender a realizar audiovisual experimentando em coletivo”, ambas com seus projetos financiados pela Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG. Os dados aqui apresentados foram coletados por meio de observação direta e entrevistas. Objetivamos, com esta reflexão, compre- ender como o Sistema CooperAÇÃO - Amigos do Cinema se articula em coletivo e como realiza seus filmes em cooperação. Como resultados, pretendemos contribuir com discussões que problematizem as relações entre o realizar filmes em coletivos. 205 ara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Segundo Paim (2012, p.7), coletivos são 203-223, jul-dez 2014 de teorizá-lo como uma forma de produção e intervenção que promove questões sobre a natureza do trabalho criativo e como a história é contada, por quem e com quais finalidades. Esta obra, em que seus capítulos são assinados por diversos autores, pretende trazer a discussão a partir dos efeitos da mudança de ponto de vista sobre o coletivismo. E mesmo que seu foco seja contemporâneo, os organizadores buscaram reconhecer algu- mas distinções entre agrupamentos artísticos pré e pós-guerras mundiais. Assim, Stimson e Sholette (2007, p.10) 2 apresentam que a “coletivização da produção artística não é novidade”, mas ela “é uma ocorrência que parece não ter vida ideológica após o período pós-guerra, não ter ismos para se sustentar como um empreendimento vivo, não ter literatura crítica para lhe dar or- gulho de ter um lugar na história”. Em contrapartida, afirmam que o moderno coletivismo artístico anterior ao pós-guerra estava vinculado ao comunismo como um ideal, que ao falhar teria deixado poucas escolhas a não ser distanciar-se de gran- des ismos. Seria ingênuo, acrescentam, pensar que o coletivis- mo atrelado a grandes ideais sucumbiu, contudo, a obra busca refletir sobre as redefinições que coletivos artísticos buscaram em termos de significados, propósitos, formas de vitalidade po- lítica que tenham impacto em sua própria concepção de arte, tendo em vista este passado. Os organizadores apóiam este argumento em característi- cas de agrupamentos, que além dos já listados acima por Paim e Mesquita, citam os futuristas, surrealistas, produtivistas, muralistas, realistas sociais e outras ocorrências entre 1950 e 60 como o expressionismo abstrato, os happenings, a pop art, o minimalismo e outras categorias artísticas que se compu- seram em coletivo. Mas argumentam que nenhuma delas de- fendeu a bandeira coletiva, nem tinham a coletivização como vital ou primária para a sua arte, nem mesmo exploraram a afiliação ao grupo como medida da sua autonomia artística. Stimson e Sholette (idem) argumentam, portanto, que o coletivismo artístico pode e deve ser estudado em seus pe- ríodos distintos ao longo da história, pois podemos ganhar em precisão e definição se assim o fizermos. Ainda escla- recem que nós estamos em uma posição diferenciada para olhar as novas oportunidades que emergem dos coletivismos contemporâneos. Segundo Paim (2012, p.7), coletivos são Segundo Paim (2012, p.7), coletivos são Agrupamentos de artistas ou multidisciplinares que, sob um mesmo nome, atuam propositalmente de forma conjunta, criativa, autoconsciente e não hierárquica. O processo de cria- ção pode ser inteira ou parcialmente compartilhado e buscam a realização e visibilidade de seus projetos e proposições. Os coletivos podem ser mais ou menos fechados. Alguns possuem uma formação fixa e determinada internamente, outros, um núcleo central em torno do qual se agregam distintos parceiros de acordo com os projetos em execução. A autora propõe uma categorização distinguindo coletivo de iniciativas coletivas, que define por “projetos com autoges- tão de equipes de trabalho constituídas por artistas ou mistas, que se formam para um determinado fim e que não pretendem estabelecer vínculos como nos coletivos nem tem o propósito de formar um coletivo” (idem, p. 8). De acordo com esta dis- tinção os coletivos tem uma formação fixa e, simultaneamen- te, móvel, isto é, possuem um núcleo fixo e parceiros que são mobilizados a partir de determinados projetos. Já as iniciativas coletivas são sempre móveis e de articulação efêmera. Mesquita (2008), que investiga uma produção coleti- va com enfoque em arte ativista, coletiva e intervencionista, aprofunda a discussão ao argumentar que o coletivismo com este enfoque artístico inicia-se na metade do século XIX. Pos- teriormente, pode ser observado nas vanguardas artísticas eu- ropéias, em manifestações do pós-guerra, de teatro de rua, de grupos militantes e vivências engajadas da Arte Conceitual de 1960 e 1970, bem como o ativismo cultural voltado à comuni- dade e aos movimentos sociais europeus, norte-americanos, argentinos e brasileiros. O autor relata a impossibilidade de listar em sua completude, mas observa que o coletivismo ar- tístico do pós-guerra é expressivo também no Japão, Leste Eu- ropeu, México, Cuba, África do Sul, Oriente Médio e Rússia. A pesquisa de ambos os autores acima dialogam, direta ou indiretamente, com Collectism After Modernism: The Art of So- cial Imagination After 1945 cujos organizadores Stimson e Sho- lette (2007) afirmam ser um livro que abre as portas do estudo sobre coletivismo artístico, compreendendo-o não como re- presentação de uma prática artística genial, mas com o intuito 206 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Segundo Paim (2012, p.7), coletivos são Citam, por exemplo, o trabalho do coleti- vo Temporary Services, de Chicago, cujas imagens (figuras 1, 2 e 3) sugerem uma provocação ao fenômeno público do descarte, por isso, o título da compilação é Public Phenome- na. No Flickr do grupo, ela está legendada como “Fenômeno 207 ara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Público – Objetos GRATUITOS deixados no lado da calça- da em Urbana, Illionis”3 (grifos do autor). A proposta destas imagens está em um booklet, uma espécie de livro-panfleto, disponibilizado no site do grupo, cujo título é Fenômeno Pú- blico – Modificações Informais dos Espaços Compartilhados d d l d b Figura 1 henomena. ry Services. Figura 2 henomena. ry Services. Figura 3 henomena. ry Services. Figura 1 Free_1 for Public Phenomena. Cortesia de Temporary Services. Público – Objetos GRATUITOS deixados no lado da calça- da em Urbana, Illionis”3 (grifos do autor). A proposta destas imagens está em um booklet, uma espécie de livro-panfleto, disponibilizado no site do grupo, cujo título é Fenômeno Pú- blico – Modificações Informais dos Espaços Compartilhados e produzem sentido pela sua reunião em torno do objetivo descrito como “Desde a origem do Temporary Services em 1998, nós assumimos o ativo interesse na cultura vernácula encontrada publicamente no espaço transitável”4. O sentido de cultura vernácula se revela pela singularidade das inter- venções expressas nos espaços por aqueles que os usam, as quais são captadas em imagens por um grupo que despreten- siosamente transita nestes espaços, à deriva. Figura 1 Free_1 for Public Phenomena. Cortesia de Temporary Services. Figura 1 Free_1 for Public Phenomena. Cortesia de Temporary Services. Figura 2 Free_2 for Public Phenomena. Cortesia de Temporary Services. Figura 3 Free_8 for Public Phenomena. Cortesia de Temporary Services. Público – Objetos GRATUITOS deixados no lado da calça- da em Urbana, Illionis”3 (grifos do autor). A proposta destas imagens está em um booklet, uma espécie de livro-panfleto, disponibilizado no site do grupo, cujo título é Fenômeno Pú- blico – Modificações Informais dos Espaços Compartilhados e produzem sentido pela sua reunião em torno do objetivo descrito como “Desde a origem do Temporary Services em 1998, nós assumimos o ativo interesse na cultura vernácula encontrada publicamente no espaço transitável”4. Figura 3 Free_8 for Public Phenomena. Cortesia de Temporary Services. Segundo Paim (2012, p.7), coletivos são O sentido de cultura vernácula se revela pela singularidade das inter- venções expressas nos espaços por aqueles que os usam, as quais são captadas em imagens por um grupo que despreten- siosamente transita nestes espaços, à deriva. Público – Objetos GRATUITOS deixados no lado da calça- da em Urbana, Illionis”3 (grifos do autor). A proposta destas imagens está em um booklet, uma espécie de livro-panfleto, disponibilizado no site do grupo, cujo título é Fenômeno Pú- blico – Modificações Informais dos Espaços Compartilhados e produzem sentido pela sua reunião em torno do objetivo descrito como “Desde a origem do Temporary Services em 1998, nós assumimos o ativo interesse na cultura vernácula encontrada publicamente no espaço transitável”4. O sentido de cultura vernácula se revela pela singularidade das inter- venções expressas nos espaços por aqueles que os usam, as quais são captadas em imagens por um grupo que despreten- siosamente transita nestes espaços, à deriva. 208 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Retomando o argumento de Stimson e Sholette (idem), di- ferentemente dos modernistas, o texto do booklet apresenta que a compilação e sua concepção são do coletivo como se todas as etapas da construção artística fossem de todos os três membros do grupo, que são responsabilizados sem distinção de autoria. Além disso, a própria intervenção não é obra do coletivo, é antes do público, que ao se expressar publicamente interfere nos es- paços em que transita, de modo que o coletivo observa, registra e dialoga visualmente com ele, sem deixar claro quem é respon- sável pelas imagens, pela ideia da compilação, pelo booklet ou mesmo pelo sítio onde estão. Por isso, parece-nos que além de se afirmarem enquanto coletivo, há nestes agrupamentos atuais a opção por não individualizar, expressa na temática da obra - a intervenção de um coletivo maior, chamado de público, nos seus espaços de trânsito - e também na recusa de uma autoria indivi- dual por quem poetiza tal intervenção. A pesquisa de Paim (2009) tem como foco modos de fazer coletivo e iniciativas coletivas artísticas como posição política, observadas a partir de 1990, em alguns países da América do Sul. Segundo Paim (2012, p.7), coletivos são Assim apresenta que espaços artísticos autogestionados recebem impulso neste contexto devido à retração de mercado de artes praticado solitariamente e destinado às galerias; ao fim das ditaduras militares na América Latina e aos nascentes mo- vimentos de redemocratização que possibilitam microassocia- ções; a crise econômica de países latino-americanos promove o sucateamento de incentivos estatais à cultura; o aumento de cursos de artes fomenta a convivência e a crítica da atuação; a popularização dos meios de comunicação em rede, bem como a globalização dos mercados e flexibilidade do trabalho. Já Hollanda (2013) formula outra compreensão dos cole- tivos na atualidade. Para a autora a atuação dos coletivos de artistas plásticos apresenta-se como um segmento surpreen- dente no campo da produção cultural contemporânea. Argu- menta que surgem no final dos anos 1990 e tem como foco de intervenção no espaço urbano. Sobre este fenômeno, Hollan- da (2013) afirma Os coletivos, que se propagam em proporção geométrica pelo Brasil, trazem um plus de novidade. Os coletivos não se configuram por seus integrantes e sim por determinadas ações, agindo sempre num contexto de intervenção pública. Os coletivos também não são cooperativas, não são grupos, não tem número de participantes determinado, nem podem 209 ma Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema ser caracterizados como movimentos artísticos. Sua forma de organização é independente e, para cada ação ou conjunto de ações, os coletivos buscam patrocínio, oferecendo cursos, vendendo trabalhos ou realizando serviços como ilustração, design, vídeo etc. Esta auto-gestão elimina, portanto, a figura do curador, personagem cujo poder seletivo e decisório cres- ceram muito nos últimos 20 anos, adquirindo uma função de autoridade centralizadora no sistema das artes. A autora tem como referência o modo de se organizar de gru- pos e cooperativas de 1970 quando afirma que os coletivos con- temporâneos se diferenciam deles por serem “estruturalmente nômades”, por se unirem “apenas em função de projetos tópicos” e por se caracterizarem “pela reunião em torno de ações concre- tas e imediatas, rejeitando a idéia de projetos a longo e médio prazos”. Ou seja, quando a autora nega a nomeação de grupo ou de cooperativa aos coletivos dos dias atuais está considerando a mobilidade e a agilidade em que seus membros se organizam em torno de projetos curtos ou ações pontuais. VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Segundo Paim (2012, p.7), coletivos são Por exemplo, hoje um artista participa de um projeto em um coletivo X e amanhã pode participar em um coletivo Y e posteriormente ainda voltar a se envolver com outros projetos no coletivo X. Esta flexibilidade das relações entre os membros dos coletivos é que leva a autora a diferenciar os coletivos de hoje de grupos e cooperativas. Desse modo, ela os caracteriza não pelos membros do gru- po, mas pelas suas ações. São estas ações que geram, em sua opinião, a organização de um coletivo na atualidade. Por isso, ela qualifica a organização deles como rizomática e nômade. Além de uma comunicação intensa por meio de sites de serviço de redes sociais e blogs, que podem ser utilizados como espaços de troca de informação, interação e produção de conhecimen- to, retomando o argumento de Hollanda (2013), os coletivos artísticos contemporâneos caracterizam-se ainda pela auto- gestão, descentralização, flexibilidade das relações, articulação situacional e por agirem intervindo no espaço público.i Nem apenas móvel, nem apenas fixo, Mesquita (2008, p. 22) argumenta que desde os anos de 1990 no Brasil os coleti- vos artísticos tem se organizado de diversos modos e a par- tir de distintos objetivos, por isso, afirma “temos a existência de alguns coletivos trabalhando em conjunto há mais de dez anos, assim como agrupamentos efêmeros e temporários”. Sobre os modos de organização coletiva, o autor comenta que os grupos atuais optam por formações descentralizadas e heterogêneas a partir de três vetores: autoria do projeto, proces- 210 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 sos de organização e criação da obra. Neste sentido, argumenta que a pesquisa empírica sobre a organização social torna-se tão importante para tais artistas quanto à tradicional pesquisa so- bre os materiais e produtos. Mesquita (2008, p. 51) observa que, Vemos artistas trabalhando coletivamente a partir de uma única proposta ou em colaboração com indivíduos de diferentes áreas. Há também artistas que se reúnem em torno de uma ideia cole- tiva ou de um movimento, mas desenvolvem suas obras indivi- dualmente, assim como um projeto artístico com a participação do público, de uma comunidade ou de um grupo político. Desse modo, exemplifica sobre alguns modelos de práticas coletivas. Em coletivos cujos membros mantem-se fixos por um tempo, segundo o autor, a autoria expressa o modo do grupo se organizar e conviver, seu modus vivendi enquanto grupo. Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Segundo Paim (2012, p.7), coletivos são Em outros casos, projetos criados por um único artista buscam a colaboração, a participação e negociação de indivíduos, base- ando-se em situações sociais, de modo que o artista agencia a percepção social e crítica junto a comunidades e grupos, cha- mando-os a co-produzir e co-criar. Nesta direção, o diálogo, a interação, o encontro e a convivência são interesse da sua arte. Há ainda artistas que buscam a transversalidade, em oposição a uma verticalidade do hierárquico ou piramidal e também além de uma horizontalidade. Neste sentido buscam-se diversos níveis, disciplinas, grupos, movimentos e atores, assim, desa- fiam-se as noções de autoria, o culto ao artista, optando pelo anonimato do artista por meio do uso de pseudônimos ou ado- ção de nomes múltiplos, os quais podem ser usados por qual- quer pessoa em uma ação. Trata ainda de coletivos com filiações flexíveis e temporárias voltados para projetos de intervenção social e artística, chamando-as de coalizões temporárias. Paim (2009, p. 27) acrescenta à discussão outros traços nos modos de fazer de coletivos da atualidade, observando que nem todos eles são notados constantemente, - fazeres que não obedeça às decisões tomadas por um núcleo fechado; são descentralizados e compositivos de muitas falas; - não-hierarquizados; - fazeres que não obedeça às decisões tomadas por um núcleo fechado; são descentralizados e compositivos de muitas falas; - não-hierarquizados; - não-hierarquizados; - podem ter mobilidade; - são emancipatórios e positivos – propõe a saída da rigidez das ideias prontas e revelam o que elas tem de construção ideológica; - utilizam a auto-organização e são autogestionados e tam- bém são modos de fazer desburocratizados e ágeis; 211 211 Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema - apresentam tendência a operar com noções de site-specific ou oriented-site; - apresentam tendência a operar com noções de site-specific ou oriented-site; - contam com autoria coletiva em, pelo menos, alguma etapa dos projetos; - usam o ciberespaço (como espaço da prática ou como meio para sua organização e difusão); - podem ser organizados por coletivos de artistas ou com for- mação heterogênea. - podem ser organizados por coletivos de artistas ou com for- mação heterogênea. A autora, cuja pesquisa com coletivos artísticos se afina com o enfoque em arte e ativismo político, apresenta com ou- tras palavras características acima discutidas. VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 inaugura uma relação com o ambiente natural. Não mais pai- sagem a ser representada, nem manancial de forças passível de expressão plástica, a natureza é o locus onde a arte se enraíza. Focando apenas em coletivos que agem artisticamente a partir de um projeto imediato e concreto, Hollanda (2013) ar- gumenta que o coletivo se estrutura e recompõe com outros membros para ações seguintes, consequentemente, estrutu- rando outro coletivo. Portanto, tal organização estruturada em função de cada uma de suas ações requer como suporte uma comunicação intensa entre coletivos por meios blogs e listas de discussão na internet, que para Hollanda (2013), “alguns sites reúnem as informações de forma mais nodal, explicitando me- lhor a lógica de rede que rege essa produção”. Por exemplo, em São Paulo, o coletivo Horizonte Nômade iniciou uma articula- ção de artistas que trabalhavam em coletivos brasileiros a partir de um levantamento destes realizado por Flávia Vivacqua, em 2000. Desta articulação surgiu em 2002 o CORO - Colabora- dores em Redes e Organizações, com objetivo de “unir outros artistas que também trabalhavam coletivamente no Brasil”5. Vimos que embora tenhamos a surpreendente atuação ar- tística destes agrupamentos de 1990 aos dias atuais, no Brasil, eles nem são tão novos, tampouco trazem modos de fazer, de se organizar, bem como motivações originais. Todavia, a rela- ção entre arte e coletivo, seja com ênfase no ativismo político, seja no encontro afetivo, seja no profissional, ou mesmo com elas conjugadas, este fenômeno precisa ser observado, com- preendido e avaliado, pois tem se mostrado um fenômeno fértil tanto na construção de espaços comum, quanto na refle- xão sobre modos de fazer filmes em grupo, o que estudaremos melhor no próximo item. Segundo Paim (2012, p.7), coletivos são A característica da emancipação, citada por ela, decorre da desconstrução de modos de fazer ideologicamente consagrados, mas também sugere uma valorização do fazer e, consequentemente, uma contínua reflexão sobre este processo. Sennett (2012, p.18) re- flete que o fazer é um modo de pensar e um modo de aprender sobre nós mesmos, por isso, argumenta que “as pessoas podem aprender sobre si mesmas através das coisas que fazem”. Com- preendemos ser este o sentido da característica emancipatória dos coletivos para a autora, da sua possibilidade de construir e desconstruir aprendizagens sobre si mesmo em grupo. i Já a tendência a operar com noções de site-specific ou oriented-site remete à noção de arte pública que, por sua vez, expressa a ideia de uma arte realizada fora dos espaços con- sagrados a ela, como galerias, museus, salas de projeção etc. De acordo com a Enciclopédia Itaú Cultural de Artes Visuais (2010), obras ou instalações operando com tais noções podem modificar os ambientes, corriqueiramente espaços públicos e urbanos, embora também vistas em ambientes naturais, de modo permanente ou temporário. Assim, tanto site-specific quanto sítio específico Faz menção a obras criadas de acordo com o ambiente e com um espaço determinado. Trata-se, em geral, de trabalhos pla- nejados - muitas vezes fruto de convites - em local certo, em que os elementos esculturais dialogam com o meio circundan- te, para o qual a obra é elaborada. Nesse sentido, a noção de site specific liga-se à ideia de arte ambiente, que sinaliza uma tendência da produção contemporânea de se voltar para o es- paço - incorporando-o à obra e/ou transformando-o -, seja ele o espaço da galeria, o ambiente natural ou áreas urbanas. Relaciona-se de perto à chamada land art [arte da terra], que 212 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 inaugura uma relação com o ambiente natural. Não mais pai- sagem a ser representada, nem manancial de forças passível de expressão plástica, a natureza é o locus onde a arte se enraíza. Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema 3. O método CooperAÇÃO dos Amigos do Cinema Embora não exista formalmente, o Sistema CooperAÇÃO - Ami- gos do Cinema iniciou suas atividades em 1999, primeiro ano da realização do Festival Internacional de Cinema e Vídeo Am- biental (FICA), na Cidade de Goiás, Estado de Goiás. Na ocasião, Martins Muniz tinha uma produtora de filmes e alguns amigos o convidaram a realizar uma produção cinematográfica para inscrevê-la no festival e sua resposta foi “se vocês ajudarem, nós fazemos o filme […] ajuda com trabalho” (MUNIZ, 2013a). 213 Segundo ele, desde esta época o grupo mantem em suas re- alizações a proposta de “ajuda com trabalho”, que pode ser tra- duzida pela cooperação por meio de trabalho voluntário com opiniões, participação e experimentações em conjunto, em co- letivo. No trecho abaixo, Muniz (2013b) relata que o Sistema CooperAÇÃO surge de um sonho com o cinema de longa data Eu, desde o início, desde criança, tinha vontade de fazer cine- ma, mas não tinha como, de família pobre não tinha condições nenhuma de fazer filme, era inviável. A única [opção] que ti- nha na minha época era a película. Aí eu fiquei sonhando com essa história toda a vida. Aí achei que deveria ser operador de cinema para projetar filme, para poder ficar mais perto da pe- lícula, para poder manusear a película, né? Aí fui ser operador de cinema. Entrei no Cine Campinas, na época áurea do ci- nema. Cine Capri fui eu que inaugurei operando filme de 70 milímetros, Cine Ouro foi que inaugurei também projetando filmes e ninguém sabe disso, e aí fui aprendendo. Aí cansei de... [operar]. Aí estudei fotografia, estudei pintura, estudei desenho, fui estudando tudo o que no cinema precisava eu ia aprendendo, lutando para aperfeiçoar, aprender mais. Teve uma época em que eu fazia os cartazes do cinema, pintava as telonas grandes do cinema, você não lembra isso, você é meni- na, não tinha o plotter, era feito à mão, para frente do cinema... Com cartazes colados? , p , , p - Com cartazes colados? - Não, pintura. Fazia painel para frente do cinema, intei- riço. 5 metros por 10 metros de tamanho. - Não, pintura. Fazia painel para frente do cinema, intei- riço. 5 metros por 10 metros de tamanho. - Você fazia? - Fazia, era o Ti Érikes e eu. VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 3. O método CooperAÇÃO dos Amigos do Cinema Aí eu queria aprender mais e eu não tinha como, e aí eu soube da Makro Filmes, a Makro Fil- mes do Euclides Néry, você conhece, né? O pioneiro do cinema de Goiás. Aí eu entrei lá e falei “vou trabalhar com vocês aqui”, e eles “mas não temos dinheiro, não tem como te contratar”, e eu “mas eu trabalho de graça, desde que você me deixa meio pe- ríodo para eu trabalhar fora”. Aí comecei a trabalhar com eles, aí fui entrando com eles e rapidinho eu consegui um salário lá dentro, porque eu sabia fotografar, sabia fazer fotolito, sabia desenhar, eles estavam fazendo um desenho animado para um leite... Aí eu comecei a colorir o acetato do filme deles e come- cei aprender vagarosamente. Aí eu cheguei a alcançar um bom salário de Goiás. Aí fiquei empregado toda vida, empregado não tem valor, é máquina, é complemento da mão do patrão, aí fui batalhar por outros lados, mas quando eu saí da Makro 214 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 eu já sabia filmar, já manuseava o equipamento deles, aí estava pronto para trabalhar. Aí comecei a trabalhar independente. Agora o sonho do cinema grande, eu queria saber fazer cine- ma grande, porque eu entrei na publicidade querendo fazer aprender fazer cinema grande. - O que é cinema grande? - O que é cinema grande? - Mais de duas horas, uma hora de projeção. Porque o co- mercial é trinta segundos. Estourando trinta segundos, tem que contar uma historinha em trinta segundos, então é difí- cil satisfazer a gente. No cinema longo a gente faz a cena, só uma cena dá 15 minutos se precisar, né? Igual nessa gravação que você está fazendo; a gravação vai dar mais de 15 minu- tos, não é? Aí eu sonhava com cinema, procurava onde tinha uma filmagem de um longa metragem eu aproximava, mas as cabeças dos filmes já vinham tudo feito pelo diretor de fora. Eles não davam chance para as pessoas de Goiás. Como nunca deu. Quando tem uma filmagem igual essa novela que apareceu [Em família, exibida em 2014], essa gracinha aí, pega os melhores atores nossos como figurantes e olha lá se eles aparecem, é muito difícil aparecer. Aí eu sonhava com o cinema. ara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema 3. O método CooperAÇÃO dos Amigos do Cinema Aí chegou um ponto recentemente, há 10 ou 12 anos atrás, eu soube que tinham muitas pessoas que tinham esse mesmo sonho do cinema e não tinha como fazer aí fa- lou: “por que você não faz um filme para nós atuar?” (sic.), aí eu falei, “vamos fazer o seguinte, eu entro com a câmera e o material e vocês entram com a mão de obra de vocês”, aí fizemo uma união e criamos um sistema de cooperativa, Co- operAÇÃO. Aí o ator atuava, a atriz atuava, a figurinista fazia o figurino e todo mundo uniu e fez o primeiro filme que foi “A lenda do milho”, aí deu certo, aí fizemos “O Matuto” e foi fazendo filmes aí e o grupo cresceu. Hoje quando eu vou falar vou fazer um filme eu tenho que selecionar os caras e faço até inimizade porque todo mundo quer ir, mas não tem espaço para todo mundo e eles vai (sic.) pela farra, aí todo mundo trabalha no sentido de fazer uma produção independente. Aí nós pega (sic.) esse filme feito, cada um fica com uma cópia e mostra em tudo quanto é espaço, mostra para um e para outro e faz as sessões particulares e vamo (sic.) distribuindo. Esse é o sonho do cinema nosso, do Sistema CooperAÇÃO. E pretendemos fazer muito mais. Notamos que a motivação para o Sistema CooperAÇÃO – Amigos do Cinema se constituir como agrupamento emerge da combinação de um grande sonho do seu articulador, o Martins 215 Muniz, e de amigos e atores de teatro e cinema em Goiás em reação aos preconceitos que são de ordem lingüística, mas tam- bém estética, social, econômica e até de sotaque em relação às realizações fílmicas do interior do Brasil. A motivação expressa ainda críticas ao modo provinciano de conceber os festivais re- gionais e ao relacionamento ainda hierárquico e desigual que emissoras nacionais estabelecem com atores locais. Desse modo, argumentamos que a formação do Sistema CooperAÇÃO se dá pela via da produção independente assim como muitos outros grupos artísticos optam por ela. O que implica em percebermos que a existência deste grupo faz eco com muitos coletivos artísticos contemporâneos, mesmo que ele tenha optado por se denominar cooperativa, um termo que expressa uma referência organizacional distinta dos agru- pamentos atuais, como discutido acima por Hollanda (2013). 3. O método CooperAÇÃO dos Amigos do Cinema Além disso, compreendemos que embora o CooperAÇÃO não faça uso do termo coletivo, utilizado como sinônimo de agrupamento urbano atual, sua formação se dá por projeto, ou seja, como Muniz apresenta acima, a cada novo filme, um grupo distinto se configura, de modo a trabalhar como gru- po apenas durante o desenvolvimento do filme. Avançando um pouco mais nesta perspectiva e considerando a experiên- cia das filmagens de O Capitão do Mato (MARTINS MUNIZ, 2013), que foi uma das poucas captações fragmentadas em fi- nais de semana ao longo de dois meses, argumentamos que a cada final de semana um novo agrupamento se formava, tor- nando o projeto um desafio ao continuísmo, à capacidade de improvisação do grupo e à coerência narrativa. Este dado é certamente um dos mais importantes de se considerar ao assistirmos os filmes do Sistema CooperAÇÃO; uma vez que o grupo filma nos finais de semana, pois segundo Muniz (2013b) não há remuneração para a equipe envolvida, vivencia imprevistos de diversas naturezas. Era 7h23 e dois dos atores principais não tinham chegado e não atendiam ao celular. Tínhamos marcado para sair às 7h. Na por- ta da casa do Muniz, local de encontro para saída em direção à locação daquele domingo, Muniz me pergunta: “São três pre- tos fujões na nossa história, se eu deixar só um, a história fica ruim?” (SATLER, Diário de campo, em 24 de fevereiro de 2013). Desse modo, as circunstâncias geradas pela efemeridade do agrupamento do Sistema CooperAÇÃO requisitam uma 216 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 capacidade de ir e vir na narrativa fílmica das suas realizações, o que se traduz por uma flexibilidade na coerência e no con- tinuísmo, bem como na necessidade de refazer o filme con- tinuamente. É como se o roteirista e diretor, que é também o articulador do grupo, tivesse o filme na memória, mas devido às necessidades e imprevistos substituísse partes, adicionasse camadas, recortasse trechos e assim procedesse mentalmente durante todo o processo de captações que envolvem o coletivo. Contudo, não é apenas isso que parece acontecer. No caso específico de O Capitão do Mato (MARTINS MUNIZ, 2013), todo esse processo de ruminação mental da realização fílmica acon- tece entremeado de diálogos constantes com quem está no set de filmagem, seja este ator, equipe técnica ou diletante do grupo. 3. O método CooperAÇÃO dos Amigos do Cinema Depois de gravadas as cenas, ele as assiste e confere se fica- ram boas. Todos assistem numa tela de quatorze polegadas e comentam. [...] O Fábio, o Eurípedes e o Muniz comentam entre si e perguntam uns aos outros se as cenas assistidas es- tão boas ou precisam regravação. Também os participantes comentam, interferem, criticam, elogiam (SATLER, Diário de campo, em 17 de fevereiro de 2013). Deste método de fazer e refazer continuamente, estimulan- do como os presentes observam o que foi filmado e se merece ser refilmado é argumentamos que o sentido de CooperAÇÃO no nome deste grupo se afina mais com o conceito de coope- ração do quem com a ideia de cooperativa discutida acima por Hollanda (2013). Assim, para compreender sobre o método dos Amigos do Cinema e sua articulação em coletivo dialogamos com o conceito de cooperação de Piaget (1973, p.64), que a de- fine como “um sistema de operações executadas em comum ou por reciprocidade entre as [regras racionais] de seus parceiros”. Para a Piaget (1998, p. 118, grifos do autor), a cooperação é um processo “constituído pela ação dos indivíduos uns sobre os outros quando a igualdade (de fato e de direito) suplanta a autoridade”, por isso “a coerção desaparece dando lugar à cooperação, e o respeito torna-se mútuo”. Neste sentido Piaget (idem, ibidem) acrescenta Embora a cooperação nunca se desvencilhe completamente de toda coerção e embora o respeito não possa chegar a uma total reciprocidade, o segundo processo [o da cooperação] define um ideal cujos efeitos são qualitativamente diferentes 217 Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema dos da coerção. Enquanto que esta última tem por resultado essencial impor regras e verdades já elaboradas, a cooperação (ou as tentativas de cooperação) provoca, ao contrário, a cons- tituição de um método que permite ao espírito superar a si mesmo incessantemente e situar as normas acima dos estados de fato. Do ponto de vista moral, a cooperação leva não mais à simples obediência às regras impostas, sejam elas quais fo- rem, mas a uma ética da solidariedade e da reciprocidade. 3. O método CooperAÇÃO dos Amigos do Cinema Durante as gravações, observamos que a relações de hie- rarquia, presentes em certos modos de produção cinemato- gráfica tradicional, são organizados dentro do próprio gru- po, representando o que Piaget mencionou acima por regras autônomas de conduta, fundamentadas no respeito mútuo. Assim, observamos que a hierarquia representa o nível de res- ponsabilidade do membro do grupo, em detrimento de uma figura de autoridade irrevogável ou inacessível. i Muniz foi definido pelos membros do Sistema CooperA- ÇÃO como o diretor dos filmes desde o momento em que foi convidado pelos amigos a realizar a primeira produção neste grupo. Sua responsabilidade concentra-se em articular o gru- po a produzir, reunir atores, produzir roteiros, conseguir loca- ções, contatar com músicos, figurinistas, diretores de arte etc. Apresenta-se como figura que está no centro de todos os atores e equipe técnica articulando-os com o objetivo de fazer cinema em coletivo. Por isso, ocupa-se em estar no meio das relações, articulando-as. Ao invés de estar em outro patamar, observan- do e dirigindo o grupo à distância, ele parece interessar-se por agregar uns aos outros pelo prazer de fazer cinema junto. Outro ponto que observamos é o processo de integração de novatos ao grupo: acontece muito mais a partir da sua disponi- bilidade em agir em comum. Explicamos: durante as gravações das cenas, vimos diletantes que visitavam despretensiosamente o estúdio ou as locações e, que, ao final do dia, já tinham se maquiado, vestido e atuado. Assim, o método do Sistema Coo- perAÇÃO vai incorporando quem se aproxima, ou seja, quem visita o set de filmagem, pode agir, atuar de algum modo, sendo convidado a cooperar também. Por isso, é um método aberto de agremiação e cooperação. Sobre isso, Muniz (2013b) responde - Muniz, você me disse que quando seus amigos te chamaram para fazer o primeiro filme você propôs fazê-lo em cooperação, de onde você tirou esta idéia de fazer junto, o que te motivou a isso? 218 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 3. O método CooperAÇÃO dos Amigos do Cinema 203-223, jul-dez 2014 - Porque eu não podia pagar eles (sic.), um câmera hoje ganha cinco mil reais em uma filmagem, um iluminador ganha tanto, e assim por diante, um maquinista ganha tanto e nosso filme tem o salário alto, nosso filme é o mais caro do Estado de Goiás, porque eu pego os melhores profissionais do Estado de Goiás, só que eu não pago eles e eles não me pagam, cada um fica com a cópia do filme como uma vitrine nossa, do nosso trabalho. Então quando eu tive esta idéia da cooperativa foi por- que eu sabia que muita gente tinha vontade de mostrar as coisas que sabia, mas não tinha espaço em Goiás, como não tem. Toda cidade é feita de panela. Pode notar em Goiânia e Goiás que tem uma panela do teatro e tem outro grupo que está fora da panela que eles recusam e dão cotoveladas constantes nos que estão fora da panela deles. A mesma coisa é no cinema, tem um grupo de cinema em Goiânia, que nós sabemos quem é que é muito zoiudo (sic.), eles fazem as produções deles e são as mesmas pessoas e nunca deixa outro grupo que não está ligado a eles entrar então nós tinha (sic.) necessidade de fazer também um grupo nosso. Só que esse grupo ficou aberto para quem quiser, ele não ficou fechado como os outros grupos que são fechados, e tem feito vários trabalhos por isso. Está com uns quinze filmes já feitos. E com dois roteiros bolados já, fazer mais dois e vamos fazendo, enquanto tiver força vamos fazendo. Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema Notas 1. Conferir títulos em filmografia. 2. Todas as traduções presentes nesta reflexão são livres. 2. Todas as traduções presentes nesta reflexão são livres. 3. FREE URBANA - Public Phenomena. 3. FREE URBANA - Public Phenomena. 4. Public Phenomena – Informal Modifications of Shared Spaces. 5. Histórico Coro Coletivo. 5. Histórico Coro Coletivo. 4. Considerações Argumentamos que o Sistema CooperAÇÃO – Amigos do Ci- nema se comporta mais como um coletivo artístico contempo- râneo, dada a sua efemeridade e contínua formação grupal, do que como uma cooperativa de atores e técnicos realizadores de filmes. Assim, apresentamos que o método cooperação, neste agrupamento, resulta na reciprocidade das ações, ligando-se di- retamente ao equilíbrio de regras construídas em conjunto, em oposição às coações. Desse modo, podemos afirmar que, dentro do grupo, a distribuição das tarefas não se encerra na especia- lidade ou na expertise de cada membro. Como o grupo produz pela via da diversão e do voluntariado, seus membros não se en- volveram, até hoje, com as produções apenas pelo cachê, nem foram escolhidos exclusivamente pela competência individual. l Obviamente, o resultado estético das realizações refletem estas escolhas, o que implica em serem elas constituintes do estilo fílmico do Sistema CooperAÇÃO. Desse modo, perce- bemos que tão importante quanto produzir filmes em Goiás, 219 Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema no interior do Brasil, em condições adversas e à margem dos grupos locais, é produzi-lo em um coletivo que coopera entre si. Desse modo, a via da produção independente faz sentido se, e somente se, em um conjunto aberto ao método CooperA- ÇÃO. Assim o método de realizar filmes deste coletivo é a Co- operAÇÃO, que se fundamenta no voluntariado, na ausência do cachê que suprime certa obrigatoriedade, pois não apre- senta uma moeda de troca além do prazer de estar ali para se divertir como Amigos do Cinema. Ao mesmo tempo o voluntariado gera imprevistos garante o sentido do método CooperAÇÃO: que é a autonomia dos atores e a reciprocidade das relações. Assim, argumentamos que as realizações deste coletivo artístico precisam ser consi- deradas à luz do seu método, ou seja, a CooperAÇÃO que põe em risco a continuidade da realização, a coerência e o conti- nuísmo da narrativa, mas é ela mesma a estética da autonomia e da solidariedade que o grupo interessa-se por vivenciar. versidade de São Paulo. MUNIZ, Martins. Entrevista concedida a Lara Lima Satler. Goiânia. 24.fev.2013a. MUNIZ, Martins. Entrevista concedida a Lara Lima Satler. Goiânia. 24.fev.2013a. _______. Entrevista concedida a Lara Lima Satler. Goiânia. 13.nov.2013b. _______. Entrevista concedida a Lara Lima Satler. Goiânia. 13.nov.2013b. PAIM, Claudia. Táticas de artistas na América Latina: coleti- vos, iniciativas coletivas e espaços autogestionados. Porto Alegre: Panorama Crítica Ed, 2012. 200 p. _______. Coletivos, iniciativas coletivas: modos de fazer na América Latina contemporânea. Porto Alegre, 2009. 294 p. Tese (Doutorado em Artes Visuais). Instituto de Artes, Universidade Federal do Rio Grande do Sul. PAIM, Claudia. Táticas de artistas na América Latina: coleti- vos, iniciativas coletivas e espaços autogestionados. Porto Alegre: Panorama Crítica Ed, 2012. 200 p. _______. Coletivos, iniciativas coletivas: modos de fazer na América Latina contemporânea. Porto Alegre, 2009. 294 p. Tese (Doutorado em Artes Visuais). Instituto de Artes, Universidade Federal do Rio Grande do Sul. PIAGET, Jean. Estudos Sociológicos. Rio de Janeiro: Forense, 1973. 234 p. _______. Sobre a pedagogia. São Paulo: Casa do Psicólogo, 1998. 262 p.i Public Phenomena – Informal Modifications of Shared Spaces. Disponível em:< http://www.temporaryservices.org/pu- blic_phenomena.pdf>. Acesso em: 27. jan. 2014.i SENNETT, Richard. O artífice. Rio de Janeiro: Record, 2012. 360 p.i Site Specific. Enciclopédia Itaú Cultural de Artes Visuais (Ver- bete atualizado em 04.nov.2010). Disponível em: <http:// www.itaucultural.org.br/aplicExternas/enciclopedia_IC/ index.cfm?fuseaction=termos_texto&cd_verbete=5419>. Acesso em: 11.jan.2014. STIMSON, Blake; SHOLETTE, Gregory (Editors). Collectism After Modernism: The Art of Social Imagination After 1945. Minneapolis, London: University of Minnesota Press, 2007. 312 p. Referências CRUSCO, Sérgio. A vida em um coletivo. Maire Claire, São Pau- lo, n. 273, p. 94-104, dez.2013. FREE URBANA - Public Phenomena. Disponível em: <http://www. flickr.com/photos/15564781@N04/sets/72157602574603077/ with/1783067400/>. Acesso em: 27. jan. 2014. Histórico do Coro Coletivo. Disponível em:<http://coroco- letivo.org/historico-de-formacao-do-coro/>. Acesso em: 10.jan.2014. HOLLANDA, Heloisa Buarque de. Coletivos. (Artigo publi- cado em 10.set.2013).Disponível em: < http://www.he- loisabuarquedehollanda.com.br/coletivos/>. Acesso em: 06.jan.2014. MESQUITA, A. L. Insurgências poéticas: arte ativista e ação coletiva (1990-2000). São Paulo, 2008. 429 p. Dissertação (Mestrado em História). Departamento de História da Uni- 220 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Filmografia do Sistema C AÇÃO A i d Lara Lima Satler satlerlara@gmail.com Doutoranda em Arte e Cultura Visual (FAV/UFG). Professora na Universidade Federal de Goiás (UFG), na Faculdade de Comunicação e Informação (FIC), onde ministra as disciplinas Teorias da Imagem I e II e participa do grupo de pesquisa Cultura Visual e Educação (PPCACV/ FAV/UFG/ Cnpq). Lara Lima Satler satlerlara@gmail.com VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Filmografia do Sistema C AÇÃO A i d O CAPITÃO do Mato. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: 50 min. Brasil. 2013.i FORA de padrão: o filme. Direção: Martins Muniz. Realiza- ção: Sistema CooperAÇÃO Amigos do Cinema. Duração: 40 min. Brasil. 2012. GARROTE, No. Direção: Martins Muniz. Realização: Sistema Co- operAÇÃO Amigos do Cinema. Duração: 45 min. Brasil. 2010. PÉ de pano, O. Direção: Martins Muniz. Realização: Sistema Co- operAção Amigos do Cinema. Duração: 26 min. Brasil. 2011. 221 221 Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema MATUTO, O – ou Dois dias e meio. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Du- ração: 56 min. Brasil (sem referência do ano). MEDALHÃO e a fé, O. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: 45 min. Brasil (sem referência do ano). PEDRO Ludovico: herói ou vilão. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Du- ração: 60 min. Brasil (sem referência do ano). SUVACO de cobra. Direção: Martins Muniz. Realização: Sis- tema CooperAÇÃO Amigos do Cinema. Duração: 35 min. Brasil (sem referência do ano). FRAGMENTOS. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: (sem referên- cia de duração). Brasil (sem referência do ano). LIGAÇÃO, A. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: (sem referên- cia de duração). Brasil (sem referência do ano). SUPER Homem, O. Direção: Martins Muniz. Realização: Sis- tema CooperAÇÃO Amigos do Cinema. Duração: (sem re- ferência de duração). Brasil (sem referência do ano). DIABO Velho, o Anhanguera. Direção: Martins Muniz. Reali- zação: Sistema CooperAÇÃO Amigos do Cinema. Duração: (sem referência de duração). Brasil (sem referência do ano). CACHORRO Louco. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: (sem referência de duração). Brasil (sem referência do ano). LONA Preta. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: (sem referên- cia de duração). Brasil (sem referência do ano). LENDA do Milho, A. Direção: Martins Muniz. Realização: Sistema CooperAÇÃO Amigos do Cinema. Duração: (sem referência de duração). Brasil. 1999. Recebido em: 20/10/14 Aceito em: 10/11/14 222 VISUALIDADES, Goiânia v.12 n.2 p. 203-223, jul-dez 2014 Lara Lima Satler satlerlara@gmail.com Doutoranda em Arte e Cultura Visual (FAV/UFG). Professora na Universidade Federal de Goiás (UFG), na Faculdade de Comunicação e Informação (FIC), onde ministra as disciplinas Teorias da Imagem I e II e participa do grupo de pesquisa Cultura Visual e Educação (PPCACV/ FAV/UFG/ Cnpq). Alice Fátima Martins p f f g Pós-Doutora em Estudos Culturais (PACC/UFRJ), doutora em Sociolo- gia (UnB). Professora na Faculdade de Artes Visuais (FAV/ UFG), curso de Licenciatura em Artes Visuais e no PPG em Arte e Cultura Visual. Autora dos livros Catadores de Sucata da Indústria Cultural, pela Edi- tora da UFG (2013) e Saudades do Futuro: a ficção científica no cinema e o imaginário social sobre o devir, pela Editora da UnB (2013). 223 Lara Lima Satler e Alice Fátima Martins . O método cooperação dos Amigos do Cinema
https://openalex.org/W3028094623	https://bnrc.springeropen.com/track/pdf/10.1186/s42269-019-0260-1.pdf	English	null	Bacterial vaccines in poultry	Bulletin of the National Research Centre/Bulletin of the National Research Center	2,020	cc-by	7,237	REVIEW Open Access Abstract Background: Poultry bacterial pathogens are mainly controlled by using high-cost sanitary measures and medical treatment. However, the drug-resistant strains of pathogens continuously emerge, and medical treatments are often ineffective. Moreover, there is increasing public objections to drug residues in poultry products. The other important type of control is the vaccination which depends on immunity. This immunological control is the major practical alternative to chemotherapy. Success of vaccines in combating poultry diseases depends mainly on the choice of the proper type of vaccines, correct time of its usage, and method of administration. Background: Poultry bacterial pathogens are mainly controlled by using high-cost sanitary measures and medical treatment. However, the drug-resistant strains of pathogens continuously emerge, and medical treatments are often ineffective. Moreover, there is increasing public objections to drug residues in poultry products. The other important type of control is the vaccination which depends on immunity. This immunological control is the major practical alternative to chemotherapy. Success of vaccines in combating poultry diseases depends mainly on the choice of the proper type of vaccines, correct time of its usage, and method of administration. The types of vaccines include attenuated live vaccines, and these vaccines were shown to be effective in inducing protection. The second type is killed vaccine or whole bacteria extracts which is less successful in providing protection compared to live vaccines. The metabolic product vaccine (toxoids) is the third type of vaccine. The recombinant DNA technique was adopted to produce the protective antigens in a sufficient amount and in cost- effective ways. The types of vaccines include attenuated live vaccines, and these vaccines were shown to be effective in inducing protection. The second type is killed vaccine or whole bacteria extracts which is less successful in providing protection compared to live vaccines. The metabolic product vaccine (toxoids) is the third type of vaccine. The recombinant DNA technique was adopted to produce the protective antigens in a sufficient amount and in cost- effective ways. Conclusions: Protection studies against bacterial diseases were performed by using several trials: living vaccines (live attenuated vaccines; live, non-pathogenic microorganisms; live, low virulence microorganism), inactivated (killed) vaccines (heat-inactivated, chemical inactivates, radiation), metabolic product vaccines (toxoids), subunit vaccines (whole cell proteins, outer membrane proteins, purified flagellar proteins (flagellin), fimbrial proteins, pilus proteins, lipopolysaccharides), vaccines produced by recombinant deoxyribonucleic acid (DNA) technology, and DNA vaccines. Keywords: Poultry, Bacterial vaccines, Live vaccines, Inactivated vaccines, Subunit vaccines, DNA vaccines Bacterial vaccines in poultry Nagwa S. Rabie* and Zeinab M. S. Amin Girh * Correspondence: nagwasrabie@hotmail.com Department of Poultry Diseases, National Research Centre, 33 Bohouth St, Dokki, Giza, Egypt © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Bulletin of the National Research Centre Bulletin of the National Research Centre Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 https://doi.org/10.1186/s42269-019-0260-1 Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 https://doi.org/10.1186/s42269-019-0260-1 Introduction typhimurium live vaccine strain resulted in the same change in T cell composition as seen after infection with the non- attenuated salmonella wild-type strain, but at a lower level except an increase of CD8+ TCR1+ (gamma delta) double-positive cells which have an important role in the immunological defense of chickens against salmon- ella exposure (Berndt and Methner 2001). S. enteritidis live attenuated vaccine was more effective in increasing T lymphocyte proliferation than killed vaccine in laying hens (Babu et al. 2003). Vaccination of layer chickens with a live attenuated S. gallinarum 9 R strain reduced S. enterica infections and the vaccine could not spread to the egg content (Faberwee et al. 2001). The live Myco- plasma gallisepticum (M. gallisepticum) vaccine strains ts-11 and 6/85 could be transmitted from vaccinated layer pullets to unvaccinated pullets, broiler breeders, turkey breeders, or meat turkeys which were in indirect contact with them (Ley et al. 1997). bacteria. Live attenuated vaccines against several poultry diseases provided protection and were commercially available. Some of these diseases are mycoplasmosis in turkeys and chickens caused by Mycoplasma gallisepti- cum (M. gallisepticum) (Ley et al. 1997) or Mycoplasma synoviae (M. synoviae) (Morrow et al. 1998), fowl typhoid caused by Salmonella enteritidis (S. entertidis) (Babu et al. 2003) or Salmonella gallinarium (S. galli- narum) (Barrow et al. 2000). Paratyphoid caused by Salmonella typhimurium (S. typhimurium) (Bachtiar et al. 2003), colibacillosis caused by Escherichia coli (E. coli) (Peighambari and Gyles 1998), and fowl cholera caused by Pasteurella multocida (P. multocida) (Scott et al. 1999). Some of the disadvantages of live attenuated vaccines are that they are based on living organisms which face problems in preparation (as contamination) and batch uniformity. They provide limited immunity if severely attenuated and may cause diseases if insuffi- ciently attenuated. Other forms of living vaccines prepared from non- pathogenic microorganisms as in salmonellosis (Hassan and Curtiss 1997), colibacillosis (Frommer et al. 1994), and low virulent microorganisms as in campylobacterio- sis caused by Campylobacter jejuni (C.jejuni) (Ziprin et al. 2002) gave no protection. The pathogenic live bacteria was attenuated by mutagenesis Chemical mutagenesis of bacteria by using N-methyl N′-nitro-N nitroguanidine (NTG) for production of clones with a temperature sensitive (ts) and used as a vaccine candidate in Mycoplasma syno- viae (M. synoviae) in chickens (Morrow et al. 1998; Markham et al. 1998) and Ornithobacterium rhinotra- cheale (O. rhinotracheale) in turkeys (Lopes et al. 2002). Introduction Broiler chicken breeders were vaccinated with a temperature sensitive mutant of M. gallisepticum; the vaccine prevented infection in tracheas and infra orbital sinuses of these breeders and in vitelline membrane of their embryos. In addition, the broiler offsping of the vaccinated breeders had better produc- tion performance (Barbour et al. 2000). Inactivated (killed) vaccines are prepared from whole bacterial preparation combined with an adjuvant. They are inactivated by either heat at 60 °C for 1 h as in cam- pylobacteriosis (Widders et al. 1998) or chemicals as in samonellosis (Duchatel et al. 1998) and in fowl cholera (Khafagy et al. 1999) or radiation as in Pseudomonas in- fection (Mohamed et al. 2002). Metabolic product vaccines as toxoids (Fukutome et al. 2001) and subunit vaccines are prepared from outer membrane proteins (Abd-Aty and Rabie 2003) whole-cell proteins and flagellin (Rabie and Zou El Fakar, 2004), fimbrial and pilus proteins, and lipopoly- saccharids (Shujian et al. 1996). Intramuscular administration of the aro A-ser C (aro- matic dependent mutants) of the lysogenic strain of S. gal- linarium induced protection against experimental fowl typhoid (Barrow et al. 2000). An aro A-attenuated mutant of S. Typhimurium vaccine was used as heterologous anti- gen delivery and prevent salmonellosis in chickens (Bach- tiar et al. 2003). Attenuated live vaccine was prepared from respiratory chain muations (nuoG, 47GyoA, atpB, and at pH) of three Salmonella enterica servoars Typhi- murium, Gallinarum and Dublin in chickens and mice (Turner et al. 2003). Attenuated derivatives (Delta Cya Delta crp mutants) of O2 and O78 avain septisemic Escherichia coli (E. coli) strain were used to immunized broiler chickens by spraying. The mutant O2 strain pro- vided moderate protection against airsacculitis but not the O78 strain (Peighambari et al. 2002). Live attenuated streptomycin-dependent (str-dependent) mutants derived Recombinant DNA technology as in salmonellosis (Dueger et al. 2003) and DNA vaccines are used mainly in viral infections. Introduction cells, which give rise to antibodies and cooperate with eosinophils, macrophages and neutrophils) and cell- mediated immunity (mainly T cells, which produce lym- phokines). Bursa of Fabricius and the thymus serve as the primary lymphoid organs of the immune system. B cells use surface immunoglobulins as antigen receptors and differentiate into plasma cells to secrete antibodies. Three classes of antibodies are produced: IgM, IgG (also called IgY), and IgA. Successful vaccinal response in a flock is often monitored by demonstrating a rise in anti- body titer within a few days of vaccination. ELISA is used most commonly for serologic monitoring. T cells are the principal effector cells of specific cellular im- munity. T cells differentiate into alpha beta and gamma delta cells. In adult birds, gamma delta cells may consti- tute up to 50% of the circulating T cells. Functionally, CD4+ cells serve as helper cells and CD8+ cells as cyto- toxic/suppressor cells (Sharma 1999). Bacterial infections of poultry are a worldwide important factor in terms of their economic losses and public health. The control of these diseases depends on high- cost sanitary measures and medical treatment. However, the appearance of bacterial strains resistant to these drugs may be due to the overuse of antibiotics as well as due to care about the effect of drugs and their residues on poultry products. Moreover, some organisms are nat- urally resistant to most antibiotics (Nakae et al. 1997), and all these reasons led to searching for other immuno- logical means of control. One of which was vaccination which was found very effective in providing protection against bacterial diseases. The protection effect of bac- terial vaccines depends on the immune response of the host towards different antigenic components of the bac- teria. Antigens stimulate humoral immunity (mainly B The development of bacterial vaccines depends on dif- ferent techniques, live or inactivated (killed) forms of Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 Page 2 of 7 Live attenuated bacteria simulate natural infection which increases cell-mediated immune response. Immunization of day-old chicks with the attenuated S. Traditional techniques of bacterial vaccine production Conventional methods of bacterial vaccine development is based on whole bacteria, and they are divided into two groups: living vaccines and inactivated (killed) vaccines. Chemical inactivants h d A live, non-pathogenic piliated strain of E. coli was re- ported to be effective when broilers were vaccinated by the oral or intramuscular route at 14 or 21 days of age and challenged by the intramuscular route 1 week later with virulent avian pathogenic E. coli while vaccination by spraying did not induce adequate protection (From- mer et al. 1994). They are protein denaturants as formaldehyde, acetone, and alcohol or alkalyating agents as ethylene oxide, ethy- leneimine acetylmethylimine, and β-propriolactone. Chickens were vaccinated with formalized antigen of C. jejuni with or without immunodulator reduced reisola- tion of C. jejuni from internal organs but did not protect chicks (Rabie and Kutkat 2002). Pigeons were vaccinated with killed vaccines containing whole cell formaldehyde- inactivated S. typhimurium Var. Copenhagen. The vac- cine could not induce protection against challenge but only reduced fecal shedding (Vereechen et al. 2000). Vaccination of rabbits with a saponin killed vaccine of Mycoplasma mycoids resulted in increased humoral im- mune response (Sunder et al. 2001). Inactivated S. enter- itidis phagotype four vaccines (emulsified in light mineral oils or adsorbed in aluminum hydroxide) were used in 1-day-old chicks; no reisolation of S. enteritidis from cloacal swabs could be abtained after challenge. The vaccines emulsified in mineral oils produced greater antibodies compared to that prepared by adsorption into aluminum hydroxide (Fernchini et al. 1997). Autogenous in activated tissue vaccine (from the liver and spleen of diseased chickens) to be an approach to the prevention of ascites syndrome in broilers in which four bacterial strains were isolated and the E. coli was the most com- monly isolated strain (Shuxia et al. 1999). Avian Salmon- ella oil vaccine was used in day-old Japanese quail by S/ C and I/M injection. No deaths were observed and weak immunological reaction was detected. The egg produc- tion was lower only during the period of 6–10 weeks after vaccination (Ito et al. 2000). The comparative effi- cacy of oil-based and gel-based vaccine adjuvants has been studied by other researchers. Some studies demon- strated that an oil-based vaccine induced a higher anti- body level and provided better protection against field strains (Jacobs et al. 1992; Fukanoki et al. 2000; Chukiat- siri et al. 2010; Gong et al. 2014). Oral immunization with Haemophilus somnus vaccine protected broilers from infection with virulent strains of S. enteritidis (Wieliczko et al. 2000). Heat inactivated (60 °C for 1 h) Heat inactivated (60 °C for 1 h) Vaccination of broilers and layers with Tribacto- pulvis heat inactivated vaccine which was prepared from Salmonella, E. coli, and P. aeruginosa resulted in reduction in death by 30–50%, increasing in weights 100–200 g and less coccidiosis and infectious Chemical inactivants h d Attenuated or avirulent bacteria can be used as vehi- cles for the effective delivery of vaccine candidates (Rap- puoli et al. 2011). Attenuated Salmonella strains are often used in poultry for the control of salmonellosis and they can serve as safe and effective oral carrier vac- cines to prevent NE by expressing heterologous antigens (Jiang et al. 2015). Live attenuated vaccines In this type of vaccine, the living microorganisms are ei- ther avirulent or rendered avirulent by attenuation; this means that these pathogens are capable of multiplication within the host but are incapable of causing diseases. Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 Page 3 of 7 Page 3 of 7 from a virulent APEC did not cause mortality in chal- lenged birds and systemic lesions were significantly re- duced when birds were given three vaccinations on days 1 (aerosol), 14 (oral), and 28 (oral) (Amoako et al. 2004). bronchitis in vaccinated birds (Cambir 1999). Chick embryos were orally immunized at day 16 of incuba- tion by injection of heat-killed C. jejuni organisms into the amniotic fluid which increased antibody re- sponse in hatched chicks (Noor et al. 1995) which was higher than that of embryos immunized with sol- uble C. jejuni antigen (Noor 1998). Effective inacti- vated E.coli vaccines against serotypes including O2:Kl and O78:K80 have been produced and provide protec- tion against the homologous serogroups not against heterologous serogroups (Saif et al. 2003). Vaccination of fattening turkey flock with live com- mercial S. enteritidis at day 1 of age via spray and boostered at 6 and 11 weeks of age via drinking water did not reduce shedding or colonization of internal organs when birds were challenged with S. enteritidis PT4 (Krüger et al. 2008). Live, low virulence microorganism Intravenous infection of pigeons with the low virulence Streptococcus gallolyticus (S. gallolyticus) serotype I strain PDH827 did not induce clinical protection against challenge with high virulence serotype I strain STR357 (Kimpe et al. 2002). The treatment of chicks with viable non-colonizing strains of C. jejuni intramuscularly as a possible vaccine with or without adjuvant was failed to induce protective immunity (Ziprin et al. 2002). Inactivated (killed) vaccines Inactivated vaccines are generally whole bacterial prepa- rations combined with an adjuvant and the cultured pathogens are rendered non-infectious by killing and are used for immunization. Killed bacterial vaccine are called bacterin, and killing microbial pathogens is achieved by using one of the following methods: Radiation Immunization of chicks with gamma irradiated (cobalt 60) bivalent Pseudomonoas aeruginosa (Ps. aeruginosa) Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 Page 4 of 7 (2020) 44:15 Page 4 of 7 dose while double dose vaccine increased signs of botu- lism and the vaccine can be used to wild birds during botulism epizootics (Rock et al. 2000). Samonella toxins (enterotoxin plus cytotoxins) were the main virulent products of the organisms formalized (FT) and carbon- ated (CT) toxoids were prepared from partially purified toxins of S. enterica ser. weltevreden and Gallinarum. Complete protection could be obtained in birds vacci- nated with FT of S. weltevreden plus Freund’s complete adjuvant (FCA) following homologous or heterologous (S. gallinarum and S. typhimurium) challenges while protection ranged from 50 to 83.3% in the groups immu- nized with other preparations of S. weltevreden, i.e., with FT without FCA or with CT with or without FCA. Galli- narum toxins (FT) given with FCA afforded 100% pro- tection against homologous challenge, but not against heterologous serovars (Mishra and Sharma 2001). Chicks received vit. E adjuvant salmonella toxoid; high lympho- cyte stimulation was recorded and the vaccinated chicks were protected against Salmonella challenge (Barman et al. 2000). Several trials have shown that chickens could be protected against C. perfringens-induced nec- rotic enteritis (NE) by injection with inactive and active toxins (Jang et al. 2012; Kulkarni et al. 2007) and anti- genic proteins (Jiang et al. 2015). vaccine recorded protection by 100%, 96%, and 90% post challenge intramuscular, subcutaneously and orally vaccinated chicken groups, respectively; also, the vaccin- ation of layers with the same vaccine stimulated the for- mation and concentration of P. aeruginosa-specific Igy in the egg yolk (Mohamed et al. 2002). Chickens were immunized intraocular with liposome associated S. enteritidis antigen; the antigen was prepared by ultraso- nicated whole cell extract of the bacteria. The vaccine induced increase in the specific antibody producing lymphocytes in the intestinal tract and immunoglobulin secreted in the intestine inhibited the adherence of the bacteria to intestinal epithelial cells suppressing the spread of the bacterial infection in the host (Fukutome et al. 2001). Combined bacterins Two mixed bacterins from E. coli, Staphylococcus aureus, and Clostridium perifringens (C. perifringens) or Clostridium septicum (C. Radiation septicum) were used for immunization of chickens against gangrenous dermatitis; the vaccines were found to be safe and they protected the birds against challenge with live cultures of the bacteria without any untoward reactions (Kaul et al. 2001) Both the heat and formalin inactivated aluminum pre- cipitated vaccines prepared with the virulent E. coli iso- lates was effective to protect chickens of different age against various forms of avian colibacillosis (Rashid et al. 2001). A developed combined vaccine was prepared from trivalent E. coli vaccine (serotypes O1, O2, and O78) and an inactivated Newcastle disease vaccine in- duced a high degree of protection in layers and chicks (Erganis et al. 2002). Whole-cell proteins Immunization of chickens with surface antigens proteins of E. coli induced highly systemic and mucosal antibody responses (Kariyawasam et al. 2002). Ammonum sulfate perceptible protein (ASPP) of Pasteurella multocida serotype 6 B yielded three protein fractions, which can be used to develop a subunit vaccine against haemor- rhagic septicemia in rabbits (Srivastava 1999). S. enteriti- dis OMPs of 75.6 and 82.3 KDa were effective in reducing colonization of S. enteritidis on intestinal mu- cosa in chickens (Khan et al. 2003). Chicks were immu- nized with C. jejuni OMPs vaccine (44–80 KDa); it reduced the infection after challenge and increased the serum antibody titer (Abd-Aty and Rabie 2003). Simultaneous use of inactivated and live vaccines The use of a live P. multocida vaccine followed by a killed P. multocida vaccine, two live vaccines, or a killed vaccine followed by live vaccine provides almost equal immunity when measured by enzyme-linked immuno- sorbent assay (ELISA) titers (Hofacre et al. 1987). Parent chickens were vaccinated with live S. typhimurium and inactivated S. enteritidis induced an increase in antibody concentration in sera and jejunum of the chicks (Math- ner et al. 2002). Laying hens should be vaccinated with live and killed vaccines to stimulate mucosal and sys- temic immunity and reduce the prevalence of S. enteriti- dis-contaminated eggs (Davies and Breslin 2004). Subunit vaccines They are prepared from one or few immunogenic epi- topes that are found an infectious agent. Among the sur- face epitopes of an entigen molecule, few epitopes are important in stimulating protective immunity. Metabolic product vaccines (toxoids) Forty-five kilodaltons protein is considered to be a major OMP of C. jejuni and has immunogenic effect in chick- ens (Lam 1992). The immunodominant protein antigen of C. jejuni is subunit molecular weight of 59 to 61 KDa (Dubreuil et al. 1990). Chicks were immunized with C. jejuni OMPs vaccine (44–80 KDa); it reduced the These are soluble toxins that are rendered harmful (non-toxin) by addition of formalin or by gentle heating; this way does not affect the immunogenic properties of the toxin. Ducks were immunized with a type of botu- linum toxin; it induced partial protection but in a single Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 Page 5 of 7 Page 5 of 7 Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 Vaccines produced by recombinant DNA technology infection after challenge and increased the serum anti- body titer (Abd-Aty and Rabie 2003). S. enteritidis OMPs of 75.6 and 82.3 KDa were effective in reducing colonization of S. enteritidis on intestinal mucosa in chickens (Khan et al. 2003). These vaccines depend upon identification or isolation of antigenic-coding gene. Then using recombinant tech- nology transgenic implementation of the isolated gene in a bacterial vector like E. coli or yeast cells is performed. The expressed gene products of the grown culture is purified and used for immunization. Messenger RNA, which codes for the chosen proteins, is copied to pro- duce a complementary DNA (cDNA) strand. This DNA strand can also be copied to produce a second strand. The double-stranded form of cDNA is then ligated to a cloning vector (plasmid) which is ready to be cloned using cloning host. The cloned recombinant DNA (rDNA) is then expressed by transformation into bacter- ial vector usually E. coli or yeast cells which act as pro- duction factories for the selected protein. The produced recombinant antigen is identified using selectable markers. These identified recombinant proteins could be injected into birds or animals as vaccine candidate (Babiuk et al. 2003). The immunization of chickens with a temperature-sensitive mutant E/1/3 of S. enteritidis in- duced strong protection against virulent S. entritidis strain after oral challenge and reduced the caecal and spleen colonization and the number of faecal shedding. (Cerquetti and Gheradi 2000). Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by homologous and heterologous sero- vars (Dueger et al. 2003). Gene E leads to emptying pas- terulla cell envelops which are called bacterial ghosts. P. multocido and Pasteurella haemolytica (P. haemolytica) ghosts produced by expression of phage phi X174 lysis gene E are used as a vaccine for immunization of rabbits producing 100% protection (Marchart et al. 2003). Three Campylobacter jejuni 72/D2/92 genes (CjaA (omph), cjac (hisj), and cjaD (omp18)) encoding immunodomi- nant proteins are considered to be potential chicken vac- cine candidates (Pawelec et al. 2000). DNA vaccines DNA encoding the gene of antigen protein is ligated to a plasmid. Direct inoculation of this plasmid DNA into the host tissues which is able to cause ex- pression of the encoded antigen protein within the transfected cells. The expressed protein stimulates the host immune system to produce specific immune responses. DNA vaccine offers many advantages over the previously mentioned vaccines; there is no risk of infection, no purification costs, or antigen de- naturation during preparation. Also, the endogenous synthesis of microbial antigen strongly enhances the cell mediated immunity; its strong stability reduces the costs of cold chains requirement by 80%. DNA vaccines do not interfere with the maternal Purified flagellar proteins (flagellin) Immunization of broiler chicks with purified native flagellin or combined heat killed C. jejuni and fla- gellin induced reduction in the number of Campylo- bacter in caecal contents. Flagellin (61–63 KDa) and possibly the 67 KDa antigen may be valuable for immunological control of C. jejuni and used as vac- cine candidates. (Widders et al. 1998). Flagella and whole cell extraction were used as antigens for de- tection of antibodies to S. enteritidis in serum and egg yolk of infected hens by agar gel preciptin test while SEF14 (a 14-KDa fimbrial protein) was not re- active (Holt et al. 2000). Immunization of chicks with P. aeruginosa whole cell proteins of strain D and E (20–205 KDa for each strain) and flagellin (53.277 KDa and 54.184 KDa, respectively) revealed high immunological responses and reduced infection in chicks but the whole cell oil adjuvant vaccine re- corded best results than the flagellar oil adjuvant vaccine (Rabie and Zou El Fakar 2004). Fimbrial proteins Immunization of hens with Sef A and Fim A fimbrial proteins of S. enteritidis induced strong humoral im- mune response similar to that obtained with live bac- teria. Sef A and Fim A can be considered as components of subunit vaccines (Kisiela et al. 2003) Pilus proteins l Intranasal vaccination of broiler chickens with four avian pathogenic E. coli surface antigens, F pilus adhesin, P pilus adhesion, aerobactin receptor pro- tein, and lipopolysaccharide (LPS) induced high im- mune response (high titer of IgG, IgA, and IgM) and did not induce the disease after challenge. They ap- pear to be suitable candidates for a vaccine (Kariya- wasam et al. 2002). Lipopolysaccharides Capsular polysaccharide subunit vaccines for E. coli can be prepared by extraction of capsular polysaccharide and soluble bacterial protein through water–bath inactiva- tion and used for immunization of chickens and pro- vided high protection (Shujian et al. 1996). Page 6 of 7 Rabie and Amin Girh Bulletin of the National Research Centre (2020) 44:15 Page 6 of 7 (2020) 44:15 immunity and single dose can induce long term im- munity (Oshop et al. 2002). Babu U, Scott M, Myers MJ, Okamura M, Gaines D, Yancy HF, LillehoJ H, Heckert RA, Raybourne RB (2003) Effects of live attenuated and .killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens. Vet Immunol and Immunopathology 91(1):39–44 Conclusion Bachtiar EW, Sheng K, Fifis T, Gamvrellis V, Plebanski M, Coloe PJ, Smooker PM (2003) Delivery of a heterologous antigen by a registered Salmonella vaccine (STM1). FEMS Microb Lett 277(2):211–217 Protection studies against bacterial diseases were per- formed by using several trials: living vaccines (live atten- uated vaccines; live, non-pathogenic microorganisms; live, low virulence microorganism) Barbour EK, Hamadeh SK, Eidt A (2000) Infection and immunity in broiler chicken breeders vaccinated with a temperature-sensitive mutant of Mycoplasma gallisepticum and impact on performance of off spring. Poult Sci 79:1730– 1735 Inactivated vaccines (heat-inactivated, chemical inacti- vates, radiation) are metabolic product vaccines (Tox- oids), subunit vaccines (whole cell proteins, outer membrane proteins, purified flageller proteins (flagellin), fimbrial proteins, pilus proteins, lipopolysaccharides), vaccines produced by recombinant DNA technology, and DNA vaccines. The chicken farms must be care for using bacterial vaccines. Barman, T.K. . Sharma, VD and .Subodh, KU. (2000): Optimization of dose of Salmonella toxoid vaccine in poultry. Ind Vet J.79, (2)106-110 Barrow PA, Lovell MA, Stocker BAD (2000) Protection against experimental fowl typhoid by parenteral administration of live SL5828, an aro A-ser C (aromatic dependent) mutant of a wild-type S. Gallinarum Strain Made Lysogenic For p22 Sie Avian Path. 29(5):243–431 Berndt A, Methner U (2001) Gamma\ delta T cell response of chickens after oral administration of attenuated and non attenuated Salmonella typhimurium strains. Vet Immunol Immunopathol 78(2):143–161 Berndt A, Methner U (2001) Gamma\ delta T cell response of chickens after oral administration of attenuated and non attenuated Salmonella typhimurium strains. Vet Immunol Immunopathol 78(2):143–161 Cambir S (1999) Avian colibacillosis, Salmonellosis and Pseudomonas prevention and control by Tribacto-pulvis inactivated vaccine. Revista Romana –de- Medicina- veterinara 9(4):383–396 Cambir S (1999) Avian colibacillosis, Salmonellosis and Pseudomonas prevention and control by Tribacto-pulvis inactivated vaccine. Revista Romana –de- Medicina- veterinara 9(4):383–396 Recommendations Cerquetti MC, Gheradi MM (2000) Orally administered attenuated salmonella enteritidis reduces chicken cecal carriage of virulent salmonella challenge organisms. Vet Microbiol 76(2):185–192 Bacterial vaccines need more investigations and re- searches because most farms depend on the use antibi- otics for treatment when spread of bacterial diseases. Chukiatsiri, K, Chotinun, S. and N. Chansiripornchai, 2010 An Outbreak of Avibacterium paragallinarum serovar B in a Thai Layer FarmThe Thai veterinary medicine 40(4):441-444 · December 2010. Consent for publication Not applicable Fukutome K, Watarai S, Mukamoto M, Kodama H (2001) Intestinal mucosal immune response in chickens following intraocular immunization with liposome-associated S. Enterica servor enteritidis antigen Developmental and comparative Immunology 25(5-6):475–484 Acknowledgements Dueger EL, House JK, Heithoff DM, Mahan MJ (2003) Samonella DNA adenine methylase mutants prevent colcnization of newly hatched chickens by homologous and heterologous serovars. Int. J. of food Microb 80(2):153–159 Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Gong Y, Zhang P, Wang H et al (2014) Safety and efficacy studies on trivalent inactivated vaccines against infectious coryza. Vet Immunol Immunopathol 158:3–7 Received: 19 August 2019 Accepted: 18 December 2019 Hassan JO, Curtiss RI (1997) Efficacy of live aviruelnt S. typhimurium vaccine in preventing coionization and invasion of lyaing hens by S. typhimurium and S. enteritidis. Avian Dis 41(4):783–791 Hofacre CL, Glisson JR, Kleven SH (1987) Comparsion of vaccination protocols of broiler breeder hens for Pasteurella mutocida utilizing enzyme-linked immunosorbent assay and virulent challenge. Avian Dis. 31:260–263 Holt PS, Stone HD, Gast PK, Geen CR (2000) Application of the agar gel precipitin Hassan JO, Curtiss RI (1997) Efficacy of live aviruelnt S. typhimurium vaccine in preventing coionization and invasion of lyaing hens by S. typhimurium and S. enteritidis. Avian Dis 41(4):783–791 Abbreviations Davies R, Breslin M (2004) Observations on Salmonella contamination of eggs from infected commercial laying flocks where vaccination for Salmonella enterica serovar Enteritidis has been used. Avian Pathol. 33:133–144 Abbreviations C. jejuni: Campylobacter jejuni; E. coli: Escherichia coli; M. gallisepticum: Mycoplasma gallisepticum; M. synoviae: Mycoplasma synoviae; NTG: Nitroguanidine; O. rhinotracheale : Ornithobacterium rhinotracheale; P. multocida: Pasteurella multocida; Ps. aeruginos: Pseudomonoas aeruginosa; S. enterica: Salmonella enterica; S. entertidis: Salmonella enteritidis; S. gallinarum: Salmonella gallinarium; S. gallolyticus: Streptococcus gallolyticus; S. typhimurium: Salmonella typhimurium Dubreuil JD, Kostostrzynska M, Logan SM, Harris LA, Austin JW, Trust TJ (1990) Purification, characterization and localization of protein antigen shared by thermophilic Campylobacters. J Clin Microbiol June:1321–1328 Dubreuil JD, Kostostrzynska M, Logan SM, Harris LA, Austin JW, Trust TJ (1990) Purification, characterization and localization of protein antigen shared by thermophilic Campylobacters. J Clin Microbiol June:1321–1328 gallinarum: Salmonella gallinarium; S. gallolyticus: Streptococcus gallolyticus; S. typhimurium: Salmonella typhimurium Duchatel JP, Ree JM, Vindevogel H (1998) Evaluation of the optimal vaccine for vaccination trials of pigeons against paratyphoid by subcutaneous injection de. Medecine Veterinaire 142(1):35–38 Duchatel JP, Ree JM, Vindevogel H (1998) Evaluation of the optimal vaccine for vaccination trials of pigeons against paratyphoid by subcutaneous injection de. Medecine Veterinaire 142(1):35–38 Authors’ contributions Erganis O, Hadimli H, Solmaz H (2002) Vaccine development from serotypes O1 , O2 and O78 of E.Coli against avian colibacillosis: layer chickens. Turk Ve Hayvancilik DEGISI 26(6):1213–1221 NSR and ZMSAG contributed to the data collection in addition to participating in writing the manuscript. Both authors read and approved the final manuscript. 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J Antibiot 50(2):187–197 Noor SM (1998) The mucosal and systemic immune responses in chickens orally immunized with C.jejuni antigen enterapped in polylactide-co-glycolide microparticles. J Immu-Ternak-den-Veteriner 3(4):264–269 Noor SM, Husband AJ, Widders RP (1995) An ova oral vaccination with C.jejuni established early development of intestnal immunity in chickens. Br Poultry Sci 36:563–573 Oshop GL, Elankumaran S, Heckert RA (2002) DNAvaccination in the avian. Vet Imm Immunopathol 89(1-2):1–12 Mohamed M, El-Jakee J, Abo-Alyazeed H, Mossa I, Zahran AM (2002) R. efficacy of Ps. aeruginosa irradiated vaccine in chickens. Vet Med J Giza 50(2):709–719 Morrow CJ, Markham JE, Whithear KG (1998) production of temperature-sensitine clones of M.synoviae for evaluation as live vaccines. Avian Dis. 42:667–670 Nakae M, Sugahara Y, Yasui H, Imai C, Hasegwa Y, Osaka K, Shibasaki K (1997) Serotypes and drug susceptibility of Ps.aeruginosa isolated from clinical specimens. Japan. 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https://openalex.org/W4285131624	https://cedr.tsue.uz/index.php/journal/article/download/440/413	Kirghiz, Kyrgyz	null	MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN	Economics and education	2,022	cc-by-sa	5,060	O‘ZBEKISTON RESPUBLIKASIDA TASHQI IQTISODIY FAOLIYAT O‘ZGARISHLARI MONITORINGI Tursunova Munisxon Rahimdjanovna, Тoshkent kimyo-texnologiya instituti, Sanoat iqtisodiyoti va menejmenti kafedrasi, v.b. dotsenti Tursunova Munisxon Rahimdjanovna, Тoshkent kimyo-texnologiya instituti, Sanoat iqtisodiyoti va menejmenti kafedrasi, v.b. dotsenti https://doi.org/10.55439/ECED/vol23_iss2/a17 Annotatsiya. Mamlakatimizning chet davlatlar bilan iqtisodiy aloqalarini yanada mustahkamlash va xorijda respublikamizning iqtisodiy imkoniyatlarini keng targ‘ib qilish orqali uning xalqaro nufuzini yanada oshirish, investitsiyalarni jalb qilishni jadallashtirish, turizm sohasini yanada rivojlantirish iqtisodiy sohadagi ustuvor vazifalarimizdan biri hisoblanadi. O‘zbekiston iqtisodiyotini rivojlantirish strategiyasi diversifikatsiyalangan va raqobatbardosh iqtisodiyotni yaratishga, innovatsion texnologiyalar va bozorni boshqarishning yangi usullaridan foydalangan holda milliy resurslardan maksimal darajada foydalanishga yo‘naltirilgan. Mamlakatning tashqi iqtisodiy faoliyati, xususan, tashqi savdo sohasida amalga oshirilayotgan islohotlarning pirovard natijasi aholining turmush darajasiga bevosita o‘z ta’sirini ko‘rsatadi. Mamlakatimizda amalga oshirilayotgan ijtimoiy-iqtisodiy islohotlar iqtisodiyotning barcha tarmoqlari va sohalarida ishlab chiqarish samaradorligini oshirish orqali uzoq muddatli barqaror iqtisodiy o‘sishni ta’minlashga qaratilgan. Respublikamizda investitsiya siyosatini takomillashtirish va xorijiy investitsiyalarni jalb etish bo‘yicha qator ijobiy ishlar amalga oshirildi. Jalb etilgan xorijiy investitsiyalarning asosiy qismi real sektor korxonalarini rivojlantirish va ularni texnik-texnologik yangilashga yo‘naltirilgan. g g g Kalit so‘zlar: iqtisodiyot, rivojlantirish, iqtisodiy soha, tashqi iqtisodiy faoliyat, tashqi savdo, investitsiya, raqobat, eksport, import, xorijiy davlatlar, ekspert, kredit, tashqi savdo aylanmasi, xalqaro bozor, yuridik yoki jismoniy shaxs, faol tashqi savdo balansi, eksportyor, importyor. MENEJMENT VA MARKETING MENEJMENT VA MARKETING Турсунова Мунисхон Рахимджановна, и.о доцента кафедры «Экономика и менеджмент промышленности» Ташкентского химико- технологического института, Аннотация. Одним из наших приоритетов в экономической сфере является дальнейшее укрепление экономических связей нашей страны с зарубежными странами и повышение ее международного престижа, ускорение привлечения инвестиций, дальнейшее развитие туристической отрасли за счет продвижения экономического потенциала нашей страны за рубежом. Стратегия экономического развития Узбекистана направлена на создание диверсифицированной и конкурентоспособной экономики, максимальное использование национальных ресурсов с использованием инновационных технологий и новых методов управления рынком. Внешнеэкономическая деятельность страны, особенно в сфере внешней торговли, оказывает прямое влияние на уровень жизни населения в результате реформ. Проводимые в нашей стране социально-экономические реформы направлены на обеспечение долгосрочного устойчивого экономического роста за счет повышения эффективности производства во всех секторах и отраслях экономики. В стране был предпринят ряд позитивных шагов по совершенствованию инвестиционной политики и привлечению иностранных инвестиций. Основная часть иностранных инвестиций направляется на развитие предприятий реального сектора и их техническую и технологическую модернизацию. Ключевые слова: экономика, развитие, сфера экономики, внешнеэкономическая деятельность, внешняя торговля, инвестиции, конкуренция, экспорт, импорт, зарубежные страны, эксперт, кредит, внешнеторговый оборот, международный рынок, юридическое или физическое лицо, активный внешнеторговый баланс, экспортёр, импортёр. Iqtisodiyot va ta'lim / 2022-yil 2-son MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN muvozanatli va oʻzaro manfaatli aloqalar yuritish, siyosiy ishonch va xalqaro huquqqa rioya etilishi asosidagi oʻzaro hamkorlikka intilish – bizning xori- jiy sheriklarimiz Oʻzbekiston Prezidenti Shavkat Mirziyoyev olib borayotgan tashqi siyosiy kursni aynan ana shunday tavsiflamoqda [11]. Bugungi kunda tashqi savdo respublikamiz iqtisodiy faoliyatining asosiy qismi bo‘lib xizmat qil- moqda. Ushbu sohada davom ettirilayotgan islohot- lar mahalliy va xalqaro bozorlarni mahsulot va xiz- matlar bilan ta’minlash imkonini bermoqda. Yurti- mizda tashqi iqtisodiy faoliyatni rivojlantirish bora- sida amalga oshirilayotgan keng qamrovli islohotlar va dunyo mamlakatlari bilan olib borilayotgan mus- tahkam aloqalar natijasida mamlakatimizning eks- port salohiyati oshib bormoqda. Soʻnggi yillarda Prezidentimiz Shavkat Miromonovich Mirziyoyev tashabbusi bilan Oʻzbe- kiston hayotining ijtimoiy-siyosiy, iqtisodiy, mada- niy-maʼrifiy va boshqa sohalarida muhim va dolzarb ulkan islohot hamda oʻzgarishlar izchil amalga oshi- rilmoqda. 2020-yilda pandemiya oqibatida yuzaga kelgan xalqaro miqyosdagi iqtisodiy va ijtimoiy qiyinchiliklar, ayrim mintaqalardagi umumiy ahvol va davlatlararo munosabatlarning keskinlashishi tashqi siyosatimizdan yangi maqsad va yondashuv- larni talab etmoqda[7]. Shuningdek, hukumatimiz tomonidan bir qa- tor soliq, bojxona imtiyozlarining yaratilishi, ishlab chiqarishni modernizatsiya qilish va raqobatbar- dosh mahsulotlarning ishlab chiqarilishi milliy mah- sulotlarimizning tashqi bozorlarda mustahkam o‘rin egallashlari uchun zamin yaratmoqda. Mavzuga oid adabiyotlar tahlili. Jahon iqti- sodiyotidagi inqiroz hodisalari bilan tavsiflangan globallashuvning zamonaviy bosqichi davlatlar o‘r- tasidagi tashqi iqtisodiy aloqalarning tabiati va shakllariga sezilarli ta’sir ko‘rsatadi. Rivojlangan va ayrim rivojlanayotgan davlatlarning jadal rivojlani- shi bilan bir qatorda, mamlakatlar o‘rtasida nisba- tan kichik davlatlar guruhi doirasida xizmatlar, ka- pital, mehnat va axborot mahsulotlarining xalqaro oqimlari o‘rtasida farqlanish kuchaymoqda. Rivojla- nayotgan mamlakatlar so‘nggi o‘n yil ichida xalqaro tizimda o‘z pozitsiyalarini kuchaytirgan bo‘lsa-da, shu bilan birga, rivojlangan davlatlar o‘zlarining ilmiy va texnologik afzalliklariga tayanib, ishlab chi- qarishda innovatsiyalarni rag‘batlantirib, jahon iqti- sodiyotida yetakchilar bo‘lib, ular xalqaro tovar va xizmatlar savdosida bir tomonlama afzalliklarga ega bo‘lishga imkon beradi [1]. Oʻzbekiston Respublikasi davlat mustaqilligi- ning 30 yilligini izchil ichki islohotlar va yangilan- gan tashqi siyosat bilan kutib olmoqda. Mamlakat ichki hayotida kuzatilayotgan ochiqlik, innovatsiya va yangilanishlar, shuningdek, iqtisodiy liberallash- tirish, bozorni rivojlantirish, tadbirkorlik muhitini yaxshilash, raqamlashtirish, yangi texnologiyalarni joriy qilish, taʼlim va maʼrifat, fuqarolar faolligi va inson huquqlari himoyasiga eʼtibor – bularning bar- chasi, shubhasiz, mamlakatimizning tashqi siyosati- ga taʼsir oʻtkazmoqda. Aksariyat xalqaro ekspertlar va xorijiy siyo- satchilar fikricha, bugun Oʻzbekiston oʻzining misli koʻrilmagan ochiqligi va jadalligi bilan ajralib turgan izchil, aniq va konstruktiv siyosat yuritmoqda. MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN Tursunova Muniskhon Rakhimdjanovna, Acting Associate Professor of the Department “Economics and Management of Industry” Tashkent Institute of Chemical Technology Tursunova Muniskhon Rakhimdjanovna, Acting Associate Professor of the Department “Economics and Management of Industry” Tashkent Institute of Chemical Technology Abstract. One of our priorities in the economic sphere is to further strengthen the economic ties of our country with foreign countries and increase its international prestige, accelerate the attraction of investments, and further develop the tourism industry by promoting the economic potential of our country abroad. The economic development strategy of Uzbekistan is aimed at creating a diversified and competitive economy, the maximum use of national resources using innovative technologies and new methods of market management. The country’s foreign economic activity, especially in the field of foreign trade, has a direct impact on the standard of living of the population as a result of reforms. Iqtisodiyot va ta'lim / 2022-yil 2-son Iqtisodiyot va ta'lim / 2022-yil 2-son 110 MENEJMENT VA MARKETING The socio-economic reforms carried out in our country are aimed at ensuring long-term sustainable economic growth by increasing production efficiency in all sectors and sectors of the economy. The country has taken a number of positive steps to improve investment policy and attract foreign investment. The bulk of foreign investment is directed to the development of enterprises in the real sector and their technical and technological modernization. Key words: economy, development, economic sphere, foreign economic activity, foreign trade, investment, competition, export, import, foreign countries, expert, credit, foreign trade turnover, international market, legal entity or individual, active foreign trade balance, exporter, importer. “Iqtisodiyotimizning barqaror sur’atlar bilan o‘sishini ta’minlashdek muhim tamoyil va ustuvor vazifani amalga oshirish, davlat rahbari sifatida men uchun strategik vazifa bo‘lib qoladi. Biz, avvalo, iqtisodiyotni isloh qilish va erkinlashtirish borasidagi ishlarimizni yanada chuqurlashtirish, uning soha va tarmoqlarini tarkibiy jihatdan o‘zgartirish bo‘yicha boshlangan ishlarni jadallashtirishimiz kerak”[9]. O‘zbekiston Respublikasi Prezidenti Sh.M. Mirziyoyev Kirish. Bugungi kunda mamlakatimizdagi islohotlar, eng avvalo, dunyoda, shu jumladan, jahon iqtisodiyoti konyunkturasida yuz berayotgan turli o‘zgarishlarga mos holda amalga oshirilmoqda. Tan- lab olgan yo‘limiz global inqirozdan keyin jahon iqtisodiyoti rivojlanish sur’atlari pasaygan bir sha- roitda o‘zining barqarorligi va hayotiyligini ko‘rsa- tayotir. Mamlakatimizda keyingi yillarda tashqi siyosat sohasida amalga oshirilayotgan islohotlar mintaqamizda tinchlik va barqarorlikni mustah- kamlash, mamlakatimizning xalqaro maydondagi salohiyatini kengaytirish va xorijiy davlatlar bilan keng qamrovli hamda oʻzaro manfaatli aloqalarini rivojlantirishga xizmat qilib kelmoqda. Iqtisodiyot va ta'lim / 2022-yil 2-son MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN Mamlakatimizning iqtisodiy ochiqligi hamda inves- titsiyalar va biznes uchun jozibadorligi, barcha muammoli masalalarni yakdillik, oʻzaro hurmat va hamjihatlik asosida hal etishga intilish, dunyoning yetakchi davlatlari va boshqa mamlakatlari bilan Xeksher-Olinning savdo nazariyasi sifatida mashhur bo‘lgan nazariya birinchi o‘ringa ishlab chiqarish chiqimlarini emas, balki qanday resurslar ana shu chiqimlarga kirishini qo‘yadi, chunki mam- lakatlar chiqimlarida ularda ortiqcha mavjud bo‘l- Iqtisodiyot va ta'lim / 2022-yil 2-son 111 MENEJMENT VA MARKETING gan resurslardan foydalanilgan tovarlarni eksport qilishga intiladi. iqtisodiy yordamning turli shakllarini o‘z ichiga ola- di, ilmiy-texnik hamkorlik, mutaxassislik, ishlab chi- qarish, xizmat ko‘rsatish va qo‘shma tadbirkorlikdir [3]. Boshqa nazariy qarashlar ham mavjud. Lekin ular orasidan, birinchidan, olimlar va amaliyotchilar o‘rtasida keng tarqalganini, ikkinchidan, makroiqti- sodiy tusdaligi ifodalangan va uchinchidan, eng umumlashgani bilan ajralib turadiganlarini olamiz. Yu.M.Rostovskiy ta’kidlashicha, barcha tashqi savdo aylanmasi asoslari hali ham davom etmoqda, turli xil tovarlarni moddiy shaklda eksport qilish va import qilish, tovar ayirboshlash operatsiyalari, tur- li xizmatlarni ko‘rsatish, xorijiy pudratchilar bilan hamkorlik qilishda muayyan ishlarni bajarish kabi tashqi savdo hisoblanadi [4]. Xalqaro savdo mazmuniga doir biz bayon etgan barcha yondashuvlarni ancha osonlik bilan dastlabki ikki qarashga kiritsa bo‘ladi. Zero, rav- shanki, mamlakatda ortiqcha bo‘lgan resurslar sarf- lanadigan mahsulot ham, tovarning hayot davri ham, miqyos samaralari ham − ularning barchasi ishlab chiqarish chiqimlariga bevosita aloqadordir. Shuning uchun dastlabki ikki qarash eng universal doktrinalar bo‘lib, qolganlari esa ularni aniqlashti- rish hisoblanadi. Tashqi iqtisodiy faoliyatni amalga oshirish bojxona qonunchiligiga muvofiq amalga oshiriladi. Iqtisodiyotning o‘sishi sharoitida katta va kichik biznesning ko‘plab rus korxonalari jahon bozoriga kirishmoqda. Har yili ko‘plab xalqaro shartnomalar tuziladi, nafaqat tovarlar, balki uskunalar ham sotib olinadi va eksport mavzusi nafaqat xomashyo, balki o‘z mahsulotlarini ham o‘z ichiga oladi. Bunday fao- liyatni amalga oshirish orqali Rossiya kompaniya- lari tashqi iqtisodiy faoliyat ishtirokchilari bo‘- ladi[20]. Shuni ta’kidlash joizki, xalqaro savdo tashqi iqtisodiy faoliyat barcha qolgan shakllari va turlari- ning boshlang‘ich, muvofiqlashtiruvchi va ko‘payti- ruvchi negizi hisoblanadi. Tashqi iqtisodiy faoliyat- ning xorijiy sarmoyani jalb etishdek shaklining sa- maradorligi ham uning darajasiga bog‘liq. Savdo- dagi qonun hujjatlari tomonidan belgilangan va boshqa cheklashlar investitsiya jarayonlarida aks etadi [2]. [ ] Tadqiqot metodologiyasi. Tashqi iqtisodiy Tadqiqot metodologiyasi. Tashqi iqtisodiy aloqalar haqida ilmiy maqola yozish uchun tashqi iqtisodiy faoliyat bo‘yicha barcha adabiyotlar o‘rga- nib chiqildi. Tashqi iqtisodiy faoliyat bo‘yicha amal- ga oshirilayotgan islohotlar o‘rganildi. O‘rganilgan adabiyotlar natijasida mavzu to‘liq yoritib berildi. Mavzuni yoritish jarayonida guruhlash, sintez qilish, statistik ma’lumotlarni tahlil qilish, analiz qilish me- todlaridan foydalanildi. Iqtisodiyot va ta'lim / 2022-yil 2-son MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN O‘zbekiston Res- publikasidan tashqaridagi investitsiya faoliyati O‘zbekiston Respublikasidan tashqaridagi tadbir- korlik va boshqa faoliyat obyektlariga moddiy va nomoddiy ne’matlarni hamda ularga bo‘lgan huquq- larni qo‘yish bilan bog‘liq tashqi iqtisodiy faoliyat subyektlari harakatlarining yig‘indisi O‘zbekiston Respublikasidan tashqaridagi investitsiya faoliyati deb e’tirof etiladi[8].  tashqi iqtisodiy faoliyat subyektlarining tengligi;  tashqi iqtisodiy faoliyat subyektlarining tengligi;  tashqi iqtisodiy faoliyat subyektlarining tengligi;  savdo-iqtisodiy munosabatlarni amalga oshirishda kamsitishlarga yo‘l qo‘yilmasligi;  tashqi iqtisodiy faoliyatni amalga oshirish- dan o‘zaro manfaatdorlik;  tashqi iqtisodiy faoliyat subyektlarining hu- quqlari va qonuniy manfaatlari davlat tomonidan himoya qilinishi. Oldi-sotdi yoki ayirboshlash obyekti bo‘lgan tovarlar, ishlar (xizmatlar), har qanday mol-mulkka, shu jumladan, qimmatli qog‘ozlar, valyuta qimmat- liklariga, elektr, issiqlik energiyasi va boshqa turda- gi energiyaga, transport vositalariga, intellektual mulk obyektlariga nisbatan tashqi iqtisodiy faoliyat- da foydalanilishi qonun hujjatlari bilan ta’qiqlan- ganlarini istisno etganda, tashqi iqtisodiy faoliyat amalga oshirilishi mumkin[9]. Oʻzbekiston Respublikasida tovarlar tashqi savdosi statistik koʻrsatkichlarini shakllantirishda, Tashqi iqtisodiy faoliyat tovar nomenklaturasi (ke- yingi oʻrinlarda TIF TN) bilan birgalikda, Birlashgan Millatlar Tashkilotining Xalqaro standartlashgan savdo tasniflagichi (keyingi oʻrinlarda XSST) qoʻlla- niladi. Bu, oʻz navbatida, tahliliy maqsadlarda eks- port va import qilinadigan tovarlarni 10 ta katta qismga guruhlash uchun imkon beradi. Tovarlarni XSST boʻyicha kodlashtirish TIF TN va XSST oʻrtasi- da oʻtish kalitlari yordamida amalga oshiriladi[18]. Tashqi iqtisodiy faoliyatning asosiy yo‘nalish- lari quyidagilardir: Tashqi iqtisodiy faoliyatning asosiy yo‘nalish- lari quyidagilardir: 1. Xalqaro iqtisodiy va moliyaviy hamkorlik. 2. Tashqi savdo faoliyati. 2. Tashqi savdo faoliyati. Oʻzbekiston jahonning 174 ta mamlakati bilan savdo aloqalarini amalga oshirib kelmoqda. 2021-yilning yanvar-avgust oylari yakuni bilan respublikaning tashqi savdo aylanmasi (matnda TSA) 24 963,7 mln. AQSh dollarini tashkil etib, 2020-yilga nisbatan 383,1 mln. AQSh dollariga yoki 1,6 %ga koʻpaydi. 3. Chet el investitsiyalarini jalb qilish. 4. O‘zbekiston Respublikasidan tashqaridagi investitsiya faoliyati. Tashqi savdo faoliyati xalqaro tovarlar, ishlar (xizmatlar) savdosi sohasidagi tadbirkorlik faoliya- tidir. Tashqi savdo faoliyati tovarlar, ishlarni (xiz- matlarni) eksport va import qilish yo‘li bilan amalga oshiriladi [10]. TSAda eksport hajmi 9 277,9 mln. AQSh dol- lariga (20,1 %ga kamaydi) va import hajmi 15 685,8 mln. AQSh dollariga (20,9 %ga koʻpaydi) yetdi. Hisobot davrida 6 407,9 mln. AQSh dollari qiymati- da passiv tashqi savdo balansi qayd etildi [15]. Tovarlarning O‘zbekiston Respublikasi bojxo- na hududidan ularni qayta olib kirish to‘g‘risidagi majburiyatni olmasdan olib chiqib ketilishi, agar qonun hujjatlarida boshqacha qoida belgilanmagan bo‘lsa, tovarlar eksportidir. MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN Xorijiy mamlakatlar bilan hamkorlikda olib borilayotgan islohotlar ko‘rib chi- qildi. Jadvallarda tegishlicha shakllantirildi va so- lishtirildi. Mavzuni yoritishda “Oʻzbekiston Respub- likasi Davlat statistika qo‘mitasi ma’lumotlari”, O‘zbekiston Respublikasi “Tashqi iqtisodiy faoliyat to‘g‘risida”gi qonuni asos qilib olindi. Tashqi iqtisodiy faoliyat turli mamlakatlar- dan kelgan korxonalarning savdo bitimlari bilan bog‘liq. Tashqi iqtisodiy faoliyatga xalqaro ko‘rgaz- malar, investitsiyalar va qo‘shma ishlab chiqarish kiradi. Lekin tashqi savdo asosiy qismi – xalqaro savdo. Savdo qilish uchun kompaniyalar tovarlarni davlatlarning bojxona chegaralari orqali ko‘chirish- lari kerak. Tashqi iqtisodiy faoliyat mutaxassislari bunday bitimlar uchun javobgardir [19]. Tashqi iqtisodiy faoliyat tovarlar va xizmat- larni eksport qilish yoki import qilish bilan bog‘liq iqtisodiy faoliyatdir. Boshqacha aytganda, bu chet el bilan milliy vakilning savdosi. Bu holatda savdo-so- tiq nafaqat tovarlarni sotib olish va sotish, balki xal- qaro loyihalarni amalga oshirish, shuningdek, inves- titsiyalar va kreditlar bilan bog‘liq faoliyatni ham tushunish mumkin. Tashqi iqtisodiy faoliyat ishti- rokchilari davlat hokimiyati organlari, tijorat va davlat korxonalari hamda jismoniy shaxslar bo‘lishi mumkin. Tahlil va natijalar. O‘zbekiston Respublikasi “Tashqi iqtisodiy faoliyat to‘g‘risida”gi qonunining 3-moddasiga binoan Tashqi iqtisodiy faoliyat de- ganda, O‘zbekiston Respublikasi yuridik va jismoniy shaxslarining xorijiy davlatlarning yuridik va jismo- niy shaxslari, shuningdek, xalqaro tashkilotlar bilan o‘zaro foydali iqtisodiy aloqalarni o‘rnatish va rivoj- lantirishga qaratilgan faoliyati tushuniladi. O‘zbekiston Respublikasida ro‘yxatga olingan yuridik shaxslar, shuningdek, O‘zbekiston Respubli- kasining hududida doimiy yashash joyiga ega bo‘l- gan va yakka tartibdagi tadbirkor sifatida ro‘yxatga olingan jismoniy shaxslar tashqi iqtisodiy faoliyat bilan shug‘ullanishga haqlidir. O‘zbekiston Respub- likasining davlat organlari, agar qonun hujjatlarida boshqacha qoida belgilanmagan bo‘lsa, tashqi iqti- sodiy faoliyatni amalga oshirishlari mumkin [16]. А.Ю.Кнобель, А.Н.Спартак, М.А.Баева, Ю.К.Зайцев, А.Д.Левашенко, А.Н.Лощенкова, О.В.Пономарева, К.А.Прока o‘z ilmiy ma’ruzalari- da: “… tashqi iqtisodiy faoliyat va eksportni rivoj- lantirish, xususan, o‘z-o‘zidan yetarli emas, asosiy maqsad uzoq muddatli, barqaror iqtisodiy o‘sishni ta’minlashdir”, − deb ta’kidlab o‘tishgan [12]. Xuddi shunday pozitsiyani E.F.Prokushev egallaydi, bu esa quyidagilarni ko‘rsatadi: “tashqi iqtisodiy faoliyat – amalga oshirish jarayoni, tashqi iqtisodiy aloqa” va “tashqi iqtisodiy aloqa xalqaro iqtisodiy, savdo, siyosiy aloqalar, tovar almashinuvi, Tashqi iqtisodiy faoliyatning asosiy prinsip- lari quyidagilardan iborat:  tashqi iqtisodiy faoliyat subyektlarining erkinligi va iqtisodiy mustaqilligi;  tashqi iqtisodiy faoliyat subyektlarining erkinligi va iqtisodiy mustaqilligi; Iqtisodiyot va ta'lim / 2022-yil 2-son 112 MENEJMENT VA MARKETING O‘zbekiston Respublikasi hududida chet el investitsiyalarini amalga oshirish shakllari va tartibi qonun hujjatlari bilan belgilanadi. MONITORING OF CHANGES IN FOREIGN ECONOMIC ACTIVITY IN THE REPUBLIC OF UZBEKISTAN Tashqi iqtisodiy faoliyat boʻyicha 20 ta yirik hamkor-davlatlar orasidan toʻrtta davlatda faol tashqi savdo balansi kuzatilgan, xususan, Afgʻonis- ton, Qirgʻiziston Respublikasi, Tojikiston va Turkiya shular jumlasidandir. Qolgan 16 ta davlat bilan passiv tashqi savdo balansi saqlanib qolmoqda. O‘zbekiston Respublikasining yuridik yoki jismoniy shaxsi tomonidan xorijiy davlatning yuri- dik yoki jismoniy shaxsi uchun ishlar bajarish (xiz- matlar ko‘rsatish), ularning bajarilish (ko‘rsatilish) joyidan qat’i nazar, ishlar (xizmatlar) eksportidir. TSAning nisbatan salmoqli hissasi Xitoy Xalq Respublikasida (19,2 %), Rossiya Federatsiyasida (17,9 %), Qozogʻistonda (9,9 %), Turkiyada (8,6 %), Koreya Respublikasida (4,8 %), Qirgʻiziston Res- publikasi (2,4 %) va Germaniyada (1,9%) qayd etil- gan. Tovarlarning O‘zbekiston Respublikasi boj- xona hududiga ularni qayta olib chiqib ketish to‘g‘- risidagi majburiyatni olmasdan olib kirilishi tovar- lar importidir. Xorijiy davlatning yuridik yoki jismo- niy shaxsi tomonidan O‘zbekiston Respublikasining yuridik yoki jismoniy shaxsi uchun ishlar bajarish (xizmatlar ko‘rsatish), ularning bajarilish (ko‘rsati- lish) joyidan qat’i nazar, ishlar (xizmatlar) importi- dir. 2021-yil yanvar-avgust oylarida Oʻzbekiston Respublikasi tashqi savdo aylanmasining uchdan bir qismi yoki 37,9 % MDH davlatlariga toʻgʻri kelib, tashqi savdo aylanmasidagi 2020-yilning mos davriga nisbatan ulushi 7,8 %ga koʻpaygan. Boshqa xorijiy davlatlarning TSAsi 2021-yilning yanvar- avgust oylarida 2020-yilning mos davriga nisbatan 4,7 %ga kamaydi va jami TSAdagi ulushi 62,1 %ni tashkil etdi. Chet ellik investorlar tomonidan tadbirkorlik faoliyati obyektlariga hamda qonun hujjatlarida ta’- qiqlanmagan faoliyatning boshqa turlariga kiritila- yotgan moddiy va nomoddiy ne’matlarning barcha turlari hamda ularga bo‘lgan huquqlar, shu jumla- dan, intellektual mulkka bo‘lgan huquqlar, shuning- dek, chet el investitsiyalaridan olinadigan har qan- day daromad O‘zbekiston Respublikasi hududidagi chet el investitsiyalari deb e’tirof etiladi [14]. Oʻzbekiston Respublikasining MDH mamla- katlari bilan tashqi savdo aylanmasi 2021-yil yanvar-iyul holatiga koʻra 9 467,1 mln. AQSh dolla- Iqtisodiyot va ta'lim / 2022-yil 2-son 113 MENEJMENT VA MARKETING rini tashkil etdi. Shundan eksport 3 280,4 mln. AQSh dollariga yetgan boʻlsa, import 6 186,7 mln. AQSh dollari qiymatida qayd etildi. %) davlatlari bilan qayd etildi. Oʻzbekiston Respub- likasining Yevroosiyo iqtisodiy ittifoqiga a’zo dav- latlar bilan tashqi savdo aylanmasi 7 812,7 million AQSh dollarini tashkil etdi. Shundan eksport hajmi 2 541,3 mln. AQSh dollari, import hajmi esa 5 271,4 mln. AQSh dollariga yetdi [5]. Oʻzbekiston Respublikasining MDH mamla- katlari bilan tashqi savdo aylanmasining eng yuqori hajmlari Rossiya Federatsiyasi (47,2 %), Qozogʻis- ton (26,2 %) hamda Qirgʻiziston Respublikasi (6,3 1-jadval 1 jadval O‘zbekiston Respublikasi tashqi savdo aylanmasi va balansi (yanvar-avgust, mln. AQSh dollari) Ko‘rsatkichlar 2020-yil 2021-yil O‘sish sur’ati, %da Jamiga nisbatan, %da Tashqi savdo aylanmasi 24 580,6 24 963,7 101,6 x Eksport 11 607,9 9 277,9 79,9 100,0 Tovarlar 4 496,3 6281,2 139,7 67,7 Xizmatlar 1 307,2 1 610,2 123,2 17,4 Oltin 5 804,4 1 386,5 23,9 14,9 Import 12 972,7 15 685,8 120,9 100,0 Tovarlar 12 207,5 14 664,6 120,1 93,5 Xizmatlar 765,2 1 021,2 133,5 6,5 Saldo -1 364,8 -6 407,9 x x Tovarlar -1 906,8 -6 996,9 x x Oltinsiz -7 711,2 -8 383,4 x x Xizmatlar 542,0 589,0 x x Eksport (oltinsiz hajm) 5 803,5 7 891,4 136,0 x O‘zbekiston Respublikasi tashqi savdo aylanmasi va balansi (yanvar-avgust, mln. AQSh dollari) O‘zbekiston Respublikasi tashqi savdo aylanmasi va balansi (yanvar-avgust, mln. AQSh dollari) kimyoviy vositalar va boshqa toifalarga kiritilmagan shunga oʻxshash mahsulotlar (7,2 %) hissasiga toʻgʻ- ri kelmoqda. kimyoviy vositalar va boshqa toifalarga kiritilmagan shunga oʻxshash mahsulotlar (7,2 %) hissasiga toʻgʻ- ri kelmoqda. Joriy yil yanvar-avgust oylarida mamlakat- ning eksportyorlari soni 5 527 tani tashkil etib, ular tomonidan 7 891,4 mln. AQSh dollari (oltin eks- portidan tashqari) qiymatidagi ( 2020-yilning mos davriga nisbatan 36,0 %ga koʻpaydi) tovar va xiz- matlar eksport qilinishi taʼminlandi. 2021-yil yanvar-avgust oylari yakunida eks- port boʻyicha asosiy hamkor davlatlar tarkibida XXR (eksport umumiy hajmining 17,0 %) va Rossiya Federatsiyasi (13,7 %) davlatlari yetakchilik qildi. Mazkur yetti yirik davlatlarning jami eksportdagi ulushi 64,0 %ni tashkil etdi. Eksport tarkibida tovarlar ulushi 82,6 %ni tashkil etib, ular sanoat tovarlari (30,0 %), oziq- ovqat mahsulotlari va tirik hayvonlar (8,7 %) va 2-jadval Iqtisodiyot va ta'lim / 2022-yil 2-son 114 2 jadval Oʻzbekiston Respublikasining asosiy hamkor davlatlarga amalga oshirgan eksporti hajmi va oʻsish sur’atlari (yanvar-avgust, yillar kesimida) Davlatlar Mln. AQSh dollari Oʻsish sur’ati, %da O‘rin 2020-y. 2021-y. 2020-y. 2021-y. 2020-y. 2021-y. Xitoy Xalq Respublikasi 1 187,9 1 577,6 68,4 132,9 1 1 Rossiya Federatsiyasi 933,0 1 274,7 59,2 136,6 2 2 Turkiya 553,5 1 120,0 71,6 202,3 4 3 Qozogʻiston 556,9 736,7 62,2 132,3 3 4 Qirgʻiziston Respublikasi 478,6 490,2 99,4 102,4 5 5 Аfgʻoniston 464,3 441,9 128,3 95,2 6 6 2021-yil yanvar-avgust oylarida import haj- mi 15 685,8 mln. AQSh dollarini va 2020-yilga nisbatan 120,9 %ni tashkil etdi. Import tarkibida eng katta ulush mashinalar va transport asbob- uskunalari (33,6 %), sanoat tovarlari (18,3 %) hamda kimyoviy vositalar va boshqa toifalarga kiri- tilmagan shunga oʻxshash mahsulotlar (14,0 %) hi- sobiga toʻgʻri keldi. Tovarlar importi dinamikasi tahliliga koʻra, 2021-yil yanvar-avgustida import qilingan tovarlar hajmi, oʻtgan yilning mos davriga nisbatan 2 457,1 mln. AQSh dollariga koʻpaydi va 14 664,6 mln. AQSh dollarini tashkil etdi. Xizmatlar importi esa 1 021,2 mln. AQSh dollariga yetdi. 2021-yil yanvar-avgust oylari yakuni boʻyicha Oʻzbekiston Respublikasi tomonidan 153 ta davlatdan tovar va xizmatlar im- porti amalga oshirildi. 2 jadval Oʻzbekiston Respublikasining asosiy hamkor davlatlarga amalga oshirgan eksporti hajmi va oʻsish sur’atlari (yanvar-avgust, yillar kesimida) Davlatlar Mln. AQSh dollari Oʻsish sur’ati, %da O‘rin 2020-y. 2021-y. 2020-y. 2021-y. 2020-y. 2021-y. Xitoy Xalq Respublikasi 1 187,9 1 577,6 68,4 132,9 1 1 Rossiya Federatsiyasi 933,0 1 274,7 59,2 136,6 2 2 Turkiya 553,5 1 120,0 71,6 202,3 4 3 Qozogʻiston 556,9 736,7 62,2 132,3 3 4 Qirgʻiziston Respublikasi 478,6 490,2 99,4 102,4 5 5 Аfgʻoniston 464,3 441,9 128,3 95,2 6 6 2021-yil yanvar-avgust oylarida import haj- mi 15 685,8 mln. AQSh dollarini va 2020-yilga nisbatan 120,9 %ni tashkil etdi. Import tarkibida eng katta ulush mashinalar va transport asbob- k l i (33 6 %) t t l i (18 3 %) Tovarlar importi dinamikasi tahliliga koʻra, 2021-yil yanvar-avgustida import qilingan tovarlar hajmi, oʻtgan yilning mos davriga nisbatan 2 457,1 mln. AQSh dollariga koʻpaydi va 14 664,6 mln. AQSh d ll i i t hkil tdi Xi tl i ti 1 021 2 j Oʻzbekiston Respublikasining asosiy hamkor davlatlarga amalga oshirgan eksporti hajmi va oʻsish sur’atlari (yanvar-avgust, yillar kesimida) Davlatlar Mln. AQSh dollari Oʻsish sur’ati, %da O‘rin 2020-y. 2021-y. 2020-y. 2021-y. 2020-y. 2021-y. 2-jadval Xitoy Xalq Respublikasi 1 187,9 1 577,6 68,4 132,9 1 1 Rossiya Federatsiyasi 933,0 1 274,7 59,2 136,6 2 2 Turkiya 553,5 1 120,0 71,6 202,3 4 3 Qozogʻiston 556,9 736,7 62,2 132,3 3 4 Qirgʻiziston Respublikasi 478,6 490,2 99,4 102,4 5 5 Аfgʻoniston 464,3 441,9 128,3 95,2 6 6 Oʻzbekiston Respublikasining asosiy hamkor davlatlarga amalga oshirgan eksporti hajmi va oʻsish sur’atlari (yanvar-avgust, yillar kesimida) Tovarlar importi dinamikasi tahliliga koʻra, 2021-yil yanvar-avgustida import qilingan tovarlar hajmi, oʻtgan yilning mos davriga nisbatan 2 457,1 mln. AQSh dollariga koʻpaydi va 14 664,6 mln. AQSh dollarini tashkil etdi. Xizmatlar importi esa 1 021,2 mln. AQSh dollariga yetdi. 2021-yil yanvar-avgust oylari yakuni boʻyicha Oʻzbekiston Respublikasi tomonidan 153 ta davlatdan tovar va xizmatlar im- porti amalga oshirildi. 2021-yil yanvar-avgust oylarida import haj- mi 15 685,8 mln. AQSh dollarini va 2020-yilga nisbatan 120,9 %ni tashkil etdi. Import tarkibida eng katta ulush mashinalar va transport asbob- uskunalari (33,6 %), sanoat tovarlari (18,3 %) hamda kimyoviy vositalar va boshqa toifalarga kiri- tilmagan shunga oʻxshash mahsulotlar (14,0 %) hi- sobiga toʻgʻri keldi. Iqtisodiyot va ta'lim / 2022-yil 2-son 114 MENEJMENT VA MARKETING 3-jadval Oʻzbekiston Respublikasining asosiy hamkor davlatlarga amalga oshirgan importi hajmi va oʻsish sur’atlari (yanvar-avgust, yillar kesimida) Davlatlar Mln. AQSh dollari Oʻsish sur’ati, %da O‘rin 2020-y. 2021-y. 2020-y. 2021-y. 2020-y. 2021-y. Xitoy Xalq Respublikasi 2 833,9 3 213,9 87,7 113,4 1 1 Rossiya Federatsiyasi 2 652,9 3 197,9 104,8 120,5 2 2 Qozogʻiston 1 212,9 1 746,0 90,3 144,0 4 3 Koreya Respublikasi 1 419,8 1 166,3 79,4 82,1 3 4 Turkiya 676,8 1 029,8 79,3 152,1 5 5 Germaniya 436,4 434,8 70,2 99,6 6 6 Oʻzbekiston Respublikasining asosiy hamkor davlatlarga amalga oshirgan importi hajmi va oʻsish sur’atlari (yanvar-avgust, yillar kesimida) investitsiyalar va kafolatlanmagan kreditlar 23 189,9 mlrd. so‘mni tashkil etdi. Xorijiy investitsiya va kreditlarning o‘tgan yilning mos davriga nisbatan o‘sish sur’ati 96,8 %ni tashkil etdi [17]. 2021-yil yanvar-iyun oylarida O‘zbekiston y y y y Respublikasida iqtisodiy va ijtimoiy sohalarni rivojlantirish uchun jami moliyalashtirish manbala- ri hisobidan 103,1 trln. so‘m o‘zlashtirildi. Dollar ek- vivalentida 9,8 mlrd. AQSh doll. o‘zlashtirilgan bo‘- lib, o‘tgan yilning mos davriga nisbatan 105,9 %ni tashkil etdi [6]. Qayta ishlash sanoatida xorijiy investitsiya va kreditlar salmoqli hajmda o‘zlashtirilib, 17 835,6 mlrd. so‘mni yoki jami investitsiyalarning 40,8 %ini tashkil etdi. Shuningdek, uning tarkibida to‘qima- chilik va kiyim-kechak mahsulotlarini ishlab chiqa- rish faoliyat turida 11,2 % yoki 4 853,7 mlrd. 2-jadval so‘m, boshqa nometall mineral mahsulotlar ishlab chiqa- rishda 8,9 % yoki 3 878,3 mlrd. so‘m, metallurgiya sanoatida 8,5 % yoki 3 700,6 mlrd. so‘m investitsi- yalar o‘zlashtirildi. [ ] 2021-yil yanvar-iyunda 43 685,5 mlrd. so‘m (dollar ekvivalentida 4,2 mlrd. AQSh doll.) yoki 2020-yilning yanvar-iyuniga nisbatan 96,8 % asosiy kapitalga xorijiy investitsiya va kreditlar o‘zlashti- rildi. Xorijiy investitsiya va kreditlarning jami asosiy kapitalga o‘zlashtirilgan investitsiyalardagi ulushi 42,4 %ni tashkil etdi. Xorijiy investitsiya va kredit- lar tarkibida xorijiy investitsiyalar 14756,4 mlrd. so‘mni va mos ravishda xorijiy kreditlar 28 929,1 mlrd. so‘mni tashkil qildi. O‘zlashtirilgan jami xorijiy investitsiya va kreditlarning 11 338,6 mlrd. so‘mini to‘g‘ridan to‘g‘ri xorijiy investitsiyalar tashkil etdi, O‘zbekiston Respublikasi davlat kafolati ostidagi xorijiy kreditlar 9 157,0 mlrd. so‘mni, boshqa xorijiy Asosiy kapitalga o‘zlashtirilgan xorijiy inves- titsiyalarning asosiy qismi, ya’ni 58,6 %i to‘rtta in- vestor mamlakat hissasiga to‘g‘ri keladi: Xitoy – 22,3 %, Rossiya – 21,5 %, Turkiya – 7,5 % va Ger- maniya – 7,3 %. Quyidagi diagramma orqali inves- tor mamlakatlar bo‘yicha jalb qilingan investitsiya- larning salmog‘ini ko‘rish mumkin: Diagramma INVESTOR MAMLAKATLAR BO‘YICHA TO‘G‘RIDAN TO‘G‘RI XORIJIY INVESTITSIYALAR, jamiga nisbatan %da Boshqa investor mamlakatlar Boshqa investor mamlakatlarXitoy XitoyRossiya RossiyaTurkiya TurkiyaAQSH AQSHKipr KiprGermaniya GermaniyaFilipinn Filipinn INVESTOR MAMLAKATLAR BO‘YICHA TO‘G‘RIDAN TO‘G‘RI XORIJIY INVESTITSIYALAR, jamiga nisbatan %da Boshqa investor mamlakatlar jamiga nisbatan %da Boshqa investor mamlakatlar jamiga nisbatan %da Boshqa investor mamlakatlar Diagramma Xulosa va takliflar. O‘zbekistonda tarixiy jihatdan qisqa bir davrda xalqaro munosabatlarni yo‘lga qo‘yish va rivojlantirish bobida asrlarga ar- ziydigan ishlar amalga oshirildi. O‘zbekiston o‘zi- ning tinchliksevar, yaxshi qo‘shnichilik, o‘zaro foy- dali hamkorlikka qaratilgan siyosati va faoliyati bi- lan butun dunyoga tanildi, jahon hamjamiyatida o‘zining munosib o‘rnini egalladi, uning mavqeyi yil- dan yilga mustahkamlanib bormoqda. Tashqi iqtiso- diy faoliyat respublikamiz iqtisodiy faoliyatining asosiy qismi sifatida xizmat qilmoqda. Ushbu sohada davom ettirilayotgan islohotlar mahalliy va xalqaro bozorlarni mahsulot va xizmatlar bilan ta’minlash imkonini bermoqda. Iqtisodiyot va ta'lim / 2022-yil 2-son 115 MENEJMENT VA MARKETING Bugungi kunda O‘zbekiston xalqaro hamjami- yatda o‘z ovoziga ega bo‘lib, nufuzli xalqaro tashki- lotlar a’zosi hisoblanadi. Yurtimizda tashqi iqtisodiy faoliyatni rivoj- lantirish borasida amalga oshirilayotgan keng qam- rovli islohotlar va dunyo mamlakatlari bilan olib bo- rilayotgan mustahkam aloqalar natijasida mamlaka- timizning eksport salohiyati oshib bormoqda. Shu- ningdek, hukumatimiz tomonidan bir qator soliq, bojxona imtiyozlarining yaratilishi, ishlab chiqarish- ni modernizatsiya qilish va raqobatbardosh mahsu- lotlarning ishlab chiqarilishi milliy mahsulotlarimiz- ning tashqi bozorlarda mustahkam o‘rin egallashi uchun zamin yaratmoqda. 2-jadval j , y y q , 9. O‘zbekiston Respublikasi Prezidentining 2019-yil 23-apreldagi PQ-4297-sonli “Tashqi iqtisodiy faoliyatni amalga oshirishda ma’muriy tartib-taomillarni yanada takomillashtirish chora-tadbirlari to‘g‘risida” qarori. . Эскиндаров М.А., Шаркова А.В., Меркулина И.А. Экономика и финансы ТЭК. Учебник. – М.: КноРус. 2019. р , р , р у ф у . O‘zbekiston Respublikasining “Investitsiyalar va investitsiya faoliyati to‘g‘risida”gi qonuni, 2019-yil 9-dekabr. 12. Внешнеэкономическая деятельность как источник экономического роста. / А.Ю.Кнобель, А.Н.Спартак, М.А.Баева, Ю.К.Зайцев, А.Д.Левашенко, А.Н.Лощенкова, О.В.Пономарева, К.А.Прока. – М.: Издательский дом «Дело» РАНХиГС, 2019. – 60 с. (Научные доклады: экономика). ISBN-5-7749-1446-3. 12. Внешнеэкономическая деятельность как источник экономического роста. / А.Ю.Кнобель, А.Н.Спартак, М.А.Баева, Ю.К.Зайцев, А.Д.Левашенко, А.Н.Лощенкова, О.В.Пономарева, К.А.Прока. – М.: Издательский дом «Дело» РАНХиГС, 2019. – 60 с. (Научные доклады: экономика). ISBN-5-7749-1446-3. 13. Mirziyoyev Sh.M. 2017-2021-yillarda O‘zbekiston Respublikasini rivojlantirishning beshta ustivor yo‘nalishi bo‘yicha Harakatlar strategiyasining “Xalq bilan muloqot va inson manfaatlari yili”da amalga oshirishga oid Davlat dasturini o‘rganish bo‘yicha ilmiy uslubiy-risola. – T.: O‘zbekiston, 2017-y. 13. 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Oʻzbekiston Respublikasi tashqi savdo aylanmasi. 2021-yil yanvar-avgust oylari uchun dastlabki maʼlumot. 6. Oʻzbekiston Respublikasi Davlat statistika qo‘mitasi ma’lumotlari. 2021-yil yanvar-iyun oylari uchun dastlabki 5. Oʻzbekiston Respublikasi tashqi savdo aylanmasi. 2021-yil yanvar-avgust oylari uchun dastlabki maʼlumot. 6 Oʻzbekiston Respublikasi Davlat statistika qo‘mitasi ma’lumotlari 2021-yil yanvar-iyun oylari uchun dastlabk 6. Oʻzbekiston Respublikasi Davlat statistika qo‘mitasi ma’lumotlari. 2021-yil yanvar-iyun oylari uch aʼlumot. 7. Tursunova M.R., Abduqodirova N.A. “Indicators of foreign economic activity in Uzbekistan”, “9th INTERNATIONAL CONFERENCE ON CULTURE AND CIVILIZATION” full texts book, march 15-16, 2021. p. 661-666, Tashkent Chemical- Technological Institute. 7. Tursunova M.R., Abduqodirova N.A. “Indicators of foreign economic activity in Uzbekistan”, “9th INTERNATIONAL CONFERENCE ON CULTURE AND CIVILIZATION” full texts book, march 15-16, 2021. p. 661-666, Tashkent Chemical- Technological Institute. 8. Xujamkulov D.Yu., Ismailov D.A. Investitsiya loyihalarini boshqarish. – T., 2019. 9. O‘zbekiston Respublikasi Prezidentining 2019-yil 23-apreldagi PQ-4297-sonli “Tashqi iqtisodiy faoliyatni amalga oshirishda ma’muriy tartib-taomillarni yanada takomillashtirish chora-tadbirlari to‘g‘risida” qarori. ТОВАР-МОДДИЙ РЕСУРСЛАР ҲАРАКАТИНИ ТАКОМИЛЛАШТИРИШДА МАРКЕТИНГ ЛОГИСТИКАСИНИНГ ЎРНИ ВА АҲАМИЯТИ Пардаев Шерзод Холмуродович, Самарқанд иқтисодиёт ва сервис институти таянч докторанти Пардаев Шерзод Холмуродович, Самарқанд иқтисодиёт ва сервис институти таянч докторанти https://doi.org/10.55439/ECED/vol23_iss2/a18 https://doi.org/10.55439/ECED/vol23_iss2/a18 Аннотация. Ушбу мақолада товар-моддий ресурсларни истеъмолчиларига етказиб бериш жараёнида маркетинг ва логистиканинг ўрни ва аҳамияти, товар-моддий ресурслар ҳаракатини ташкил этиш ва бошқариш алгоритмини ишлаб чиқишнинг оддий ва анъанавий кўриниши, уни амалга оширишда улгуржи савдо объектларидаги тегишли бўлимлар ва уларнинг вазифалари ҳақида илмий хулосалар келтирилган. Калит сўзлар: маркетинг логистикаси, “ишлаб чиқарувчи-воситачи-истеъмолчи” занжири, тарқатиш логистикаси, товар-моддий ресурслар, алгоритм, логистик тизим, улгуржи истеъмолчи. Iqtisodiyot va ta'lim / 2022-yil 2-son Iqtisodiyot va ta'lim / 2022-yil 2-son 116 116
https://openalex.org/W2095516934	https://revistas.ucm.es/index.php/ESMP/article/download/40953/39203	Spanish; Castilian	null	La publicidad en España como elemento de marketing: límites	Estudios sobre el mensaje periodístico	2,012	cc-by	6,567	La publicidad en España como elemento de marketing: límites Fernando RAMOS FERNÁNDEZ Universidad de Vigo ferramos@uvigo.es Teresa PIÑEIRO OTERO Universidad de A Coruña teresa.pineiro@udc.es David CALDEVILLA DOMÍNGUEZ Universidad Complutense de Madrid davidcaldevilla@ccinf.ucm.es Recibido: 24/07/2012 Aceptado: 22/10/2012 Recibido: 24/07/2012 Aceptado: 22/10/2012 Abstract h The new Criminal Code offense advertising includes among its provisions, noting the growing risk gen- erated by the creative audacity of some advertising, for those limits, socially accepted, hype, do not stop at what misleading, creating confusion or the good faith of consumers. Advertising self-regulation through the application of codes of ethics is a palliative to avoid judicial remedy, while denoting that conscious deception or breach of the rules governing advertising or lawful commerce is accepted by some without the least scruple. Hiding behind freedom of speech is introduced into the market much information con- taminated. If not detected early, the risks to consumers are deceived many, the same as for the fair trader. Keywords: Unfair and deceptive advertising. Consumer rights. Advertising creativity. Lawful commerce. Introducción El nuevo Código Penal incluye el delito publicitario entre sus previsiones, al observar el creciente riesgo que genera la osadía creativa de algunos publicitarios, para quienes los límites de la, aceptada socialmente, exageración publicitaria, no se detienen ante lo engañoso, la creación de confusión o la buena fe de los consumidores. La autorregulación publicitaria, mediante la aplicación de códigos deontológicos es un pa- liativo para evitar el remedio judicial, al tiempo que denota que el engaño consciente o el incumplimiento de las reglas que regulan la publicidad o el lícito comercio es aceptada por algunos sin el menos escrú- pulo. Escudándose en la libertad de expresión se introduce en el mercado mucha información contami- nada. Si no se detecta a tiempo, los riesgos para el consumidor engañado son muchos, los mismos que para el comerciante leal. Palabras clave: Autocontrol. Publicidad desleal y engañosa. Derechos de los consumidores. Creativi- dad publicitaria. Comercio lícito. ISSN: 1134-1629 http://dx.doi.org/10.5209/rev_ESMP.2012.v18.40953 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviem. (2012) 741-751 741 La publicidad en España como elemento de marketing: límites Fernando RAMOS FERNÁNDEZ Universidad de Vigo ferramos@uvigo.es Teresa PIÑEIRO OTERO Universidad de A Coruña teresa.pineiro@udc.es David CALDEVILLA DOMÍNGUEZ Universidad Complutense de Madrid davidcaldevilla@ccinf.ucm.es La publicidad en España como elemento de marketing: límites Fernando RAMOS FERNÁNDEZ Universidad de Vigo ferramos@uvigo.es Teresa PIÑEIRO OTERO Universidad de A Coruña teresa.pineiro@udc.es David CALDEVILLA DOMÍNGUEZ Universidad Complutense de Madrid davidcaldevilla@ccinf.ucm.es 1. La publicidad y sus efectos sobre los consumidores McLuhan (1996: 237) afirma que los anuncios no están hechos para ser consumidos conscientemente. Son concebidos como píldoras subliminales para el inconsciente con el fin de producir un trance hipnótico. Es cierto que la publicidad contribuye al bienestar del consumidor dándole información. La doctrina económica tradicional gusta de distinguir entre publicidad informativa y persuasiva, atribuyendo a la se- gunda alcance o efectos comerciales o mercantiles. En este sentido precisaron que un mismo bien, publicitado o no, es objetivamente una cosa distinta. La publicidad es una vía de transmisión de parámetros de comportamientos socia- les, vinculada a la cultura del consumo (Carrillo, 97: 11). De la Cuesta Rute (1985: 47), al afirmar que “los actos publicitarios, desde el punto de vista jurídico, se caracteri- zan por su finalidad mercantil más que por su estructura comunicativa”, nos enseña que, al provocar efectos de orden patrimonial que el Derecho conoce, generan res- ponsabilidades no sólo de orden civil y mercantil, sino también, y ahora, penales. Hoy ya no bastan las medidas administrativas, mercantiles o civiles. La reflexión fundamental nos lleva a considerar que el objetivo original de la pu- blicidad ha sido persuadir para consumir el producto anunciado. Martínez Rodrigo y Sánchez Martín (2011: 469) centran la discusión en el concepto de finalidad de la pu- blicidad, aseverando que “en sus inicios, el discurso publicitario mostraba las carac- terísticas básicas del objeto, sus atributos propios y sus bondades. Sin embargo, esta narrativa ha ido evolucionando hasta la actualidad, en donde predomina un discurso centrado en la venta de emociones y experiencias. […] No se vende el artículo de forma directa, sino que se apela a significados concretos, universalmente conocidos y, por tanto, fácilmente identificables, que son los deseados por el receptor. Estos ele- mentos simbólicos en venta son emociones asociadas al consumo del producto”. Este proceso desde lo demostrativo a la aspiración sirve de base a los publicistas para ela- borar su mensaje, centrándolo en el pathos por encima del ethos y del logos. Cuando se transmite un mensaje, el sujeto activo desarrolla una forma específica de libertad de expresión. El hecho de que lo haga con ánimo de lucro determina el con- tenido y la responsabilidad que del mismo se desprende. Referencia normalizada Á RAMOS FERNÁNDEZ, Fernando; PIÑEIRO OTERO, Teresa y CALDEVILLA DOMÍNGUEZ, David (2012): “La publicidad en España como elemento de marketing: límites”. Estudios sobre el mensaje pe- riodístico. Vol. 18, núm. especial noviembre, págs.: 741-751. Madrid, Servicio de Publicaciones de la Uni- versidad Complutense. Sumario: 1. La publicidad y sus efectos sobre los consumidores. 2. Libertad de expresión y creatividad publicitaria. 3. La autorregulación publicitaria en España. 4. El efecto de los anuncios sobre los consu- midores. 5. Los estándares del estilo de vida que promueve la publicidad. 6. Las nuevas herramientas para la defensa de los consumidores. 7. La eficacia del Jurado de la Publicidad. 8. Conclusiones. 9. Referencias bibliográficas. 10. Legislación. ISSN: 1134-1629 http://dx.doi.org/10.5209/rev_ESMP.2012.v18.40953 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviem. (2012) 741-751 741 La publicidad en España como elemento de marketing: límites Fernando Ramos Fernández et al. Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 1. La publicidad y sus efectos sobre los consumidores Tribunal Constitucional es- pañol, y el Supremo de los Estados Unidos, negó, en el primer caso, y matizó severamente en el segundo, que la libertad de expresión (tal y como la formula el ar- tículo 20 CE o la Primera Enmienda de la de los Estados Unidos) pudiera ser invocada indistintamente a la hora de emitir informaciones y opiniones, o reclamos publicita- rios, cosa esencialmente distinta. Los norteamericanos dicen que cuando se redactó la Primera Enmienda, “no estaban precisamente pensando en los vendedores de puche- ros”. La Ley 26/1984, de 19 de julio, General de la Defensa de los Consumidores, mo- dificó la vieja concepción de que la publicidad trataba simplemente de una informa- ción inocua, que no exigía garantía de cumplimiento o responsabilidad como consecuencia de sus contenidos. Las normas de protección de los consumidores esta- blecen que éstos pueden exigir el cumplimiento efectivo de las prestaciones o conte- 742 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 do Ramos Fernández et al. La publicidad en España como elemento de marketing: límites nidos de los anuncios o reclamos publicitarios, aun cuando no figuren expresamente en el contrato celebrado (art. 8.1)1. 2. Libertad de expresión y creatividad publicitaria 2. Libertad de expresión y creatividad publicitaria El Tribunal Europeo de Derechos Humanos ha extendido, por igual, la protección del artículo 20 CE a la información periodística y a la publicidad. Los publicitarios se aprovechan de este hecho; pero la doctrina insiste en preguntarse si el concepto de li- bertad de expresión, tal y como lo formula nuestra Constitución o la Primera En- mienda de la de los Estados Unidos, es aplicable, en toda su extensión a la creación publicitaria. Esta preocupación está motivada, en ocasiones, por el escándalo social (tan rentable, por otro lado, desde el propio punto de vista publicitario), provocado por algunas conocidas campañas. El Tribunal Constitucional entendió que la Constitu- ción, en este sentido, no debía interpretarse en sentido amplio. En principio no apre- ciaron que la libertad de expresión del artículo 20 pudiera hacerse extensiva a la información persuasiva o comercial, precisamente por este carácter y finalidad eco- nómicos de la misma. Pero el Tribunal Europeo de Derechos Humanos impuso otro criterio, la libertad ante todo. La responsabilidad, después. p p La directiva 84/450 CEE otorgó a los estados un amplio margen de maniobra para que, a través de las respectivas legislaciones nacionales, siempre en armonía con aque- lla directiva, se establecieran mecanismos judiciales y/o administrativos que permitan combatir eficazmente la competencia desleal tan vinculada al concepto mismo de pu- blicidad engañosa. El Tratado de la Unión Europea (Tratado de Maastricht de 1992) introduce en el documento constitutivo de la Comunidad Europea, dentro del Título XI, la protección de los consumidores, en artículo 129 a., indicando que la Comuni- dad contribuirá a que se alcance un alto nivel de protección de los consumidores.2 3. La autorregulación publicitaria en España 3. La autorregulación publicitaria en España Desantes (1973: 213) definía la deontología como el conjunto de las normas éticas objetivas para una colectividad profesional, independientemente del grado de positi- vización a que hayan llegado, bien sean normas de rango legal, estatutario de las or- ganizaciones profesionales; normas convencionales, incorporadas o no a los convenios, o meros principios, aceptados comúnmente. 1 Los productos deben incorporar de forma cierta y objetiva información veraz, eficaz y sufi- ciente (art.13). Lo que diga un anuncio tiene que cumplirse. De ahí que los publicitarios huyan de decir cosas concretas, de las que se desprendan denotaciones determinadas. Se mueve en el terreno de lo simbólico, buscando connotaciones sublimes, pero que no dicen realmente nada. Suscitan o tratan de suscitar emociones. 2 a) Medidas de aproximación de las disposiciones legales, reglamentarias y administrativas de los Estados miembros que tengan por objeto el establecimiento y funcionamiento del mercado interior (art.100 a); b) Acciones concretas que apoyen y complementen la política de los Estados miembros a fin de proteger la salud, la seguridad y los intereses económicos de los consumidores, de garantizarles una información adecuada. Las acciones concretas no obstarán a que los Estados miembros adopten medidas de mayor protección. 743 Ramos Fernández et al. La publicidad en España como elemento de marketing: límite Fernando Ramos Fernández et al. La publicidad en España como elemento de marketing: límites En España la Asociación para la Autorregulación de la Comunicación Comercial (Autocontrol) es una asociación sin ánimo de lucro compuesta por anunciantes, agen- cias, medios de comunicación y otras empresas de servicios a la comunicación co- mercial, que en conjunto representan más del 90% de la inversión publicitaria española, y por diversas asociaciones empresariales. Su objetivo es prevenir y resol- ver eventuales controversias publicitarias, estableciendo un marco ético donde se des- envuelva una comunicación comercial responsable y veraz. Este proyecto colectivo, nacido el 13 de junio de 1995, ha logrado dotar al sector de un instrumento de resolución extrajudicial de controversias en materia publicitaria, así como de un sistema voluntario de consulta previa (“copy advice” e informes) que beneficia tanto a los consumidores como a la propia industria publicitaria. 3. La autorregulación publicitaria en España Autocon- trol es miembro de la Alianza Europea para la Ética Publicitaria (EASA, European Advertising Standards Alliance), con sede en Bruselas, en cuyo seno se reúnen los sistemas de autorregulación publicitaria de los 15 Estados miembros de la Unión Eu- ropea y de otros países. El Código de Conducta Publicitaria de Autocontrol fue probado en la Asamblea General Extraordinaria celebrada el 19 de diciembre de 1996. La última versión, con las modificaciones aprobadas por la Asamblea General Ordinaria, data del día 26 de abril de 2011. Estas normas deontológicas se aplican a toda actividad de comunica- ción publicitaria tendente a fomentar, de manera directa o indirecta y sean cuales fue- ren los medios empleados, la contratación de bienes o servicios, o el potenciamiento de marcas y nombres comerciales3. 3 Códigos deontológicos y convenios en los que participa Autocontrol: - Publicidad Cervezas: Convenio con Cerveceros de España (2003) p ( ) - Publicidad Bebidas Espirituosas: Convenio con FEBE (2003) Publicidad Bebidas Espirituosas: Convenio con FEBE (2003) - Publicidad Televisiva: Convenio AUTOCONTROL – AEA - operadores de TV (junio 2002). ) - Convenio SETSI (Ministerio Industria)-Autocontrol (2003) ( ) ( ) - Publicidad Interactiva y Comercio Electrónico: Convenio AUTOCONTROL – AECE para cogestionar CONFIANZA ONLINE. 3 Códigos deontológicos y convenios en los que participa Autocontrol: - Publicidad Cervezas: Convenio con Cerveceros de España (2003) 3. La autorregulación publicitaria en España (2002) g ( ) - Promoción de Medicamentos: Convenio con Farmaindustria (2002) - Defensa de la Marca: Convenio con ANDEMA (2004) - Publicidad de Alimentos y Bebidas dirigida a niños, Código PAOS: Convenio FIAB- Mi- nisterio Sanidad-Autocontrol (2005) - Publicidad de Videojuegos: Convenio con ADESE (2005) j g ( ) - Publicidad de Tecnología Sanitaria: Convenio FENIN (2006) - Autorregulación del Mercado de Investigación: Convenio con - Autorregulación del Mercado de Investigación: Convenio con ANEIMO (2006) bli id d d d d i i l i i i l - Publicidad de Productos de Nutrición Enteral: Convenio con Asociación Española de Fa- bricantes y Distribuidores de Productos de Nutrición Enteral - (AENE) (2006) - Publicidad de Productos de Nutrición Enteral: Convenio con Asociación Española de Fa- bricantes y Distribuidores de Productos de Nutrición Enteral - (AENE) (2006) - Convenio firmado con Red.es para la resolución de controversias en la asignación de do- minios “.es” (2006) - Publicidad sobre alimentos especiales, dietéticos, y plantas medicinales: Convenio con AFEPADI (2007) - Publicidad de medicamentos dirigida al público: Convenio con la Asociación para el Au- tocuidado de la Salud (ANEFP) (2007) - Publicidad de medicamentos dirigida al público: Convenio con la Asociación para el Au- tocuidado de la Salud (ANEFP) (2007) Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 744 do Ramos Fernández et al. La publicidad en España como elemento de marketing: límites Constituido en octubre de 1995, el Jurado de Ética Publicitaria es un órgano espe- cializado, e independiente, creado en el seno de la asociación, dedicado a la observa- ción y seguimiento de las pautas deontológicas en el ámbito de la publicidad. Está compuesto por diez miembros, incluido su presidente. Todos son nombrados por la junta directiva. Cuatro componentes, entre ellos el presidente, son juristas de recono- cida competencia, y el resto expertos en materias publicitarias. Su mandato es de tres años. Desde el inicio de su actividad ha conocido más de 2.500 casos. Ofrece un ser- vicio de consulta previa, no vinculante, para que los anunciantes sometan a revisión sus anuncios antes de emitirlos. Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 3. La autorregulación publicitaria en España El profesor De la Cuesta Rute (1985: 47 y ss.) sostiene que, desde el punto de vista jurídico, los anuncios deben ser evaluados por el efecto que causan en el patrimonio de los ciudadanos, al promover el tráfico mercantil, y no solamente por su belleza u originalidad. Los anuncios no son obras de arte, sino mecanismos de marketing y pro- moción de la venta, y cuando promueven la confusión y el quebranto económico pue- dan llegar a incurrir en supuestos de ilicitud penal. Es en la televisión donde hallamos más publicidad contaminada, que en el cómputo global de los anuncios controverti- dos puede llegar a cuatro de cada diez reclamos cuyo contenido ha merecido el re- proche de los consumidores, las administraciones o la competencia, puesto que no conviene olvidar que la publicidad engañosa es al mismo tiempo desleal, pues que- branta las reglas de juego del lícito comercio. En 2011, Autocontrol recibió casi 18.000 consultas diversas sobre los anuncios a emitir, lo que refleja que los anunciantes y los publicitarios quieren curarse en salud ante de emitir anuncios que puedan contravenir la legislación protectora de los con- sumidores. La extensión de una postura crítica frente a las agresiones de la publicidad es una muestra de la madurez de los ciudadanos.4 - Publicidad del vino: Convenio con la Federación Española del Vino (FEV) (2009) p ( ) ( ) - Publicidad Medioambiente: Acuerdo con el Ministerio de Medio Ambiente y Medio Rural y Marino, y empresas del sector energético y de automoción (2009) 4. El efecto de los anuncios sobre los consumidores William Meyers, (1994: 24-31) ex publicista, experto en temas de publicidad, cola- borador del New York Times, afirma que para conseguir que el público no deserte de su deber de consumir, la avenida de la Publicidad (se refiere a Madison Square, donde se encuentran las principales agencias de publicidad del mundo) creó una nueva téc- nica de marketing: la transformación de la imagen. Dice Meyers que una campaña que ofrece a la una recompensa emocional aumenta su percepción con respecto a algo. Los escrúpulos morales quedan en segundo plano. - Publicidad Medioambiente: Acuerdo con el Ministerio de Medio Ambiente y Medio y Marino, y empresas del sector energético y de automoción (2009) - Publicidad de Juguetes: Convenio con la Asociación Española de Fabricantes de Juguetes (AEFJ) y el Instituto Nacional de Consumo (INC) (2010) ( ) y ( ) ( ) - Publicidad de Servicios de búsqueda de pareja y amistad: Convenio con eDarling, Meetic y Parship (2011) 4 Después de la televisión, es ya Internet el ámbito donde la publicidad engañosa crece a ritmo exponencial. 745 La publicidad en España como elemento de marketing: límites Fernando Ramos Fernández et al. “La gente ya sabe que la publicidad exagera y cada uno se cree lo que quiera”, se es- cudan cínicamente. El profesor Lema Devesa (2007: 387) afirma al respecto: “La exageración encie- rra una alegación concreta y comprobable, ya que la frase que integra esta modalidad publicitaria posee un contenido determinado, cuya veracidad o falsedad puede fijarse de acuerdo con las pautas generales”. Entiende que en toda exageración pueda ha- llarse un núcleo de verdad, pero no es menos cierto que también puede ser interpre- tada como “una alabanza de tono altisonante, concreta y comprobable que posee un núcleo verdadero y que no es tomada en serio por el público”. En los mensajes publicitarios de nuestros días se superponen diversas funciones. Es decir, que no solamente se nos anuncia un producto (y/o una marca), sino que se predican sus cualidades, su simbología cultural, las consecuencias que de su uso se desprende y hasta el rol social que asumimos cuando consumimos una determinada marca. (Sánchez Guzmán, 1976: 139). 5 La publicidad comercial dispone de recursos muy superiores a los de los individuos o los grupos que no están de acuerdo con una campaña de ventas; y esos recursos rebasan incluso a menudo los de los poderes públicos. La mayoría de los países imponen controles y garan- tías diversas, tales como códigos de conducta para los anunciantes, normas legislativas en- caminadas a comprobar la veracidad de las afirmaciones y requisitos de acuerdo previo. Pero no siempre se cumplen. Desde la psicología de la comunicación, se afirma que la pu- blicidad constituye un intento de influir sobre las personas con un fin determinado y sin co- acción (Kagelmann y Wenninger, 1996: 34 y ss.). Se trata de una influencia intencionada. El principal objetivo publicitario es el aumento de las ventas (objetivo económico). 4. El efecto de los anuncios sobre los consumidores Y si damos el salto cualitativo desde el producto hasta la marca, coincidimos con Dafonte (2011: 45) en que “el conocimiento de los consumidores y con el contexto so- ciocultural no figuran en ninguno de los otros modelos clásicos; sin embargo es obvio que en la creación de cualquier empresa, hoy en día, se tienen muy en cuenta las de- mandas –latentes o manifiestas– del mercado, para detectar las oportunidades de ne- gocio, y también la influencia del contexto sociocultural en cuanto a valores y actitudes […]aspectos que influirán en todas y cada una de las decisiones en torno a la identi- dad”. Se trata, en resumen de ofrecer lo que se está dispuesto a admitir. Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 nuevas herramientas para la defensa de los consumidores p En el caso de España, en cumplimiento de los objetivos fijados en las directivas eu- ropeas, se han ido introduciendo progresivas medidas para la defensa de los consu- midores, pero al mismo tiempo para acabar con las frecuentes prácticas de publicidad desleal que tanto perjudican al comerciante y al publicitario, honrados. En este sen- tido, la Ley 29/2009, de 30 de diciembre, por la que se modifica el régimen legal de la competencia desleal y de la publicidad para la mejora de la protección de los con- sumidores y usuarios está siendo un instrumento adecuadamente eficaz. y Especialmente relevante es el concepto de «consumidor medio», acuñado por la ju- risprudencia del Tribunal de Justicia de las Comunidades Europeas como la reacción típica del consumidor normalmente informado, razonablemente atento y perspicaz, teniendo en cuenta los factores sociales, culturales y lingüísticos. En consecuencia, no es un término que la ley haya de definir, sino que han de ser los tribunales los que van a efectuar su concreción en cada caso concreto. La relevancia de la publicidad en el proceso de toma de decisiones de los ciuda- danos es cada vez mayor. Por ello, cobra especial sentido la pervivencia del concepto de publicidad ilícita en el ámbito de la Ley General de Publicidad garantizando las ac- ciones y remedios que posibilitan su represión, especialmente frente a la publicidad que atente contra la dignidad de la persona o vulnere los derechos y valores recono- cidos en la Constitución, significativamente en lo que se refiere a la infancia, la ju- ventud y la mujer.6 6 El problema surge en ocasiones por el concurso entre la Ley General de Publicidad y la Ley de Competencia Desleal, que hace necesario, y así lo recoge la jurisprudencia, la introduc- ción de mecanismos de coordinación. Se modifica la Ley General de Publicidad, para dis- poner de un mismo cuerpo de acciones y remedios contra todas las prácticas comerciales que perjudiquen los intereses económicos de los consumidores, sin renunciar a la regulación es- pecífica de la publicidad y sin menoscabo de la legitimación especial que en la Ley Gene- ral de Publicidad se establece frente a la publicidad ilícita por utilizar de forma vejatoria o discriminatoria la imagen de la mujer. 5. Los estándares del estilo de vida que promueve la publicidad McBride (1980: 194) afirma que por centrarse fundamentalmente en la venta de bienes y servicios que la evalúan en términos monetarios, la publicidad tiende a promover unas actitudes y estilos de vida que exaltan la compra y el consumo de bienes en de- trimento de los demás valores. Chomsky y Herman (2000: 11) opinan que la publici- dad condiciona al medio, pues quiere atraerse para sí audiencias lucrativas que les proporcionen beneficios económicos y no audiencias que no posean fuerza econó- mica para consumir5. Si el lenguaje publicitario se encuentra instalado en el campo de la comunicación es por su capacidad para trasladar los significados a la sintonía de las afinidades, en un proceso intercambiable y continuo que va de la naturaleza de las cosas a la natu- raleza de las gentes, con todos sus códigos de interpretación y de inducción 5 La publicidad comercial dispone de recursos muy superiores a los de los individuos o los grupos que no están de acuerdo con una campaña de ventas; y esos recursos rebasan incluso a menudo los de los poderes públicos. La mayoría de los países imponen controles y garan- tías diversas, tales como códigos de conducta para los anunciantes, normas legislativas en- caminadas a comprobar la veracidad de las afirmaciones y requisitos de acuerdo previo. Pero no siempre se cumplen. Desde la psicología de la comunicación, se afirma que la pu- blicidad constituye un intento de influir sobre las personas con un fin determinado y sin co- acción (Kagelmann y Wenninger, 1996: 34 y ss.). Se trata de una influencia intencionada. El principal objetivo publicitario es el aumento de las ventas (objetivo económico). Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 746 Ramos Fernández et al. La publicidad en España como elemento de marketing: límites “La publicidad se mueve especialmente cómoda dentro del universo de la metáfora. Estimula, desde su lenguaje natural el juego comparativo, las subs- tancias adjetivas de las palabras más en su simbolismo que en su literalidad, entre lo enunciable y lo anunciable. El verbo es, en la publicidad radiada el eje substancial del mensaje radiofónico. Es el elemento que da coherencia a todo el mensaje. La publicidad radiofónica gusta de apoyarse en formas imperativas: «ven, mire, compre, haga, pruebe, tome»” (Ferrer, 1995: 43 y ss.). 7 Desde su creación, el Jurado de la Publicidad de Autocontrol ha resuelto más de 2.500 casos, en un tiempo medio de 14 días en primera instancia; en torno un 30% se resolvieron por “mediación” o “aceptación” de la reclamación por el anunciante en un plazo medio de 5 días, con el consiguiente cese de la publicidad. Concretamente en 2011 se tramitaron 146 casos. La mayor parte de las reclamaciones presentadas ante Autocontrol procedían de con- sumidores u otras organizaciones ciudadanas (en torno al 65%), siendo las presentadas por empresas o asociaciones empresariales el 31,5% aproximadamente. La Administración tam- bién utiliza esta vía extrajudicial en muchas ocasiones; el 1,4% de los casos tramitados en 2011 eran consecuencia de requerimientos de diferentes organismos de la Administración. Desde hace unos años el número de reclamaciones recibidas anualmente se ha estabilizado, gracias a la labor preventiva desarrollada por la Asociación, con un aumento considerable del uso de los servicios de consulta previa. Autocontrol cuenta con más de 428 miembros di- rectos y 4.000 indirectos en estos momentos, lo que prácticamente representa la totalidad de los actores que intervienen en la publicidad en España. 7. La eficacia del Jurado de la Publicidad Las decisiones del Jurado de Autocontrol son de obligado cumplimiento para los so- cios y voluntaria para el resto de casos sobre los que recaigan sus resoluciones, pero 6 El problema surge en ocasiones por el concurso entre la Ley General de Publicidad y la Ley de Competencia Desleal, que hace necesario, y así lo recoge la jurisprudencia, la introduc- ción de mecanismos de coordinación. Se modifica la Ley General de Publicidad, para dis- poner de un mismo cuerpo de acciones y remedios contra todas las prácticas comerciales que perjudiquen los intereses económicos de los consumidores, sin renunciar a la regulación es- pecífica de la publicidad y sin menoscabo de la legitimación especial que en la Ley Gene- ral de Publicidad se establece frente a la publicidad ilícita por utilizar de forma vejatoria o discriminatoria la imagen de la mujer. 747 Fernando Ramos Fernández et al. La publicidad en España como elemento de marketing: límites se supone que siempre tendrán repercusión social. No obstante, los socios, sean me- dios, anunciantes o agencias se comprometen a acatar las decisiones del Jurado7. La propia memoria anual de “Autocontrol”, correspondiente al año 2011, sobre esta controversia, señala: , “Pese a la voluntariedad del sistema, que sólo puede vincular estatutaria- mente a las entidades asociadas, es innegable la fuerza moral de la que gozan los pronunciamientos del Jurado de la Publicidad de Autocontrol en todo el sec- tor, adheridos y no adheridos al sistema. La autoridad técnica y la imparciali- dad demostrada por sus resoluciones han generado una alta credibilidad y confianza, tanto entre la industria publicitaria, como ante la Administración y la Sociedad en general. Buena muestra de ello es que, hasta la fecha, en menos de un 5% del total de casos resueltos por el Jurado de la Publicidad, la empresa reclamada no ha cumplido la resolución dictada al efecto por el Jurado. En al- gunos de esos casos, cuando, tras haber sometido el asunto al Jurado de la Pu- blicidad, las partes han acudido posteriormente a los Tribunales de Justicia, los pronunciamientos judiciales, hasta la fecha, han coincidido sustancialmente con el contenido de las resoluciones del Jurado previamente dictadas (excepto en un solo caso)”. Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 7. La eficacia del Jurado de la Publicidad (Autocontrol, 2011) Con respecto al ejercicio de 2011, los tres motivos principales de las reclamacio- nes presentadas por los consumidores, otras empresas o entidades mercantiles o la propia Administración que incluso usa esta vía fueron: la publicidad engañosa sin más (92 casos), la infracción del principio de legalidad en la publicidad de alimentos (cosa gravísima, 41 casos) o la infracción de códigos deontológicos sectoriales (43 casos; es decir, publicidad de productos milagro, publicidad de horario restringido, protec- ción de la infancia, etc.). Otros casos notables son aquellos en que los anuncios deni- gran a la competencia o explotan el miedo. Sobre un total de 148 casos, en 39; es decir, el 26 por ciento, el anunciante se avino con el reclamante a modificar o retirar el anuncio; pero el estimó totalmente las razo- 7 Desde su creación, el Jurado de la Publicidad de Autocontrol ha resuelto más de 2.500 casos, en un tiempo medio de 14 días en primera instancia; en torno un 30% se resolvieron por “mediación” o “aceptación” de la reclamación por el anunciante en un plazo medio de 5 días, con el consiguiente cese de la publicidad. Concretamente en 2011 se tramitaron 146 casos. La mayor parte de las reclamaciones presentadas ante Autocontrol procedían de con- sumidores u otras organizaciones ciudadanas (en torno al 65%), siendo las presentadas por empresas o asociaciones empresariales el 31,5% aproximadamente. La Administración tam- bién utiliza esta vía extrajudicial en muchas ocasiones; el 1,4% de los casos tramitados en 2011 eran consecuencia de requerimientos de diferentes organismos de la Administración. Desde hace unos años el número de reclamaciones recibidas anualmente se ha estabilizado, gracias a la labor preventiva desarrollada por la Asociación, con un aumento considerable del uso de los servicios de consulta previa. Autocontrol cuenta con más de 428 miembros di- rectos y 4.000 indirectos en estos momentos, lo que prácticamente representa la totalidad de los actores que intervienen en la publicidad en España. Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 748 do Ramos Fernández et al. La publicidad en España como elemento de marketing: límites Fernando Ramos Fernández et al. La publicidad en España como elemento de marketing: límites nes de 40 reclamaciones; es decir, el 27 por ciento, parcialmente en 23 casos, esto es casi el 16 por ciento y desestimó totalmente 36 reclamaciones; es decir, el 24 por ciento. 7. La eficacia del Jurado de la Publicidad Nada nuevo bajo el sol al analizar la causa de las resoluciones de Jurado de la Publici- dad. La publicidad engañosa sigue siendo la causa más repetida de reclamaciones y decisiones del jurado, con una peligrosa evidencia de las infracciones que se producen en torno a la publicidad de productos alimentarios o de los casos de publicidad sujeta a regulaciones específicas, de carácter sectorial, que asimismo se vulneran. Lo que está teniendo gran éxito es el servicio de Consulta Previa (Copy advice®) que fue puesto en marcha por la industria publicitaria, a través de Autocontrol, como complemento del sistema de control post-emisión del anuncio y con el objetivo de re- ducir el riesgo de incumplimiento de las normas que regulan la publicidad. Se trata de un servicio de asesoramiento no vinculante sobre la corrección legal y ética de un pro- yecto de campaña o anuncio, antes de su difusión al público. Tal es el grado de acep- tación de este servicio que, actualmente, es más solicitado que las propias intervenciones del Jurado, con más 62.700 consultas previas atendidas desde su puesta en marcha8. Resulta especialmente llamativo, en torno a lo pertinaz del engaño, según los pro- pios datos de Autocontrol, que entre los motivos que, tras la consulta previa, aconse- jaron la modificación de determinados anuncios, casi medio millar se debiera a publicidad engañosa (a juicio del propio Jurado de la Publicidad); que hubiera caso mil anuncios, no dirigidos a menores con limitaciones horarias o de medio; además de otros 323 casos de publicidad engañosa dirigida a menores o anuncios perniciosos para los menores y sus relaciones familiares hasta 62. p y En el recorrido histórico desde que existe Autocontrol, los anuncios emitidos en te- levisión representan el mayor volumen de contaminación engañosa, seguida por la prensa y por un preocupante crecimiento de los casos registrados en Internet, donde cada vez se engaña más. No obstante son significativos los datos del engaño e mar- keting directo, publicidad exterior y etiquetado Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 8 El Jurado de la Publicidad de Autocontrol también se encarga de la resolución extrajudicial de controversias sobre publicidad interactiva de CONFIANZA ONLINE. Este organismo nació a finales de 2002 por acuerdo de Autocontrol y Adigital (Asociación Española de la Economía Digital), antes AECEM. Su objetivo principal es aumentar la confianza de los consumidores en los nuevos medios interactivos. Actualmente, alrededor de 1.800 webs de empresas ya están adheridas. 8. Conclusiones La proliferación de Códigos de Conducta Ética, abundantísimos en la publicidad pa- rece más una estrategia de marketing e imagen que un sincero deseo de que la publi- cidad sea honesta y leal. El engaño y el incumplimiento de las normas que la afectan siguen siendo una herramienta de marketing, especialmente odiosa cuando se vulnera la buena fe de los consumidores. Aunque la organización Autocontrol presume de su eficacia, no todos creen en la sinceridad de sus objetivos, sino más bien que es un recurso para tener buena prensa y evitar el remedio judicial. 8 El Jurado de la Publicidad de Autocontrol también se encarga de la resolución extrajudicial de controversias sobre publicidad interactiva de CONFIANZA ONLINE. Este organismo nació a finales de 2002 por acuerdo de Autocontrol y Adigital (Asociación Española de la Economía Digital), antes AECEM. Su objetivo principal es aumentar la confianza de los consumidores en los nuevos medios interactivos. Actualmente, alrededor de 1.800 webs de empresas ya están adheridas. 749 ndo Ramos Fernández et al. La publicidad en España como elemento de marketing: límite La publicidad en España como elemento de marketing: límites La justificación de que la exageración publicitaria está socialmente aceptada y que los públicos no se creen los anuncios ha sido corregida por la Unión Europea, en el sentido de que los reclamos deben ser interpretados como lo haría “El ciudadano medio”; es decir, el hombre de la calle. Si la publicidad no se corrige, el Estado se ha armado con una serie de medidas que pueden imponer el cumplimiento efectivo de las afirmaciones de los reclamos. De ahí que los publicitarios huyan de decir cosas con- cretas, de las que se desprendan denotaciones determinadas. Se mueve en el terreno de lo simbólico, buscando connotaciones sublimes, pero que no dicen realmente nada. Suscitan o tratan de suscitar emociones. 9. Referencias bibliográficas. AUTOCONTROL (Asociación para la Autorregulación de la Comunicación Comer- cial) (2011): “Trabajamos por una publicidad responsable. Memoria y balance de actividad 2011”. Madrid, ediciones de la Asociación para la Autorregulación de la Comunicación Comercial. AUTOCONTROL (Asociación para la Autorregulación de la Comunicación Comer- cial) (2011): “Trabajamos por una publicidad responsable. Memoria y balance de actividad 2011”. Madrid, ediciones de la Asociación para la Autorregulación de la Comunicación Comercial. CARRILLO, Marc, (1997): Prólogo a BABÓ i BALBÉ. María Josep (1997): A pu- blicidade ilícita e a defensa dos consumidores. Santiago de Compostela, Lea. CHOMSKY, Noam y EDWARD S. Herman (2000): Los guardianes de la libertad. Barcelona, Planeta. DAFONTE GÓMEZ, Alberto (2011): “Evolución de los rasgos culturales del formato televisivo Operación triunfo en España desde la perspectiva de la identidad de marca (2001-2011). Revista de Comunicación de la SEECI, nº 25 de julio. Dispo- nible en: www.seeci.net/seeci/Numeros/Numero%2025/Alberto.pdf. Consultado el 12 de julio de 2012. DE LA CUESTA RUTE, José María (1985): Lecciones de Derecho de la Publicidad, Madrid. Madrid, Ediciones de la Universidad Complutense. DESANTES GUANTER, José María (1973): El autocontrol de la actividad infor- mativa. Madrid, Edicusa. FERRER, Eulalio (1995): El lenguaje de la publicidad. México, Fondo de Cultura Económica. KAGELMANN, H. Jürgen y WENNINGER, Gerd (1996): Psicología de los medios de comunicación. Barcelona, Herder. LEMA DEVESA, Carlos (2007): Problemas jurídicos de la publicidad. Madrid, Mar- cial Pons. MARTÍNEZ RODRIGO, Estrella y SÁNCHEZ MARTÍN, Lourdes (2012): “Publici- dad en Internet: nuevas vinculaciones en las redes sociales”. Revista de Comuni- cación Vivat Academia, nº especial de febrero. Disponible en: www.ucm.es/info/vivataca/numeros/n117E/PDFs/Varios13.pdf. Consultado el 15 de julio de 2012. Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 750 do Ramos Fernández et al. La publicidad en España como elemento de marketing: límites McBRIDE, Sean et al. (1980): Un solo mundo, voces múltiples. Informe sobre la Co- municación e Información en nuestro tiempo. México, Fondo de Cultura Econó- mica. McBRIDE, Sean et al. (1980): Un solo mundo, voces múltiples. Informe sobre la Co- municación e Información en nuestro tiempo. México, Fondo de Cultura Econó- mica. McLUHAN, Marshall (1996): Comprender los medios de comunicación. Las exten- siones del ser humano. Barcelona, Paidós Comunicación. McLUHAN, Marshall (1996): Comprender los medios de comunicación. Las exten- siones del ser humano. Barcelona, Paidós Comunicación. SÁNCHEZ GUZMÁN, José Ramón (1976): Breve historia de la publicidad. 9. Referencias bibliográficas. Madrid. Ediciones Pirámide. 10. Legislación Ley 29/2009, de 30 de diciembre, modifica el régimen legal de la competencia des- leal y de la publicidad para la mejora de la protección de los consumidores y usua- rios. Ley 29/2009, de 30 de diciembre, modifica el régimen legal de la competencia des- leal y de la publicidad para la mejora de la protección de los consumidores y usua- rios. Ley de Defensa de Consumidores y Usuarios es el Real Decreto Legislativo 1/2007, aprobado el 16 de noviembre de 2007 y publicado en el Boletín Oficial del Estado el 30 de noviembre. Ley de Defensa de Consumidores y Usuarios es el Real Decreto Legislativo 1/2007, aprobado el 16 de noviembre de 2007 y publicado en el Boletín Oficial del Estado el 30 de noviembre. Ley 43/2007, de 13 de diciembre, de protección de los consumidores en la contrata- ción de bienes con oferta de restitución del precio. Ley 43/2007, de 13 de diciembre, de protección de los consumidores en la contrata- ción de bienes con oferta de restitución del precio. Ley 43/2007, de 13 de diciembre, de protección de los consumidores en la contrata- ción de bienes con oferta de restitución del precio. Ley 23/2003, de 10 de julio, de Garantías en la Venta de Bienes de Consumo Ley 23/2003, de 10 de julio, de Garantías en la Venta de Bienes de Consumo. Ley 7/1998, de 13 de abril, sobre condiciones generales de la contratación. Ley 7/1998, de 13 de abril, sobre condiciones generales de la contratación. Ley 34/1988, de 11 de noviembre, General de Publicidad Ley 34/1988, de 11 de noviembre, General de Publicidad Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 Fernando RAMOS FERNÁNDEZ Universidad de Vigo ferramos@uvigo.es Teresa PIÑEIRO OTERO Universidad de A Coruña teresa.pineiro@udc.es David CALDEVILLA DOMÍNGUEZ Universidad Complutense de Madrid davidcaldevilla@ccinf.ucm.es Fernando RAMOS FERNÁNDEZ Universidad de Vigo ferramos@uvigo.es Fernando RAMOS FERNÁNDEZ Universidad de Vigo ferramos@uvigo.es Teresa PIÑEIRO OTERO Universidad de A Coruña teresa.pineiro@udc.es Teresa PIÑEIRO OTERO Universidad de A Coruña teresa.pineiro@udc.es David CALDEVILLA DOMÍNGUEZ Universidad Complutense de Madrid davidcaldevilla@ccinf.ucm.es 751 751 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751 Estudios sobre el Mensaje Periodístico Vol. 18 Núm. especial noviembre (2012) 741-751
https://openalex.org/W4304807477	https://repositorio.unican.es/xmlui/bitstream/10902/30151/3/SpinUpTime.pdf	English	null	Spin-up time and internal variability analysis for overlapping time slices in a regional climate model	Research Square (Research Square)	2,022	cc-by	12,882	Abstract Long-term regional climate simulations are computationally very costly. One way to improve their computational efficiency is to split them into overlapping time slices, which can then be run in parallel. Although this procedure reduces the cost, suf- ficient spin-up must be left at the start of each slice. In any case, discontinuities will occur due to internal variability where two different slices join. In this study, we explore the relative role of spin-up time and internal variability in the discontinui- ties of overlapping time slice simulations and their effect on the simulated climate. This analysis has implications also for non-overlapping time slices, commonly used in very high resolution climate modelling, where long transient simulations cannot be afforded. We show that discontinuities are negligible for surface and upper-air variables, but they are noticeable in variables with long response times, such as soil moisture or snow depth. For these variables, differences between the slices are mainly attributed to internal variability, but also to insufficient spin-up time, depending on the region. In general, the results show that the overlapping time slice approach is valid to accomplish long term regional climate simulations. Keywords Regional climate model · Time slice · Spin-up time · Internal variability · CORDEX Climate Dynamics (2023) 61:47–64 https://doi.org/10.1007/s00382-022-06560-2 Climate Dynamics (2023) 61:47–64 https://doi.org/10.1007/s00382-022-06560-2 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate model A. Lavin‑Gullon1 · J. Milovac1 · M. García‑Díez2 · J. Fernández1 Received: 27 August 2021 / Accepted: 21 October 2022 / Published online: 8 November 2022 © The Author(s) 2022 1 Introduction at kilometer-scale grid spacing) can only be afforded for time slices of about a decade (Coppola et al. 2020; Pichelli et al. 2021). This approach considers a decadal simulation driven by a future scenario and a reference decade driven by historical conditions to explore changes in climate. Such practice limits the climate analyses to periods well below the minimal climate standard of 30 years (WMO 2017). In the near future, centennial simulations of a kilometer-scale RCM will be feasible, especially if the RCM community adopts the latest advances in computing (Leutwyler et al. 2016). However, time slices will still be required for the ever-increasing model resolution, complexity and coupling with other demanding model components. In this work, we consider the use of a set of overlapping time slices to accom- plish multi-decadal RCM simulations and we explore the effects of this approach on the simulated climate. Climate model resolution has always increased hand in hand with the available computer power. As an example, 30 years ago, the computational demand of the first regional climate models (RCMs; Giorgi 2019) limited their use to 60 km grid spacing for a month-long simulation (Dickinson et al. 1989; Giorgi and Bates 1989). Currently, centennial RCM simulations at ca. 10 km grid spacing are routinely carried out at different research centers (Jacob et al. 2020). Still, the experiments with the highest spatial resolution (currently * J. Fernández jesus.fernandez@unican.es A. Lavin‑Gullon alvaro.lavin@unican.es J. Milovac milovacj@unican.es M. García‑Díez garciam@predictia.es 1 Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain 2 Predictia Intelligent Data Solutions, Santander, Spain * J. Fernández jesus.fernandez@unican.es A. Lavin‑Gullon alvaro.lavin@unican.es J. Milovac milovacj@unican.es M. García‑Díez garciam@predictia.es 1 Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain 2 Predictia Intelligent Data Solutions, Santander, Spain * J. Fernández jesus.fernandez@unican.es A. Lavin‑Gullon alvaro.lavin@unican.es J. Milovac milovacj@unican.es M. García‑Díez garciam@predictia.es f The idea of splitting a climate simulation into pieces is nearly as old as regional climate modelling (Pan et al. 1999). There are different reasons for doing so, though. A common reason to re-initialize a climate simulation is to keep it close to the observations. For this purpose, a frequent re-initial- ization is advocated (Pan et al. 1999; Qian et al. 2003; Lo et al. 2008). 1 Introduction The frequent cold-start from reanalysis initial conditions constrains the weather trajectory of the model to 1 Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain 2 Predictia Intelligent Data Solutions, Santander, Spain (0121 3456789) 3 48 A. Lavin‑Gullon et al. Due to internal variability in the RCM (Christensen et al. 2001; Lucas-Picher et al. 2008), a perfect match of the weather trajectory in two consecutive time slices is not possible. Internal variability is unavoidable and is triggered by the different initial conditions in the time slices, so there will always be a ’weather jump’ at the joints. On top of the internal variability, the coarse initial conditions from the driving GCM or reanalysis need some time (spin-up time) to be assimilated by the RCM dynamics. This spin-up time depends on the variable. It is quite short for atmospheric variables, but it can extend for several months or even years for other slow-varying variables (Christensen 1999; Cos- grove et al. 2003; Jerez et al. 2020).f be close to the observed one. This approach can introduce discontinuities in the weather events and, more importantly for climate analyses, may disrupt the proper evolution of variables with long response times, such as deep soil vari- ables. To prevent this disruption, the so-called poor man’s reanalysis approach (Berg and Christensen 2008; Stahl et al. 2011; Lucas-Picher et al. 2013) keeps the soil variables across the different re-initializations, and updates only the atmospheric initial conditions from reanalysis data. In this latter approach, the simulation pieces are not independent of each other and there is no computational advantage in the re-initialization. The computational advantage is a second reason to split an RCM simulation into pieces, which can then be run in parallel (Jimenez et al. 2010; Menendez et al. 2014). This form of parallelism can be more efficient than standard high- performance parallel computing paradigms such as Open Multi-Processing (OpenMP) or Message Passing Interface (MPI) (Jerez et al. 2009). Using these standard parallel com- puting approaches, computing time typically scales well with the number of processors up to a limit. This limit is usually much lower than the number of processors available. Even for a reasonable scaling, there is always a loss in using an increasing number of processors. Therefore, computing time is used more efficiently when splitting the simulation and running the pieces on a smaller amount of processors. 1 Introduction For their use in climate studies, the initial part of each sim- ulation piece must be disregarded as model spin-up. This spin-up period is, typically, at least one year (Christensen 1999), although a few months might suffice depending on the season when the pieces are initialized (Jerez et al. 2020). This computing time trade-off between the gain by a more efficient use of the processors and the waste due to spin up of each simulation piece, can be used to optimize the length of the pieces (Jerez et al. 2009). The objective of this study is to show the effect of the overlapping time slice approach on the regional climate sim- ulated by an RCM. For this purpose, we used state-of-the- art simulations carried out under the COordinated Regional climate Downscaling EXperiment (CORDEX; Giorgi and Gutowski 2015) framework using the Weather Research & Forecasting (WRF) modelling system (Sect. 2.1). Analy- ses were carried out for variables with different response time, using split and continuous simulations for different regions, at different resolutions and with time slices in split simulations initialized in different seasons. We studied the weather jumps in split simulations, locating their occurrence (Sect. 3.1), analysing their evolution in time (Sect. 3.2) and their geographical location (Sect. 3.3). Finally, we analysed the potential effect of splitting the simulations into time slices on the simulated climate (Sect. 3.4). 3 2.1 Data This CP simulation was nested into the EUR-15 domain and centered on the Alpine region (ALP- 3). The model setup is the same as EUR-15, except for the cumulus parameterization, which was deactivated (Ban et al. 2021).fi version and linked to a different version of the parallel com- puting (OpenMPI) libraries. Therefore, the differences between the EUR-15 continuous run and the first time slice of the split simulation (EUR-15 S1) will be due to different numerical round-off in the model executable. These differ- ences are expected to grow and evolve with the flow as those caused by perturbing initial conditions, i.e. internal variabil- ity (Geyer et al. 2021). The continuous runs are considered as a reference to investigate possible inhomogeneities caused by the time slicing of the split simulations. It is worth noting that these sets of simulations were not specifically designed for this study. We use them as an ensemble of opportunity to study the spin-up length and the role of internal variability in the climate simulated by overlapping time slices. As such, we can only explore the variables available for each simulation, and the initialization seasons of the slices used in these multi-year simulations. A designed, systematic exploration of the required spin-up time has been recently carried out by Jerez et al. (2020) over Europe for a 1-year test period. Our approach extends this work by considering domains in different climates, different spatial resolutions, longer spin-up lengths, and the role of interannual and internal variability. Two different model configurations were used in terms of physical parameterizations. Namely, EURO-CORDEX WRF configuration WRF341I (Manzanas et al. 2018) was used in EUR-44 and SAM-44, while an updated model ver- sion and configuration, WRF381BI (Ban et al. 2021), was used in EUR-15. The most important difference concern- ing our results is the different land surface model (LSM). WRF381BI used the new Noah with multi-parameteriza- tion options (Noah-MP v1.1) LSM (Niu et al. 2011), while WRF341I was coupled to its predecessor Noah LSM (Chen and Dudhia 2001). All simulations were carried out with prescribed sea surface temperature and sea ice, evolving as provided by the driving global model or reanalysis. The three sets of simulations were performed by splitting the runs into several time slices. In order to allow for the required spin-up time, adjacent time slices were overlapped for at least one year (Fig. 1). 2.1 Data In this study we analyse three sets of simulations using the WRF model (Skamarock et al. 2008), with different param- eterization schemes, spatial domains, horizontal resolutions and time periods. Simulations were performed over three model domains as defined within CORDEX. One set of sim- ulations was carried out over the CORDEX South American domain at the standard 0.44◦ horizontal resolution (SAM- 44), regular on a rotated latitude-longitude projection (Falco et al. 2019; Solman and Blázquez 2019). These simulations were carried out for the historical period 1951–2005 and for the future scenarios RCP 4.5 and RCP 8.5 for the period 2002–2100, all driven by the Canadian Earth System Model (CanESM2; Arora et al. 2011). The other two sets consisted of evaluation simulations over Europe at 0.44◦ (EUR-44) and ∼ 15 km (EUR-15) horizontal grid spacing, driven by the ERA-Interim reanalysis (Dee et al. 2011). EUR-44 simula- tions span the period 1979–2010 (Vautard et al. 2013), while the EUR-15 simulations were generated under the CORDEX RCM simulation splitting is hardly avoidable for very long simulations, such as those for the last millennium (Gómez-Navarro et al. 2011). This procedure can also allevi- ate the computational burden for research groups to perform centennial climate change RCM simulations. As an example, in this work, we analyze RCM simulations carried out in the last decade at Universidad de Cantabria (UCAN) as split runs and also as continuous simulation (Sect. 2.1). A form of simulation splitting is also used for the most computa- tionally demanding RCM simulations (Coppola et al. 2020; Pichelli et al. 2021), where only a couple of decadal time slices can be afforded. Time slicing is just a simulation split into pieces, but with pieces that do not usually overlap. Here, we analyse the effect that overlapping a set of time slices would have on the simulated regional climatology. Note that this analysis cannot be carried out on standard time slice experiments, where the slices do not overlap and the proper initialization of the simulation cannot be assessed. 1 3 49 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… Flagship Pilot Study on convective phenomena at high reso- lution over Europe and the Mediterranean (FPS-CONV), covering the period 1999–2009 (Coppola et al. 2020). An additional convection-permitting (CP) simulation at ∼ 3 km horizontal resolution, is also analysed to evaluate the effect of the resolution. 2.1 Data Additionally, the EUR-15 and EUR-44 simulations were also performed continuously. The continuous runs were produced in two different ways. For EUR-44, the first time slice (S1) was extended to cover the full period. For EUR-15, the full period was simulated again, but the model was compiled with a different compiler We focus our analyses on three types of variables. Initially, we consider slow-varying variables as their accurate initializa- tion and representation are of key importance for weather and climate modelling. They require significant spin-up times as their initial conditions, taken from the driving model, usually differ greatly from the conditions generated by the RCM (Jerez et al. 2020). For this purpose, among the available variables, here we analyze total soil moisture and snow depth. These variables control energy partitioning at the land surface and, 1 3 1 3 Fig. 1 Schematic representation of the analysed simulations. Time slice simulations (S1, S2, etc.) are used to compose split simulations for each domain (switching time between slices is indicated by arrows). Continuous simulations are represented either as independ- ent simulations (EUR-15) or by extending the S1 slice (EUR-44). Overlapping periods are shaded in grey shades: light grey for the overlap of two simulations, and dark grey for three overlapping simulations 1 3 A. Lavin‑Gullon et al. 50 through land-atmosphere feedbacks, they influence the evolu- tion of the atmospheric conditions in the planetary bound- ary layer. In such a way, these variables impact local weather and regional climate (Seneviratne et al. 2006). Furthermore, we considered near-surface temperature and precipitation, two fundamental variables that characterize the regional cli- mate and are often considered in climate and impact studies. Both are highly variable in time and space. The atmospheric circulation was analysed as well, since it has also a strong but non-local impact on the regional climate (Zappa 2019). Atmospheric circulation shows the shortest response time, as compared to surface or subsurface fields. We characterize the circulation by means of the geopotential height at 850 hPa. The analysis was carried out for daily mean values for all variables. (7) X 휏(s, n) = 1 T(휏) ∑ t∈휏 X(s, n, t) (7) with T(휏) representing the number of time steps in 휏 . This is done to use intra-seasonal variability as reference, thus preventing the annual cycle variability to mask large differ- ences for a given season. 2.2 Methodology (8) RMSDtX(s, t) = √ √ √ √1 N N ∑ n=1 DtX(s, n, t)2 (8) The analysis of discrepancies across the simulation slices is based on simple differences. For a given variable X(s, n, t), taken from a simulation slice s at grid point n and time step t, we define the following differences: Inter-slice differences ( RMSDsX ) are employed to quantify the differences between two slices along the overlapped period (see Sect. 3.3): (1) DtX(s, n, t) = X(s, n, t) −X(s, n, t −1) (2) DsX(s, n, t) = X(s, n, t) −X(s −1, n, t) (3) DstX(s, n, t) = X(s, n, t) −X(s −1, n, t −1) (1) (9) RMSDsX(s, t) = √ √ √ √1 N N ∑ n=1 DsX(s, n, t)2 (9) (2) (3) We also consider the quadratic average of DstX ( RMSDstX ), which arises in the context of split simulations; it is the RMSDtX at the joint of time slices. This measure quantifies the inhomogeneity introduced at the joint for different vari- ables (see Sect. 3.1): for consecutive time steps t −1 and t, and consecutive simu- lation slices s −1 and s. Note that the meteorological jump ( Dst ) in variable X, occurring at the joint between time t −1 in simulation slice s −1 and time t in slice s, can be decom- posed as the difference between slices Ds at time t −1 plus the variable tendency in time ( Dt ) within slice s: for consecutive time steps t −1 and t, and consecutive simu- lation slices s −1 and s. Note that the meteorological jump ( Dst ) in variable X, occurring at the joint between time t −1 in simulation slice s −1 and time t in slice s, can be decom- posed as the difference between slices Ds at time t −1 plus the variable tendency in time ( Dt ) within slice s: (10) RMSDstX(s, t) = √ √ √ √1 N N ∑ n=1 DstX(s, n, t)2 (10) (4) DstX(s, n, t) = DsX(s, n, t) + DtX(s −1, n, t) = DsX(s, n, t −1) + DtX(s, n, t), (4) Finally, as a reference for natural variability we also estimate transient eddy variability (Caya and Biner 2004; Lucas- Picher et al. 2008; Lavin-Gullon et al. 3 Results (6) 2.2 Methodology 2021): In order to have a relative measure, we consider non-dimen- sional differences in terms of standard deviation units, by dividing each of them by the standard deviation in time at each grid point: (11) TEV(s, 휏) = √ √ √ √1 N N ∑ n=1 [ X(s, n, t) −X 휏(s, n) ]2휏 (11) (5) dstX(s, n, t) = DstX(s, n, t) sdDtX(s, n) (5) where 휏 represents in this case all days in a calendar month, in order to have a monthly TEV estimate. where the standard deviation sdDtX(s, n) is calculated as (6) sdDtX(s, n) = √ 1 T(휏)−1 ∑ t∈휏 [ DtX(s, n, t) −DtX 휏(s, n) ]2 2.1 Data These time period averages have also been used to assess the long-term impact of time slicing on the climatology of a given variable (Sect. 3.4).fi Differences (D) are spatio-temporal fields. We summa- rize them by means of spatial root mean squared differences (RMSD), to avoid compensation of opposite differences across the domain. For each slice, the intra-slice daily ten- dency ( RMSDtX ) summarizes the differences between con- secutive time steps: 3.1 Detection of meteorological inhomogeneities from a 45-day time period ( 휏 ) prior to the target time t. The overline represents time average over a given time period 휏: Unlike continuous regional climate simulations, split simulations contain meteorological inhomogeneities, i.e. 1 3 1 3 3 3 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… 51 the season when joining occurs. The SAM-44 domain spans mid-latitude as well as tropical regions and, thus, geopoten- tial heights show a smaller range of change (20–80 m) and a much weaker seasonal cycle. However, daily inhomoge- neities go unnoticed. Geopotential height is strongly driven by the lateral boundary conditions and the pass of weather systems through the domain. Moreover, the 1-year spin-up period considered in the time slices is expected to be long enough for upper atmospheric variables, such as geopoten- tial height, to reach physical equilibrium within the model. Therefore, these variables do not suffer from noticeable inhomogenities.l unphysical changes in the state of the system at the joints of the time slices. This is unavoidable, given that an exact match of two climate simulations is impossible due to the chaotic nature of the climate system. For RCMs, the con- straint exerted by the lateral boundary conditions make the inhomogeneities much smaller than in global models. Still, substantial internal variability develops in RCMs (Lucas- Picher et al. 2008; Bassett et al. 2020; Lavin-Gullon et al. 2021), preventing a smooth transition between simulation slices. The ability to detect these meteorological inhomogenei- ties depends on the simulation sampling frequency (i.e. out- put frequency) used. The inhomogeneity will pass unnoticed if it is smaller than the change between consecutive out- put times. And this change is larger as sampling frequency decreases. Intra-slice daily tendencies ( RMSDt ) quantify the changes between consecutive time steps. At the slice joints in a split simulation, RMSDt becomes RMSDst and quanti- fies the size of the inhomogeneity along with the variable tendency. The same result applies for variables that are influenced to a greater or lesser extent by the lateral boundary forc- ing, such as near-surface temperature, precipitation or snow depth (not shown). For snow depth, which varies slowly, regional inhomogeneities are apparent, but the domain-wide summary in RMSDt masks the differences in the relatively small snow-covered regions in the domains considered. Soil variables (e.g. total soil moisture in Fig. 3.1 Detection of meteorological inhomogeneities 3), however, show large discontinuities at the slice joints. RMSDt shows inho- mogeneities in all three sets of simulations, clearly unveiling the time when two slices join. Daily tendencies in total soil moisture range between 2 and 10 kg∕m2 , except for peaks on certain days with values beyond 30 kg∕m2 , corresponding to the joints of the time slices. The order of magnitude of these peaks is not sensitive to the season in which the adjacent slices join (winter in EUR-44, summer in SAM-44, winter Of course, the relevance of the inhomogeneity depends also on the variable. For geopotential height (Fig. 2), inho- mogeneities go unnoticed. Average daily geopotential ten- dencies in mid-latitudes (EUR-15, EUR-44) range between 20 and 100 m, with a prominent annual cycle. The geo- potential change remains within this range when passing from one time slice on one day to the next time slice on the next day (indicated by arrows in Fig. 2), regardless of Fig. 2 RMSD between consecutive days ( RMSDt ) for geopotential height at 850 hPa (m) in the EUR-15, EUR-44 and SAM-44 split simulations. Joints of time slices are indicated by arrows. In SAM-44, light gray shading refers to the historical run while dark gray shading corresponds to RCP 8.5 forced run light gray shading refers to the historical run while dark gray shading corresponds to RCP 8.5 forced run Fig. 2 RMSD between consecutive days ( RMSDt ) for geopotential height at 850 hPa (m) in the EUR-15, EUR-44 and SAM-44 split simulations. Joints of time slices are indicated by arrows. In SAM-44, 1 3 52 A. Lavin‑Gullon et al. Fig. 3 As Fig. 2, but for the total soil moisture content ( kg∕m2 ). The inset in the lower panel shows the whole 1950-2100 SAM-44 historical (grey) plus RCP 8.5 scenario period. Numbers at the top of the each panel represent maximum values (out of scale) at the joints of the slices Fig. 3 As Fig. 2, but for the total soil moisture content ( kg∕m2 ). The inset in the lower panel shows the whole 1950-2100 SAM-44 historical (grey) plus RCP 8.5 scenario period. Numbers at the top of the each panel represent maximum values (out of scale) at the joints of the slices and spring in EUR-15). 3.2.1 Total soil moisture Total soil moisture intra-slice daily tendencies (Fig. 4a, top panel) differ between the two time slices at the beginning of the overlapping period. At this time, total soil moisture in S2 is mainly provided by the initial conditions from ERA-Interim, while in S1 the soil state is generated by the RCM itself. In EUR-15, the overlap period is initiated in late summer (September) so that the difference in the daily tendencies between S1 and S2 decreases rapidly. Day-to-day variability is mainly determined by the variability in the top soil layer, which in turn depends on precipitation. Therefore, there is evident correlation between day-to-day variability of total soil moisture and precipitation (see Fig. 4a and d, top panels). Thus, under drier conditions like it is the case in late summer, daily soil moisture tendencies from the driving 3.1 Detection of meteorological inhomogeneities These peaks also show low interan- nual variability, clearly standing out from the background variable tendency for every joint in different years (see inset in Fig. 3). Despite the strong signal in RMSDt , these peaks in the differences are still one order of magnitude smaller than the variable itself; e.g. the quadratic mean of total soil moisture in SAM-44 is about 600 kg∕m2. shows RMSDs for the 1.5-year overlap between S1 and S2 time slices for EUR-15 domain, covering the period from Sep., 2003 to Feb., 2005. The EUR-15 continuous simula- tion is also included (black line in the Fig. 4) as reference for RMSDs . Figure 5 shows another example for a 1-year overlap period between S1 and S2 for the SAM-44 domain, covering the complete year 2006. In this case, no reference continuous run was available, but we included TEV as a reference for natural variability (black line in the Fig. 5). These strong discontinuities at the joints indicate that there are high discrepancies in soil moisture between two time slices. This could be due to two reasons: (1) the spin- up period considered is not long enough for soil moisture to balance within the model or/and (2) soil moisture internal variability is larger than the daily tendency. This is investi- gated next. 3.2 Meteorological inhomogeneities in time In a split simulation, a set of time-sliced simulations are concatenated after removing an initial spin-up period. In principle, the longer the spin-up period, the better. A given slice enters the split simulation just after the previous slice has finished (as depicted in Fig. 1). However, the switch between the slices can be chosen to occur at any time dur- ing the overlapping period (grey-shaded areas in Fig. 1). We can quantify the size of the discontinuity by means of inter-slice differences ( RMSDs , Eq. 9). As an example, Fig. 4 1 3 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… 53 53 Spin up time and internal variability analysis for overlapping time slices in a regional climate… model adapt faster to the soil moisture conditions in the long necessary for the two adjacent slices S1 and S2 to start Fig. 4 RMSD for a total soil moisture, in kg∕m2 , b snow depth, in m, c near-surface temperature, in K, d daily accumulated precipitation, in mm, and e geopotential height at 850 hPa, in m, for EUR-15 S1, S2 and continuous simulations; see Fig. 1. For each variable, intra-slice daily tendencies ( RMSDtX ) for each simulation (top panel) and inter- slice differences ( RMSDsX ) between S1 and S2, and between S1 and continuous (bottom panel) Fig. 4 RMSD for a total soil moisture, in kg∕m2 , b snow depth, in m, c near-surface temperature, in K, d daily accumulated precipitation, in mm, and e geopotential height at 850 hPa, in m, for EUR-15 S1, S2 and continuous simulations; see Fig. 1. For each variable, intra-slice daily tendencies ( RMSDtX ) for each simulation (top panel) and inter- slice differences ( RMSDsX ) between S1 and S2, and between S1 and continuous (bottom panel) model adapt faster to the soil moisture conditions in the long term RCM run (i.e. slice S1 here). necessary for the two adjacent slices, S1 and S2, to start evolving coherently in time. It is interesting to notice that the two slices tend to diverge again at the end of the overlap- ping period, which also corresponds to the austral summer. This can be associated to the discrepancies in summertime precipitation between the two slices (see Fig. 5d top panel). 3.2 Meteorological inhomogeneities in time These results show that, for this specific simulation, joining the slices at the end of summer would minimize the inhomogeneity of the snow depth, while not affecting significantly the inhomogeneity of soil moisture at the S1–S2 joint. Moreover, this is valid also for shorter overlapped periods initialized in June (for EUR- 15 for S2–S3 slices) or in March (for EUR-15 for S3–S4 slices), which can be seen in the Supplementary Material in Figures ESM1 and ESM2, respectively. Minimal snow depth inhomogeneities across time slices are obtained by switching slices at the end of late summer (September) in all cases, although for the S2–S3 slices when the overlapped period starts in June (just 3 months before) 1 year long overlap period is insufficient for soil moisture to spin up. In SAM-44, minimum values also appear in austral summer for RMSDt (Fig. 5a) and in late summer for RMSDs (Fig. 5b), when it would be most convenient to join the slices. Although snow in SAM-44 is scarce, covering only some areas in the south- ern Andes, selecting austral summer to start the overlapping in SAM-44 is beneficial. EUR-15, RMSDs stabilizes at 40 kg∕m2 after about one year, while in SAM-44 the minimum value of 70 kg∕m2 is reached after just a few months. At these points internal variability becomes the major cause of the differences between S1 and S2, which leads to a conclusion that spin-up in SAM-44 is shorter than in EUR-15. This highlights that not only the season determines the spin-up time, but also the synoptic regimes specific for the region. In SAM-44, the overlapped period starts in the aus- tral summer (DJF) but, unlike in EUR-15, summer is the wet season in Central South America (Liebmann and Mechoso 2011), which largely contributes to the annual precipitation (and soil moisture) in the whole SAM-44 domain. Shorter spin-up in moist regimes (SAM-44) than in drier regimes (EUR-15) may be related to the parameterization of vertical water transport within the soil. It is based on Richards’ equa- tion in all LSMs used in the analysed simulation sets, which is more efficient under moist conditions in the wet season (Khodayar et al. 2015). It is worth comparing the scales of inter- and intra-slice differences for soil moisture, which differ by about one order of magnitude. 3.2 Meteorological inhomogeneities in time In SAM-44, the overlapped period starts in austral sum- mer, which in this region corresponds to the rainy season. In this case, intra-slice daily tendencies for soil moisture (Fig. 5a, top panel) show that more than 3 months are 1 3 1 A. Lavin‑Gullon et al. 54 54 A. Lavin‑Gullon et al. Fig. 5 As Fig. 4, but for the SAM-44 domain, and overlapping time slices S1 and S2 Fig. 5 As Fig. 4, but for the SAM-44 domain, and overlapping time slices S1 and S2 spin-up, we can also use the continuous run as the reference when available (i.e. EUR-15 domain). In EUR-15 case, we computed the inter-slice difference between S1 (initialized 5 years before the overlapping period, see Fig. 1) and the continuous simulation (black line in Fig. 4a). These differ- ences are controlled by internal variability only, and they set the lower limit for inter-slice differences. RMSDs between S1 and S2 during the overlapped period are initially very high in both domains (bottom panels of Figs. 4a and 5a). In Daily soil moisture inter-slice differences ( RMSDs , bot- tom panels of Figs. 4a and 5a) are more controlled by the moisture state of deeper soil layers, therefore we can con- sider these differences to be strongly influenced by soil mois- ture spin-up . These differences will reach a minimum and never a zero value due to internal variability (Lavin-Gullon et al. 2021). When RMSDs is stabilized, this denotes that internal variability had overcome the initial spin-up tran- sient. To distinguish internal variability from insufficient 3 3 55 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… minimum. Afterwards, as the snow depth starts to increase, RMSDs increases again following the seasonality pattern of internal variability. However, RMSDs never reaches the first winter minimum again. This leads to a conclusion that the occurrence of the initial drop to the first winter minimum does not mean the balancing of the snow. When the RMSDs values drop to the overall minimum, which is limited by the inter-slice differences between slice S1 and the continuous run (black line on the bottom panel in Fig. 4b), snow depth spin-up can be considered as completed. 3.2 Meteorological inhomogeneities in time This scale difference causes the RMSDt peaks in split simulations denoting the soil moisture inhomogenei- ties at the slice joints shown in the previous section. In par- ticular, the first peak in Fig. 3 (top panel) has a contribution from both RMSDt(S2) and RMSDs(S1 −S2) lines (Fig. 4a) at the end of the overlapping period, when slice S1 switches to S2 in the EUR-15 split simulation. This shows that an earlier switch from S1 to S2 would have not led to smaller inhomogeneities. i In EUR-44 (Figs. ESM 3a and b in the Supplementary Material) a 2-year overlap period is available. This allows for better assessment of the annual cycles and variability of RMSDt , inter-slice differences ( RMSDs ) between S2 and S3, and the internal variability, estimated by RMSDs between S2 and the continuous run. This time slice overlap (S2-S3) confirms all the results previously shown for EUR-15: (1) late summer is a good season to choose to switch slices for snow cover, (2) the seasonal cycle in soil moisture internal variability, peaks also in late summer, which is clearer here. However, for this particular year, soil moisture seems not to be completely spun up. This highlights the need for an interannual assessment of soil moisture spin-up times. 3.2.2 Snow depth Snow depth shows a different behavior than soil moisture in EUR-15 (Fig. 4b, top panel). Initial RMSDt values in S2 are significantly higher than those in S1, but they get close to each other in just a few days. In SAM-44 (Fig. 5b, top panel), this initial difference is not evident as the snow depth is insignificant there during the austral summer, when the overlapped period starts. After balancing, RMSDt evolves coherently in time for both slices in both domains, with an evident seasonal cycle. The variability of day-to-day RMSDt is higher in winter and early spring, when snow depth changes due to snowfalls and snow melting, and lower values occur in summer when snow coverage is small and limited to areas with permanent snow. Inter-slice differences during the overlapped period in EUR-15 (bottom panel in Fig. 4b) show an interesting behavior. After just a few days, RMSDs for S1 and S2 slices reaches a winter minimum that remains almost constant until the end of April 2004. After that time, it starts to drop again. In summer, since the snow coverage is minimal, inter-slice differences reach the overall 3.2.3 Other variables The other variables (Fig. 4c–e) show no initial discrepancies as a hint of spin-up period, with an evolution of day-to-day changes ( RMSDt ) similar for both slices, and inter-slice dif- ferences ( RMSDs ) consistent with internal variability. The order of magnitude of both RMSDs is the same, therefore there is no apparent inhomogeneities at the joints (e.g. see Fig. 2). 1 3 56 A. Lavin‑Gullon et al. Geopotential height (Fig. 4c top panel) exhibits seasonal cycle with larger day-to-day changes ( RMSDt ) in winter than summer, which is typical mid-latitude variability in Europe (Caya and Biner 2004). On the other hand, this seasonality is not evident in SAM-44 (Fig. 5c top panel). The SAM- 44 domain is not limited only to mid-latitudes, but it also covers large tropical areas showing no variable seasonal- ity, which smooths the final results. Nevertheless, the larger summer internal variability is apparent in the RMSDs . Inter- slice differences ( RMSDs ) between S1 and S2 slices do not show strong deviations from internal variability (i.e. RMSDs between the continuous simulation and S1 in EUR-15), therefore most of the differences between time slices can be explained by the internal variability itself. For precipita- tion intra-slice daily tendencies evolve more coherently in SAM-44 than in EUR-15, especially in the austral winter. In both domains, differences in RMSDt between the two slices does not change along all the overlapped period, regardless of the season. On the other hand, a seasonal cycle appears in inter-slice differences, with higher (lower) differences between the slices in the austral summer (winter), following the seasonal pattern observed for geopotential height at 850 hPa. This seasonal behaviour is more apparent in SAM-44 than in EUR-15. the forcing between the RCP 4.5 and 8.5 scenarios are so small in 2006 that the two global climate realizations can be considered as resulting from the GCM internal variability. At least, regarding the atmospheric fields fed to the RCM. In a few weeks, the slight forcing differences make the GCM circulation diverge and the synoptic situation of correspond- ing days in the two global model realizations will be as dif- ferent as two random days in the corresponding season. Note that no inhomogeneity occurs, since both GCM realizations are started from the same final state of the historical run at the end of 2005. 3.2.4 Horizontal resolution We bilinearly remapped all EUR-15 variables to the ALP-3 domain (not shown). Apart from a slightly higher initial state of the soil moisture in ALP-3, all RMSDt and RMSDs time series during the overlap periods are virtually identical in the remapped EUR-15 and ALP-3 resolutions. Horizontal resolution does not seem to play any major role on the model spin-up and inhomogeneities of split simulations. This small sensitivity to the change in horizontal resolution can be seen also in Figs. 2 and 3, with very similar RMSDt evolution in both EUR-44 and EUR-15, regardless of the time slice considered and despite the differences in model version and configuration. This decorrelation time to reach the GCM internal vari- ability level sets a minimum response time for the spin- up time. The RCM starts the adjustment process from an internally consistent state, unlike in the spin-up process, which needs to bring the initial state into line with the RCM dynamics. Therefore, spin-up times should be longer than the smooth adjustment time to decorrelated synoptic situ- ations. This is illustrated in Fig. 5, where the monthly √ 2 TEV lines have been included as reference. As expected, surface and upper air variables adjust in a few weeks. In fact, geopotential height will adjust almost immediately, since the 2-week delay shown in Fig. 5e is likely the time taken by the GCM circulation to decorrelate. Soil moisture takes about 3 months to reach decorrelation ( √ 2TEV line in Fig. 5a). Note that the Figure includes Dec-2005, which still represents RCM internal variability levels. 3.2.3 Other variables Synoptic conditions depart smoothly as slight changes in the forcing introduce small perturbations which are amplified by the model dynamics to become finite perturbations. From the point of view of the RCM, soil variables will evolve smoothly, with the land surface model responding in a physically consistent manner to the new atmospheric con- ditions. Snow cover should also adjust smoothly to the new synoptic conditions fed through the boundaries. This adjust- ment process is similar to the spin-up, since the RCM inter- nal fields need to adjust to the new driving fields. Unlike the spin-up process, model states are physically consistent dur- ing the whole process; no tendencies develop in the model to account for the mismatch between the initial conditions and a balanced model state. The expected RMSDs(S1 −S14) value after the adjustment is not the RCM internal variabil- ity limit in this case (since the driving fields differ), but the GCM internal variability. This can be estimated from the transient eddy variability (TEV, Eq. 11). In particular, for uncorrelated fields from two GCM realizations, RMSDs should reach √ 2TEV (Caya and Biner 2004). The results for near-surface temperature follow the simi- lar pattern as those for the geopotential height at 850 hPa. 3 3.2.5 Another view on spin‑up time However, by the end of the spring, the strength of the boundary forcing decreases and internal variability increases, allowing for larger discrepancies between time slices. Thus, the discrepancies between the two slices are larger at the joint on June 1st, 2006 (Fig. ESM 4), as it can be observed over northern Europe Precipitation also gets close to the decorrelation limit dur- ing the austral summer months. In this case, summer con- vective precipitation is weakly forced by the boundaries and precipitation centers are likely mislocated between slices, even if forced by the same boundary conditions. 3.2.5 Another view on spin‑up time The SAM-44 simulation setup does not allow to estimate the internal variability limit, since the only year with two long-term overlapping simulations (S14 and S1) is 2006. In this year, the GCM boundary conditions driving S14 and S1 bifurcate into two different global climate realizations forced by the RCP 4.5 (S14) and 8.5 (S1 and S2) concen- tration scenarios. As a result, differences between S14 and S1 in 2006 do not represent RCM internal variability, but different global driving fields. In particular, the changes in Snow depth takes longer to decorrelate, since the adjust- ment starts in austral summer, with no snow, and RMSDs keeps low until April (mid-autumn). Then, decorrelates rela- tively fast, growing along with the TEV line. This is different from the spin-up process, which efficiently uses the summer months to reset the snow cover fields. 3 57 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… full equilibrium and internal variability starts to dominate (Fig. 4). This can vary with the region, since the length of the spin-up depends on the soil characteristic and the climate conditions. Therefore, these inhomogenities can be contrib- uted to the internal variability for sure, but regionally they may contain also the effects of the spin-up due to the differ- ent climate conditions (Yang et al. 2011; Lim et al. 2012). On the other hand, high differences in northern Africa are the consequence of our standardization - this area is very dry and absolute changes of soil moisture are very small, which leads to large (i.e. above three standard deviation) relative changes in standard deviation units. The results for snow depth are qualitatively similar. Due to its long response time, most of the differences occur between the slices, except for an elongated area north of the Black Sea. Since the spin-up was sufficient (Fig. 4b), we may attribute the discrepancies between both slices to the internal variability. These snow accumulation differences between slices (Fig. 6f) have typi- cal depths from individual misplaced snowfall events, such as the one north of the Black Sea, occurring on the ana- lysed day (Fig. 6g). As such, unlike soil moisture, which is relatively stable at this joint (Fig. 4), the specific spatial pattern can fully differ from one joint to another for snow depth, even considering the same season. As an example, on March 1st, 2008 (Fig. 3.2.5 Another view on spin‑up time ESM 5), the synoptic conditions over Europe barely provided any snow and, as a result, inho- mogenities in snow depth are very small. This emphasizes the role of interannual variability at the joint, which in turn may increase or decrease the inhomogeneities. Four areas with differences up to two standard deviations stand out in the 850 hPa geopotential height (Fig. 6i, l). Unlike for the previous slow-varying variables, these differences are almost exclusively due to changes in synoptic conditions between consecutive days (Fig. 6k). Along the day, two lows develop north of France and Scandinavia, and a third low moves and deepens from the west to the north of the Black Sea. Only west of the Black Sea the inhomogeneities have a slightly larger contribution of the changes between between the two slices (Fig. 6j), which weakens and slightly shifts the low northwards. The season is again an important factor. In winter (Figs. 6 and ESM 5), when boundary forcing at mid latitudes is dominant, the differences are mainly attrib- uted to the day-to-day natural variability of the atmosphere (e.g. low pressure systems entering or moving across the domain). However, by the end of the spring, the strength of the boundary forcing decreases and internal variability increases, allowing for larger discrepancies between time slices. Thus, the discrepancies between the two slices are larger at the joint on June 1st 2006 (Fig ESM 4) as it can Interestingly, for soil moisture, inter-slice differences between S2 and S1 stabilize at the √ 2TEV level. This means that soil moisture differences between time slices subject to the same boundary conditions are as different as those in two random days in this month (note that TEV was computed considering interannual variability). This may indicate a generally low departure from average conditions in this vari- able. It could also be result of insufficient spin-up. Since the low internal variability level ( RMSDs(S1 −S14) ) from Dec- 2005 is not reach by the end of the overlapping period, this suggest that the soil moisture spin-up for RMSDs(S1 −S2) may have not finished in Dec-2006, despite the apparent stabilization. full equilibrium and internal variability starts to dominate (Fig. 4). This can vary with the region, since the length of the spin-up depends on the soil characteristic and the climate conditions. 3.2.5 Another view on spin‑up time Therefore, these inhomogenities can be contrib- uted to the internal variability for sure, but regionally they may contain also the effects of the spin-up due to the differ- ent climate conditions (Yang et al. 2011; Lim et al. 2012). On the other hand, high differences in northern Africa are the consequence of our standardization - this area is very dry and absolute changes of soil moisture are very small, which leads to large (i.e. above three standard deviation) relative changes in standard deviation units. The results for snow depth are qualitatively similar. Due to its long response time, most of the differences occur between the slices, except for an elongated area north of the Black Sea. Since the spin-up was sufficient (Fig. 4b), we may attribute the discrepancies between both slices to the internal variability. These snow accumulation differences between slices (Fig. 6f) have typi- cal depths from individual misplaced snowfall events, such as the one north of the Black Sea, occurring on the ana- lysed day (Fig. 6g). As such, unlike soil moisture, which is relatively stable at this joint (Fig. 4), the specific spatial pattern can fully differ from one joint to another for snow depth, even considering the same season. As an example, on March 1st, 2008 (Fig. ESM 5), the synoptic conditions over Europe barely provided any snow and, as a result, inho- mogenities in snow depth are very small. This emphasizes the role of interannual variability at the joint, which in turn may increase or decrease the inhomogeneities. Four areas with differences up to two standard deviations stand out in the 850 hPa geopotential height (Fig. 6i, l). Unlike for the previous slow-varying variables, these differences are almost exclusively due to changes in synoptic conditions between consecutive days (Fig. 6k). Along the day, two lows develop north of France and Scandinavia, and a third low moves and deepens from the west to the north of the Black Sea. Only west of the Black Sea the inhomogeneities have a slightly larger contribution of the changes between between the two slices (Fig. 6j), which weakens and slightly shifts the low northwards. The season is again an important factor. In winter (Figs. 6 and ESM 5), when boundary forcing at mid latitudes is dominant, the differences are mainly attrib- uted to the day-to-day natural variability of the atmosphere (e.g. low pressure systems entering or moving across the domain). 3.3 Meteorological inhomogeneities: spatial distribution In the previous sections we quantified day-to-day changes and changes between time slices with spatial root mean squared differences, which masked the spatial distribution of these changes. Therefore, in this section, we analyse how the inhomogeneities in split simulations are spatially dis- tributed. As an example we show the differences in EUR15 (Fig. 6) that cause the RMSDt peak on March 1st, 2005, shown in Fig. 3 (top panel). The results for other joints are qualitatively similar (not shown). The first column shows the observed change at the time-slice joint ( DstX ) for the different variables. The changes shown will stand out as a noticeable inhomogeneity (i.e. peaks in Figs. 2 or 3) if (and where) DsX (second column) is significantly larger (for an order of magnitude) than DtX (third column). In order to have a relative measure to compare different variables, in the fourth column we show the differences in standard deviation units ( dstX).i For soil moisture content (Fig. 6, first row), as already inferred from the RMSDt time series, the change between consecutive days ( DtX ) is negligible as compared to change between slices ( DsX ). The relatively high differences at joints are spread across the whole domain (Fig. 6d), with highest values (i.e above three standard deviations) in east- ern and northeastern parts of Europe, as well as in north- ern Africa. In the European regions, these high discrepan- cies can be explained only by changes between the slices ( DsX ) at the specific joint, since soil moisture has very long response time so the changes between consecutive days ( DtX ) are negligible. The two slices are joined within the transitional time during which soil moisture reaches the Even though differences found in the geopotential height are small, they may affect the results in other vari- ables, especially those that are dependent on the synoptic 1 58 A. Lavin‑Gullon et al. Fig. 6 Spatial distribution of differences (Equations 1–3 and 5 ) for EUR-15 at the joint from 28th February ( t −1 ) to 1st March, 2005 (t) between slices S2 (s) and S1 ( s −1 ). From left to right: DstX(s, n, t) , DsX(s, n, t −1) , DtX(s, n, t) , and dstX(s, n, t) . 3.3 Meteorological inhomogeneities: spatial distribution Note that the latter is non-dimensional and the same colorbar is used for all variables (from top to bottom): total soil moisture ( kg∕m2 ), snow depth (m), geo potential height at 850 hPa (m), daily accumulated precipitation (mm and near-surface temperature (K) non-dimensional and the same colorbar is used for all variables (from top to bottom): total soil moisture ( kg∕m2 ), snow depth (m), geo- potential height at 850 hPa (m), daily accumulated precipitation (mm) and near-surface temperature (K) Fig. 6 Spatial distribution of differences (Equations 1–3 and 5 ) for EUR-15 at the joint from 28th February ( t −1 ) to 1st March, 2005 (t) between slices S2 (s) and S1 ( s −1 ). From left to right: DstX(s, n, t) , DsX(s, n, t −1) , DtX(s, n, t) , and dstX(s, n, t) . Note that the latter is 1 3 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… 59 is consistent with the relatively colder region over northern Africa (row e and column 4 in Fig. 7).f circulation, such as precipitation. Discrepancies between the slices ( DsX ) in the low pressure area, west of the Black Sea (Fig. 6f), drive precipitation changes there, causing inhomo- geneities to increase up to 50 mm in northern Bulgaria. This is due to a northward shift in precipitation which accompa- nies the corresponding shift in the low pressure system. This is observed in the other domains, as well. For example, in SAM-44 (not shown), the joint on January 1st, 2015 exhibits extended precipitation inhomogeneities east of Brazil, driven by time slice differences in simulating a low pressure system over the Atlantic. In order to check whether these differences are consist- ent with low frequency internal variability or a side effect of the splitting, we computed the seasonal climatology differences for two different 10-year periods. The years 1991–1995 and 2001–2005 (columns 2 and 5 in Fig. 7) are considered as years during which the differences could potentially be affected by a long term spin-up transient, since they were initialized less than 6 years before the sim- ulation start (see Fig. 1). On the other hand, we consider the years 1996–2000 and 2006–2010 (columns 3 and 6 in Fig. 3.4 Simulated climate In general, no significant difference can be ascribed par- ticularly to the period with a potential spin-up transient regime. Seasonal differences in the two 10-year periods show similar magnitudes for all variables. Moreover, some features in the 20-year climatology differences, such as summer differences in geopotential height or tempera- ture, correspond to stronger differences during the inter- nal variability dominated 10-year period. More or less co-located differences during the first 10-year period lead to significant differences during the full 20-year period. Patchy summer precipitation differences in the 20-year period also correspond to constructive averages with even patchier differences in the 10-year periods. The same is true for winter snow depth. This points to no spin-up tran- sient effect in the first 10-year period, and to differences compatible with internal variability, even if statistically significant.f In previous sections we have shown that discontinuities in split simulations can be relatively large, especially for slow- varying variables. Since these discontinuities occur on indi- vidual time steps, it is expected that the simulated climate should not be affected by the splitting procedure, especially when the differences are within the range of model’s internal variability. In this section, we compare the climatology of the split and continuous simulations. For this purpose, we used the EUR-44 set of simulations, which has the longest simulated overlapping period (20 years) among all of our simulation sets (see Fig. 1). Figure 7 shows the winter (DJF) and summer (JJA) dif- ferences in the seasonal climatology for the full overlapping period 1991–2010 (first and fourth columns). All variables considered are shown in different rows and differences use a common non-dimensional scale of seasonal standard devia- tion units. In winter, soil moisture and snow depth show significant differences, while upper air and surface variables show much lower, non-significant differences. Differences are spatially smooth for all variables except snow depth, which shows patchy differences over snow-dominated areas in the domain. In summer (Fig. 7, fourth column), snow depth differences vanish due to lack of snow. Somewhat larger differences arise in upper air and surface variables, especially in the summer season. Significant precipitation differences (Fig. 7, row d, column 4) are concentrated mostly over Mediterranean, having a very patchy pattern which is consistent with a weak mid-latitude lateral boundary forc- ing in summer months, and the mainly convective origin of precipitation. 3.3 Meteorological inhomogeneities: spatial distribution 7) as periods when the differences are dominated by the model internal variability, since the RCM initialization occurred at least 6 years after the simulation start. Note that the 20-year seasonal climatology differences (columns 1 and 4 in Fig. 7) are the average of these two 10-year cli- matology differences. In this way, differences in the long- term climatology can be ascribed to periods dominated either by potential spin-up or internal variability.if Inter-slice differences for near-surface temperature (Fig. 6r) are generally small over the domain. There are only isolated locations with notable differences, especially in Ukraine, but still these are within one standard deviation (Fig. 6t). Therefore, inhomogeneities that appear at the joint can be mostly explained with the day-to-day natural change of the variable (Fig. 6s). 3.4 Simulated climate A relative low geopotential height develops in the split simulation over northern Africa/central Mediterra- nean Sea in summer (row c and column 4 in Fig. 7), which i No systematic effect is apparent in both periods, except for the soil moisture differences over northern Africa, which are quite robust in all periods and seasons and reach several standard deviations. This is likely related to the extremely dry soils and low variability there, which would need further research. Unfortunately, for this simulations soil moisture at different depths is not available to inves- tigate properly the source of this difference between the continuous and split runs. Over continental Europe these differences are also significant, reaching about half the standard deviation. However, they are compatible with internal variability since there is no systematic location of differences across the 10-year periods. The differences found in the full 20-year period are the result of partly overlapping positive and negative differences in the other two time periods. 1 3 3 A. Lavin‑Gullon et al. 60 Fig. 7 Seasonal climatology differences for different periods (in col- umns) and variables (in rows) between the EUR-44 split and EUR- 44 continuous. Differences are in non-dimensional standard deviation units. Black contours show statistically significant differences accord- ing to a two-sample t-test with 95% confidence. Snow depth (snd) dif- ferences are masked out where the variability is below 0.001 m Fig. 7 Seasonal climatology differences for different periods (in col- umns) and variables (in rows) between the EUR-44 split and EUR- 44 continuous. Differences are in non-dimensional standard deviation units. Black contours show statistically significant differences accord- ing to a two-sample t-test with 95% confidence. Snow depth (snd) dif- ferences are masked out where the variability is below 0.001 m 4 Summary and conclusions As an example, unlike in Europe, the warm season in South America is also the wet season, due to deep convective events which lead to greater pre- cipitation internal variability and, thus, more uncertain initial soil moisture conditions. The optimal starting point should therefore be found for each region to minimize the contents of slow-varying reservoirs (e.g. snow and soil water), thus avoiding gross errors in their initial levels. We also found that mismatches at the slice joints are also minimized during the warm/dry season. Therefore, a mini- mal overlapping time slice setup could be a 1-year spin-up period initialized at the end of the warm/dry season and entering the split simulation one year later. Our results are robust to interannual variability regarding the detection of meteorological inhomogeneities. Spin-up times for slow-varying variables can depend on the specific conditions of the initialization year. We showed examples with a long plateau in snow depth differences across time slices in EUR-15 in 2003 (Fig. 4), which did not occur in other overlapping years (Figs. ESM1 or ESM2). Also, the soil moisture spin-up time, as represented by the time to reach the internal variability limit, differs from year to year. While one year is usually enough, there are instances (e.g. 1999 in EUR-44, Figure ESM3) when over two years seem necessary to reach equilibrium. All in all, this work shows that the use of overlapping time slices to accomplish long term regional climate sim- ulations is a valid approach. This procedure can largely improve the efficiency of regional climate simulations, both for computationally heavy simulation (e.g. kilometer-scale simulation) or for a faster accomplishment of lower reso- lution runs, which do not scale efficiently to a large num- ber of processors. Modelling workflow managers, such as WRF4G (Fernández-Quiruelas et al. 2015) —used in our simulations—, can help in the extra design, job submission and monitoring burden of this approach.i The largest and more spatially extended differences were found in total soil moisture, both regarding mete- orological inhomogeneities and long term climatologies. This agrees with previous studies warning on the very long spin-up required by soil variables, and especially soil mois- ture (Christensen 1999; Cosgrove et al. 2003; Yang et al. 2011; Jerez et al. 2020). We found that significant differ- ences in the climatology over continental Europe might be ascribed to internal model variability. 4 Summary and conclusions of overlapping time slices on the regional climate was assessed.f We presented a post hoc analysis of several regional cli- mate simulation experiments carried out with the WRF RCM as a set of overlapping time slices. The simulations span different domains, boundary conditions, horizontal grid spacing and overlapping periods. Overlapping time slices were joined to build split simulations. Continuous simulations are available as reference to evaluate split simulations for some of these experiments. We evalu- ated the discontinuities in time and space introduced by this procedure at the joints of the time slices, devising a methodology to discern between insufficient spin-up and RCM internal variability effects. Finally, the effect The analysis was carried out on variables with different response times and, at the same time, variables typically saved in coordinated dynamical downscaling initiatives, such as CORDEX. For this purpose, we also focused on daily model output, commonly available in public reposi- tories. An analysis at higher temporal frequencies would likely ease the location of meteorological discontinuities for the variables with the shortest response times (geo- potential height, temperature, precipitation), which went unnoticed in our study. However, the output frequency does not affect spin-up times or seasonal climatology analyses. 1 3 3 61 Spin‑up time and internal variability analysis for overlapping time slices in a regional climate… We avoided spatial averaging and used root mean squared differences to highlight any mismatch between time slice simulations. The relative size of time slice switch differences with respect to daily variable tendencies only led to noticeable inhomogeneities in soil moisture. Locally, snow depth can also be used to reveal slice joints. Surface and upper air variables show larger day-to-day variations than across time slices. to other slow interactive components such as a regional ocean (Ho-Hagemann et al. 2020). For other variables, the discrepancies between the cli- matology in split simulations and continuous simulations can be ascribed to internal variability, even if statistically significant (e.g. for snow depth). No special effect on the climatology was found for periods closer to the initialization with respect to those farther away. Jerez et al. (2020) showed that the optimal spin-up period is not always the longest, recommending an ini- tialization in the warm season. We found that this depends on the region, though. 4 Summary and conclusions AL-G acknowledges financial support from grant BES-2016-078158 and MINECO/FEDER co-funded project MULTI-SDM (CGL2015-66583-R). JM is funded by the Unidad de Excelencia María de Maeztu (MDM-2017-0765), funded by MCIN/AEI/10.13039/501100011033. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work is part of projects INSIG- NIA (CGL2016-79210-R) and CORDyS (PID2020-116595RB-I00), funded by MCIN/AEI/10.13039/501100011033. AL-G acknowledges financial support from grant BES-2016-078158 and MINECO/FEDER co-funded project MULTI-SDM (CGL2015-66583-R). JM is funded by the Unidad de Excelencia María de Maeztu (MDM-2017-0765), funded by MCIN/AEI/10.13039/501100011033. Bassett R, Young PJ, Blair GS, Samreen F, Simm W (2020) A large ensemble approach to quantifying internal model variabil- ity within the WRF numerical model. J Geophys Res: Atmos 125(7):e2019JD031286. https://doi.org/10.1029/2019JD031286 Berg P, Christensen J (2008) Poor man’s re-analysis over Europe. Tech Rep, WATCH Technical 5 Report No. 2 Caya D, Biner S (2004) Internal variability of RCM simulations over an annual cycle. Clim Dyn 22(1):33–46. https://doi.org/ 10.1007/s00382-003-0360-2 Availability of data and material The simulation data used is fully available upon request to the corresponding author. Sample data for the analyzed overlapping periods over the CORDEX-SAM domain along with the model configuration files are available at https://doi. org/10.5281/zenodo.5012560. Split simulation data for SAM-44 and EUR-44 are freely available through the Earth System Grid Federation (ESGF). EUR-15 and ALP-3 data will also be made available through ESGF in the future. Chen F, Dudhia J (2001) Coupling an advanced land surface-hydrol- ogy model with the penn state-NCAR MM5 modeling system. Part i: Model implementation and sensitivity. Mon Weather Rev 129(4):569–585. https://doi.org/10.1175/1520-0493(2001)129< 0569:caalsh>2.0.co;2 Christensen OB (1999) Relaxation of soil variables in a regional climate model. Tellus A 51:674–685. https://doi.org/10.1034/j. 1600-0870.1999.00010.x Christensen OB, Gaertner MA, Prego JA, Polcher J (2001) Internal variability of regional climate models. Clim Dyn 17:875–887. https://doi.org/10.1007/s003820100154f Code availability The WRF source code is freely available through GitHub at https://github.com/wrf-model/WRF. 4 Summary and conclusions Coppola E, Sobolowski S, Pichelli E, Raffaele F, Ahrens B, Anders I, Ban N, Bastin S, Belda M, Belusic D, Caldas-Alvarez A, Cardoso RM, Davolio S, Dobler A, Fernandez J, Fita L, Fumiere Q, Giorgi F, Goergen K, Guttler I, Halenka T, Heinzeller D, Hodnebrog O, Jacob D, Kartsios S, Katragkou E, Kendon E, Khodayar S, Kun- stmann H, Knist S, Lavin-Gullon A, Lind P, Lorenz T, Maraun D, Marelle L, van Meijgaard E, Milovac J, Myhre G, Panitz HJ, Piazza M, Raffa M, Raub T, Rockel B, Schär C, Sieck K, Soares PMM, Somot S, Srnec L, Stocchi P, Tolle MH, Truhetz H, Vau- tard R, de Vries H, Warrach-Sagi K (2020) A first-of-its-kind multi-model convection permitting ensemble for investigating convective phenomena over Europe and the Mediterranean. Clim Dyn 55:3–34. https://doi.org/10.1007/s00382-018-4521-8 Declarations Conflict of interest We confirm that this work is original and has not been published elsewhere, nor is it currently under consideration for publication elsewhere. Furthermore, we have no conflicts of interest to disclose. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Cosgrove BA, Lohmann D, Mitchell KE, Houser PR, Wood EF, Schaake JC, Robock A, Sheffield J, Duan Q, Luo L, Higgins RW, Pinker RT, Tarpley JD (2003) Land surface model spin-up behavior in the North American Land Data Assimilation System (NLDAS). J Geophys Res Atmos. https://doi.org/10.1029/2002J D003316 Dee DP, de Rosnay P, Poli P, Kobayashi S, Andrae U, Balmaseda M, Balsamo G, Van de Berg L, Bidlot J, Bormann N, Dragani R, Fuentes M, Vitart F (2011) The ERA-interim reanalysis: con- figuration and performance of the data assimilation system. Q J Royal Meteorol Soc 137:553–597. https://doi.org/10.1002/qj.828 4 Summary and conclusions However, differ- ences over low-precipitation, non-vegetated areas (northern Africa) present systematic differences which persist along the simulated period. This is likely due to initial soil mois- ture inconsistencies between the forcing reanalysis and the RCM equilibrium soil state over these areas. The land sur- face model takes a very long time to restore the equilibrium, especially from extremely dry initial conditions (Cosgrove et al. 2003). The situation is likely exacerbated due to the lack of precipitation and deep roots. A dedicated study with long-term, continuous simulations would be necessary to properly disentangle the internal variability and spin-up of soil variables in coupled simulations. Most studies on soil spin-up rely on off-line land surface model simulations (Cosgrove et al. 2003; Yang et al. 2011), where there is a target equilibrium soil moisture that is consistent with the prescribed atmospheric forcing. Coupled simulations, with an active soil-atmosphere feedback, could develop greater internal variability with persistent anomalies in the slowest components. There are examples, though, of the opposite effect (reduced internal variability) for simulations coupled As a final cautionary remark, note that we considered RCM simulations with prescribed sea surface tempera- tures. RCM simulations coupled to a regional ocean pose an even greater spin-up challenge due to the strong ther- mal and dynamic inertia of the ocean surface. The results shown could also be sensitive to the model parameteriza- tions and other model components (e.g. land surface model) used. Multi-model uncertainty was also not explored in this study and would require coordination of different modelling groups. Given the uncertainties, in order to be on the safe side of soil spin-up, longer slices and spin-up times could be considered. For example, centennial scenario simulations could be safely split into 30-year slices (e.g. near-, mid-, and far-future) with a 5-year spin-up each, especially if soil variables are initialized from an RCM soil climatology (Cos- grove et al. 2003; Rodell et al. 2005; Jerez et al. 2020). Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00382-022-06560-2. Acknowledgements All simulations have been carried out on the Altamira Supercomputer at the Instituto de Física de Cantabria (IFCA- CSIC), member of the Spanish Supercomputing Network. 1 3 3 62 A. Lavin‑Gullon et al. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. 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W2554777523.txt	https://www.ssoar.info/ssoar/bitstream/document/60033/2/ssoar-zpth-2016-1-schulz-Die_Unbestimmtheit_der_Demokratie_ein.pdf	de		„Die Unbestimmtheit der Demokratie“. Ein Gespräch mit Pierre Rosanvallon	Zeitschrift für politische Theorie	2,016	cc-by-sa	7,971	www.ssoar.info "Die Unbestimmtheit der Demokratie": ein Gespräch mit Pierre Rosanvallon Schulz, Daniel Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Zur Verfügung gestellt in Kooperation mit / provided in cooperation with: Verlag Barbara Budrich Empfohlene Zitierung / Suggested Citation: Schulz, D. (2016). "Die Unbestimmtheit der Demokratie": ein Gespräch mit Pierre Rosanvallon. ZPTh - Zeitschrift für Politische Theorie, 7(1), 105-119. https://doi.org/10.3224/zpth.v7i1.09 Nutzungsbedingungen: Dieser Text wird unter einer CC BY-SA Lizenz (NamensnennungWeitergabe unter gleichen Bedingungen) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by-sa/4.0/deed.de Terms of use: This document is made available under a CC BY-SA Licence (Attribution-ShareAlike). For more Information see: https://creativecommons.org/licenses/by-sa/4.0 Diese Version ist zitierbar unter / This version is citable under: https://nbn-resolving.org/urn:nbn:de:0168-ssoar-60033-5 Interview „Die Unbestimmtheit der Demokratie“ Ein Gespräch mit Pierre Rosanvallon1 Daniel Schulz* Schulz: Pierre Rosanvallon, in Ihrem letzten Buch unterscheiden Sie vier Begriffe der Demokratie: Die Bürger-Demokratie, die Demokratie als Regime, die Demokratie als Form der Gesellschaft und die Demokratie als Regierung. Was bedeuten diese vier Begriffe der Demokratie und wie sind sie miteinander verbunden? Rosanvallon: In meiner Arbeit gibt es zwei große Dimensionen. Die erste Dimension besteht in der Suche nach etwas, was man ein erweitertes Verständnis der Demokratie nennen könnte. Tatsächlich beschränken die Politikwissenschaft und auch die Geschichtswissenschaft ihr Studium der Demokratie zu häufig auf eine politische Form. Wenn man sich beispielsweise die Literatur der „democratization studies“ anschaut, dann hat sich die Untersuchung der Demokratisierung alleine auf die Frage nach den Bedingungen für ein politisches Regime konzentriert, das dann durch das allgemeine Wahlrecht und den Rechtsstaat definiert wird. In dieser Weise definieren sich die minimalistischen Demokratiebegriffe. Diesem Feld der Demokratisierungsforschung liegt im weitesten Sinne das Schumpetersche Konzept der Demokratie zugrunde. Was mir dagegen sehr wichtig erscheint und woran es sich zu erinnern lohnt: In ihrer eigenen Entwicklung wurde die Demokratie auf ganz andere Weise verstanden. Man kann sogar sagen, dass in der Amerikanischen und in der Französischen Revolution der Begriff der Demokratie zunächst verwendet wurde, um ein gesellschaftliches Regime zu definieren. Der Begriff „Demokratie“ gehört übrigens nicht zum Vokabular der Französischen Revolution und er gehört auch nicht zum Vokabular der Amerikanischen Revolution. Ein im Jahre 1788 veröffentlichtes Wörterbuch beschreibt Demokratie ein Jahr vor der Revolution folgendermaßen: „Veraltetes Wort, wird nur noch zur Bezeichnung der antiken Republiken oder einiger kleiner Schweizer Kantone gebraucht“.2 Der Begriff wurde zu dieser Zeit also in seinem ursprünglichen Sinne verstanden: die Regierung einer Stadt durch * PD Dr. Daniel Schulz, LMU München Kontakt: daniel.schulz@gsi.uni-muenchen.de 1 2 Das Gespräch mit Pierre Rosanvallon wurde am 16. Dezember 2015 im Collège de France geführt. Vgl. dazu Rosanvallon, Pierre, 2007: L’universalisme démocratique: histoire et problèmes, in: http://www. laviedesidees.fr/L-universalisme-democratique.html. Daniel Schulz: „Die Unbestimmtheit der Demokratie“, ZPTh Jg. 7, Heft 1/2016, S. 105–119 106 Zeitschrift für Politische Theorie, Heft 1/2016 eine Volksversammlung. Und tatsächlich ist es daher die Idee der repräsentativen Regierung gewesen, die auf eine Gesellschaft wie die während der französischen Revolution angewandt werden konnte. Frankreich hatte damals 25 Millionen Einwohner – die USA dagegen nur drei Millionen. Man vergisst häufig, dass die Französische Revolution in dem am stärksten bevölkerten Land Europas stattfand, während Amerika dagegen viel kleiner war. Daher wurde der Begriff der Demokratie zu dieser Zeit in Frankreich nicht verwendet. Es gibt übrigens fast fünftausend Zeitschriftenausgaben und Flugschriften, die während der Französischen Revolution veröffentlicht wurden – und in keiner einzigen taucht das Wort „Demokratie“ auf. Nur in zwei von diesen fünftausend erscheint das Adjektiv „demokratisch“. Sogar in den Vereinigten Staaten wurde das Wort zumeist in einem pejorativen Sinne gebraucht, um etwa die demagogische Situation einer Pöbelherrschaft zu bezeichnen. Es handelte sich demnach um eine politische Beleidigung: Jemanden als Demokraten zu behandeln hieß, ihn als Demagogen oder, wie wir heute sagen würden, als Populisten zu behandeln. In der politischen Sprache begann der Begriff „Demokratie“ erst zu Beginn der 1820er Jahre in Frankreich in einem positiven Sinn gebraucht zu werden – aber in diesem Moment wurde damit eine Gesellschaft bezeichnet, in der gleiche Rechte herrschten. Demokratie bezeichnete also eine Gesellschaftsform. Es kommt im Übrigen zu dieser Zeit in Frankreich häufig vor, dass gesagt wird: Wir befinden uns in einer Demokratie – soll heißen: in einem Land, in dem keine Stände, Ränge oder Privilegien mehr existieren, eine Gesellschaft also, in der die Gleichheit der Rechte herrscht – allerdings sind wir noch nicht in der Republik, also: wir haben noch kein universales Wahlrecht. In den Jahren um 1820 wird in Frankreich der Begriff Demokratie daher nicht gebraucht, um das universale Wahlrecht zu bezeichnen. Als Tocqueville von der Demokratie in Amerika spricht, wird er den Akzent auf die Demokratie als Gesellschaftsform legen – er nennt es die Gesellschaft der einander Ähnlichen. Dabei handelt es sich um eine Definition, die sehr viel reicher und sensibler ist als lediglich von einer Gesellschaft der gleichen Rechte zu sprechen. Und diese „originellere“ Dimension des Demokratiebegriffes als eine von der Gleichheit definierte Gesellschaftsform – eine Gleichheit, die weitreichender ist als eine einfache Gleichheit der Rechte, mit allen Mehrdeutigkeiten und Perspektiven, die der Begriff der Gesellschaft der einander Ähnlichen bei Tocqueville eröffnet – diese Dimension ist etwas, das mir zunehmend vergessen zu werden scheint. Man tut so, als ob die Fragen der ökonomischen Ungleichheit, des Vermögens oder auch kultureller Ungleichheit allein unter dem Blickwinkel der sozialen Gerechtigkeit behandelt werden sollten. Mir scheint, dass diese Fragen jenseits der sozialen Gerechtigkeit auch die Idee der Demokratie charakterisieren. Aus diesem Grunde habe ich das Buch Die Gesellschaft der Gleichen geschrieben: um diese Definition der Demokratie wieder herzustellen.3 Aber in seiner Geschichte seit 1848 ist der Demokratiebegriff auch zu einem Banner geworden, zu einem Wort, mit dem das Ziel eines Kampfes umschrieben wird, welcher der vollen Verwirklichung der aktiven Staatsbürgerschaft über das gleiche Wahlrecht hinaus gewidmet ist. Die aktive Staatsbürgerschaft, also die aufgeklärten und gebildeten, zur Beratung befähigten Staatsbürger, die am Leben des Gemeinwesens teilhaben – diese Dimension der zugleich aktiven und kritischen Partizipation wollte ich mit meinem Buch zur Gegen-Demokratie ausleuchten.4 Ziel der Gegen-Demokratie war es, den Be3 4 Rosanvallon, Pierre, 2013: Die Gesellschaft der Gleichen, Hamburg. Rosanvallon, Pierre, 2006: La contre-démocratie, Paris. Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 107 griff der aktiven Staatsbürgerschaft anzureichern und zu modernisieren. Zugleich hat das Konzept der Gegen-Demokratie an die zentrale Vorstellung anzuknüpfen versucht, nach der in der Demokratie immer ein aktives und ein negatives Prinzip wirksam ist. Das aktive Prinzip ist dasjenige der Formen der Macht, das negative Prinzip ist dasjenige der Formen der Kritik und der Kontrolle. Die Demokratie ist ein Regime, das strukturell unabgeschlossen ist in dem Sinne, dass es stets auf der Suche nach sich selbst ist. Die Demokratie ist immer eine Regimeform, eine Gesellschaftsform, die sich zu ihrer eigenen Unvollständigkeit befragt und versucht, sie zu bekämpfen. Das Wort „Gegen-Demokratie“ ist zwiespältig, weil es zum einen das Gegenteil der Demokratie meinen kann, und zum anderen – und in diesem Sinne habe ich es verwendet – die Stütze, das Fundament der Demokratie, dasjenige, was die Demokratie festigt, indem es jenseits der Institutionen zeigt, auf welche Weise es ein besonderes Regime, eine kritische Suche nach sich selbst darstellt. Schließlich habe ich zuletzt auch die Demokratie als eine Regierungsform vorgestellt.5 Ich habe dies aus zwei Gründen getan: zum ersten, weil jede moderne Idee der repräsentativen Demokratie auf einer Grundlage gedacht wurde, nach der die gestaltende Macht der Politik in der legislativen Gewalt, im Parlament läge. Alle Fragen der Repräsentation wurden vor dem Hintergrund einer Überlegung gestellt, wie sich eine stellvertretende Versammlung zusammensetzen müsste; alle Fragen des allgemeinen Wahlrechts wurden mit Blick auf dieses legislative Ideal betrachtet. Heute ist es aber so, dass seit einiger Zeit die Exekutive zur wirklich zentralen Gewalt geworden ist. Wenn man übrigens im 18. Jahrhundert den Begriff der exekutiven Gewalt verwendete, dann sollte „exekutiv“ eine nachrangige Bedeutung verdeutlichen. Es handelte sich um die Gewalt, die dem Gesetz nachgeordnet war. Heute dagegen leben wir in Gesellschaften, die zwar vom Gesetz, von Normen regiert werden, in denen aber die Sphäre der Entscheidung immer wichtiger geworden ist. In einer globalisierten, sich permanent verändernden Welt kann man sagen, dass die Entscheidung im Bezug zur Norm einen immer größeren Raum einnimmt und dass die Entscheidung das spezifische Merkmal der nicht nur exekutiven, sondern der regierenden Sphäre darstellt. Ich wollte daher über diese unreflektierte Grauzone der Demokratie nachdenken, die das Regierungshandeln darstellt. Das Thema meines letzten Buches ist also die Frage, wie man die demokratische Regierung zum Gegenstand der Demokratietheorie machen könnte. Die Demokratie ist zugleich ein Regime, weil sie auf Institutionen beruht, aber auch eine Art und Weise, die Regierung zu führen. Die Dimension der exekutiven Gewalt ist daher zentral. Die Demokratie ist eine Gesellschaftsform ebenso wie eine Beziehung der Bürger zum Leben des Gemeinwesens. Aus diesem Grund spreche ich von den vier Demokratien – gegen jede Engführung, gegen alle permanenten Versuche, Demokratie auf ein Wahlregime plus Rechtsstaat zu reduzieren. Beides ist sicherlich sehr wichtig, aber wir sehen heute viele Regime, in denen das universale Wahlrecht den Souverän stellt, die aber eher – in Lateinamerika – Volksdemokratien oder populistischen Demokratien sind, oder – schauen wir auf das Russland Putins oder die Türkei Erdoğans – autoritäre Demokratien. Schulz: Nach einer ersten Trilogie über die Entwicklung der Demokratie in Frankreich haben Sie einen zweiten Zyklus vorgelegt, der die Demokratie noch stärker unter dem 5 Rosanvallon, Pierre, 2015: Le bon gouvernement, Paris. (deutsche Fassung: (2016) Die gute Regierung, Hamburg). 108 Zeitschrift für Politische Theorie, Heft 1/2016 Gesichtspunkt der politischen Theorie behandelt: Warum erschien Ihnen ein solcher Perspektivwechsel notwendig? Rosanvallon: Ich muss zunächst sagen, dass die erste Trilogie zwar im Wesentlichen der französischen Demokratiegeschichte gewidmet war, ich aber zugleich versucht habe, diese Geschichte mit einer gewissen Anzahl von vergleichenden Einsichten zu verbinden. Das Beste wäre gewesen, ein vergleichendes Buch über die Geschichte der Demokratie schreiben zu können. Ich habe dazu viel gelesen und kenne die amerikanische Geschichte recht gut, die englische und die deutsche dagegen etwas weniger. Ich konnte damit immerhin an gewissen Punkten eine vergleichende Perspektive einbeziehen. Aber es übersteigt die Fähigkeit einer einzelnen Person, eine allgemeine vergleichende Geschichte der Demokratien schreiben zu wollen. Man hätte es tun müssen. Ich hatte nicht die Mittel dazu und bin daher von dem Fall ausgegangen, den ich am besten kannte: den französischen Fall. Dabei habe ich dennoch in den drei Bänden einige vergleichende Einsichten untergebracht, vor allem die Vereinigten Staaten und England. Man muss aber auch sehen, dass man eine solche Arbeit nicht beginnt, indem man sagt: „Ich werde zuerst eine Trilogie machen, danach eine Tetralogie“, so funktioniert das nicht. Ich habe zuerst begonnen, mich für die Fragen des Funktionierens der Demokratie zu interessieren. Nach und nach habe ich dann gesehen, dass man für ein echtes, politiktheoretisches Verständnis der Demokratie die Geschichte der Demokratie kennen muss. Die Geschichte der Demokratie zu kennen heißt nicht nur, ihre Entwicklung zu kennen. Für mich ging es darum, die Demokratie von ihren Aporien und ihren Problemen, von ihren Spannungen und ihren Widersprüchen aus zu verstehen. Daher ist für mich die Geschichte der Demokratie untrennbar von einer theoretischen Reflexion der Natur der Demokratie. Die Demokratie weist Formen der Unbestimmtheit auf und die Geschichte erlaubt uns, diese Formen der Unbestimmtheit zu erhellen. Erst die Geschichte kann die großen Aporien beleuchten, um die herum die Demokratie konstituiert ist: die Aporien über die Definition der Gleichheit zwischen sozialer und rechtlicher Gleichheit, die Aporien über den Begriff der Repräsentation zwischen einer prozeduralen und einer mehr substantiellen Definition. Im Deutschen gibt es zwei Begriffe – Stellvertretung und Repräsentation. Im Französischen gibt es nur einen Begriff, um beides zu sagen. Zwischen beiden Elementen herrscht eine permanente Spannung. Eine Geschichte der Demokratie zu betreiben bedeutet also für mich nicht, eine kontinuierliche Entwicklung zu suggerieren, sondern die Demokratie als eine Erfahrung zu verstehen: eine soziale Erfahrung und eine politische Erfahrung. In der Trilogie habe ich anhand der Fragen der Staatsbürgerschaft, der Repräsentation und des Wahlrechts versucht, die Wirkung der großen demokratischen Aporien aufzuzeigen. Was mich angeht – und wir kommen ja vielleicht noch auf den Ansatz von Quentin Skinner zu sprechen –, so stelle ich keineswegs einen normativen Ansatz der Demokratie gegen einen historischen Ansatz. Ich glaube, das Problem liegt darin, die Frage der Normativität anders zu verstehen. Man kann einen normativen Ansatz a priori entwerfen. Oder man kann einen normativen Ansatz entwerfen, der durch diese Definition der Aporien angereichert ist. Und wenn man ein auf diese Weise angereichertes Verständnis der Norm hat, dann scheint mir der historische Umweg nicht lediglich ein Umweg über die Geschichte zu sein, sondern ein Begreifen der eigentlichen Definition demokratischer Unbestimmtheit mit anderen Mitteln. Genau dies ist die Definition meiner Arbeit. Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 109 Schulz: In einer frühen, habermasianisch geprägten Rezeption Ihrer Arbeit in Deutschland hat man Sie als Vertreter einer „narrativen“ Demokratietheorie eingestuft und diesen Ansatz von einer normativen Demokratietheorie unterschieden. Was würden Sie auf diese Unterscheidung antworten? Rosanvallon: Ich glaube, dass meine Arbeit jenseits dieser Debatte angesiedelt ist. Sie geht über den Gegensatz zwischen historischem Ansatz und normativem Ansatz hinaus und ist zudem breiter angelegt als die normativen Theorien, die sich sehr auf das Niveau der Deliberation fokussiert haben. Ich glaube, dass die Fragen der Deliberation lediglich eine Dimension dessen darstellen, was man demokratische Aktivitäten nennen könnte. Hinter meinem Ansatz steht der Wille, einen sehr viel umfassenderen Begriff der Demokratie zu entwickeln. Die Deliberationstheorien blenden Fragen der Gleichheit vollkommen aus, oder sie nähern sich ihnen über den Umweg der Gerechtigkeitstheorie. Auf diese Weise haben wir Theorien der Gerechtigkeit, die neben der Theorie demokratischer Deliberation angesiedelt sind und die sich berühren. Ich denke aber, man sollte einen allgemeinen intellektuellen Rahmen konstruieren, um die Demokratie in all ihren Dimensionen erfassen zu können. Die Demokratie in all ihren Dimensionen zu erfassen stellt heute die Bedingung dar, um den von uns gelebten demokratischen Moment zu reflektieren – auch mit seinen Versuchen der Regression, seinen Versuchen des Rückschritts, oder den Sackgassen, in welchen er sich mitunter befindet. Ich sehe nicht, wie die klassischen angelsächsischen Theorien der Deliberation erlauben, diesen Moment zu denken. Was haben diese Theorien zum Problem des Populismus zu sagen? Was haben sie zum Problem der autoritären Demokratien zu sagen? Was haben sie zu den Effekten wachsender Ungleichheit zu sagen? Ich glaube, dass die Theorien der Deliberation eine Grenze aufweisen, weil sie die Demokratie lediglich über eine partizipative Dimension begreifen. In dieser einen Hinsicht sind sie sehr interessant und relevant, weil ich selbstverständlich auch denke, dass Fragen der Deliberation sehr wichtig sind. Aber es gibt nicht nur eine Krise der Deliberation, es gibt auch eine Krise der Repräsentation, und aus diesem Grund habe ich den kleinen Band Das Parlament der Unsichtbaren geschrieben:6 um eine Idee der narrativen Demokratie einzuführen, aber als eine Erweiterung des Repräsentationsbegriffs. Ich würde also sagen, dass ich aus zwei Gründen versuche, mich ein wenig jenseits dieser deliberativen Theorien zu verorten: zum Ersten, weil ich die demokratische Idee und die Vorstellungen der Demokratie auf eine breitere Weise begreife; und zweitens, weil ich denke, dass dieses erweiterte Verständnis es erlaubt, die theoretische Reflexion mit einer Intervention in der gegenwärtigen Wirklichkeit zu verbinden. Schulz: Glauben Sie, dass die gegenwärtige Diskussion über die „Postdemokratie“ aus einer Enttäuschung geboren ist, welche auch durch die idealistischen Theorieentwürfe genährt wurde? Rosanvallon: Nicht nur die Theorien der Postdemokratie lassen sich darauf zurückführen, sondern eine grundsätzlich pessimistische Feststellung, die darin besteht zu sagen: Die Wahl-Demokratie ist dabei, zugrunde zu gehen. Aber das ist nur eine punktuelle Feststellung. Es ist interessant zu sehen, wie das Verkümmern der Wahl-Demokratie auch an andere demokratische Erwartungen gebunden ist, beispielsweise – und dies war der Gegen6 Rosanvallon, Pierre, 2015: Das Parlament der Unsichtbaren, Wien. 110 Zeitschrift für Politische Theorie, Heft 1/2016 stand meines Buches über die Legitimität7 – an die gesellschaftliche Erwartung an Institutionen, in denen die Allgemeinheit auf andere Weise repräsentiert wird. Wie entwickeln sich Institutionen der Unparteilichkeit? Die Theorie der Postdemokratie sieht diese Fragen überhaupt nicht – auf dieselbe Weise, wie sie die neuen Beziehungen zur konstitutionellen Ordnung und zu den Verfassungsgerichten nicht sieht, die sich gerade entwickeln. Das Problem dieser Theorien der Postdemokratie ist im Grunde, dass sie selbst von einer eher minimalistischen Definition der Demokratie ausgehen. Ihr Pessimismus rührt daher von einer Verengung der demokratischen Idee. Schulz: Ihre Arbeit widmet sich der Genealogie der Demokratie und des Liberalismus. Welche Bedeutung besitzt für Sie die republikanische Tradition? Welche Verbindungen sehen Sie zwischen der republikanischen Tradition in Frankreich und dem angelsächsischen Republikanismus in der politischen Theorie, wie er vor allem von Philip Pettit und Quentin Skinner vertreten wird? Rosanvallon: Darauf gibt es zwei Dinge zu sagen: Zunächst einmal können die Republikanismustheorien von Pettit und Skinner in Frankreich nicht auf die gleiche Weise unter dem Begriff „Republikanismus“ aufgenommen werden. Der Republikanismus ist ein zentrales Element der französischen Geschichte und die Republik ist hier die Geschichte des Rechtsstaats und auch, so kann man sagen, des Sozialstaats. Der Begriff der Republik kann daher nicht im gleichen Sinne in Frankreich benutzt werden wie bei Philip Pettit und Quentin Skinner. Ich denke, man muss zuerst auch ein wenig über die besondere Vorgehensweise von Skinner sprechen. Was war die grundlegende Idee? Bei Skinner ging es darum zu sagen: Wenn wir uns nur für die liberal-demokratische Idee interessieren, dann verlieren wir aus dem Blick, dass die Moderne auch von einem anderen Verständnis des Gemeinwohls geprägt wurde, welches nicht einfach eine institutionelle Tradition darstellte, sondern eine auf substantiellen Elementen beruhende Tradition war. Seine große Idee, die er in Liberty before Liberalism erklärt,8 war es zu sagen: Vor dem Liberalismus gab es die kritische Idee einer Republik, die zwischen dem Ideal einer Volksrepublik und dem Ideal einer aristokratischen Republik schwankte. In der angelsächsischen Welt war es daher sehr wichtig zu zeigen, dass die demokratische Moderne nicht einfach vom liberalen Moment aus verstanden werden konnte, sondern dass sie auch von einer sehr viel breiteren Geschichte des Widerstandes gegen Unterdrückung her verstanden werden musste – und einer breiteren Geschichte der Suche nach der guten Regierung. Die republikanische Idee bei Skinner ist folgende: eine Anreicherung, oder ich würde sogar sagen, eine Überwindung der liberalen Tradition als einziger Referenz. Diese liberale Tradition ist in Frankreich weniger stark, und ich gehöre zu denjenigen, die den Versuch unternommen haben, diese Tradition wiederzubeleben – weil sie es war, die vergessen worden ist. Man kann sagen, die republikanische Tradition, oder die Tradition des Kampfes gegen den Absolutismus, besteht zugleich aus einer aristokratischen Definition der Freiheit, aber auch aus einer gewissen idealen Vorstellung des Gemeinwohls und des öffentlichen Nutzens. Eine weitere Dimension bei Skinner ist die methodische. Diese methodische Dimension muss vor dem Hintergrund einer besonderen Situati7 8 Rosanvallon, Pierre, 2010: Demokratische Legitimität. Unparteilichkeit – Reflexivität – Nähe, Hamburg. Skinner, Quentin, 1997: Liberty before Liberalism, Cambridge. Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 111 on der Ideengeschichte in England in den 1950er und Beginn der 1960er Jahre verstanden werden: Zu der Zeit, als Skinner studierte, war die politische Ideengeschichte extrem traditionell ausgerichtet, stellvertretend durch das Journal of the History of Ideas von Lovejoy. In dieser sehr traditionellen Ideengeschichte ging man von einer Art fortschreitender Entwicklung aus, einer Idee der Freiheit, die sich nach und nach entfaltet. Skinner wollte daran erinnern, dass man die Geschichte dieses Denkens nicht in einer abgeschlossenen Welt der Ideen verstehen konnte, die sich von Buch zu Buch, von Denker zu Denker bereichert, sondern dass man dagegen von geschichtlichen Momenten ausgehen muss, durch welche diese Ideen Gestalt bekommen. Aber was seine Definition betrifft, die er „ideas in context“ genannt hat, so glaube ich genau das in meiner Arbeit entwickelt zu haben. Damit ist nicht einfach gemeint zu zeigen, auf welche Weise Rousseau ein Sohn des 18. Jahrhunderts ist oder was Machiavelli mit der Geschichte der Bürgerkriege oder den Kämpfen zwischen den großen Familien und dem Volk in Italien verbindet. Im Prinzip kann man das eine kontextualistische Ideengeschichte nennen, die für sich genommen sehr interessant ist. Aber ich denke, jenseits der kontextualistischen Ideengeschichte war ich eher daran interessiert, eine Geschichte der Aporien, eine Geschichte der demokratischen Erfahrungen zu betreiben. Ich habe also nur Sympathie für Quentin Skinner – den ich sehr gut kenne, mit dem ich gearbeitet habe, den ich nach Paris eingeladen habe und dessen Gast ich in Großbritannien gewesen bin. Aber so ist das, wir arbeiten beide in unseren politischen und historischen Feldern, und ich glaube, bei aller Anerkennung für sein Werk, dass meine Arbeit einen erweiterten Ansatz hat. Schulz: Welche Bedeutung besitzt für Sie das Werk von Reinhart Koselleck? Rosanvallon: Reinhart Koselleck habe ich sehr gut gekannt, weil er recht häufig zu Besuch in das Maison de Science de l’Homme kam. Er kannte François Furet sehr gut, er hatte auch Fernand Braudel sehr gut gekannt und kam oft nach Paris. Das von ihm geleitete Wörterbuch der Begriffsgeschichte ist eine großartige Arbeit, die ich selbst häufig benutzt habe. Auch in diesem Fall habe ich große Sympathie für seine Arbeit, und vor allem auch für seine Arbeiten über Erfahrungsräume und Erwartungshorizonte, die in Frankreich sehr stark rezipiert worden sind, auch von mir. Aber der Grund, warum ich meinen eigenen Weg entwickelt habe liegt auch darin, dass mein zentraler Gegenstand immer die Demokratie selbst gewesen ist. Mir ging es nicht darum, einen Gegenstand der allgemeinen Politikgeschichte oder der allgemeinen Ideen zu behandeln. Die Tatsache, dass ich als wirklich zentralen Gegenstand die Demokratie behandle, hat meiner Arbeit auch ihre spezifische Besonderheit gegeben – das betrifft die Natur der Werke, die ich veröffentlicht habe ebenso wie ihre Konzeptualisierung. Ein Unterschied, so muss ich sagen, rührt auch von der Tatsache her, dass ich das Glück hatte, zwei doch etwas außergewöhnlichen intellektuellen Institutionen anzugehören: Der École des hautes études en sciences sociales und dem Collège de France. Als ich an die École des hautes études en sciences sociales berufen wurde, hatte ich gerade eine Dissertation über Guizot geschrieben – aber ich bin, wenn man das so sagen kann, in das akademische System quasi durch das Fenster eingestiegen. Viele Leute dachten damals, ich hätte eine politische Karriere machen sollen – na ja. Ich bin eher davon angezogen, Bücher zu lesen und zu schreiben. Ich war 26, vielleicht 27 Jahre alt und sehr mit Michel Foucault verbunden; ich war sehr mit François Furet verbunden, mit Claude Lefort, Cor- 112 Zeitschrift für Politische Theorie, Heft 1/2016 nelius Castoriadis und mit Jacques Le Goff, und voilà: Ich hatte ganz einfach große Lust, mit diesen Leuten zusammen zu sein. Ich fand es viel interessanter als im politischen Leben, aber vor allem hatte man mich, als ich an die École des hautes études gekommen bin, nie nach meinem disziplinären Ausweispapier gefragt. Man hat mich nie gefragt: Sind Sie Philosoph oder Historiker? Man hat mich, so könnte man sagen, meine Arbeit von der Bedeutung meines Gegenstandes her entwickeln lassen – und das ist ein großer Glücksfall. Als ich dann später an das Collège de France gekommen bin, war es genauso: Das Collège de France interessiert sich nicht für die disziplinären Ausweise der Leute, sondern für ihre Arbeit. Aber aus diesem Grund hat meine Rezeption in der angelsächsischen Welt manchmal ein wenig gelitten. Die Leute sagten: Sind Sie ein politischer Philosoph? Sind Sie Politikwissenschaftler? Und tatsächlich sind es manchmal die politikwissenschaftlichen Institute, die mich einladen, manchmal die historischen Institute, je nach Land und je nach Universität. Mich kümmern aber solche Fragen nicht. Ich glaube, dass es eine Einheit der Sozialwissenschaften gibt – und diese Einheit der Sozialwissenschaften war ja nun auch genau der historische Anspruch der École des hautes études und vor allem der Anspruch einer Person, die für mich intellektuell sehr viel gezählt hat und der ich persönlich sehr nahe war. Diese Person war Albert Hirschman, den man damals ja als einen Ökonomen bezeichnete. Hirschman habe ich sehr gut gekannt, weil ich eine historische Dissertation über Le capitalisme utopique geschrieben habe – ungefähr zur selben Zeit, als er The Passions and the Interests veröffentlichte.9 Der Gegenstand war fast der gleiche, aber mit einem etwas anderen Ansatz. Und er, damals schon sehr bekannt, war sehr großzügig mit dem jungen, gerade erst beginnenden Intellektuellen, der ich war. Wir haben viel zusammen diskutiert und wir waren sogar sehr verbunden, weil er zufälligerweise seine Ferien in den Alpen immer neben einem Ort verbrachte, wo ich auch Urlaub machte. Wir sind also viel zusammen gewandert und ich konnte viel mit Hirschman diskutieren. Ich erinnere mich, dass er sogar der Vorsitzende der American Economic Association gewesen ist – was man sich heute nur schwer vorstellen kann – und er hatte eine Position am Institute for Advanced Study in Princeton. Und weil er ebenfalls diese Dimension des politischen Philosophen besaß, sagte er eines Tages zu mir: „Ich bin ein Mann des neunzehnten Jahrhunderts, ein Mann der sciences morales et politiques – der allgemeinen Sozialwissenschaften“. Ich fand das ziemlich treffend und würde mich ebenfalls so bezeichnen wollen – in jedem Fall kann ich mir das leisten, weil ich niemals einer Institution angehörte, die in disziplinäre Fächer unterteilt war. Als ich an der École des hautes études war, haben wir eine Gruppe gebildet, der Historiker ebenso wie politische Philosophen und Soziologen angehörten. Als zu Beginn der neunziger Jahre das Centre Raymond Aron gegründet wurde, das ich geleitet habe, hat François Furet sich gewünscht, die junge Generation möge sich um diese Institution kümmern. Neben den Historikern François Furet und Mona Ozouf gab es Philosophen wie Vincent Descombes, Marcel Gauchet oder Pierre Manent, und auch Politikwissenschaftler wie Bernard Manin oder Soziologen. Dieses Zentrum war ein Zentrum derjenigen Leute, die sich für die Konzeption und die Erforschung des Politischen interessierten. Und als ich einen Promotionsstudiengang an der École des hautes études gegründet und 9 Rosanvallon, Pierre, 1979: Le capitalisme utopique. Critique de l’idéologie économique, Paris (Neuausgabe 1999 unter dem Titel: Le capitalisme utopique. Histoire de l’idée de marché, Paris); Hirschman, Albert O., 1977: The Passions and the Interests. Political Arguments for Capitalism before its Triumph, Princeton, NJ. Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 113 geleitet habe, nannten wir das „politische Studien“, zu denen auch Projekte aus der politischen Soziologie, der Geschichte und der Philosophie zugelassen wurden. Schulz: Ihr jüngstes Buch haben Sie der Frage des guten Regierens gewidmet. Warum haben Sie sich entschieden, die Bedeutung der Exekutive in ihrer Demokratietheorie stark zu machen? Rosanvallon: Weil ganz einfach die Art und Weise, in der die Bürger mit politischen Handlungen konfrontiert sind, zuerst über das Handeln der Regierung läuft – sehr viel mehr, als über das Handeln eines Parlamentes. Zunächst einmal sind es die Bürger, die regiert werden. Eine Reflexion der politischen Herrschaft, eine Reflexion politischer Handlungen und des Stellenwerts des Politischen ist heute notwendigerweise eine Reflextion der Kategorie der Regierung. Es war ein Fehler, dass der Begriff der Regierung lange Zeit in einem sehr besonderen Sinne verwandt wurde. Wenn man von der repräsentativen Ordnung spricht, dann spricht man eigentlich nicht von einer repräsentativen Regierung – eine Regierung kann im strengsten Sinne des Wortes nie repräsentativ sein. Ein Präsident der Republik kann nicht repräsentativ sein, eine Versammlung dagegen kann repräsentativ sein. Wenn man von repräsentativer Regierung spricht, dann war damit zumeist das repräsentative System gemeint. Es ging um eine Theorie des parlamentarischen Systems und eine Theorie der politischen Repräsentation, es ging nicht um eine Theorie der Regierung. Die ersten, die eine Theorie der Regierung gemacht haben, sind natürlich die antiken Theoretiker der Regierungskunst oder die Theoretiker des Machiavellismus, die Theoretiker der Staatsräson. Die ersten Regierungstheoretiker waren zugleich Regierungsberater, keine Theoretiker des Gemeinwohls – oder solche wie Machiavelli, die die Regierten vor den Künsten der Regierenden warnten. Im zwanzigsten Jahrhundert waren dann in der Tat die Theoretiker des Dezisionismus die ersten, die sich wieder für die Theorie des Regierens interessiert haben, und zwar in einer nicht-demokratischen Perspektive. Man kann von Carl Schmitt als einem großen Theoretiker des Regierens sprechen – aber eben nur als Theoretiker der Regierung und gerade nicht des demokratischen Regierens. Er ist ein Theoretiker der Entscheidung und – aus historischen Gründen ebenso wie aus intellektuellen und philosophischen Gründen – ein theoretischer Kritiker des Parlamentarismus, und deshalb ist er ein Theoretiker der Regierung. Aber niemals hat er die Frage des Regierens mit der Demokratie verbunden, es sei denn über seine Theorie der Akklamation. Heute würde man wohl von einer Theorie des Populismus sprechen. Sicherlich hat Hugo Chávez niemals Carl Schmitt gelesen, aber die Idee des „hommepeuple“, die Idee also, dass das Volk über die Figur eines Anführers regiert wird, die entspricht vollkommen der Vorstellung von Carl Schmitt. Ich weiß übrigens nicht, ob Carl Schmitt den ersten großen Theoretiker des Populismus in Lateinamerika kannte, einen Kolumbianer namens Jorge Eliécer Gaetán. Er hat seine juristische Doktorarbeit in Italien bei einem großen Rechtsprofessor geschrieben, der ein Intimus Mussolinis war – und er war in der Tat der erste in Lateinamerika, der die Theorie des „homme-peuple“ entwickelt hat, und die man heute als Theorie des Populismus bezeichnen würde. Er wurde übrigens auch von Fidel Castro gefeiert, ebenso wie von Chávez und von Perón. Er besaß ganz sicher nicht die theoretische Macht Carl Schmitts, aber ich würde sagen, das politische Modell hinter seiner Vision ist von derselben Natur. Heute ist mein Ziel, die Demokratie mit der Frage des Regierens zu verbinden. 114 Zeitschrift für Politische Theorie, Heft 1/2016 Schulz: Warum haben Sie sich für die traditionelle Semantik der Politik als Regierung entschieden? In welchem Maße lässt sich Ihr Werk als eine Kritik der „governance“Vokabel lesen, wie sie vor allem in der Politikwissenschaft verwendet wird? Rosanvallon: Aus zwei Gründen: Ich denke, dass alle Governance-Theorien versucht haben, die Qualität des Regierungshandelns zu reflektieren. Wie kann die Qualität des Regierungshandelns verbessert werden? Im Grunde unterscheiden sich diese Theorien also nicht von betriebswirtschaftlichen Management-Theorien. Das sind Theorien des guten Managements. Was macht aber den Unterschied zwischen einer Theorie des guten Managements und einer Theorie der guten Regierung aus? Es gibt natürlich gemeinsame Punkte: Schon zu Beginn des 20. Jahrhunderts wurde von denjenigen, die über die moderne Regierung nachdachten, die Führung großer Unternehmen mit der Führung des Staates verglichen. Wie sie wissen, wurde Taylor im politischen Milieu sehr intensiv gelesen – zum Beispiel von Lenin, jeder weiß das, aber er wurde auch von Léon Blum gelesen. Léon Blum hat ein Buch geschrieben, in dem Taylor für seine Ideen der rationalen Organisation gefeiert wird und dafür, das Regieren als Beruf zu betrachten.10 Diese Theorien der Governance gehören für mich in die Kategorie der Theorien effizienter Führung. Was kann man nun darüber hinaus von einer Theorie der Regierung erwarten? Sie unterscheidet sich von einer Theorie der Governance in zwei Punkten: Der erste Punkt ist, dass eine Theorie der Regierung der good Governance Kategorien des Eingreifens der Bürger hinzufügt. Die Theorien der good Governance setzen den Akzent auf den Faktor der Information, aber nicht auf den Faktor der Intervention. Die Theorien der good Governance würden beispielsweise keinen Akzent auf Bürgerjurys legen, was eine Theorie der Regierung tun würde. Eine der ersten Unterscheidungen wäre also daher die Art und Weise, wie das Zusammenspiel zwischen Regierenden und Regierten zu verstehen ist. Die Theorien der Governance sehen das einfach unter dem Aspekt der Kommunikationspolitik. Eine Theorie der Regierung wird sie dagegen in einer sehr viel weitereichenden Weise betrachten und die eingreifende Rolle der Bürger hervorheben. Das Gleiche gilt mit Blick auf die Institutionen: Eine Theorie der Governance wird beispielsweise auf der Notwendigkeit der Evaluation beharren, auf der Notwendigkeit eines Kriteriums für den Rechenschaftsbericht, wird auf den Begriff der Transparenz pochen, der in der Tat sehr wichtig erscheint. Aber eine Theorie der Regierung in der Demokratie wird im Gegensatz zu einer Theorie der Governance verstehen, dass die Art und Weise der Rechenschaftsprüfung nicht einfach durch interne Institutionen erledigt werden kann, sondern ebenfalls auf Praktiken der Bürger angewiesen ist. Schließlich läuft die erste Theorie des Parlaments darauf hinaus zu sagen, Parlamente haben die Funktion, Transparenz zu kontrollieren und die Rechenschaft zu überprüfen. Diese Funktionen sind noch erweitert worden, es gibt „general accounting offices“, Rechnungshöfe sowie andere mit Evaluationsaufgaben betreute Institutionen, aber die meisten Theorien sehen das unter dem Gesichtspunkt funktionaler Notwendigkeit und nicht als typisch demokratische Notwendigkeit – hier liegt also wirklich ein Unterschied. Der zweite Unterschied ist für mich der wichtigere: Es gibt eine Besonderheit des Regierungsbegriffs, die ihn vom Begriff des Managements und der Leitung abhebt. Dem Begriff der Leitung ist es eigen, eine Organisation oder eine Gemeinschaft in einem relativ horizontalen Funktionsverhältnis zu begreifen. Das Besondere der Regierung ist es, 10 Blum, Léon, 1918: Lettres sur la réforme gouvernementale, Paris. Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 115 eine soziale Reflexionsfunktion zu besitzen. Um auf einen Ausdruck von Hobbes zurückzugreifen: Die Funktion der Regierung ist es, die Vielheit zu einer Einheit zu bringen. Die Funktion der Regierung ist es, jegliche Form des Antagonismus, des Unverständnisses, des Missverständnisses in der Gesellschaft zu überwinden oder ihnen eine auf Dauer gestellte Form zu verleihen. Es geht darum, vom sozialen Körper zum politischen Körper überzugehen. In der Welt des Managements dagegen gibt es nur einen horizontalen Körper, die Welt der Arbeit oder die Welt des Konsums. Wir befinden uns hier in einer rein funktionalen Beziehung zur Institution. Mit der Regierung dagegen befinden wir uns nicht lediglich in einer funktionalen Beziehung. Das Regierungshandeln besitzt eine eigene Dimension der Reflexion. Schulz: In diesem Buch erweitern Sie auch Ihre Überlegungen zur demokratischen Repräsentation mit Blick auf die Rolle der Regierenden. In welchem Sinne gestehen Sie ihnen – verglichen mit anderen Akteuren und Institutionen – eine besondere Funktion zu, wenn es um die Herstellung der Lesbarkeit des demokratischen Prozesses geht? Rosanvallon: Sie besitzen die Besonderheit, direkt gewählt worden zu sein. Ich gehe von dem Prinzip aus, dass der Präsidentialismus das politische Modell ist, das sich universalisiert hat. Der Präsidentialismus besitzt unterschiedliche konstitutionelle Ausdrucksformen, besonders in Europa: Aus der Ferne betrachtet könnte man meinen, in Europa hätten sich die politischen Formen des neunzehnten Jahrhunderts konserviert. In Europa existieren noch sieben Monarchien – auf keinem anderen Kontinent gibt es noch eine solche Zahl. Aber neben diesen Monarchien gibt es noch einen verdeckten Präsidentialismus: Die Deutschen stimmen für oder gegen Merkel, die Engländer stimmen für oder gegen den Vorsitzenden von Labour oder der Torys, das Gleiche gilt für die Monarchie Spaniens oder auch für Italien. Man kann also sagen, dass sich das präsidentialistische Modell jenseits der konstitutionellen Unterschiede als politische Praxis durchgesetzt hat, auch wenn die jeweiligen Verfassungstraditionen variieren können. Die Besonderheit dieses Modells liegt darin, dass die Wahlen zur Regierung eine sehr polarisierende Wirkung auf die Debatten ausüben – in einem solchen Maße, dass damit zwangsläufig auch der Unterschied zwischen dem Wahlkampfdiskurs vor der Wahl und dem Regierungsdiskurs nach der Wahl maximiert wird. Der Wahlkampfdiskurs funktioniert nach der umgekehrten Logik des ökonomischen Wettbewerbs. Der wirtschaftliche Wettbewerb führt zu sinkenden Preisen, der politische Wettbewerb führt zu steigenden Versprechen. Man hat hier also eine strukturelle Spannung in der Demokratie. Während der Französischen Revolution hat man das sehr gut verstanden, denn es war dort verboten, sich als Kandidat selbst zu bewerben. Dahinter stand die Annahme, wenn sich Personen selbst zum Kandidaten erklären, dann wird ein Wettbewerb des Ehrgeizes daraus hervorgehen – und ein Wettbewerb des Ehrgeizes wird eine Art strukturelle Perversion mit der Wahl verbinden. Man hat dann diese Frage auf eine sehr spezielle Art gelöst, die sich nicht einfach übernehmen lässt: Da es verboten war, sich als Kandidat zu präsentieren, fanden während der Französischen Revolution Wahlen ohne vorher festgelegte Kandidaten statt, das ging zehn Jahre so. Dabei wurde die Annahme zugrunde gelegt, dass die Wähler ohne Kandidaten spontan die Talentiertesten, die Tugendhaftesten auswählen würden – und das Ganze passierte in kleinen Versammlungen von etwa tausend Personen. Ich erinnere an dieses historische Beispiel um zu zeigen, dass man sofort das durch den Kandidatenwettbewerb erzeugte Problem bei Wahlen erkannt hat. Der Wahlkampf als 116 Zeitschrift für Politische Theorie, Heft 1/2016 Wettbewerb ist nicht einfach ein Pfand des Liberalismus, eine durch die Öffnung des Möglichkeitsraumes entstehende Hypothek – der Wahlkampf ist ein typischer Beleg der perversen Effekte, die durch einen Wettbewerb des Ehrgeizes und der Versprechen hervorgebracht werden. Dieser Wettbewerb besteht nicht lediglich in einer Auseinandersetzung der Werte und Programme, vor allem dort nicht, wo die Politik in einer unsicheren Welt immer stärker personalisiert wird. In den 1950er Jahren hatte man den Eindruck, dass die Politik, die gemacht werden würde, bereits in den vorliegenden Programmen enthalten war. Heute weiß man genau, dass die Politik, die gemacht werden wird, nicht in den Programmen enthalten ist, die man hat. Der einfache Grund hierfür ist die enorme Zahl exogener Elemente, durch welche die durchzuführende Politik bestimmt wird – Elemente internationaler Krisen und Situationen, die Gesundheit der chinesischen Wirtschaft oder ein irgendwo in der Welt ausbrechender Bürgerkrieg, all dies ist wichtiger für die ökonomische Lage eines europäischen Landes als seine internen Entscheidungen. Unter diesen Bedingungen braucht man sicher etwas anderes: eine Verbindung zwischen dem Moment der Wahl und dem Moment der Regierung. Und wenn Programme diese Verbindung nicht mehr herstellen können, dann muss dies heute eben durch Personen geleistet werden, denen man vertraut. Vertrauen wird zum zentralen Wert demokratischer Politik, während es vorher die Programme waren. Es gibt immer noch Programmelemente, die sehr wichtig sind – es gibt Divergenzen und verschiedene Philosophien, sicher. Aber das Vertrauen in Personen hat eine wachsende Bedeutung gewonnen. Dieses Vertrauen beruht darauf, inwieweit die Bürger diese Personen mit dem Gemeinwohl identifizieren. Und genau das nenne ich die Integrität und das „wahr sprechen“. Schulz: Sie versuchen, die Figur des guten Regierenden für unsere Gegenwart zu beschreiben und greifen dafür auf die Idee der „parrhesia“ von Michel Foucault zurück. In welchem Maße gefährdet sich ein Politiker durch dieses „wahre Sprechen“ nicht auch selbst? Rosanvallon: Der Begriff der Wahrheit ist vieldeutig. Über alles zu sprechen bedeutet nicht einfach, die Wahrheit zu sagen. „Parler vrai“ (also „wahres Sprechen“), ist die Übersetzung der „parrhesia“, die dem Begriff im Französischen üblicherweise gegeben wird. Es ist eher die offene Rede, eine Sprache, die zugleich durch das Engagement der Person und durch eine gewisse Stetigkeit geprägt ist. Wenn die Sprache den Anspruch hätte, eine definitive Wahrheit zu geben, dann wäre das eine ans Totalitäre grenzende Definition. Aber wenn die Sprache über verschiedene Momente der Politik hinweg beständig bleibt, wenn diese Sprache mit einem persönlichen Engagement verbunden ist, in diesem Moment kann man nicht den Begriff der Wahrheit verwenden, sondern den der Beständigkeit der Sprache und des Engagements. Schulz: Auch wenn es nicht um das Aussprechen unmittelbarer Wahrheiten geht: Inwieweit kann dieses „parler vrai“ in einem Spannungsverhältnis zu der grundlegenden Idee von Claude Lefort stehen, der zufolge die Demokratie ein politisches Regime ist, in dem die Idee der Wahrheit als politische Qualität suspendiert wird? Rosanvallon: Um darauf zu antworten, muss man die Unterschiede zwischen der Idee der Unbestimmtheit in der Demokratie bei Claude Lefort und bei mir erklären. Die Unbestimmtheit bei Lefort ist vollkommen an seine Wahrnehmung des Totalitarismus gebunden, das heißt: Claude Lefort hat einer Generation angehört, für die das Denken des Poli- Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 117 tischen zuallererst ein Kritik des Totalitarismus ist. Dieses Denken ging von der Feststellung aus, dass der Marxismus als begrifflicher Horizont der fortschrittlichen Intellektuellen keine Theorie der Demokratie besaß. Zudem gab es im Marxismus keine politische Theorie. Genau diese Abwesenheit der politischen Theorie im Marxismus hat in den Augen von Intellektuellen wie Claude Lefort das Abdriften in den Totalitarismus erklärt. Für ihn lag der Totalitarismus in genau diesem Anspruch auf den Besitz einer wissenschaftlichen Wahrheit, die dann auch noch die Gesellschaft führen soll. Wenn man also sagt, die Demokratie ist unbestimmt, dann bedeutet das vor allem, dass niemand behaupten kann, ihm gehöre die Geschichte oder die Wahrheit. Das ist seine Definition von der Demokratie als leerer Ort. In der Demokratie muss die Macht ein leerer Ort sein, das heißt: die Macht darf nicht auf dem Anspruch beruhen, Wissenschaft zu sein oder die Totalität der Gesellschaft zu verkörpern. Heute würden wir von der populistischen Versuchung sprechen, dem Anspruch einer Person oder einer Gruppe, die Gesellschaft zu verkörpern. Die demokratische Macht als leeren Ort zu entwerfen heißt daher, sowohl diesen sozialen Verkörperungsanspruch als auch die wissenschaftliche Vision der Macht zurückzuweisen – die Idee also, das Recht zu Herrschen leite sich aus dem Besitz eines vollkommen wissenschaftlichen Wissens über die Gesellschaft ab. Meine Konzeption der politischen Unbestimmtheit dagegen ist eine etwas andere – auch, weil ich einer anderen Generation angehöre und auch wenn ich Claude Lefort intellektuell sehr nahe war und sogar für eine bestimmte Zeit das Büro mit ihm geteilt habe. Ich betone eine andere Dimension der Unbestimmtheit – die Unbestimmtheit ist eine konstitutive Kategorie der Demokratie selbst. Lefort hat dagegen die Unbestimmtheit gegen die Determiniertheit gestellt, die für ihn den Totalitarismus ausmachte. Ich habe Unbestimmtheit mit Bezug auf die Determiniertheit gedacht, die als eine Engführung der demokratischen Idee erscheint, eine Engführung durch die minimalistischen Theorien der Demokratie, in denen Demokratie auf eine einfache prozedurale Vorstellung reduziert wird. Von der demokratischen Unbestimmtheit zu sprechen heißt also zu zeigen, dass die Demokratie nicht einfach ein Modell ist, das sich angemessen definieren und dann auch entsprechend vollenden lässt. Die Demokratie ist ein Regime, das auf der permanenten Suche nach sich selbst ist. Es ist durch seine strukturelle Unvollständigkeit und Unvollendetheit gekennzeichnet, durch die permanente Rückkehr seiner Pathologien und Probleme. Darin liegt wiederum etwas, das ich mit den Theoretikern des Totalitarismus teile: Man muss ein Regime von seinen Pathologien aus verstehen, oder zumindest von der drohenden Rückkehr seiner Pathologien. Schulz: Was die aktuelle Diskussion über die Zukunft der Demokratie betrifft, so kann in Ihrem Werk eine Leerstelle ausgemacht werden: Europa, oder in allgemeinerer Hinsicht, der Kosmopolitismus als politische Perspektive. Diese Perspektive wurde gerade in Deutschland besonders stark gemacht. Muss man angesichts der Krise der demokratischen Gleichheit den Nationalstaat verteidigen? Rosanvallon: Zunächst einmal stimmt es, dass ich meine Konzeption des demokratischen Kosmopolitismus nie voll entwickelt habe. Idealerweise wäre es sicher wichtig gewesen, dies zu tun, und wenn ich einen fünften Band zu schreiben hätte, dann würde ich es über diese Frage tun. Warum habe ich das bisher nicht getan? Aus einem einfachen Grund: Die Definition, die ich der Demokratie gegeben habe, setzt ihre besondere Entwicklung im Rahmen des Nationalstaats voraus. Die erste Voraussetzung besteht in der Konzeption des 118 Zeitschrift für Politische Theorie, Heft 1/2016 Demos, und die zweite in der Bedeutung der Umverteilung. Jede Theorie des Kosmopolitismus wird eine Theorie sein, welche die Menschheit als impliziten Demos annimmt, eine Theorie, die sich auf eine Theorie der Menschenrechte beschränkt, also eine Theorie der good Governance, die sehr reichhaltig sein kann, aber die niemals eine Theorie der Redistribution beinhaltet. Europa ist das beste Beispiel dafür, und deshalb ist Europa sehr wichtig.11 Warum ist der europäische Fall so wichtig? Weil Europa verstanden werden kann als ein Versuch des effizienten Kosmopolitismus. Europa kann verstanden werden als ein Versuch, einen limitierten Universalismus zu organisieren. Man kann sagen, dass Europa das Laboratorium des Kosmopolitismus hätte sein sollen. Was hat die europäische Erfahrung gezeigt? Europa kann als Erfahrung eines solchen limitierten Universalismus beschrieben werden, aber die europäische Konstruktion wurde zuallererst durch die Austreibungserfahrung seiner Vergangenheit bestimmt. Europa war die Erfahrung eines Exorzismus, ein Exorzismus der deutsch-französischen Konfrontation, des Zweiten Weltkrieges. Für die Gründerväter war das fundamental. Ich gehöre zu einer Generation, in der fast jeder in der Schule Deutsch lernte – ich habe zwölf Jahre Deutsch gelernt und spreche es heute nur sehr schlecht, aber für meine Eltern war das sehr wichtig. Zweitens war es die historische Austreibung der gerade überwundenen Diktaturen. Jeder wusste, dass es ökonomisch nicht den geringsten Sinn macht, Griechenland, Spanien oder Portugal aufzunehmen, aber es handelte sich um eine abzutragende Schuld angesichts der Geschichte, um aus dem zweiten europäischen Unglück zu entkommen. Und das dritte europäische Unglück war der Kommunismus. Auch hier gab es eine große Debatte darüber, ob man die osteuropäischen Länder aufnehmen sollte. Aber es war politisch nahezu unmöglich, es nicht zu tun – denn es bedeutete, die fundamentale Teilung zwischen Demokratie und Kommunismus zu beenden. Europa hat aber seine Rezepte nur anhand einer gewissen Zahl begrenzter Institutionen entwickelt. Europa war die Konstruktion eines gemeinsamen Marktes, was gut funktioniert hat. Es war auch die Konstruktion eines Rechtsraums, was ebenfalls ziemlich gut funktioniert hat und ein Erfolg war. Man kann sagen, dass die WTO niemals wirklich einen funktionierenden gemeinsamen Markt zu Wege gebracht hat. Man kann sagen, dass der Rechtsraum der Vereinten Nationen keinen vergleichbaren Erfolg hatte und auch niemals Elemente einer kulturellen oder intellektuellen Gemeinschaft entwickelte. Ganz sicher war das Erasmus-Programm für viele Leute, die heute zwischen 25 und 45 Jahre alt sind, eine spürbare Erfahrung. Das hat sehr viel ausgemacht. Was den Rest betrifft, so hat Europa sich nie gemeinsam entscheiden können, weiter zu gehen, und zwar aus einem einfachen Grund: Wenn man von Europa spricht, dann muss man immer eine Zahl im Kopf behalten. Das europäische Budget hat sich zwischen den Römischen Verträgen und heute kaum verändert. Die Wahrheit über Europa ist, dass das europäische Budget ein Prozent des BNE beträgt: 0,1 Prozent für die Institutionen, 0,4 Prozent für die gemeinsame Agrarpolitik und 0,4 Prozent für die sogenannten Strukturfonds. Diese 0,4 Prozent sind wichtig, weil sie für kleine Länder wie Griechenland viel Geld bedeuten – viele Autobahnen. Wenn man nach Griechenland oder Portugal gereist ist, konnte man überall Schilder sehen, auf denen stand: „Finanziert von der Europäischen Union“. Aber gab es jemals irgendwo ein einziges Projekt, eine öffentliche Entwicklung, die fast siebzig Jahre stagnierte? Nein, überall, in Frankreich, in Deutschland, hat man gesehen, wie die Orga11 Vgl. dazu Rosanvallon, Pierre, 2015: The Nature, Political Model and Future Prospects of Europe. In: Luuk van Middelaar, Philippe Van Parijs (Hg.), After the Storm. How to Save Democracy in Europe, Tielt. Daniel Schulz: „Die Unbestimmtheit der Demokratie“ 119 nisation des Gemeinwesens sich entwickelt hat, wie man neue Instrumente gefunden hat – in Europa nicht. Man ist bei einem Prozent geblieben. Daher ist Europa ein liberaler Raum gewesen, der erfolgreich war, und der als ein Laboratorium für das betrachtet werden kann, was der Kosmopolitismus sein sollte. Aber Europa konnte nicht den Schritt zu einem demokratischen Europa gehen, weil dieser Schritt zu einem demokratischen Europa bedeutet hätte, einen Raum der Umverteilung zu betreten. Nun ist in den einzelnen Ländern die Umverteilung enorm. Die Umverteilung in Europa – ich nehme die gemeinsame Agrarpolitik davon aus, die eine eigene Geschichte ist – und auch die Industriepolitik in verschiedenen Bereichen machen sehr wenig aus. Umverteilungspolitik bedeutet, dass der Arbeiter des deutschen Ruhrgebiets für den Beamten in Ostdeutschland bezahlt hat. Genau das war die Wiedervereinigung. Hier ging es nicht einfach darum zu sagen: Wir sind endlich alle Deutsche. Sondern vielmehr hat man eine gemeinsame Haushaltskasse geteilt, und man weiß, was dies das Land gekostet hat. In Europa hat man dagegen nie einen gemeinsamen Haushalt geteilt, und das Paradox ist im Gegenteil, dass die europäischen Länder seit den achtziger Jahren angesichts des sinkenden Wachstums nach und nach ihre nationale Ohnmacht auf Europa projiziert haben und man Europa als den Raum der Verwirklichung dieser Ohnmacht definierte. Man hat es in Frankreich nicht mehr geschafft, ein neues wohlfahrtsstaatliches Regime zu finden. Man hat gesagt: Das soziale Europa wird uns retten. Aber das soziale Europa war nur ein leeres Wort. Es gab in Frankreich Probleme mit der Industriepolitik und man hat gesagt: Wir machen eine europäische Industriepolitik. Als man dann gesehen hat, dass das Wort „Europa“ zunehmend als Container für unsere nationale Ohnmacht eingesetzt wurde, als deutlich zu werden begann, dass Europa nichts als ein liberaler Raum war, im positiven Sinne des Wortes „liberal“ des achtzehnten Jahrhunderts – und das war bereits eine enorme Leistung –, in diesem Moment begannen die Schwierigkeiten des Vertrags von Maastricht und die negativen Voten über den Verfassungspakt, den man übrigens zu Unrecht als Verfassung bezeichnet hat. Eine Verfassung wäre ein echter Schritt nach vorne in eine gemeinsame Welt gewesen, aber hier war das nicht der Fall. Mit dem Euro ist man dann noch weiter gegangen, aber man ist sich sehr schnell der Schwierigkeiten bewusst geworden, die damit verbunden sind: den Euro zu machen, ohne zugleich Elemente der Abstimmung und einen gemeinsamen Kontext zu besitzen. Daher: Kosmopolitismus ja, aber das beste Beispiel für den Kosmopolitismus heute ist die Europäische Union, und man sieht ihre Grenzen – Grenzen im positiven Sinne, weil ich glaube, dass es sich bereits um eine großartige Erfahrung handelt. Die kosmopolitische Demokratie kann man als einen sehr wertvollen Horizont betrachten, aber sie stellt sich nicht die Fragen nach dem, was grundlegend für die Konstituierung des Gemeinwesens ist, und zwar die Redistribution. Für mich liegt hier das große Problem.
https://openalex.org/W2014554703	http://dspace.stir.ac.uk/bitstream/1893/17359/1/Payne%20and%20Pates%202009%20Wetlands%20Ecology%20and%20Management.pdf	English	null	Vertical stratification of testate amoebae in the Elatia Mires, northern Greece: palaeoecological evidence for a wetland response to recent climatic change, or autogenic processes?	Wetlands ecology and management	2,008	public-domain	9,789	WETL234 1 2 Vertical stratification of testate amoebae in the Elatia Mires, northern Greece: 3 Palaeoecological evidence for a wetland response to recent climatic change, or 4 autogenic processes? 5 6 Richard J. Payne1,2 and Jacqueline M. Pates3 7 8 1 The Fitch Laboratory, British School at Athens, Odhós Souidhías 52, GR-10676 Athens, 9 Greece. 10 2 Geography, School of Environment and Development, The University of Manchester, 11 Oxford Road, Manchester, M13 9PL, UK.* 12 3Department of Environmental Science, Lancaster Environment Centre, Lancaster 13 University, Lancaster, LA1 4YQ, UK. 14 * To whom correspondence should be addressed. E-mail: r.j.payne@manchester.ac.uk 15 16 ABSTRACT 17 18 The Elatia Mires of northern Greece are unique ecosystems of high conservation value. 19 The mires are climatically marginal and may be sensitive to changing hydroclimate, 20 while northern Greece has experienced a significant increase in aridity since the late 20th 21 century. To investigate the impact of recent climatic change on the hydrology of the 22 mires, the palaeoecological record was investigated from three near-surface monoliths 23 WETL234 1 2 Vertical stratification of testate amoebae in the Elatia Mires, northern Greece: 3 Palaeoecological evidence for a wetland response to recent climatic change, or 4 autogenic processes? 5 6 Richard J. Payne1,2 and Jacqueline M. Pates3 7 8 1 The Fitch Laboratory, British School at Athens, Odhós Souidhías 52, GR-10676 Athens, 9 Greece. 10 2 Geography, School of Environment and Development, The University of Manchester, 11 Oxford Road, Manchester, M13 9PL, UK.* 12 3Department of Environmental Science, Lancaster Environment Centre, Lancaster 13 University, Lancaster, LA1 4YQ, UK. 14 * To whom correspondence should be addressed. E-mail: r.j.payne@manchester.ac.uk 15 16 ABSTRACT 17 18 The Elatia Mires of northern Greece are unique ecosystems of high conservation value. 19 The mires are climatically marginal and may be sensitive to changing hydroclimate, 20 while northern Greece has experienced a significant increase in aridity since the late 20th 21 century. To investigate the impact of recent climatic change on the hydrology of the 22 mires, the palaeoecological record was investigated from three near-surface monoliths 23 1 1 extracted from two sites. Testate amoebae were analysed as sensitive indicators of 1 hydrology. Results were interpreted using transfer function models to provide 2 quantitative reconstructions of changing water table depth and pH. AMS radiocarbon 3 dates and 210Pb suggest the peats were deposited within the last c.50 years, but do not 4 allow a secure chronology to be established. Results from all three profiles show a 5 distinct shift towards a more xerophilic community particularly noted by increases in 6 Euglypha species. Transfer function results infer a distinct lowering of water tables in this 7 period. A hydrological response to recent climate change is a tenable hypothesis to 8 explain this change; however other possible explanations include selective test decay, 9 vertical zonation of living amoebae, ombrotrophication and local hydrological change. It 10 is suggested that a peatland response to climatic change is the most probable hypothesis, 11 showing the sensitivity of marginal peatlands to recent climatic change. 12 13 KEYWORDS: Mires, Peatlands, Climate Change, Testate Amoebae, Palaeohydrology 14 extracted from two sites. Testate amoebae were analysed as sensitive indicators of 1 hydrology. Results were interpreted using transfer function models to provide 2 quantitative reconstructions of changing water table depth and pH. KEYWORDS: Mires, Peatlands, Climate Change, Testate Amoebae, Palaeohydrology 14 KEYWORDS: Mires, Peatlands, Climate Change, Testate Amoebae, Palaeohyd INTRODUCTION 2 Such a case may be the Mediterranean region, 9 where peatlands are rare but are often climatically marginal and are likely to be sensitive 10 to comparatively minor climatic changes. Mediterranean peatlands are relatively 11 unimportant in terms of their carbon reserve but are extremely important in terms of 12 regional biodiversity. 13 Climate change may lead to changes in peatland carbon sequestration (Belyea and 3 Malmer, 2004; Lavoie et al., 2005), gas flux (Chapman and Thurlow, 1998; Regina et al., 4 1999; Keller et al., 2004), plant communities (Weltzin et al., 2003; Robroek et al., 2007) 5 and permafrost melting (Camill, 2005; Turetsky et al., 2007). In many regions of the 6 world impacts on peatland hydrology have been noted (e.g. Klein et al., 2005). The 7 peatlands likely to show the greatest impacts are the most marginal sites in regions which 8 experience the greatest climate change. Such a case may be the Mediterranean region, 9 where peatlands are rare but are often climatically marginal and are likely to be sensitive 10 to comparatively minor climatic changes. Mediterranean peatlands are relatively 11 unimportant in terms of their carbon reserve but are extremely important in terms of 12 regional biodiversity. 13 Meteorological records for northern Greece show a general trend of reduced 14 precipitation over the last c.50 years. Climatic impacts on the hydrology of northern 15 Greece have already been noted and are modelled to increase in severity over coming 16 decades with consequent impacts for human populations (Mimikou, 1993; Mimikou et 17 al., 1999; 2000; Bürger, 2002; Baltas and Mimikou, 2005). These changes may have 18 affected peatlands in the region. Most peatlands in Greece have been heavily impacted by 19 human activity primarily through drainage for agriculture in the 20th Century, and often 20 have low conservation value (Bouzinos et al., 1994; 1997; Christanis, 1996). An 21 exception to this is the Elatia Mires of northern Macedonia where remoteness and Cold 22 War access restrictions have prevented major impacts. These mires are the most 23 Meteorological records for northern Greece show a general trend of reduced 14 precipitation over the last c.50 years. WETL234 1 2 Vertical stratification of testate amoebae in the Elatia Mires, northern Greece: 3 Palaeoecological evidence for a wetland response to recent climatic change, or 4 autogenic processes? 5 6 Richard J. Payne1,2 and Jacqueline M. Pates3 7 8 1 The Fitch Laboratory, British School at Athens, Odhós Souidhías 52, GR-10676 Athens, 9 Greece. 10 2 Geography, School of Environment and Development, The University of Manchester, 11 Oxford Road, Manchester, M13 9PL, UK.* 12 3Department of Environmental Science, Lancaster Environment Centre, Lancaster 13 University, Lancaster, LA1 4YQ, UK. 14 * To whom correspondence should be addressed. E-mail: r.j.payne@manchester.ac.uk 15 16 ABSTRACT 17 18 The Elatia Mires of northern Greece are unique ecosystems of high conservation value. 19 The mires are climatically marginal and may be sensitive to changing hydroclimate, 20 while northern Greece has experienced a significant increase in aridity since the late 20th 21 century. To investigate the impact of recent climatic change on the hydrology of the 22 mires, the palaeoecological record was investigated from three near-surface monoliths 23 AMS radiocarbon 3 dates and 210Pb suggest the peats were deposited within the last c.50 years, but do not 4 allow a secure chronology to be established. Results from all three profiles show a 5 distinct shift towards a more xerophilic community particularly noted by increases in 6 Euglypha species. Transfer function results infer a distinct lowering of water tables in this 7 period. A hydrological response to recent climate change is a tenable hypothesis to 8 explain this change; however other possible explanations include selective test decay, 9 vertical zonation of living amoebae, ombrotrophication and local hydrological change. It 10 is suggested that a peatland response to climatic change is the most probable hypothesis, 11 showing the sensitivity of marginal peatlands to recent climatic change. 12 13 15 2 1 1 INTRODUCTION 2 INTRODUCTION 2 INTRODUCTION 2 Climate change may lead to changes in peatland carbon sequestration (Belyea and 3 Malmer, 2004; Lavoie et al., 2005), gas flux (Chapman and Thurlow, 1998; Regina et al., 4 1999; Keller et al., 2004), plant communities (Weltzin et al., 2003; Robroek et al., 2007) 5 and permafrost melting (Camill, 2005; Turetsky et al., 2007). In many regions of the 6 world impacts on peatland hydrology have been noted (e.g. Klein et al., 2005). The 7 peatlands likely to show the greatest impacts are the most marginal sites in regions which 8 experience the greatest climate change. Such a case may be the Mediterranean region, 9 where peatlands are rare but are often climatically marginal and are likely to be sensitive 10 to comparatively minor climatic changes. Mediterranean peatlands are relatively 11 unimportant in terms of their carbon reserve but are extremely important in terms of 12 regional biodiversity. 13 Meteorological records for northern Greece show a general trend of reduced 14 precipitation over the last c.50 years. Climatic impacts on the hydrology of northern 15 Greece have already been noted and are modelled to increase in severity over coming 16 decades with consequent impacts for human populations (Mimikou, 1993; Mimikou et 17 al., 1999; 2000; Bürger, 2002; Baltas and Mimikou, 2005). These changes may have 18 affected peatlands in the region. Most peatlands in Greece have been heavily impacted by 19 human activity primarily through drainage for agriculture in the 20th Century, and often 20 have low conservation value (Bouzinos et al., 1994; 1997; Christanis, 1996). An 21 exception to this is the Elatia Mires of northern Macedonia where remoteness and Cold 22 War access restrictions have prevented major impacts. These mires are the most 23 Climate change may lead to changes in peatland carbon sequestration (Belyea and 3 Malmer, 2004; Lavoie et al., 2005), gas flux (Chapman and Thurlow, 1998; Regina et al., 4 1999; Keller et al., 2004), plant communities (Weltzin et al., 2003; Robroek et al., 2007) 5 and permafrost melting (Camill, 2005; Turetsky et al., 2007). In many regions of the 6 world impacts on peatland hydrology have been noted (e.g. Klein et al., 2005). The 7 peatlands likely to show the greatest impacts are the most marginal sites in regions which 8 experience the greatest climate change. INTRODUCTION 2 20 Vegetation of the mires includes Juncus effusus, Carex spp., Eriophorum latifolium, 21 Myosotis spp., Geum spp., Ranunculus spp., Mentha spicta, Plagiomnium elatum, 22 oligotrophic peatlands in Greece and are probably the only location in the southern 1 Balkans where many wetland endemics typical of more nutrient-poor conditions may be 2 found. These are the only peatlands in Greece where Sphagnum is a permanent presence. 3 Due to their unique nature the Elatia Mires have been a focus of recent conservation 4 attention. This study uses a palaeoecological approach based on testate amoebae analysis 5 to test the hypothesis that the hydrology of the Elatia mires is responding to recent 6 climatic changes. 7 oligotrophic peatlands in Greece and are probably the only location in the southern 1 Balkans where many wetland endemics typical of more nutrient-poor conditions may be 2 found. These are the only peatlands in Greece where Sphagnum is a permanent presence. 3 Due to their unique nature the Elatia Mires have been a focus of recent conservation 4 attention. This study uses a palaeoecological approach based on testate amoebae analysis 5 to test the hypothesis that the hydrology of the Elatia mires is responding to recent 6 climatic changes. 7 10 The Elatia Mires lie in the Elatia Forest, approximately 70 km north of the city of 11 Drama and 5 km south of the Bulgarian frontier at around 1500m asl. (41˚29’N, 24˚19’E; 12 Fig. 1). Four small peatlands are situated in clearings within a natural coniferous forest 13 dominated by Picea abies, the only such forest in Greece (Papazisimou et al., 2002). A 14 comparatively cool and moist climate combined with impermeable granite bedrocks have 15 allowed peat formation. Peat deposits are shallow, probably not exceeding 1 m of 16 continuous peat, although deeper, buried, peat deposits are also found in one site. The 17 mires have been termed ‘transitional’ due to their mix of features typical of fens and bogs 18 (Papazisimou et al 2002). Samples were extracted from the two most oligotrophic sites: 19 Dexameni mire (site DE; mean pH 6.5) and Krya Vrissi mire (site KB; mean pH 6.4). INTRODUCTION 2 Climatic impacts on the hydrology of northern 15 Greece have already been noted and are modelled to increase in severity over coming 16 decades with consequent impacts for human populations (Mimikou, 1993; Mimikou et 17 al., 1999; 2000; Bürger, 2002; Baltas and Mimikou, 2005). These changes may have 18 affected peatlands in the region. Most peatlands in Greece have been heavily impacted by 19 human activity primarily through drainage for agriculture in the 20th Century, and often 20 have low conservation value (Bouzinos et al., 1994; 1997; Christanis, 1996). An 21 exception to this is the Elatia Mires of northern Macedonia where remoteness and Cold 22 War access restrictions have prevented major impacts. These mires are the most 23 3 3 oligotrophic peatlands in Greece and are probably the only location in the southern 1 Balkans where many wetland endemics typical of more nutrient-poor conditions may be 2 found. These are the only peatlands in Greece where Sphagnum is a permanent presence. 3 Due to their unique nature the Elatia Mires have been a focus of recent conservation 4 attention. This study uses a palaeoecological approach based on testate amoebae analysis 5 to test the hypothesis that the hydrology of the Elatia mires is responding to recent 6 climatic changes. 7 8 SITES and METHODS 9 10 The Elatia Mires lie in the Elatia Forest, approximately 70 km north of the city of 11 Drama and 5 km south of the Bulgarian frontier at around 1500m asl. (41˚29’N, 24˚19’E; 12 Fig. 1). Four small peatlands are situated in clearings within a natural coniferous forest 13 dominated by Picea abies, the only such forest in Greece (Papazisimou et al., 2002). A 14 comparatively cool and moist climate combined with impermeable granite bedrocks have 15 allowed peat formation. Peat deposits are shallow, probably not exceeding 1 m of 16 continuous peat, although deeper, buried, peat deposits are also found in one site. The 17 mires have been termed ‘transitional’ due to their mix of features typical of fens and bogs 18 (Papazisimou et al 2002). Samples were extracted from the two most oligotrophic sites: 19 Dexameni mire (site DE; mean pH 6.5) and Krya Vrissi mire (site KB; mean pH 6.4). INTRODUCTION 2 20 Vegetation of the mires includes Juncus effusus, Carex spp., Eriophorum latifolium, 21 Myosotis spp., Geum spp., Ranunculus spp., Mentha spicta, Plagiomnium elatum, 22 The Elatia Mires lie in the Elatia Forest, approximately 70 km north of the city of 11 Drama and 5 km south of the Bulgarian frontier at around 1500m asl. (41˚29’N, 24˚19’E; 12 Fig. 1). Four small peatlands are situated in clearings within a natural coniferous forest 13 dominated by Picea abies, the only such forest in Greece (Papazisimou et al., 2002). A 14 comparatively cool and moist climate combined with impermeable granite bedrocks have 15 allowed peat formation. Peat deposits are shallow, probably not exceeding 1 m of 16 continuous peat, although deeper, buried, peat deposits are also found in one site. The 17 mires have been termed ‘transitional’ due to their mix of features typical of fens and bogs 18 (Papazisimou et al 2002). Samples were extracted from the two most oligotrophic sites: 19 Dexameni mire (site DE; mean pH 6.5) and Krya Vrissi mire (site KB; mean pH 6.4). 20 Vegetation of the mires includes Juncus effusus, Carex spp., Eriophorum latifolium, 21 Myosotis spp., Geum spp., Ranunculus spp., Mentha spicta, Plagiomnium elatum, 22 4 Sphagnum flexuosum, Aulacomnium palustris and Climacium dendroides (Papazisimou et 1 al 2002). 2 Peat monoliths between 16 and 26 cm in length were extracted by cutting down 3 from the peat surface. Two monoliths were removed from central areas of the Krya Vrissi 4 mire and one from Dexameni; denoted KB1, KB2 and DE respectively. Peat stratigraphy 5 was noted and humification recorded on the Von Post scale (Von Post, 1924). Contiguous 6 1cm-deep samples were taken through the length of the monoliths. 7 Testate amoebae analysis was used to reconstruct changes in hydrology. Testate 8 amoebae are shell-forming unicellular microorganisms that are abundant in peatlands and 9 sensitive to peatland hydrology. By analysing the changing community composition 10 down the length of a peat core and interpreting the results with a transfer function model 11 it is possible to reconstruct how mire wetness has varied over time (Charman, 2001; 12 Mitchell et al., 2008). Such reconstructions have been validated by comparison with 13 instrumental data and independent proxy-climatic records (Charman and Hendon, 2000; 14 Charman et al., 2004; Schoning et al., 2005). INTRODUCTION 2 15 Peat sub-samples for testate amoebae analysis were boiled in deionised water, 16 filtered at 250 μm and then back-filtered at 15 μm with the 15>250 μm fraction retained 17 (Hendon and Charman, 1997). Slides were made up with glycerol and amoebae identified 18 Sphagnum flexuosum, Aulacomnium palustris and Climacium dendroides (Papazisimou et 1 al 2002). 2 Sphagnum flexuosum, Aulacomnium palustris and Climacium dendroides (Papazisimou et 1 al 2002). 2 Peat monoliths between 16 and 26 cm in length were extracted by cutting down 3 from the peat surface. Two monoliths were removed from central areas of the Krya Vrissi 4 mire and one from Dexameni; denoted KB1, KB2 and DE respectively. Peat stratigraphy 5 was noted and humification recorded on the Von Post scale (Von Post, 1924). Contiguous 6 1cm-deep samples were taken through the length of the monoliths. 7 Testate amoebae analysis was used to reconstruct changes in hydrology. Testate 8 amoebae are shell-forming unicellular microorganisms that are abundant in peatlands and 9 sensitive to peatland hydrology. By analysing the changing community composition 10 down the length of a peat core and interpreting the results with a transfer function model 11 it is possible to reconstruct how mire wetness has varied over time (Charman, 2001; 12 Mitchell et al., 2008). Such reconstructions have been validated by comparison with 13 instrumental data and independent proxy-climatic records (Charman and Hendon, 2000; 14 Charman et al., 2004; Schoning et al., 2005). 15 Peat sub-samples for testate amoebae analysis were boiled in deionised water, 16 filtered at 250 μm and then back-filtered at 15 μm with the 15>250 μm fraction retained 17 (Hendon and Charman, 1997). Slides were made up with glycerol and amoebae identified 18 following the taxonomic scheme described in Payne and Mitchell (2007); a count of 150 19 tests was aimed for. Amoebae diagrams were constructed using C2 ver. 1.4 (Juggins, 20 2003) and zoned using optimal sum of squares partitioning (Birks and Gordon, 1985) in 21 ZONE ver. 1.2 (Juggins, 1992). Quantitative environmental reconstruction was carried 22 out using the transfer function models developed by Payne and Mitchell (2007). Depth to 23 Peat sub-samples for testate amoebae analysis were boiled in deionised water, 16 5 5 water table (DWT) was reconstructed using a maximum likelihood model (RMSEPboot 1 1.9cm) and pH using a weighted average model (RMSEPboot 0.4). INTRODUCTION 2 The reconstructed 2 values are termed testate amoebae inferred depth to water table (TI-DWT) and testate 3 amoebae inferred pH (TI-pH). Bootstrapped error estimation with 1000 cycles was used 4 to provide sample-specific error estimates. 5 6 Chronology 7 Establishing reliable chronologies for recent peat deposits has been a persistent 8 challenge for peatland palaeoecologists (Turetsky et al., 2004) and is particularly 9 problematic for these sites as they are non-ombrotrophic and include unusual plant 10 communities. A search for cryptotephras (following the method of Pilcher and Hall, 11 1992) failed to identify any shards and a search for spheroidal carbonaceous particles 12 (following the method of Rose et al, 1995) failed to find adequate concentrations. Two 13 radiometric methods, 210Pb and 14C analysis were applied to the peat profiles. 210Pb was 14 analysed by assuming equilibrium with its grand-daughter 210Po. Peat samples were 15 dissolved in strong acids with a 209Po yield tracer, plated onto copper disks and activity 16 measured by α-spectrometry. Four samples from towards the base of the monoliths were 17 AMS radiocarbon dated (Goodsite et al, 2001; Goslar et al, 2005). Bulk samples were 18 used due to the absence of Sphagnum (Nilsson et al. 2001). Samples were carefully 19 prepared to minimise risk of external contamination and rootlets were picked out. 20 21 water table (DWT) was reconstructed using a maximum likelihood model (RMSEPboot 1 1.9cm) and pH using a weighted average model (RMSEPboot 0.4). The reconstructed 2 values are termed testate amoebae inferred depth to water table (TI-DWT) and testate 3 amoebae inferred pH (TI-pH). Bootstrapped error estimation with 1000 cycles was used 4 to provide sample-specific error estimates. 5 water table (DWT) was reconstructed using a maximum likelihood model (RMSEPboot 1 1.9cm) and pH using a weighted average model (RMSEPboot 0.4). The reconstructed 2 values are termed testate amoebae inferred depth to water table (TI-DWT) and testate 3 amoebae inferred pH (TI-pH). Bootstrapped error estimation with 1000 cycles was used 4 to provide sample-specific error estimates. 5 Chronology 7 Establishing reliable chronologies for recent peat deposits has been a persistent 8 challenge for peatland palaeoecologists (Turetsky et al., 2004) and is particularly 9 problematic for these sites as they are non-ombrotrophic and include unusual plant 10 communities. Testate amoebae 3 Testate amoebae 3 Testate amoebae were found through the length of the monoliths but apparent 4 concentrations were low (counting time approximately 6-8 hours per sample). The three 5 testate amoebae diagrams show similar trends (Fig. 2). At the base of the profiles (zones 6 DE-1, KB1-1 and the lower portion of KB2-1) the community composition is noted by 7 abundant Difflugia spp., particularly Difflugia pulex type. Above this section there is a 8 decline in these taxa leading to a more diverse community noted by Centropyxis 9 aerophila (zones DE-2, KB1-2 and the upper portion of KB2-1). The next significant 10 change is a marked increase in Euglypha rotunda, and in KB1, Euglypha ciliata type. The 11 uppermost samples are different from those directly below, recognized as a separate zone 12 in KB2 (zone KB2-3) and DE (zone DE-3). 13 Overlap between the palaeoecological data and the modern training set is very 14 good. Over 99% of all amoebae counted are included in the training set; the total for 15 individual samples does not fall below 97%. Water table reconstructions show a similar 16 pattern between sites (Fig. 3). From the base of the sequences to c.6 cm depth there is low 17 amplitude variability with no obvious similarity between profiles. At 6 cm there is a rapid 18 increase in values that significantly exceeds bootstrapped error estimates, representing a 19 significant lowering of water table in all sites. At the top of the sequence there is a slight 20 decline in TI-DWT values and then a resumed increase in sites KB1 and DE, although 21 there is a continued decline in site KB2. pH reconstructions show gradual lowering of pH 22 Overlap between the palaeoecological data and the modern training set is very 14 good. Over 99% of all amoebae counted are included in the training set; the total for 15 individual samples does not fall below 97%. Water table reconstructions show a similar 16 pattern between sites (Fig. 3). From the base of the sequences to c.6 cm depth there is low 17 amplitude variability with no obvious similarity between profiles. At 6 cm there is a rapid 18 increase in values that significantly exceeds bootstrapped error estimates, representing a 19 significant lowering of water table in all sites. INTRODUCTION 2 A search for cryptotephras (following the method of Pilcher and Hall, 11 1992) failed to identify any shards and a search for spheroidal carbonaceous particles 12 (following the method of Rose et al, 1995) failed to find adequate concentrations. Two 13 radiometric methods, 210Pb and 14C analysis were applied to the peat profiles. 210Pb was 14 analysed by assuming equilibrium with its grand-daughter 210Po. Peat samples were 15 dissolved in strong acids with a 209Po yield tracer, plated onto copper disks and activity 16 measured by α-spectrometry. Four samples from towards the base of the monoliths were 17 AMS radiocarbon dated (Goodsite et al, 2001; Goslar et al, 2005). Bulk samples were 18 used due to the absence of Sphagnum (Nilsson et al. 2001). Samples were carefully 19 prepared to minimise risk of external contamination and rootlets were picked out. 20 21 Chronology 7 Establishing reliable chronologies for recent peat deposits has been a persistent 8 challenge for peatland palaeoecologists (Turetsky et al., 2004) and is particularly 9 problematic for these sites as they are non-ombrotrophic and include unusual plant 10 communities. A search for cryptotephras (following the method of Pilcher and Hall, 11 1992) failed to identify any shards and a search for spheroidal carbonaceous particles 12 (following the method of Rose et al, 1995) failed to find adequate concentrations. Two 13 radiometric methods, 210Pb and 14C analysis were applied to the peat profiles. 210Pb was 14 analysed by assuming equilibrium with its grand-daughter 210Po. Peat samples were 15 dissolved in strong acids with a 209Po yield tracer, plated onto copper disks and activity 16 measured by α-spectrometry. Four samples from towards the base of the monoliths were 17 AMS radiocarbon dated (Goodsite et al, 2001; Goslar et al, 2005). Bulk samples were 18 used due to the absence of Sphagnum (Nilsson et al. 2001). Samples were carefully 19 prepared to minimise risk of external contamination and rootlets were picked out. 20 21 23 6 6 Testate amoebae 3 At the top of the sequence there is a slight 20 decline in TI-DWT values and then a resumed increase in sites KB1 and DE, although 21 there is a continued decline in site KB2. pH reconstructions show gradual lowering of pH 22 7 7 7 values, increasing in rate above c.6 cm. The changes in pH are less pronounced than 1 those in DWT and (particularly in KB2) only marginally exceed the error estimates. 2 The main amoebae community change is a shift in the most abundant taxon to 3 Euglypha rotunda from Centropyxis aerophila type and Difflugia pulex type. C. 4 aerophila is generally regarded as typical of moderately wet conditions, while E. rotunda 5 is probably most typical of intermediate conditions (Charman et al., 2000). The indicator 6 value of D. pulex type is uncertain. The majority of transfer functions to encounter both 7 taxa show E. rotunda to have a higher DWT optimum than C. aerophila type (or 8 equivalent C. cassis type) (Payne et al., 2006; 2007; in press; Woodland et al., 1998; 9 Charman and Warner, 1997; Charman, 1997; Warner and Charman, 1994; Lamentowicz 10 and Mitchell, 2005). The transfer function results therefore agree with the known 11 autecology of the taxa in interpreting this change as a shift to drier conditions. 12 13 Chronology 14 15 The 210Pb results show no decline in activity with depth and a sequence of peaks 16 and troughs (Fig. 4). It was not possible to determine the “unsupported” component of the 17 210Pb; insufficient material was available for direct 226Ra analysis and the base of the 18 unsupported layer was not reached. The lack of a monotonic decrease in activity with 19 depth undermines the attempt to use the method for dating. There are two possible causes 20 of these results; very rapid peat accumulation, as suggested by the radiocarbon dates, and 21 movement of lead within the peat profile given the minerotrophic nature of the sites 22 (MacKenzie et al., 1998). 23 values, increasing in rate above c.6 cm. The changes in pH are less pronounced than 1 those in DWT and (particularly in KB2) only marginally exceed the error estimates. 2 The main amoebae community change is a shift in the most abundant taxon to 3 Euglypha rotunda from Centropyxis aerophila type and Difflugia pulex type. C. Testate amoebae 3 4 aerophila is generally regarded as typical of moderately wet conditions, while E. rotunda 5 is probably most typical of intermediate conditions (Charman et al., 2000). The indicator 6 value of D. pulex type is uncertain. The majority of transfer functions to encounter both 7 taxa show E. rotunda to have a higher DWT optimum than C. aerophila type (or 8 equivalent C. cassis type) (Payne et al., 2006; 2007; in press; Woodland et al., 1998; 9 Charman and Warner, 1997; Charman, 1997; Warner and Charman, 1994; Lamentowicz 10 and Mitchell, 2005). The transfer function results therefore agree with the known 11 autecology of the taxa in interpreting this change as a shift to drier conditions. 12 13 Chronology 14 15 The 210Pb results show no decline in activity with depth and a sequence of peaks 16 and troughs (Fig. 4). It was not possible to determine the “unsupported” component of the 17 210 226 values, increasing in rate above c.6 cm. The changes in pH are less pronounced than 1 those in DWT and (particularly in KB2) only marginally exceed the error estimates. 2 The main amoebae community change is a shift in the most abundant taxon to 3 Euglypha rotunda from Centropyxis aerophila type and Difflugia pulex type. C. 4 8 8 Radiocarbon dates were all returned as post-bomb and calibrated using CaliBomb 1 (http://calib.qub.ac.uk/CALIBomb/frameset.html). All dates give multimodal probability 2 distributions ranging from the mid-1950s to post-1995, indicating rapid peat 3 accumulation (Table 1). As the dates were based on bulk samples it is possible that they 4 have been contaminated by modern carbon, perhaps through penetration by sedge roots. 5 However, the samples were prepared carefully to avoid contamination and obvious roots 6 were removed. Systematic differences between dates on bulk samples and selected 7 macrofossils have not been proven (Blaauw et al. 2004). The dates are internally 8 consistent in showing the peats to be late 20th century in age and are not contradicted by a 9 date of 100±40 14C yrs BP at 31-36 cm from a neighbouring site (Papazisimou et al., 10 2002). For two samples from the KB2 monolith, the deeper sample (GdA-1178: 21- 11 24cm) has a highest probability peak more recent than the upper sample (GdA-1016: 13- 12 15cm). However this date also has a subsidiary probability peak at 1957-1958 (10.3%). Testate amoebae 3 13 As there is no stratigraphic reason to suspect a reversal it seems more likely that this 14 older peak is the correct one. 15 210 Radiocarbon dates were all returned as post-bomb and calibrated using CaliBomb 1 (http://calib.qub.ac.uk/CALIBomb/frameset.html). All dates give multimodal probability 2 distributions ranging from the mid-1950s to post-1995, indicating rapid peat 3 accumulation (Table 1). As the dates were based on bulk samples it is possible that they 4 have been contaminated by modern carbon, perhaps through penetration by sedge roots. 5 However, the samples were prepared carefully to avoid contamination and obvious roots 6 were removed. Systematic differences between dates on bulk samples and selected 7 macrofossils have not been proven (Blaauw et al. 2004). The dates are internally 8 consistent in showing the peats to be late 20th century in age and are not contradicted by a 9 date of 100±40 14C yrs BP at 31-36 cm from a neighbouring site (Papazisimou et al., 10 2002). For two samples from the KB2 monolith, the deeper sample (GdA-1178: 21- 11 24cm) has a highest probability peak more recent than the upper sample (GdA-1016: 13- 12 15cm). However this date also has a subsidiary probability peak at 1957-1958 (10.3%). 13 As there is no stratigraphic reason to suspect a reversal it seems more likely that this 14 older peak is the correct one. 15 Neither the 210Pb results nor the radiocarbon dates allow us to establish a secure 16 chronology for these profiles. However both sets of results can be taken to suggest that 17 the sediments are very recent. Most probably these peats have accumulated within the last 18 few decades but it is not possible to be more precise. Nevertheless, there is no reason to 19 suspect disturbance of the stratigraphy and it is still probable that these profiles do 20 preserve a continuous record of testate amoebae changes and may reveal recent 21 hydrological changes in the mires. 22 Neither the 210Pb results nor the radiocarbon dates allow us to establish a secure 16 chronology for these profiles. However both sets of results can be taken to suggest that 17 the sediments are very recent. Most probably these peats have accumulated within the last 18 few decades but it is not possible to be more precise. Testate amoebae 3 Nevertheless, there is no reason to 19 suspect disturbance of the stratigraphy and it is still probable that these profiles do 20 preserve a continuous record of testate amoebae changes and may reveal recent 21 hydrological changes in the mires. 22 23 9 9 DISCUSSION 1 2 The testate amoebae results show a single large change in amoebae community at 3 around 6 cm depth which can be inferred as a shift to a drier mire surface. This is entirely 4 consistent with the hypothesis of a lowered water table due to the general reduction in 5 precipitation in northern Greece. However simple attribution of the changes to a climatic 6 impact is not possible due to the presence of other factors which could also be responsible 7 for these changes. 8 9 1. Non-climatic hydrological change 10 It seems probable that the Elatia Mires have undergone hydrological change; 11 however as the sites are not ombrotrophic it is possible that this is unrelated to climate. It 12 is possible that processes such as forestry, tectonic or geomorphological change in the 13 wider area could have lead to a change in water input into the mires. Although there is no 14 particular evidence to suggest that this may be the case the hypothesis cannot be 15 discounted on the basis of the data presented here. 16 17 2. Vertical zonation of living amoebae 18 Interpretation of the uppermost testate amoebae assemblages may be complicated 19 if the amoebae are still alive below the surface. Testate amoebae have been noted to 20 exhibit vertical zonation forced by gradients in light, moisture and mineral material for 21 DISCUSSION 1 The testate amoebae results show a single large change in amoebae community at 3 around 6 cm depth which can be inferred as a shift to a drier mire surface. This is entirely 4 consistent with the hypothesis of a lowered water table due to the general reduction in 5 precipitation in northern Greece. However simple attribution of the changes to a climatic 6 impact is not possible due to the presence of other factors which could also be responsible 7 for these changes. 8 10 with xenosome tests such as Difflugia spp. and C. aerophila are commonly observed in 1 lower horizons, probably due to availability of material for test construction. However the 2 typical position of Euglypha species varies greatly between studies (Chacharonis 1956; 3 Booth 2002; Mitchell and Gilbert 2004; Mazei and Bobnova 2007). The only study in 4 transitional mires (Mazei and Bobnova 2007) found vertical zonation to be much weaker 5 than in bogs. In these sites alive or encysted amoebae were only noted in the top 2-3 cm. 6 It is probable that vertical zonation in this region could explain the unusual communities 7 in the uppermost samples, but unlikely that vertical zonation is the cause of the major 8 change at 6cm. 9 10 3. Test preservation 11 The taxa which are primarily responsible for the increase in TI-DWT in the upper 12 portions of the sequence (E.rotunda, E.ciliata, C.dubium) all have tests constructed of 13 idiosomes. Such tests (and particularly those of Euglypha) may be particularly prone to 14 decomposition in the fossil record (Lousier and Parkinson, 1981; Swindles and Roe, 15 2007; Payne 2007). Selective test loss could have led to inaccurate palaeoenvironmental 16 reconstruction in these sites (Mitchell et al. 2008). A number of strands of evidence 17 suggest this is unlikely to be the major cause of the changes: 1) during microscopy no 18 apparent reduction in overall test concentrations was noted with depth (although 19 concentrations were not enumerated), this might be expected given the high proportion of 20 idiosome tests; 2) no increase in degraded tests was noted with depth; 3) the taxa 21 concerned decline sharply at around 6 cm, however they continue through the rest of the 22 profiles in lower concentrations; 4) other closely related taxa reach their highest 23 with xenosome tests such as Difflugia spp. and C. DISCUSSION 1 aerophila are commonly observed in 1 lower horizons, probably due to availability of material for test construction. However the 2 typical position of Euglypha species varies greatly between studies (Chacharonis 1956; 3 Booth 2002; Mitchell and Gilbert 2004; Mazei and Bobnova 2007). The only study in 4 transitional mires (Mazei and Bobnova 2007) found vertical zonation to be much weaker 5 than in bogs. In these sites alive or encysted amoebae were only noted in the top 2-3 cm. 6 It is probable that vertical zonation in this region could explain the unusual communities 7 in the uppermost samples, but unlikely that vertical zonation is the cause of the major 8 change at 6cm. 9 with xenosome tests such as Difflugia spp. and C. aerophila are commonly observed in 1 lower horizons, probably due to availability of material for test construction. However the 2 typical position of Euglypha species varies greatly between studies (Chacharonis 1956; 3 Booth 2002; Mitchell and Gilbert 2004; Mazei and Bobnova 2007). The only study in 4 transitional mires (Mazei and Bobnova 2007) found vertical zonation to be much weaker 5 than in bogs. In these sites alive or encysted amoebae were only noted in the top 2-3 cm. 6 It is probable that vertical zonation in this region could explain the unusual communities 7 in the uppermost samples, but unlikely that vertical zonation is the cause of the major 8 change at 6cm. 9 3. Test preservation 11 The taxa which are primarily responsible for the increase in TI-DWT in the upper 12 portions of the sequence (E.rotunda, E.ciliata, C.dubium) all have tests constructed of 13 idiosomes. Such tests (and particularly those of Euglypha) may be particularly prone to 14 decomposition in the fossil record (Lousier and Parkinson, 1981; Swindles and Roe, 15 2007; Payne 2007). Selective test loss could have led to inaccurate palaeoenvironmental 16 reconstruction in these sites (Mitchell et al. 2008). DISCUSSION 1 A number of strands of evidence 17 suggest this is unlikely to be the major cause of the changes: 1) during microscopy no 18 apparent reduction in overall test concentrations was noted with depth (although 19 concentrations were not enumerated), this might be expected given the high proportion of 20 idiosome tests; 2) no increase in degraded tests was noted with depth; 3) the taxa 21 concerned decline sharply at around 6 cm, however they continue through the rest of the 22 profiles in lower concentrations; 4) other closely related taxa reach their highest 23 11 11 concentrations lower in the profiles (e.g. E.tuberculata in KB1); 5) the timescale under 1 consideration is very short so differential preservation is perhaps less likely than in 2 longer-term studies; 6) when Euglypha spp. are removed from the reconstructions an 3 increase in TI-DWT at 6cm remains (Fig. 3), although this is less marked and there are 4 other changes such as a large drop in TI-DWT at the top of KB1. 5 6 4. Autogenic mire development processes 7 The Elatia mires have been termed ‘transitional’ between fens and bogs; it is 8 possible that the changes in the palaeoecological record are due to ombrotrophication. 9 Ombrotrophication can be expected to lead to major amoebae community changes as fens 10 have distinctly different testate amoebae communities from bogs and nutrient status is an 11 important secondary gradient (Opravilova and Hajek, 2006). 12 Conventionally it has been assumed that an autogenic mechanism drives 13 ombrotrophication; peat gradually accumulates above the water table until it is no longer 14 reliant on groundwater and becomes acidified by leaching and the establishment of 15 Sphagnum (Hughes, 2000). This would sit well with the reconstructed increase in TI- 16 DWT and slight decrease in TI-pH. However, it seems unlikely that this autogenic model 17 could explain the suddenness of the amoebae change, the 2cm+ offset between 18 stratigraphic and amoebae community changes and the apparent synchroneity in change 19 between two sites. More recent studies have suggested allogenic forcing of 20 ombrotrophication with a lowered water table leading to the peat surface being separated 21 from groundwater (Hughes, 2000; Hughes and Dumayne-Peaty, 2002; Hughes and 22 concentrations lower in the profiles (e.g. DISCUSSION 1 E.tuberculata in KB1); 5) the timescale under 1 consideration is very short so differential preservation is perhaps less likely than in 2 longer-term studies; 6) when Euglypha spp. are removed from the reconstructions an 3 increase in TI-DWT at 6cm remains (Fig. 3), although this is less marked and there are 4 other changes such as a large drop in TI-DWT at the top of KB1. 5 concentrations lower in the profiles (e.g. E.tuberculata in KB1); 5) the timescale under 1 consideration is very short so differential preservation is perhaps less likely than in 2 longer-term studies; 6) when Euglypha spp. are removed from the reconstructions an 3 increase in TI-DWT at 6cm remains (Fig. 3), although this is less marked and there are 4 other changes such as a large drop in TI-DWT at the top of KB1. 5 4. Autogenic mire development processes 7 The Elatia mires have been termed ‘transitional’ between fens and bogs; it is 8 possible that the changes in the palaeoecological record are due to ombrotrophication. 9 Ombrotrophication can be expected to lead to major amoebae community changes as fens 10 have distinctly different testate amoebae communities from bogs and nutrient status is an 11 important secondary gradient (Opravilova and Hajek, 2006). 12 Ombrotrophication can be expected to lead to major amoebae community changes as fens 10 have distinctly different testate amoebae communities from bogs and nutrient status is an 11 important secondary gradient (Opravilova and Hajek, 2006). 12 Conventionally it has been assumed that an autogenic mechanism drives 13 ombrotrophication; peat gradually accumulates above the water table until it is no longer 14 reliant on groundwater and becomes acidified by leaching and the establishment of 15 Sphagnum (Hughes, 2000). This would sit well with the reconstructed increase in TI- 16 DWT and slight decrease in TI-pH. However, it seems unlikely that this autogenic model 17 could explain the suddenness of the amoebae change, the 2cm+ offset between 18 stratigraphic and amoebae community changes and the apparent synchroneity in change 19 between two sites. More recent studies have suggested allogenic forcing of 20 ombrotrophication with a lowered water table leading to the peat surface being separated 21 from groundwater (Hughes, 2000; Hughes and Dumayne-Peaty, 2002; Hughes and 22 12 Barber, 2003; Hughes et al., 2000). It is therefore also possible that ombrotrophication is 1 occurring, but is driven by a real allogenic change. DISCUSSION 1 2 Barber, 2003; Hughes et al., 2000). It is therefore also possible that ombrotrophication is 1 occurring, but is driven by a real allogenic change. 2 3 The results presented here are consistent with the hypothesis of a climate change 4 induced hydrological change; however other explanations cannot be excluded. It is 5 possible that multiple processes have lead to the observed patterns. If the changes are du 6 to climate then this study provides the first evidence for the impacts of recent climate 7 change on Mediterranean peatlands. Climate change is likely to be a key challenge to 8 regional peatland conservation. 9 10 11 12 Acknowledgements 13 14 This study was primarily funded by a BSA MacMillan-Rodewald fellowship to RJP. 15 Radiocarbon dates were funded by grants from the Richard Bradford McConnell Fund 16 and a Gladstone Memorial Prize. Thanks to Kimon Christanis and Stavros Kalaitzidis 17 (University of Patras) for discussion of the Elatia Mires, to Edward Mitchell (Lausanne) 18 for discussion of testate amoebae ecology and taxonomy, and to two anonymous 19 reviewers for comments on a previous draft. The map was prepared by Edward Mitchell 20 This work was carried out by permission of the Greek Institute of Geology and Mineral 21 Exploration (IGME) and the Forest Service of the Prefecture of Drama. 22 23 The results presented here are consistent with the hypothesis of a climate change- 4 induced hydrological change; however other explanations cannot be excluded. It is 5 possible that multiple processes have lead to the observed patterns. If the changes are due 6 to climate then this study provides the first evidence for the impacts of recent climate 7 change on Mediterranean peatlands. Climate change is likely to be a key challenge to 8 regional peatland conservation. 9 23 13 References 1 2 Baltas EA, Mimikou MA (2005) Climate change impacts on the water supply of 3 Thessaloniki. Water Resources Development 21:341-353 4 5 Belyea L, Malmer N (2004) Carbon sequestration in peatland: patterns and mechanisms 6 of response to climate change. Glob Change Biol 10:1043-1052 7 8 Birks HJB, Gordon AD (1985) Numerical methods in Quaternary pollen analysis. 9 Academic Press, London 10 11 Blaauw M, van der Plicht J, van Geel B (2004) Radiocarbon dating of bulk peat samples 12 from raised bogs: non-existence of a previously reported ‘reservoir effect’? DISCUSSION 1 Quaternary 13 Sci Rev 23:1537-1542 14 15 Booth RK (2002) Testate amoebae as paleoindicators of surface-moisture changes on 16 Michigan peatlands: modern ecology and hydrological calibration. 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Examining the dissolution characteristics of testate 13 amoebae (Protozoa: Rhizopoda) in low pH conditions: Implications for peatland 14 palaeoclimate studies. Palaeogeogr Palaeocl 252:486-496 15 16 Turetsky MR, Manning SW, Wieder RK (2004) Dating recent peat deposits. Wetlands 17 24:324-326 18 19 Turetsky M.R, Wieder RK, Vitt DH, Evans RJ, Scott KD (2007) The disappearance of 20 relict permafrost in boreal north America: Effects on peatland carbon storage and fluxes 21 Glob Change Biol 13:1922-1934 22 23 22 Von Post L (1924) Das genetische System der organogenen Bildungen Schwedens. DISCUSSION 1 1 Comité International de Pedologie IV Commission, Helsinki 2 3 Warner B, Charman D (1994) Holocene changes on a peatland interpreted from testate 4 amoebae (Protozoa) analysis. Boreas 23:270-280 5 6 Weltzin J, Bridgham S, Pastor J, Chen J, Harth C (2003) Potential effects of warming and 7 drying on peatland plant community composition. Glob Change Biol 9:141-151 8 9 Woodland W, Charman D, Simms P (1998) Quantitative estimates of water tables and 10 soil moisture in Holocene peatlands from testate amoebae. Holocene 8: 261-273 11 12 13 23 1 1 FIGURES and TABLE Figure 1. Location of the Elatia Mires. 4 5 5 Figure 2. Testate amoebae profiles from three monoliths. Showing peat stratigraphy, 6 major testate amoebae species (% of total), testate amoebae total count, humification 7 expressed on the Von Post scale, and testate amoebae zones. Stratigraphic columns show 8 moss-dominated peat (solid wavy lines), peat of mixed composition with mosses most 9 abundant (interrupted wavy lines), peat of mixed composition with macrofossils most 10 abundant (interrupted vertical lines) and macrofossil-dominated peat (solid vertical lines). 11 Figure 2. Testate amoebae profiles from three monoliths. Showing peat stratigraphy, 6 major testate amoebae species (% of total), testate amoebae total count, humification 7 expressed on the Von Post scale, and testate amoebae zones. Stratigraphic columns show 8 moss-dominated peat (solid wavy lines), peat of mixed composition with mosses most 9 abundant (interrupted wavy lines), peat of mixed composition with macrofossils most 10 abundant (interrupted vertical lines) and macrofossil-dominated peat (solid vertical lines). 11 24 2 25 Figure 3. Testate amoebae inferred depth to water table (TI-DWT) and testate amoebae 1 inferred pH (TI-pH) from the three peat monoliths with boot-strapped error estimates. 2 The transfer function model was based on one-off DWT and pH measurements so the 3 units of reconstruction are depth to water table (cm) and pH based on an October 2005 4 datum. Also showing TI-DWT and TI-pH reconstructions excluding Euglypha species, 5 which might be lost from the palaeoecological record. 6 Figure 3. Testate amoebae inferred depth to water table (TI-DWT) and testate amoebae 1 inferred pH (TI-pH) from the three peat monoliths with boot-strapped error estimates. 2 The transfer function model was based on one-off DWT and pH measurements so the 3 units of reconstruction are depth to water table (cm) and pH based on an October 2005 4 datum. Figure 3. Testate amoebae inferred depth to water table (TI-DWT) and testate amoebae 1 inferred pH (TI-pH) from the three peat monoliths with boot-strapped error estimates. 2 The transfer function model was based on one-off DWT and pH measurements so the 3 units of reconstruction are depth to water table (cm) and pH based on an October 2005 4 datum. Also showing TI-DWT and TI-pH reconstructions excluding Euglypha species, 5 which might be lost from the palaeoecological record. 6 DISCUSSION 1 Also showing TI-DWT and TI-pH reconstructions excluding Euglypha species, 5 which might be lost from the palaeoecological record. 6 26 Figure 4. Radiocarbon dates and 210Pb profile for monolith KB2. Figure 4. Radiocarbon dates and 210Pb profile for monolith KB2. 2 27 28 1 2 3 Table 1. Radiocarbon dates from the peat monoliths 4 5 Monolith Depth (cm) Date code Percent modern carbon (pMc) Calibrated age range (95% probability) (cal. years AD) DE 15-16 GdA-1177 120.55 ± 0.37 1958-1961 (9.3%) 1985-1988 (90.5%) KB1 21-24 GdA-1015 121.45 ± 0.36 1958-1961 (52.7%) 1984-1986 (42.3%) 1 2 3 Table 1. Radiocarbon dates from the peat monoliths 4 1 2 3 Table 1. Radiocarbon dates from the peat monoliths 4 1 2 3 Table 1. Radiocarbon dates from the peat monoliths 4 Table 1. Radiocarbon dates from the peat monoliths 4 5 Monolith Depth (cm) Date code Percent modern carbon (pMc) Calibrated age range (95% probability) (cal. years AD) DE 15-16 GdA-1177 120.55 ± 0.37 1958-1961 (9.3%) 1985-1988 (90.5%) KB1 21-24 GdA-1015 121.45 ± 0.36 1958-1961 (52.7%) 1984-1986 (42.3%) 5 28 KB2 13-15 GdA-1016 116.17 ± 0.33 1956-1958 (0.2%) 1989-1991 (94.8%) KB2 21-24 GdA-1178 110.88 ± 0.53 1957-1958 (10.3%) 1995- (85.1%) 1 29
https://openalex.org/W2883403601	http://www.scielo.br/pdf/rbmet/v33n2/0102-7786-rbmet-33-02-0247.pdf	Portuguese	null	Padrões de Chuva e de Evapotranspiração em Goiânia, GO	Revista Brasileira de Meteorologia	2,018	cc-by	6,584	Resumo A chuva (P) e a evapotranspiração potencial (ETP) são variáveis climáticas relevantes para diferentes áreas do conhecimento, tais como: agricultura, manejo de bacias, previsões de tempo e drenagem urbana. O objetivo deste trabalho foi caracterizar os padrões de chuva e de ETP para o município de Goiânia, GO. Foram utilizados dados de 36 anos para chuva e de 21 anos para ETP. A precipitação foi de 1.494,66 mm, com tendência de redução a uma taxa de 3,7 mm ano-1. Observou-se que 99% das precipitações diárias são p 50 mm dia-1. Registraram-se 292 dias sem chuva e 72 dias chuvosos, com tendência de redução dos dias chuvosos e aumento de dias secos. A ETP foi de 1.380,57 mm, sendo a maior e a menor média registrada no 3° (139,02 mm) e 35° (55,36 mm) quinquídios. Há tendência de aumento na ETP (6,37 mm ano-1). Registrou-se excedente hídrico de 607 mm ano-1 e déficit 471 mm ano-1. ETR/ETP <0,8 entre os meses de abril e outubro. Frequências predominantes de P e ETP foram de 38% ( ) P X e 31% ( ) ETP X , com periodicidades 4,0 (P) e 3,2 (ETP) anos. Observou-se r = 0,68 entre a temperatura média do ar e as taxas de desmatamento. Palavras-chave: balanço hídrico, temperatura, Cerrado. Abstract Rainfall (P) and potential evapotranspiration (ETP) are climatic variables relevant to different areas of knowledge, such as: agriculture, watershed management, weather forecasts and urban drainage. The aim of this work was to characterize the rainfall and ETP patterns for the Goiânia, GO city. Data of 36 years for rainfall and 21 years for ETP were used. The precipitation was 1,494.66 mm, with a trend of reduction at a rate of 3.7 mm year-1. It was observed that 99% of the daily precipitations are p 50 mm day-1. We recorded 292 days without rain and 72 rainy days, with a tendency to reduce rainy days and increase dry days. The ETP was 1,380.57 mm, with the highest and the lowest mean recorded in the 3rd (139.02 mm) and 35th (55.36 mm) quinquides. There is a tendency to increase in ETP (6.37 mm year-1). A water surplus of 607 mm year-1 and a deficit of 471 mm year-1 were recorded. ETR/ETP <0.8 between the months of April and October. Predominant frequencies of P and ETP were 38% ( ) P X and 31% ( ) ETP X , with periodicities 4.0 (P) and 3.2 (ETP) years. Correlation of 0.68 between the mean air temperature and Goiás deforestation rates was observed. Keywords: water balance, temperature, Brazilian savanna. years. Correlation of 0.68 between the mean air temperature and Goiás deforestation rates was observed. Keywords: water balance, temperature, Brazilian savanna. 22 °C e 23 °C. Em regiões agrícolas que têm por caracte- rística períodos de déficit hídrico, como o Estado de Goiás, estudos relacionados à oferta e demanda por recursos hídri- cos possuem elevada importância (Vivan et al., 2013). Níveis de déficit hídrico durante estádios críticos de desen- Autor de correspondência: Derblai Casaroli, derblai@ufg.br. Recebido em 17 de Outubro de 2017 – Aceito em 12 de Abril de 2018. Recebido em 17 de Outubro de 2017 – Aceito em 12 de Abril de 2018. Revista Brasileira de Meteorologia, v. 33, n. 2, 247-256, 2018 DOI: http://dx.doi.org/10.1590/0102-7786332004 Revista Brasileira de Meteorologia, v. 33, n. 2, 247-256, 2018 DOI: http://dx.doi.org/10.1590/0102-7786332004 rbmet.org.br Artigo Derblai Casaroli1 , Tallyta Ramalho Rodrigues2, Angélica Pires Batista Martins2, Adão Wagner Pêgo Evangelista1, José Alves Júnior1 1Setor de Engenharia de Biossistemas, Escola de Agronomia, Universidade Federal de Goiás, Goiânia, GO, Brasil. 2 2Programa de Pós-graduação em Agronomia, Escola de Agronomia, Universidade Federal de Goiás, Goiânia, GO, Brasil. 1. Introdução A evapotranspiração é uma variável de grande importância para a agricultura sendo utilizada na modelagem dos processos climatológicos e hidrológicos, projetos de irrigação, previsão de safras e na caracterização climática de determinada região (Blain et al., 2009). em que Kp é o coeficiente do Tanque Classe-A (adimen- sional), o qual foi utilizado um valor fixo de 0,7 (Cunha et al., 2013). Os dados diários de precipitação e evapotranspiração foram agrupados em períodos de quinquídios (cinco dias). Determinaram-se as médias e desvio-padrão para a estação seca e chuvosa, e para os quinquídios. Também foram iden- tificados os dias secos e chuvosos, seguindo a metodologia proposta por Sans et al. (2001) que considera dias chuvosos com precipitação P > 5,0 mm, e dias secos P 5,0 mm. Para a detecção dos períodos de excedente e de déficit hídrico utilizou-se o Balanço Hídrico Climatológico pro- posto por Thornthwaite e Mather (1955) considerando-se o valor inicial da capacidade de água disponível no solo (CAD) igual a 100 mm. O estudo das distribuições de variáveis climáticas no tempo, determinando seus padrões de ocorrência, sua pe- riodicidade, amplitude e sazonalidade, permitem uma maior aproximação dos valores reais pelos sistemas de previsão, tornando-se uma ferramenta de grande valor para o planejamento e gestão de inúmeras atividades agropecuá- rias e humanas. Ainda, para a detecção de frequências de ocorrências, magnitudes dos eventos, periodicidades e sazonalidades das chuvas e das evapotranspirações aplicou-se a trans- formada rápida de Fourier (FFT - Fast Fourier Transform), que representa a integral de funções sinusoidal, que neste trabalho foi em função do tempo (anos). Primeiramente foram determinadas as frequências (f) e períodos (T), sendo f f i i N t 1 1 , em que f0 = 0, N é o número total de amos- tragens, t a variação do tempo entre duas amostragens. Para a determinação do período utilizou-se T f 1. Vale lembrar que a FFT requer um número de amostras iguais a uma potência de dois (i.e. 2, 4, 8, 16, 32...), assim, foram utilizados 32 anos para a variável chuva e 16 anos para ETP. Tanto a chuva como a ETP tem como característica sua periodicidade (não linear), que trata da tendência a um período (ciclo) em relação ao tempo, ao espaço, ou a am- bos. O elemento básico das séries temporais é o ciclo, contendo frequências de todos os seus ciclos dominantes. 1. Introdução O clima predominante no Estado de Goiás é o Tropi- cal sazonal, de inverno seco, sendo a média de precipitação pluviométrica de 1529 mm ano-1 ( 400 mm) (Costa et al., 2012) e a temperatura do ar média anual fica em torno de 248 Casaroli et al. volvimento das culturas agrícolas (germinação, emergên- cia, floração e enchimento de grãos) podem acarretar em quebras de produtividade (Sans et al., 2001; Farias et al., 2008; Kron et al., 2008; Patanè et al., 2011). 2015 para evapotranspiração (21 anos). Os dados foram obtidos na estação meteorológica da Universidade Federal de Goiás, localizada no munícipio de Goiânia, GO (16°35’52,72648” S; 49°16’39,84946” W; altitude 729,846 m). Segundo Köppen, o clima é do tipo Aw, com temperatura média anual de 22,5 °C, e regime pluviomé- trico bem definido, com estação chuvosa (outubro a abril) e seca (maio a setembro), sendo a média anual de 1.460 mm. A evaporação média anual, medida pelo Tanque Classe-A, é igual a 1.938 mm (Kliemann et al., 2006). A água é dos principais fatores de produção agrícola, sendo essencial para o desenvolvimento sustentável da ati- vidade agrícola. O ciclo hidrológico de uma região é conta- bilizado pelo balanço hídrico, inferindo-se informações da cultura, como a profundidade das raízes, do solo, capaci- dade de armazenamento de água, e do clima, sobretudo quanto à precipitação pluvial (entrada de água) e evapo- transpiração (saída de água). Assim, a soma algébrica dos processos de entrada e saída de água num volume conhe- cido de solo durante um tempo pré-estabelecido é conhe- cida como balanço hídrico (Miranda e Pires, 2001). A evapotranspiração potencial (ETP, mm dia-1) foi calculada a partir da evaporação do Tanque Classe-A (ECA, mm dia-1): ETP Kp ECA (1) (1) ETP Kp ECA A precipitação pluvial é a principal forma de entrada de água em um dado sistema. O conhecimento do seu histórico auxilia no planejamento e monitoramento dos impactos causados pelo excedente ou pelo déficit hídrico em determinada região, contribuindo para tomada de deci- são em vários setores da economia, inclusive nas atividades agropecuárias (Vela et al., 2007; Pizzato et al., 2012). Por outro lado, as saídas de água são regidas, principalmente, pela evapotranspiração, pelo escoamento superficial e pela drenagem profunda. 1. Introdução Ou seja, uma maneira de especificar um sistema pode ser a constatação de possíveis frequências de um processo orga- nizado de acordo com a sua relativa importância ou magni- tude, caracterizando suas periodicidades e sazonalidades (Beecham e Chowdhury, 2008; Caloiero et al., 2011; Rashid et al., 2013). Para a determinação da FFT, primeiramente encon- trou-se uma função f(xi) yi satisfazendo uma reta: y ax b , estimando seus coeficientes a e b pelo método dos mínimos quadrados. Após isto se obteve a diferença en- tre os valores estimados (Ê) e observados (Ô), gerando uma série livre de tendências, na qual se aplicou o algoritmo FFT. A FFT gerou a magnitude dos dados em números complexos na forma y = ax + bi, havendo a necessidade de se aplicar o módulo nestes números complexos ( ) y a b 2 2 , mostrando a magnitude dos coeficientes. Desta forma, este estudo teve como objetivo carac- terizar os padrões de chuvas e de evapotranspiração poten- cial para o município de Goiânia, GO. 2. Material e Métodos Foi analisada uma série histórica de dados diários de precipitação pluvial e evapotranspiração potencial, para o período de 1979 a 2015 para precipitação (36 anos) e 1994 a Padrões de Chuva e de Evapotranspiração em Goiânia, GO 249 3. Resultados e Discussão precipitação 26,5% maiores que a média (P < 1890,9 mm ano-1), para os anos 1982, 1983 e 1989, ficando os demais valores dentro da faixa de variabilidade, dada pelo desvio- padrão médio (Fig. 1a). Vale destacar que há uma tendência geral de redução da precipitação média anual de 3,71 mm ano-1 (R2 = 53,2%) (Fig. 1a). Este decréscimo já foi verifi- cado em outros estudos para esta região, com precipitação pluviométrica anual média reduzindo em torno de 6,0 mm ano-1, sendo janeiro o mês mais afetado, podendo reduzir até 3,0 mm ano-1 (Marcuzzo, 2012). No período avaliado (1978-2015) observou-se uma média de precipitação pluvial anual P = 1.494,7 mm, com desvio-padrão médio de 187,6 mm, que representa uma variabilidade de 12,55% da média, sendo os valores de mínimo e máximo iguais a 1.044,20 mm (2007) e 2.067,20 (1982), respectivamente (Fig. 1a). Esta variabilidade tanto nas médias de precipitações temporais quanto espaciais já foi identificada em outros estudos tanto para a região cen- tro-oeste (Assad e Castro, 1991; Cardoso et al., 2014) quanto para a região metropolitana de Goiânia, GO, deter- minando valores entre 1400-1600 mm ano-1, sendo as maiores precipitações observadas no município de Pira- canjuba, GO e as menores em Inhumas, GO e Palmeiras de Goiás, GO (Cardoso et al., 2011). Ainda no Centro-oeste, Cardoso et al. (2014) regist- raram uma precipitação média anual entre 1.000 e 1.200 mm, no entanto, essa média refere-se apenas a 10 anos de observação (1989-1998). Precipitações médias anuais entre 741 e 1.335 mm foram registradas para regiões com classificação climática semelhante à deste estudo (Murta et al., 2005; Chaves e Piau, 2008; Neves et al., 2011; Pizzato et al., 2012). Foi possível observar uma variação cíclica nas mé- dias anuais de precipitação, onde ocorreram valores 24,6% menores que a média (P >1.126,5 mm), em intervalos de aproximadamente cinco anos (1984, 1990, 1993, 1999, 2007 e 2015) (Fig. 1a). Na mesma figura, observaram-se Os anos com precipitações acima (1982, 1983 e 1989) ou abaixo (1984, 1990, 1993, 1999, 2007 e 2015) da média (Fig. 1a) foram marcados pelos fenômenos oceano-atmos- Figura 1 - Distribuição da precipitação pluvial (P) anual (a) e por quinquídios (b), para o município de Goiânia, GO, no período de 1978 a 2015. As linhas pontilhadas (a) e barras (b) representam o desvio-padrão médio. 3. Resultados e Discussão O mesmo foi observado para os eventos de La Niña, que ocorreram em 1984/85 (Fraca), 1988/89 (Forte), 1998/1999 (Moderada) e 2007/08 (Mo- derada) (NOAA, 2017) (Fig. 2) . Vale ressaltar que mesmo apresentando um R2 < 0,03 e correlação r < 0,2, observa-se uma tendência de variação abaixo e acima da média de precipitação de 50 mm ano-1, para eventos moderados de El Niño e La Niña, respectivamente (Fig. 2b). Ainda na Fig. 2, observa-se que os eventos mais frequentes foram Neutros ( 42%), seguidos pelos eventos de La Niña ( 24%) e El Niño ( 34%), evidenciando periodicidades iguais a 2,4, 4,2 e 2,9 anos, respectivamente. Estes fenô- menos demonstram que podem alterar as médias de chuvas, ou potencializar outros eventos meteorológicos possuem maior influencia nos regimes pluviométricos sobre os Esta- dos do centro-oeste, como por exemplo, a Alta da Bolívia e frentes frias (Melo, 2000), além da Oscilação Decadal do Pacífico (ODP), que atua de forma significativa sobre o Estado de Goiás, ficando evidente a relação direta entre a redução nos totais anuais de precipitação e na frequência de dias com precipitação durante a fase fria da ODP, assim como aumento na precipitação anual e frequência de dias com chuva durante a sua fase quente (Souza et al., 2010). sazonalidade da precipitação pluviométrica em Goiás e no Distrito Federal, os autores determinaram que na região há cinco meses secos (maio a setembro) e sete meses úmidos (outubro a abril) (Costa et al., 2012). Observa-se também que 54% da precipitação total anual ocorre nos primeiros quinquídios do ano, de janeiro a abril (787,5 mm) e o res- tante, nos demais quinquídios da estação chuvosa (Fig. 1b). A estação chuvosa determinou uma média igual a 34,1 mm por quinquídio, e a estação seca 3,8 mm, onde foram obser- vadas as maiores variabilidades (Fig. 1b). Isso caracteriza bem o subtipo climático (w) da classificação de Köppen (Pizzato et al., 2012; Pinto et al., 2014). As maiores médias de precipitação ( 200 mm) ocor- reram entre os quinquídios 1-17 (janeiro a março) e 63-73 (novembro a dezembro) (Fig. 1b), corroborando com a literatura (Alves, 2011). A média de dias secos e chuvosos foi de 292 e 73 dias, respectivamente, demonstrando que apenas 20% do ano ocorrem precipitações pluviométricas (Fig. 3). 3. Resultados e Discussão Figura 1 - Distribuição da precipitação pluvial (P) anual (a) e por quinquídios (b), para o município de Goiânia, GO, no período de 1978 a 2015. As linhas pontilhadas (a) e barras (b) representam o desvio-padrão médio. Figura 1 - Distribuição da precipitação pluvial (P) anual (a) e por quinquídios (b), para o município de Goiânia, GO, no período de 1978 a 2015. As linhas pontilhadas (a) e barras (b) representam o desvio-padrão médio. 250 Casaroli et al. Casaroli et al. féricos ENOS-Oscilação Sul, também conhecidos como El Niño (EN) e La Niña (LN), fases quente e fria do fenômeno, respectivamente. Assim, o El Niño ocorreu nos anos 1982/83 (Forte), 1991/92 (Forte), 1997/98 e 2015/16 (For- te), não evidenciando um padrão significativo de redução ou aumento nas precipitações. O mesmo foi observado para os eventos de La Niña, que ocorreram em 1984/85 (Fraca), 1988/89 (Forte), 1998/1999 (Moderada) e 2007/08 (Mo- derada) (NOAA, 2017) (Fig. 2) . Vale ressaltar que mesmo apresentando um R2 < 0,03 e correlação r < 0,2, observa-se uma tendência de variação abaixo e acima da média de precipitação de 50 mm ano-1, para eventos moderados de El Niño e La Niña, respectivamente (Fig. 2b). Ainda na Fig. 2, observa-se que os eventos mais frequentes foram Neutros ( 42%), seguidos pelos eventos de La Niña ( 24%) e El Niño ( 34%), evidenciando periodicidades iguais a 2,4, 4,2 e 2,9 anos, respectivamente. Estes fenô- menos demonstram que podem alterar as médias de chuvas, ou potencializar outros eventos meteorológicos possuem maior influencia nos regimes pluviométricos sobre os Esta- dos do centro-oeste, como por exemplo, a Alta da Bolívia e frentes frias (Melo, 2000), além da Oscilação Decadal do Pacífico (ODP), que atua de forma significativa sobre o Estado de Goiás, ficando evidente a relação direta entre a redução nos totais anuais de precipitação e na frequência de dias com precipitação durante a fase fria da ODP, assim como aumento na precipitação anual e frequência de dias com chuva durante a sua fase quente (Souza et al., 2010). féricos ENOS-Oscilação Sul, também conhecidos como El Niño (EN) e La Niña (LN), fases quente e fria do fenômeno, respectivamente. Assim, o El Niño ocorreu nos anos 1982/83 (Forte), 1991/92 (Forte), 1997/98 e 2015/16 (For- te), não evidenciando um padrão significativo de redução ou aumento nas precipitações. 3. Resultados e Discussão A áli d t dê i t d 25 9 édi d (24 76 °C) d id d Padrões de Chuva e de Evapotranspiração em Goiânia, GO 251 Figura 4 - Distribuição de frequência da precipitação pluvial diária (a) e da evapotranspiração potencial diária (b), para o município de Goiânia, GO, no período de 1978 a 2015. Figura 3 - Ocorrência anual de número de dias secos (NDs) e chuvosos (NDc) no município de Goiânia, GO, para o período de 1978 a 2015. As linhas pontilhadas representam o ajuste de regressão linear. Padrões de Chuva e de Evapotranspiração em Goiânia, GO 251 Figura 3 - Ocorrência anual de número de dias secos (NDs) e chuvosos (NDc) no município de Goiânia, GO, para o período de 1978 a 2015. As linhas pontilhadas representam o ajuste de regressão linear. Figura 3 - Ocorrência anual de número de dias secos (NDs) e chuvosos (NDc) no município de Goiânia, GO, para o período de 1978 a 2015. As linhas pontilhadas representam o ajuste de regressão linear. Figura 3 - Ocorrência anual de número de dias secos (NDs) e chuvosos (NDc) no município de Goiânia, GO, para o período de 1978 a 2015. As linhas pontilhadas representam o ajuste de regressão linear. Figura 4 - Distribuição de frequência da precipitação pluvial diária (a) e da evapotranspiração potencial diária (b), para o município de Goiânia, GO, no período de 1978 a 2015. Figura 4 - Distribuição de frequência da precipitação pluvial diária (a) e da evapotranspiração potencial diária (b), para o município de Goiânia, GO, no período de 1978 a 2015. Figura 4 - Distribuição de frequência da precipitação pluvial diária (a) e da evapotranspiração potencial diária (b), para o município de Goiânia, GO, no período de 1978 a 2015. A análise de tendência mostrou que a cada 25,9 anos, aumenta um dia seco, e a cada 23,8 anos, diminui um dia chuvoso (Fig. 3). média do ar (24,76 °C) e uma das menores umidades relativa médias (65,85%), já o ano de 1996 obteve uma das menores temperaturas médias do ar (22,29 °C) e uma das maiores umidades relativas do ar média (87,45%) (Fig. 6). A evapotranspiração total média foi de 1.384,67 mm ano-1 (Fig. 3. Resultados e Discussão A aná- lise de tendência mostrou que a cada 25,9 anos, aumenta um dia seco, e a cada 23,8 anos, diminui um dia chuvoso (Fig. 3) . Ressalta-se que, aproximadamente, 66% dos valores de precipitação diária correspondentes a 0,0 mm, 22% ente 0,0 e 10,0 mm e 6,5% de 10,0 a 20,0 mm (Fig. 4a). Os anos de 1990 e 2007 foram os anos que apresentaram maiores frequências de dias secos, 308 dias secos cada. Já o ano de 1982 foi o mais chuvoso com 98 dias considerados chu- vosos (Fig. 3). Para municípios de mesma classificação climática Aw (Mato Grosso) foram encontrados valores médios de 305 (secos) e 60 (chuvosos) dias (Pizzato et al., 2012), e 280 (secos) e 85 (chuvosos) dias (Moreira et al., 2010). A Fig. 1b apresenta as precipitações médias por quin- quídios. Nesta figura foram separadas as estações seca (maio-setembro) e chuvosa (outubro-abril), corresponden- te aos quinquídios de 26 a 53 (seca), e do 1° ao 25°, e 54° ao 73° (chuvosa). Em um estudo sobre a espacialização e Figura 2 - Anos de ocorrência de El Niño (ION > +0,5 °C) e La Niña (ION <-0,5 °C) e suas respectivas classificações quanto à intensidade: fraco (0,5 ION 0,9), moderado (1,0 ION 1,4) e forte (ION 1,5) (a). Relação entre a diferença PE-P (estimada-observada) e o índice ION, ajuste de uma regressão linear, e suas frequências de ocorrência (%) (b). Figura 2 - Anos de ocorrência de El Niño (ION > +0,5 °C) e La Niña (ION <-0,5 °C) e suas respectivas classificações quanto à intensidade: fraco (0,5 ION 0,9), moderado (1,0 ION 1,4) e forte (ION 1,5) (a). Relação entre a diferença PE-P (estimada-observada) e o índice ION, ajuste de uma regressão linear, e suas frequências de ocorrência (%) (b). Figura 2 - Anos de ocorrência de El Niño (ION > +0,5 °C) e La Niña (ION <-0,5 °C) e suas respectivas classificações quanto à intensidade: fraco (0,5 ION 0,9), moderado (1,0 ION 1,4) e forte (ION 1,5) (a). Relação entre a diferença PE-P (estimada-observada) e o índice ION, ajuste de uma regressão linear, e suas frequências de ocorrência (%) (b). 3. Resultados e Discussão 5a), valor um pouco menor ao encontrado no município de Cáceres-MT (1.650,55 mm ano-1), que pode ser devido a menor temperatura média do município de Goiânia, GO, igual a 22,1 °C (Lobato, 2006), em relação a Cárceres-MT de 26,24 °C (Neves et al., 2011). A maior evapotranspiração foi observada no ano de 2015 (1.484,42 mm) e a menor no ano de 1996 (1.283,03 mm) (Fig. 5). Aproximadamente 38% dos valores de ETP encon- tram-se fora do intervalo de confiança, sendo 24% acima da média (2007, 2010, 2012, 2014 e 2015) e 14% abaixo (1996, 1997 e 2001). Novamente, não foi observado um padrão entre aumento ou diminuição da ETP em função das ocorrências de El Niño ou La Niña, mas pode-se inferir que para estes anos, em que houveram variações significativas na ETP, ou tiveram eventos de El Niño ou La Ninã (NOAA, 2017). Esse comportamento pode ser explicado pela tempe- ratura e umidade relativa do ar que tem efeito diretamente proporcional e inversamente proporcional, respectivamen- te (Fig. 6). O ano de 2015 apresentou a maior temperatura A média da ETP em quinquídios foi de 18,9 mm, apresentando tendências de redução entre os quinquídios 1° ao 36° (0,14 mm/quinquídio), aumento de 0,74 mm/quin- 252 Casaroli et al. Figura 5 - Evapotranspiração potencial anual média (ETP) e seus desvios-padrão (barras verticais), ETP média do período avaliado (linha cheia) e desvios-padrão (linhas pontilhadas) (a), bem como a ETP acumulada por quinquídios (b), para o município de Goiânia, GO, de 1994 a 2015. OBS: 0,408 mm dia-1 = 1 MJ m-2 dia-1. 252 Casaroli et al. Figura 5 - Evapotranspiração potencial anual média (ETP) e seus desvios-padrão (barras verticais), ETP média do período avaliado (linha cheia) e desvios-padrão (linhas pontilhadas) (a), bem como a ETP acumulada por quinquídios (b), para o município de Goiânia, GO, de 1994 a 2015. OBS: 0,408 mm dia-1 = 1 MJ m-2 dia-1. nitude, pela análise de Fourier (FFT), tanto para os valores de precipitação (Fig. 7 a, b, c) quanto para ETP (Fig. 7 d, e, f). quídio entre o 37° e 50°, e novamente decréscimo entre o 51° e 73° quinquídio (0,37 mm/quinquídio) (Fig. 5b). Fo- ram encontrados valores de ETP significativamente abaixo da média entre o 25° e 36° quinquídio, e maiores entre o 45° e 54° quinquídios (Fig. 5b). 3. Resultados e Discussão Estes valores são referentes ao final de abril e início de junho, e final de julho e início de setembro, respectivamente, correspondentes às maiores va- riações de temperatura e umidade relativa do ar em Goiâ- nia, GO (Fig. 6). Para a variável precipitação foi possível destacar duas maiores magnitudes, tanto para as frequências quanto para os períodos. A maior magnitude ocorreu na frequência 38%, correspondente a valores acima da média de precipi- tação, já a segunda magnitude foi registrada para uma frequência de 25%, referente a precipitações abaixo da média, sendo suas periodicidades de 4,0 e 2,7 anos, respec- tivamente (Fig. 7b e 7c). A frequência predominante de ETP foi 31%, correspondentes a valores acima da média, com periodicidade de 3,2 anos (Fig. 7e e 7f). A análise de frequência demonstrou que aproximada- mente 80% das lâminas de ETP encontram-se entre os valores de 0,2 e 5,0 mm dia-1 (0,49 e 12,25 MJ m-2 dia-1), sendo a maior frequência encontrada entre 3,0 e 4,0 mm dia-1 (7,35 e 9,80 MJ m-2 dia-1) (Fig. 5b). A análise do balanço hídrico climatológico demons- trou que o déficit hídrico médio anual foi de 178,18 mm, e o excedente hídrico foi de 198,54 mm. O déficit hídrico quinquienal médio foi de 26,29 mm e o excedente hídrico foi de 33,81 mm (Fig. 8). As maiores médias de déficit hídrico foram iguais a 70,73 e 71,14 mm por quinquídio, Observaram-se variações entre os valores estimados e observados de precipitação e ETP, com amplitude de apro- ximadamente -600 a 400, e de -80 a 60 mm, respecti- vamente (Fig. 7). A partir destes dados foi possível obter as frequências e períodos predominantes ou com maior mag- Padrões de Chuva e de Evapotranspiração em Goiânia, GO 253 Figura 6 - Temperatura do ar máxima (Tmax, °C), mínima (Tmin, °C) e média (Tm, °C), e umidade relativa do ar (UR, %), dadas em médias anuais (a,b) e médias por quinquídios (c,d), para Goiânia, GO, de 1994 a 2015. Figura 6 - Temperatura do ar máxima (Tmax, °C), mínima (Tmin, °C) e média (Tm, °C), e umidade relativa do ar (UR, %), dadas em médias anuais (a,b) e médias por quinquídios (c,d), para Goiânia, GO, de 1994 a 2015. 4. Conclusões O município de Goiânia, GO apresenta duas estações bem definidas, uma seca, de abril a setembro, e outra chuvosa, de outubro a abril. A média de precipitação plu- viométrica foi de 1.494,66 mm ano-1, com variabilidade de 12,6%. Há uma tendência de redução das chuvas em Goiâ- nia, GO, na ordem de 3,7 mm dia-1, com probabilidade de 99% de ocorrerem chuvas iguais ou menores que 50 mm dia-1. Há uma tendência de redução do número de dias chuvosos e aumento do número de dias secos. A evapo- transpiração potencial (ETP) média de Goiânia, GO é de 1.380,57 mm dia-1, com variabilidade anual de 3,68% e probabilidade de 80% de ocorrerem ETP iguais ou menores que 5 mm dia-1. A ETP média diária é de 3,78 mm dia-1 e por quinquídio igual a 18,9 mm. Os valores médios não podem ser utilizados em dois momentos do ano, entre os quinquídios 25° e 36°, e do 45° ao 54°, pois nestes períodos as médias de ETP é menor ou maior que a média, respecti- vamente. A lâmina de excedente hídrico é de 607 mm ano-1 e de déficit hídrico 471 mm ano-1. Evapotranspirações relativas ETR/ETP <0,8 são registradas entre a segunda quinzena de abril e segunda quinzena de outubro. Quase 40% das chuvas anuais estão acima da média, retornando a cada quatro anos, e cerca de 30% das ETP anuais também estão acima da média, repetindo-se a cada 3,2 anos. Tem-se Sabe-se também que as chuvas no Estado de Goiás são regidas por massas úmidas provenientes da Amazônia (massa equatorial continental - mEc), sendo transportadas por ventos contra-alísios no sentido noroeste-sudeste (No- bre et al., 2009; Aragão, 2012;), desta forma, o desmata- mento da Floresta Amazônica pode ter efeito tanto sobre as chuvas quanto na evapotranspiração em Goiás. Com base nas médias de desmatamento do Cerrado goiano (Machado et al., 2004; Silva e Ferreira Júnior, 2010), foi possível detectar uma correlação de 0,53 entre a taxa de desmatamento e as temperaturas médias nos perío- dos de detecção destas taxas (Fig. 9a), entretanto, não foi identificada correlação significativa (r <0,28) entre as taxas de desmatamento e as variáveis precipitação e ETP (Fig. 9b). Estas mesmas variáveis quando correlacionadas as taxas de desmatamento na Amazônia (INPE, 2016) veri- ficou-se r = -0,60 para temperatura, r = 0,04 para precipi- tação e r = -0,51 para ETP (Figs. 9c e 9d). 3. Resultados e Discussão Figura 6 - Temperatura do ar máxima (Tmax, °C), mínima (Tmin, °C) e média (Tm, °C), e umidade relativa do ar (UR, % e médias por quinquídios (c,d), para Goiânia, GO, de 1994 a 2015. ra 7 - Diferenças entre valores estimados e observados, bem como as frequências e períodos predominantes, para as variáveis precipitação pluvial (a, e evapotranspiração potencial ETP (d, e, f), para o município de Goiânia, GO. Figura 7 - Diferenças entre valores estimados e observados, bem como as frequências e períodos predominantes, para as variáveis precipitação pluvial (a, b, c) e evapotranspiração potencial ETP (d, e, f), para o município de Goiânia, GO. Figura 7 - Diferenças entre valores estimados e observados, bem como as frequências e períodos predominantes, para as variáveis precipitação pluvial (a, b, c) e evapotranspiração potencial ETP (d, e, f), para o município de Goiânia, GO. 254 Casaroli et al. observadas nos quinquídios 47 (agosto) e 50 (setembro), respectivamente. Já a menor média foi de 2,65 mm, obser- vada no quinquídio 73 (dezembro), durante o período chu- voso. Vale ressaltar que todos os quinquídios apresentaram déficit hídrico, independente da estação, seca ou chuvosa (Fig. 8). Assim, a evapotranspiração relativa ETR/ETP < 1,0 (Fig. 8). de 2015, onde ocorreram temperaturas acima da média (Fig. 6a) e precipitações abaixo da média (Fig. 1a), ETP acima da média (Fig. 5a), em ano de El Niño Moderado (Fig. 2a). Além disto, o fenômeno denominado “ilhas de calor”, dada pela diferença de temperatura do ar entre áreas urbanas e rurais de um determinado município pode ter proporcionado este aumento da temperatura, conforme já indicado na literatura (Nascimento e Oliveira, 2011). O maior excedente hídrico médio anual foi de 161,22 mm, constatado no 1° quinquídio (janeiro). Entre os quin- quídios 29° ao 32° (maio-junho), 35° ao 51° (junho-setem- bro) e no 57° (outubro) não foram observados excedentes hídricos. Em Cáceres-MT, o déficit hídrico foi de 400,30 mm e o excedente hídrico médio anual foi igual a 147,03 mm (Neves et al., 2011). 4. Conclusões Contudo, este aumento da temperatura pode ter ocorrido como um reflexo do desmatamento da área do município de Goiânia, GO, bem como suas regiões circunvizinhas. Vale destacar o ano Figura 8 - Balanço hídrico climatológico identificando o excedente (EXC) e o déficit (DEF) hídrico de períodos quinquienais, bem como a evapotranspiração relativa (ETR/ETP), para região de Goiânia, GO, no período de 1994 a 2015. Figura 8 - Balanço hídrico climatológico identificando o excedente (EXC) e o déficit (DEF) hídrico de períodos quinquienais, bem como a evapotranspiração relativa (ETR/ETP), para região de Goiânia, GO, no período de 1994 a 2015. Figura 8 - Balanço hídrico climatológico identificando o excedente (EXC) e o déficit (DEF) hídrico de períodos quinquienais, bem como a evapotranspiração relativa (ETR/ETP), para região de Goiânia, GO, no período de 1994 a 2015. Padrões de Chuva e de Evapotranspiração em Goiânia, GO 255 Figura 9 - Taxas de desmatamento em Goiás (a, b) e na Amazônia (c, d) (linha pontilhada) e suas relações com a temperatura média do ar anual (Tar, colunas brancas) e média móvel em diferentes períodos (Tar, linhas cheias), bem como com a precipitação pluvial (P, colunas brancas) e eva- potranspiração potencial (ETP, colunas cinzas). Figura 9 - Taxas de desmatamento em Goiás (a, b) e na Amazônia (c, d) (linha pontilhada) e suas relações com a temperatura média do ar anual (Tar, colunas brancas) e média móvel em diferentes períodos (Tar, linhas cheias), bem como com a precipitação pluvial (P, colunas brancas) e eva- t i ã t i l (ETP l i ) Figura 9 - Taxas de desmatamento em Goiás (a, b) e na Amazônia (c, d) (linha pontilhada) e suas relações com a temperatura média do ar anual (Tar, colunas brancas) e média móvel em diferentes períodos (Tar, linhas cheias), bem como com a precipitação pluvial (P, colunas brancas) e eva- potranspiração potencial (ETP, colunas cinzas). PROCEEDINGS OF THE WORLD ENVIRONMENTAL AND WATER RESOURCES CONGRESS, 2008, Hono- lulu, USA. Annals... Honolulu: American Society of Civil Engineers, 2008. p. 1-13. indícios de que as taxas de desmatamento em Goiás podem contribuir com o aumento da temperatura média anual em Goiânia, GO, porém não tem influencia sobre as chuvas e ETP. Maiores taxas de desmatamento na Amazônia não inferiram aumentos de temperatura do ar, maiores ETP e redução das chuvas em Goiânia, GO. BLAIN, G.C.; KAYANO, M.T.; SENTELHAS, P.C.; LULU, J. Agradecimentos CALOIERO, T.; COSCARELLI, R.; FERRARI, E.; MANCINI, M. Trend detection of annual and seasonal rainfall in Cala- bria (Southern Italy). Int. J. Climatol., v. 31, n. 1, p. 44-56, 2011. http://dx.doi.org/10.1002/joc.2055. A Escola de Agronomia da Universidade Federal de Goiás (EA/UFG) por auxiliar financeiramente a Estação Meteorológica instalada na EA/UFG, ao Prof. Engler José Vidigal Lobato (Climatologista EA/UFG), ao Técnico em Meteorologia Sr. Paulo José da Costa. CARDOSO, M.R.D.; MARCUZZO, F.F.N.; MELO, D.C.R. Ma- peamento temporal e espacial da precipitação pluviométrica da região metropolitana de Goiânia. In: SIMPÓSIO BRA- SILEIRO DE SENSORIAMENTO REMOTO, 15., 2011, Curitiba. Anais... Curitiba: INPE, 2011. p. 4594-4601. 4. Conclusões Variabilidade sazonal da evapotranspiração relativa em Campinas (SP): caracterização climática e análise de ten- dências. Bragantia, v. 68, n. 2, p. 545-553, 2009. Referências CARDOSO, M.R.D.; MARCUZZO, F.F.N.; BARROS, J.R. Classificação climática de Köppen-Geiger para o Estado de Goiás e o Distrito Federal. Acta Geográfica, v. 8, n. 16, p. 40-55, 2014. ALVES, E.D.L. Frequência e probabilidade de chuvas no mun- icípio de Iporá, GO. Caminhos de Geografia, v. 12, n. 37, p. 65-72, 2011. ARAGÃO, L.E.O.C. Environmental science: The rainforest’s wa- ter pump. Nature, v. 489, n. 7415, p. 217-218, 2012. CHAVES, H.M.L.; PIAU, L.P. Efeito da variabilidade da precipi- tação pluvial e do uso e manejo do solo sobre o escoamento superficial e o aporte de sedimento de uma bacia hidro- gráfica do Distrito Federal. Revista Brasileira de Ciência do Solo, v. 32, n. 1, p. 333-343, 2008. ASSAD, E.D.; CASTRO, L.H.R. Análise frequêncial da pluvio- metria para a estação de Sete Lagoas, MG. 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https://openalex.org/W3030528375	https://www.biorxiv.org/content/biorxiv/early/2020/05/28/2020.05.26.116046.full.pdf	English	null	Exposure to hypergravity during zebrafish development alters cartilage material properties and strain distribution	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	24,142	. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 3 Elizabeth A Lawrence1, Jessye A Aggleton1,2, Jack J. W. A. van Loon3, Josepha Godivier 4 Robert L. Harniman5, Jiaxin Pei1, N. C. Nowlan4, Chrissy L Hammond 1 5 6 1. School of Physiology, Pharmacology and Neuroscience, University of Bristol, BS8 1TD, 7 UK 8 2. School of Anthropology and Archaeology, University of Bristol, BS8 1UU 9 3. A: European Space Agency (ESA) Technology Center (ESTEC), TEC-MMG, NL-2200 10 AG, Noordwijk Netherlands. 11 B: Department Oral & Maxillofacial Surgery/Pathology, Amsterdam Movement Sciences 12 & Amsterdam Bone Center (ABC), Amsterdam University Medical Center Location 13 VUmc & Academic Center for Dentistry Amsterdam (ACTA), Amsterdam, Netherlands 14 4. Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK 15 5. School of Chemistry, University of Bristol, BS8 1TS, UK 16 17 18 Keywords: Zebrafish, musculoskeletal, gravity, finite element analysis, chondrocyte 19 maturation, extracellular matrix. 20 Running title: Zebrafish development in hypergravity 21 22 Abstract 23 24 Terrestrial vertebrates have adapted to life on Earth and its constant gravitational field, which 25 exerts load on the body and influences the structure and function of tissues. While the effects o 26 microgravity on muscle and bone homeostasis are well described, the effects of shorter 27 exposures to increased gravitational fields are less well characterized. Here, we exposed 28 zebrafish to 3 and 6g hypergravity from 3-5 days post fertilisation, when key events in jaw 29 cartilage morphogenesis occur. We did not observe changes to growth, or morphology of 30 cartilage or muscle. However, we observed altered mechanical properties of jaw cartilages. We 31 model the impact of these material property changes using Finite Element Analysis and show 32 strain distribution in the jaw is altered following hypergravity. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint In regions of predicted altered 33 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ma The copyright holder for this preprint this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint strain we observed local changes to chondrocyte morphology, suggesting altered gravity affects 34 chondrocyte maturation, ultimately leading to changes to cartilage structure and function. 35 36 Background 37 38 Mechanical loading of the skeleton occurs during physical activity through muscle contraction 39 and ground reaction forces (Lanyon et al., 1975; Usui et al., 2003). This loading builds and 40 maintains bone mass, making increased skeletal loading an area of interest in the treatment of 41 osteoporosis (Russo and MD, 2009). A physiological level of mechanical loading is beneficial to 42 cartilage function in vitro by increasing chondrocyte proliferation and anabolic processes, 43 boosting proteoglycan synthesis (Klein-Nulend et al., 1987; Lee and Bader, 1997; Soltz et al., 44 2000; Shelton, Bader and Lee, 2003; Sharma, Saxena and Mishra, 2007). In vivo experiments 45 in hamsters (Otterness et al., 1998), rats (Galois et al., 2003), and humans (Manninen, 2001), 46 have indicated that moderate exercise has a chondroprotective role, resulting in decreased risk 47 of severe osteoarthritis (OA). In contrast, overloading or reduced loading of joints has a role in 48 cartilage destruction by promoting catabolic pathways. Extreme loading (through high impact 49 sports (Arendt and Dick, 1995; Levy et al., 1996) or joint misalignment (Meireles et al., 2017)) 50 leads to extracellular matrix damage, loss of collagen, chondrocyte cell death and eventually OA 51 (Torzilli et al., 1999; Loening et al., 2000; Patwari et al., 2004). 52 53 strain we observed local changes to chondrocyte morphology, suggesting altered gravity affects 34 chondrocyte maturation, ultimately leading to changes to cartilage structure and function. 35 36 strain we observed local changes to chondrocyte morphology, suggesting altered gravity affects 34 chondrocyte maturation, ultimately leading to changes to cartilage structure and function. Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 In vivo experiments 45 in hamsters (Otterness et al., 1998), rats (Galois et al., 2003), and humans (Manninen, 2001), 46 have indicated that moderate exercise has a chondroprotective role, resulting in decreased risk 47 of severe osteoarthritis (OA). In contrast, overloading or reduced loading of joints has a role in 48 cartilage destruction by promoting catabolic pathways. Extreme loading (through high impact 49 sports (Arendt and Dick, 1995; Levy et al., 1996) or joint misalignment (Meireles et al., 2017)) 50 leads to extracellular matrix damage, loss of collagen, chondrocyte cell death and eventually OA 51 (Torzilli et al., 1999; Loening et al., 2000; Patwari et al., 2004). 52 53 As mechanical loading is exerted on the skeleton by gravitational forces (Kohrt, Barry and 54 Schwartz, 2009), many studies on the musculoskeletal system have been carried out across a 55 range of gravity levels. Microgravity has significant, well documented, effects on the skeleton 56 with decreased bone density observed in humans (Demontis et al., 2017) and fish (Chatani et 57 al., 2015), and disruptions to skeletal maturation observed in immature mice (Maupin et al., 58 2019). Studies on astronauts and cosmonauts following long duration space flight found that 59 92% had a minimum of 5% bone loss in at least one skeletal site (LeBlanc et al., 2007) with 60 weight-bearing regions most affected (LeBlanc, Shackelford and Schneider, 1998; Vico et al., 61 2000). Sarcopenia is also observed in microgravity, with decrease in muscle volume of around 62 15% following 4-6 months in microgravity (Adrian LeBlanc et al., 2000). While decreased 63 mechanical loading in microgravity has been uniformly associated with disuse bone loss, 64 exposure to hypergravity has been shown to increase or decrease bone depending on the 65 degree of hypergravity. One study exposed mice to hypergravity for 21 days and found that at 66 2g, there was an improvement in trabecular bone volume, fewer osteoclasts and an increase in 67 As mechanical loading is exerted on the skeleton by gravitational forces (Kohrt, Barry and 54 Schwartz, 2009), many studies on the musculoskeletal system have been carried out across a 55 range of gravity levels. Microgravity has significant, well documented, effects on the skeleton 56 with decreased bone density observed in humans (Demontis et al., 2017) and fish (Chatani et 57 al., 2015), and disruptions to skeletal maturation observed in immature mice (Maupin et al., 58 2019). Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 Extreme loading (through high impact 49 sports (Arendt and Dick, 1995; Levy et al., 1996) or joint misalignment (Meireles et al., 2017)) 50 leads to extracellular matrix damage, loss of collagen, chondrocyte cell death and eventually OA 51 (Torzilli et al., 1999; Loening et al., 2000; Patwari et al., 2004). 52 53 As mechanical loading is exerted on the skeleton by gravitational forces (Kohrt, Barry and 54 Schwartz, 2009), many studies on the musculoskeletal system have been carried out across a 55 range of gravity levels. Microgravity has significant, well documented, effects on the skeleton 56 with decreased bone density observed in humans (Demontis et al., 2017) and fish (Chatani et 57 al., 2015), and disruptions to skeletal maturation observed in immature mice (Maupin et al., 58 2019). Studies on astronauts and cosmonauts following long duration space flight found that 59 92% had a minimum of 5% bone loss in at least one skeletal site (LeBlanc et al., 2007) with 60 weight-bearing regions most affected (LeBlanc, Shackelford and Schneider, 1998; Vico et al., 61 2000). Sarcopenia is also observed in microgravity, with decrease in muscle volume of around 62 15% following 4-6 months in microgravity (Adrian LeBlanc et al., 2000). While decreased 63 mechanical loading in microgravity has been uniformly associated with disuse bone loss, 64 exposure to hypergravity has been shown to increase or decrease bone depending on the 65 degree of hypergravity. One study exposed mice to hypergravity for 21 days and found that at 66 2g, there was an improvement in trabecular bone volume, fewer osteoclasts and an increase in 67 Background 37 38 Mechanical loading of the skeleton occurs during physical activity through muscle contraction 39 and ground reaction forces (Lanyon et al., 1975; Usui et al., 2003). This loading builds and 40 maintains bone mass, making increased skeletal loading an area of interest in the treatment of 41 osteoporosis (Russo and MD, 2009). A physiological level of mechanical loading is beneficial to 42 cartilage function in vitro by increasing chondrocyte proliferation and anabolic processes, 43 boosting proteoglycan synthesis (Klein-Nulend et al., 1987; Lee and Bader, 1997; Soltz et al., 44 2000; Shelton, Bader and Lee, 2003; Sharma, Saxena and Mishra, 2007). Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 35 36 Background 37 38 Mechanical loading of the skeleton occurs during physical activity through muscle contraction 39 and ground reaction forces (Lanyon et al., 1975; Usui et al., 2003). This loading builds and 40 maintains bone mass, making increased skeletal loading an area of interest in the treatment of 41 osteoporosis (Russo and MD, 2009). A physiological level of mechanical loading is beneficial to 42 cartilage function in vitro by increasing chondrocyte proliferation and anabolic processes, 43 boosting proteoglycan synthesis (Klein-Nulend et al., 1987; Lee and Bader, 1997; Soltz et al., 44 2000; Shelton, Bader and Lee, 2003; Sharma, Saxena and Mishra, 2007). In vivo experiments 45 in hamsters (Otterness et al., 1998), rats (Galois et al., 2003), and humans (Manninen, 2001), 46 have indicated that moderate exercise has a chondroprotective role, resulting in decreased risk 47 of severe osteoarthritis (OA). In contrast, overloading or reduced loading of joints has a role in 48 cartilage destruction by promoting catabolic pathways. Extreme loading (through high impact 49 sports (Arendt and Dick, 1995; Levy et al., 1996) or joint misalignment (Meireles et al., 2017)) 50 leads to extracellular matrix damage, loss of collagen, chondrocyte cell death and eventually OA 51 (Torzilli et al., 1999; Loening et al., 2000; Patwari et al., 2004). 52 Background 37 38 Mechanical loading of the skeleton occurs during physical activity through muscle contraction 39 and ground reaction forces (Lanyon et al., 1975; Usui et al., 2003). This loading builds and 40 maintains bone mass, making increased skeletal loading an area of interest in the treatment of 41 osteoporosis (Russo and MD, 2009). A physiological level of mechanical loading is beneficial to 42 cartilage function in vitro by increasing chondrocyte proliferation and anabolic processes, 43 boosting proteoglycan synthesis (Klein-Nulend et al., 1987; Lee and Bader, 1997; Soltz et al., 44 2000; Shelton, Bader and Lee, 2003; Sharma, Saxena and Mishra, 2007). In vivo experiments 45 in hamsters (Otterness et al., 1998), rats (Galois et al., 2003), and humans (Manninen, 2001), 46 have indicated that moderate exercise has a chondroprotective role, resulting in decreased risk 47 of severe osteoarthritis (OA). In contrast, overloading or reduced loading of joints has a role in 48 cartilage destruction by promoting catabolic pathways. Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 Human 78 bed-rest studies, hind-limb unloading studies in rats and studies performed on mice exposed to 79 real microgravity have demonstrated that loss of mechanical forces lead to cartilage degradation 80 primarily through proteoglycan loss (Souza et al., 2012; Ganse et al., 2015; Willey et al., 2016; 81 Fitzgerald et al., 2019). Cell culture experiments carried out in microgravity also support the 82 observation of cartilage degradation under reduced loading conditions, with cytoskeletal 83 reorganization and extracellular matrix (ECM) composition altered following short exposures 84 (Van Loon et al., 1995; Freed et al., 1997; Zhang et al., 2003; Ulbrich et al., 2010; Aleshcheva 85 et al., 2013). In comparison to work on cartilage in unloading conditions, less is known about the 86 effect of hypergravity and increased mechanical loading. One study on cultured chondrocytes 87 showed downregulation of BMP4 (crucial in collagen type II and aggrecan synthesis (Reddi, 88 2001)) following very short term cyclic hypergravity exposure during parabolic flight (Wehland et 89 al., 2015), suggesting that articular cartilage health would be impaired under such loading 90 conditions. 91 92 mineralization (Gnyubkin, 2015). At 3g they found cortical thinning, more osteoclasts and a 68 reduced rate of bone formation (Gnyubkin, 2015), supporting the idea that loading is beneficial 69 to a point, after which it becomes deleterious (Yokota, Leong and Sun, 2011). Zebrafish larvae 70 have been exposed to hypergravity in a Large Diameter Centrifuge (LDC). Aceto et. al. (2015) 71 exposed zebrafish to 3g and observed increased ossification in the cranial skeleton of larvae 72 exposed to 3g between 5 and 9 days post fertilisation (dpf) (Aceto et al., 2015). 73 74 Another component of the musculoskeletal system sensitive to alterations in biomechanics is 75 cartilage, particularly the articular cartilage of synovial joints. This cartilage has a limited 76 regenerative capacity (Karuppal, 2017) and is important for absorbing load to protect the 77 underlying bone, ensuring the smooth function of joints (Fox, Bedi and Rodeo, 2009). Human 78 bed-rest studies, hind-limb unloading studies in rats and studies performed on mice exposed to 79 real microgravity have demonstrated that loss of mechanical forces lead to cartilage degradation 80 primarily through proteoglycan loss (Souza et al., 2012; Ganse et al., 2015; Willey et al., 2016; 81 Fitzgerald et al., 2019). Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 Studies on astronauts and cosmonauts following long duration space flight found that 59 92% had a minimum of 5% bone loss in at least one skeletal site (LeBlanc et al., 2007) with 60 weight-bearing regions most affected (LeBlanc, Shackelford and Schneider, 1998; Vico et al., 61 2000). Sarcopenia is also observed in microgravity, with decrease in muscle volume of around 62 15% following 4-6 months in microgravity (Adrian LeBlanc et al., 2000). While decreased 63 mechanical loading in microgravity has been uniformly associated with disuse bone loss, 64 exposure to hypergravity has been shown to increase or decrease bone depending on the 65 degree of hypergravity. One study exposed mice to hypergravity for 21 days and found that at 66 2g, there was an improvement in trabecular bone volume, fewer osteoclasts and an increase in 67 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint mineralization (Gnyubkin, 2015). At 3g they found cortical thinning, more osteoclasts and a 68 reduced rate of bone formation (Gnyubkin, 2015), supporting the idea that loading is beneficial 69 to a point, after which it becomes deleterious (Yokota, Leong and Sun, 2011). Zebrafish larvae 70 have been exposed to hypergravity in a Large Diameter Centrifuge (LDC). Aceto et. al. (2015) 71 exposed zebrafish to 3g and observed increased ossification in the cranial skeleton of larvae 72 exposed to 3g between 5 and 9 days post fertilisation (dpf) (Aceto et al., 2015). 73 74 Another component of the musculoskeletal system sensitive to alterations in biomechanics is 75 cartilage, particularly the articular cartilage of synovial joints. This cartilage has a limited 76 regenerative capacity (Karuppal, 2017) and is important for absorbing load to protect the 77 underlying bone, ensuring the smooth function of joints (Fox, Bedi and Rodeo, 2009). Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 Cell culture experiments carried out in microgravity also support the 82 observation of cartilage degradation under reduced loading conditions, with cytoskeletal 83 reorganization and extracellular matrix (ECM) composition altered following short exposures 84 (Van Loon et al., 1995; Freed et al., 1997; Zhang et al., 2003; Ulbrich et al., 2010; Aleshcheva 85 et al., 2013). In comparison to work on cartilage in unloading conditions, less is known about the 86 effect of hypergravity and increased mechanical loading. One study on cultured chondrocytes 87 showed downregulation of BMP4 (crucial in collagen type II and aggrecan synthesis (Reddi, 88 2001)) following very short term cyclic hypergravity exposure during parabolic flight (Wehland et 89 al., 2015), suggesting that articular cartilage health would be impaired under such loading 90 conditions. 91 92 Here, we show that exposure to hypergravity for 48 hours from 3 days post fertilisation (dpf) in 93 zebrafish has no substantial effect on craniofacial cartilage morphology or musculature, but 94 causes significant changes to cartilage material properties, chondrocyte morphology and ECM 95 organisation. We also demonstrate altered strain distribution across the lower jaw following 96 hypergravity exposure, providing an explanation for the cell-level changes. Altogether, this 97 shows that hypergravity exposure in zebrafish larvae between 3-5dpf can induce subtle, but 98 detrimental, changes to cartilage which could become more severe over time. 99 100 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Materials and methods 102 103 Zebrafish husbandry and mutant lines. 104 105 Zebrafish were maintained as described previously (Westerfield, 2000). Experiments were 106 approved by the European Space Agency (ESA) and performed in accordance with UK ASPA 107 regulations. 108 109 Hypergravity experiments 110 Zebrafish were exposed to hypergravity in the Large Diameter Centrifuge (LDC) (Van Loon et 111 al., 2008) at the European Space Research and Technology Centre (ESTEC) for 48 hours from 112 3dpf to 5dpf. The LDC consists of a central axis linked to 2 arms. Samples can be placed in 6 113 gondolas (which can be set to 2 different hypergravity levels) plus 1 central gondola at 1g to 114 control for rotation and possible related Coriolis accelerations (Van Loon, 2007). Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 The larvae 115 were exposed to 3g and 6g, with control larvae located at the central axis, further larvae were 116 maintained at 1g static (Supplementary Figure 1A). In each gondola, 4 petri dishes containing 117 150ml of Danieau’s solution and <35 larvae each were stacked in the centre of an incubator set 118 to a constant temperature of 28°C (Supplementary Figure 1A-D). Larvae were incubated in the 119 dark except during the recording of videos (to monitor survival and swim behaviour during the 120 experiment). Following exposure to hypergravity, 1 petri dish from each gravity condition was 121 reserved for behavioural studies, with the rest fixed in 4% paraformaldehyde (PFA) or bone fix 122 (3.5% formaldehyde in 40mM phosphate buffer) for further analysis. 123 Whole fish measurements 124 125 Larvae were mounted in glycerol and imaged on a Leica MZ10F stereo microscope. Head to tail 126 length was measured using the line function in Fiji (Schindelin et al., 2012) (Supplementary 127 Figure 1E). 128 Zebrafish were maintained as described previously (Westerfield, 2000). Experiments were 106 approved by the European Space Agency (ESA) and performed in accordance with UK ASPA 107 regulations. 108 Hypergravity experiments 110 Zebrafish were exposed to hypergravity in the Large Diameter Centrifuge (LDC) (Van Loon et 111 al., 2008) at the European Space Research and Technology Centre (ESTEC) for 48 hours from 112 3dpf to 5dpf. The LDC consists of a central axis linked to 2 arms. Samples can be placed in 6 113 gondolas (which can be set to 2 different hypergravity levels) plus 1 central gondola at 1g to 114 control for rotation and possible related Coriolis accelerations (Van Loon, 2007). The larvae 115 were exposed to 3g and 6g, with control larvae located at the central axis, further larvae were 116 maintained at 1g static (Supplementary Figure 1A). In each gondola, 4 petri dishes containing 117 150ml of Danieau’s solution and <35 larvae each were stacked in the centre of an incubator set 118 to a constant temperature of 28°C (Supplementary Figure 1A-D). Larvae were incubated in the 119 dark except during the recording of videos (to monitor survival and swim behaviour during the 120 experiment). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint rehydrated to 1 x PBST, permeabilised and bl 134 primary antibodies (Collagen type II [Abcam a 135 Hybridoma Bank] 1:500, L-plastin (Cvejic et a 136 T6793] 1:200) at 4°C overnight (o/n). Samples 137 and incubated in secondary antibodies (Goat a 138 [Thermofisher Scientific], Goat anti-chick 647 139 Where required, larvae were incubated in 5μg 140 [Invitrogen] for 1 hour and washed in PBST pr 141 low melting point agarose and imaged on a Le 142 objective. 143 144 Alcian blue and Alizarin red staining 145 146 Alcian Blue and Alizarin red whole mount larv 147 (Walker and Kimmel, 2007) on larvae fixed in 148 149 Atomic force microscopy 150 151 Atomic Force Microscopy (AFM) was conducte 152 Nanoscope V controller, operating in a PeakF 153 cartilage was investigated in a hydrated state 154 Lawrence et al., 2018). Prior to AFM investiga 155 stained with Alcian blue and Alizarin red (as a 156 glycerol in PBS to prevent structural changes 157 modulus (YM) via Quantitative Nano-mechani 158 [Bruker, Ca, USA] were utilized, having nomin 159 respectively. Cantilevers were calibrated via th 160 data fit to a DMT model accounting for the effe 161 model for indentation. Three fish were investig 162 each fish, three separate 500nm x 500nm reg 163 chondrocytes and hypertrophic chondrocytes, 164 taken per scanned region and their RMS aver 165 normalised to values from 1g static samples to 166 Nigmatullin et al., 2018, 2019; Swift et al., 201 167 rehydrated to 1 x PBST, permeabilised and blocked in 5% horse serum prior to incubation in 134 primary antibodies (Collagen type II [Abcam ab34712] 1:500, A4.1025 [Developmental Studies 135 Hybridoma Bank] 1:500, L-plastin (Cvejic et al., 2008) 1:200, Acetylated tubulin [Sigma-Aldrich 136 T6793] 1:200) at 4°C overnight (o/n). Samples were washed three times in PBST, re-blocked 137 and incubated in secondary antibodies (Goat anti-rabbit 555 [Dylight], Donkey anti-mouse 138 [Thermofisher Scientific], Goat anti-chick 647 [Thermofisher scientific] all at 1:500) o/n at 4°C. 139 Where required, larvae were incubated in 5μg/ml 4′,6-diamidino-2-phenylindole (DAPI) 140 [Invitrogen] for 1 hour and washed in PBST prior to imaging. Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 Samples were mounted in 0.5% 141 low melting point agarose and imaged on a Leica SP5II confocal microscope with a 10x or 20x 142 objective. 143 144 Alcian blue and Alizarin red staining 145 146 Alcian Blue and Alizarin red whole mount larval staining was performed as previously described 147 (Walker and Kimmel, 2007) on larvae fixed in 3.5% formaldehyde. 148 149 Atomic force microscopy 150 151 Atomic Force Microscopy (AFM) was conducted utilizing a Multi-mode VIII microscope with 152 Nanoscope V controller, operating in a PeakForce control regime [Bruker, CA, USA]. Larval 153 cartilage was investigated in a hydrated state in an ambient environment (Elizabeth A. 154 Lawrence et al., 2018). Prior to AFM investigation, larvae were fixed in 4% formaldehyde, 155 stained with Alcian blue and Alizarin red (as above), and the lower jaw was dissected in 1% 156 glycerol in PBS to prevent structural changes induced by drying. For measurement of Young’s 157 modulus (YM) via Quantitative Nano-mechanical Mapping (QNM) RTESPA-150 cantilevers 158 [Bruker, Ca, USA] were utilized, having nominal spring constant and tip radii of 5 N/m and 8 nm 159 respectively. Cantilevers were calibrated via the relative method utilizing a PDMS standard with 160 data fit to a DMT model accounting for the effect of adhesion forces in the standard Hertzian 161 model for indentation. Three fish were investigated for each level of hypergravity exposure. For 162 each fish, three separate 500nm x 500nm regions were scanned in both the immature 163 chondrocytes and hypertrophic chondrocytes, six regions in total. 65,536 measurements were 164 taken per scanned region and their RMS average recorded for comparison. Data was 165 normalised to values from 1g static samples to show the relative YMs (Melbourne et al., 2018; 166 Nigmatullin et al., 2018, 2019; Swift et al., 2018; Terry et al., 2019; Gubała et al., 2020). 167 rehydrated to 1 x PBST, permeabilised and blocked in 5% horse serum prior to incubation in 134 primary antibodies (Collagen type II [Abcam ab34712] 1:500, A4.1025 [Developmental Studies 135 Hybridoma Bank] 1:500, L-plastin (Cvejic et al., 2008) 1:200, Acetylated tubulin [Sigma-Aldrich 136 T6793] 1:200) at 4°C overnight (o/n). Samples were washed three times in PBST, re-blocked 137 and incubated in secondary antibodies (Goat anti-rabbit 555 [Dylight], Donkey anti-mouse 138 [Thermofisher Scientific], Goat anti-chick 647 [Thermofisher scientific] all at 1:500) o/n at 4°C. Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 Following exposure to hypergravity, 1 petri dish from each gravity condition was 121 reserved for behavioural studies, with the rest fixed in 4% paraformaldehyde (PFA) or bone fix 122 (3.5% formaldehyde in 40mM phosphate buffer) for further analysis. 123 Whole fish measurements 124 125 Larvae were mounted in glycerol and imaged on a Leica MZ10F stereo microscope. Head to tail 126 length was measured using the line function in Fiji (Schindelin et al., 2012) (Supplementary 127 Figure 1E). 128 129 Antibody labelling 130 131 Immunohistochemistry was performed as previously described (Hammond and Schulte-Merker, 132 2009). Briefly, larvae were fixed in 4% PFA and dehydrated to 100% MeOH for storage, then 133 Zebrafish were exposed to hypergravity in the Large Diameter Centrifuge (LDC) (Van Loon et 111 al., 2008) at the European Space Research and Technology Centre (ESTEC) for 48 hours from 112 3dpf to 5dpf. The LDC consists of a central axis linked to 2 arms. Samples can be placed in 6 113 gondolas (which can be set to 2 different hypergravity levels) plus 1 central gondola at 1g to 114 control for rotation and possible related Coriolis accelerations (Van Loon, 2007). The larvae 115 were exposed to 3g and 6g, with control larvae located at the central axis, further larvae were 116 maintained at 1g static (Supplementary Figure 1A). In each gondola, 4 petri dishes containing 117 150ml of Danieau’s solution and <35 larvae each were stacked in the centre of an incubator set 118 to a constant temperature of 28°C (Supplementary Figure 1A-D). Larvae were incubated in the 119 dark except during the recording of videos (to monitor survival and swim behaviour during the 120 experiment). Following exposure to hypergravity, 1 petri dish from each gravity condition was 121 reserved for behavioural studies, with the rest fixed in 4% paraformaldehyde (PFA) or bone fix 122 (3.5% formaldehyde in 40mM phosphate buffer) for further analysis. 123 Immunohistochemistry was performed as previously described (Hammond and Schulte-Merker, 132 2009). Briefly, larvae were fixed in 4% PFA and dehydrated to 100% MeOH for storage, then 133 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . Exposure to hypergravity during zebrafish development alters cartilage material 1 properties and strain distribution. 2 139 Where required, larvae were incubated in 5μg/ml 4′,6-diamidino-2-phenylindole (DAPI) 140 [Invitrogen] for 1 hour and washed in PBST prior to imaging. Samples were mounted in 0.5% 141 low melting point agarose and imaged on a Leica SP5II confocal microscope with a 10x or 20x 142 objective. 143 Nanoindentation 170 Nanoindentation 170 Larvae were fixed in 4% PFA and stored in 100% MeOH before rehydration to 30% sucrose in 171 PBS. Samples were submerged in 30% sucrose in PBS, diluted 1:2 in optimum cutting 172 temperature compound (OCT) until they sunk. This solution was refreshed for embedding and 173 samples were flash frozen and sectioned in a coronal orientation using an NX70 Cryostat 174 [ThermoFisher] at a thickness of 10µm. Nanoindentation was performed on sections containing 175 the jaw joint and/or Meckel’s cartilage using a Chiaro nanoindentation device [Optics11, 176 Amsterdam, The Netherlands]. Sections were kept submerged in PBS at room temperature 177 whilst measurements were taken. A spherical nanoindentation probe with an 8µm radius and 178 stiffness of 0.49N/m was used, and tissues were indented to a depth of 1µm with velocity of 179 1µm s-1, with the tip held at a constant depth for 10s. The collected curves were analysed based 180 on Hertzian contact theory for direct comparison with AFM measurements and the resultant YM 181 Ehertz were calculated assuming sample’s incompressibility. Nanoindentation was performed 182 across all sections containing the joint or Meckel’s cartilage, with one measurement collected 183 per region of interest in each section. The resulting YM were averaged for each region across 184 sections. Nanoindentation was performed on five fish from each of the 1g static and 6g spin 185 groups. 186 187 Histological staining 188 189 Fixed samples were processed into paraffin, cut in 5µm sections, deparaffinised and stained 190 with Haematoxylin & Eosin (H&E) and Alcian blue, Picrosirius Red, Safranin O/Fast Green or 191 Masson’s Trichrome. H&E Alcian blue slides were stained in Erhlic's haematoxylin for 5 192 minutes, rinsed, differentiated in acid alcohol and Scotts water, placed in eosin solution for 10 193 seconds, rinsed and immersed in Alcian blue for 30 minutes. Picrosirius Red slides were 194 immersed in picrosirius red for 1 hour and washed in 2 changes of acidified water. Safranin 195 O/Fast Green slides were stained with Weigert's iron haematoxylin for 10 minutes, washed for a 196 further 10 minutes, stained with 0.05% fast green solution for 5 minutes, rinsed in 1% acetic 197 acid and immersed in 0.1% Safranin O solution for 5 minutes. Atomic force microscopy 150 Atomic Force Microscopy (AFM) was conducted utilizing a Multi-mode VIII microscope with 152 Nanoscope V controller, operating in a PeakForce control regime [Bruker, CA, USA]. Larval 153 cartilage was investigated in a hydrated state in an ambient environment (Elizabeth A. 154 Lawrence et al., 2018). Prior to AFM investigation, larvae were fixed in 4% formaldehyde, 155 stained with Alcian blue and Alizarin red (as above), and the lower jaw was dissected in 1% 156 glycerol in PBS to prevent structural changes induced by drying. For measurement of Young’s 157 modulus (YM) via Quantitative Nano-mechanical Mapping (QNM) RTESPA-150 cantilevers 158 [Bruker, Ca, USA] were utilized, having nominal spring constant and tip radii of 5 N/m and 8 nm 159 respectively. Cantilevers were calibrated via the relative method utilizing a PDMS standard with 160 data fit to a DMT model accounting for the effect of adhesion forces in the standard Hertzian 161 model for indentation. Three fish were investigated for each level of hypergravity exposure. For 162 each fish, three separate 500nm x 500nm regions were scanned in both the immature 163 chondrocytes and hypertrophic chondrocytes, six regions in total. 65,536 measurements were 164 taken per scanned region and their RMS average recorded for comparison. Data was 165 normalised to values from 1g static samples to show the relative YMs (Melbourne et al., 2018; 166 Nigmatullin et al., 2018, 2019; Swift et al., 2018; Terry et al., 2019; Gubała et al., 2020). 167 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Nanoindentation 170 Following 201 staining, all sections were dehydrated sequentially to 100% industrial methylate spirit (IMS), 202 immersed in xylene 3 x 5 minutes and mounted using DPX mountant. Slides stained with 203 Picrosirius red were imaged under polarized light on a Leica DMI6000 inverted epifluorescence 204 microscope, all other slides were imaged on Leica MZ10F stereo microscope. 205 206 207 Measurement of staining intensity from histology slides 208 209 To measure the staining intensity of Safranin O and Masson’s trichrome, images were opened 210 in Fiji and the segmented line tool was used to draw a line through the ECM surrounding 211 immature and hypertrophic chondrocytes in the jaw region. The plot profile command was then 212 performed to extract the gray value along this line and measurements normalised to the image 213 background to remove white balance discrepancies. This was performed in ten areas of 214 immature and hypertrophic chondrocytes respectively, with measurements recorded from three 215 fish per gravity condition. 216 217 Cell circularity and area quantification 218 219 Chondrocyte morphology was measured in Fiji from brightfield images of alcian blue stained 220 lower jaw paraffin sections (5µm thick). The polygon selection tool was used to outline 221 chondrocytes at the joint and intercalation zone of the Meckel’s cartilage (Figure 2 A), the area 222 and roundness shape descriptors were collected using the measure command. Cells from the 223 middle of the Meckel’s cartilage were classified as hypertrophic and cells from the jaw joint and 224 Meckel’s symphysis (black asterisk in Figure 2 A) were classified as immature. 225 226 Jaw measurements 227 228 Confocal image stacks of the lower jaw immunostained for type II collagen were loaded in to Fiji 229 (Schindelin et al., 2012) and the line tool, followed by the measure command, were used to take 230 length and width measurements (Figure 1 A). 3D jaw renders and joint measurements were 231 executed as previously described in (Elizabeth A. Lawrence et al., 2018) (Figure 1 D) using 232 Amira 6.0 [FEI]. 233 234 stained with Aniline blue both 5 minutes and dipped in 1% acetic acid for 10 seconds. Following 201 staining, all sections were dehydrated sequentially to 100% industrial methylate spirit (IMS), 202 immersed in xylene 3 x 5 minutes and mounted using DPX mountant. Nanoindentation 170 Slides stained with 203 Picrosirius red were imaged under polarized light on a Leica DMI6000 inverted epifluorescence 204 microscope, all other slides were imaged on Leica MZ10F stereo microscope. 205 206 207 Measurement of staining intensity from histology slides 208 209 To measure the staining intensity of Safranin O and Masson’s trichrome, images were opened 210 in Fiji and the segmented line tool was used to draw a line through the ECM surrounding 211 immature and hypertrophic chondrocytes in the jaw region. The plot profile command was then 212 performed to extract the gray value along this line and measurements normalised to the image 213 background to remove white balance discrepancies. This was performed in ten areas of 214 immature and hypertrophic chondrocytes respectively, with measurements recorded from three 215 fish per gravity condition. 216 217 Cell circularity and area quantification 218 219 Chondrocyte morphology was measured in Fiji from brightfield images of alcian blue stained 220 lower jaw paraffin sections (5µm thick). The polygon selection tool was used to outline 221 chondrocytes at the joint and intercalation zone of the Meckel’s cartilage (Figure 2 A), the area 222 and roundness shape descriptors were collected using the measure command. Cells from the 223 middle of the Meckel’s cartilage were classified as hypertrophic and cells from the jaw joint and 224 Meckel’s symphysis (black asterisk in Figure 2 A) were classified as immature. 225 226 Jaw measurements 227 228 Confocal image stacks of the lower jaw immunostained for type II collagen were loaded in to Fiji 229 (Schindelin et al., 2012) and the line tool, followed by the measure command, were used to take 230 length and width measurements (Figure 1 A). 3D jaw renders and joint measurements were 231 executed as previously described in (Elizabeth A. Lawrence et al., 2018) (Figure 1 D) using 232 Amira 6.0 [FEI]. Nanoindentation 170 Masson’s Trichrome Sections 198 were re-fixed in Bouins solution, stained in Weigert’s iron haematoxylin then Ponceau Fuschin 199 (Masson’s) for 5 minutes, rinsed, immersed in phosphomolybdic acid solution and counter 200 Larvae were fixed in 4% PFA and stored in 100% MeOH before rehydration to 30% sucrose in 171 PBS. Samples were submerged in 30% sucrose in PBS, diluted 1:2 in optimum cutting 172 temperature compound (OCT) until they sunk. This solution was refreshed for embedding and 173 samples were flash frozen and sectioned in a coronal orientation using an NX70 Cryostat 174 [ThermoFisher] at a thickness of 10µm. Nanoindentation was performed on sections containing 175 the jaw joint and/or Meckel’s cartilage using a Chiaro nanoindentation device [Optics11, 176 Amsterdam, The Netherlands]. Sections were kept submerged in PBS at room temperature 177 whilst measurements were taken. A spherical nanoindentation probe with an 8µm radius and 178 stiffness of 0.49N/m was used, and tissues were indented to a depth of 1µm with velocity of 179 1µm s-1, with the tip held at a constant depth for 10s. The collected curves were analysed based 180 on Hertzian contact theory for direct comparison with AFM measurements and the resultant YM 181 Ehertz were calculated assuming sample’s incompressibility. Nanoindentation was performed 182 across all sections containing the joint or Meckel’s cartilage, with one measurement collected 183 per region of interest in each section. The resulting YM were averaged for each region across 184 sections. Nanoindentation was performed on five fish from each of the 1g static and 6g spin 185 groups. 186 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint stained with Aniline blue both 5 minutes and dipped in 1% acetic acid for 10 seconds. Nanoindentation 170 All muscle forces were calculated based on cross-sectional area of the anatomical 264 muscles, with forces reduced to 60% of maximal force (to represent jaw respiratory movement). 265 266 Transmission Electron Microscopy 267 Nanoindentation 170 233 234 stained with Aniline blue both 5 minutes a 201 staining, all sections were dehydrated s 202 immersed in xylene 3 x 5 minutes and 203 Picrosirius red were imaged under polariz 204 microscope, all other slides were imaged 205 206 207 Measurement of staining intensity from 208 209 To measure the staining intensity of Safr 210 in Fiji and the segmented line tool was 211 immature and hypertrophic chondrocytes 212 performed to extract the gray value along 213 background to remove white balance 214 immature and hypertrophic chondrocytes 215 fish per gravity condition. 216 217 Cell circularity and area quantification 218 219 Chondrocyte morphology was measured 220 lower jaw paraffin sections (5µm thick). Th 221 chondrocytes at the joint and intercalation 222 and roundness shape descriptors were co 223 middle of the Meckel’s cartilage were clas 224 Meckel’s symphysis (black asterisk in Fig 225 226 Jaw measurements 227 228 Confocal image stacks of the lower jaw im 229 (Schindelin et al., 2012) and the line tool, 230 length and width measurements (Figure 1 231 executed as previously described in (Eliza 232 Amira 6.0 [FEI]. 233 234 stained with Aniline blue both 5 minutes and dipped in 1% acetic acid for 10 seconds. Following 201 staining, all sections were dehydrated sequentially to 100% industrial methylate spirit (IMS), 202 immersed in xylene 3 x 5 minutes and mounted using DPX mountant. Slides stained with 203 Picrosirius red were imaged under polarized light on a Leica DMI6000 inverted epifluorescence 204 microscope, all other slides were imaged on Leica MZ10F stereo microscope. 205 206 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Muscle quantifications and calculation of muscle forces 235 236 Muscle forces for each muscle group were calculated using methodology from (Brunt et al., 237 2016). Nanoindentation 170 In brief, muscle fibre number and length was quantified manually in Fiji from confocal 238 images of A4.1025 stained zebrafish and the cross-sectional area of the fibers was calculated 239 using the formula: πr2. The radii of the fibres was calculated by taking a measurement across 240 each fibre and dividing it by two. To calculate the cross-sectional area of the whole muscle 241 group, the resulting value was multiplied by the number of muscle fibers. This area was 242 multiplied by the maximal force generated per unit area for larval zebrafish skeletal muscles 243 (40n N/µm2, from (Iorga et al., 2011)) to give the final force value for the muscle. 244 245 Finite Element Analysis. 246 247 Finite Element (FE) models of the lower jaw for 1g static and 6g conditions were created from a 248 confocal image stack of a representative experiment specimen. The same cartilage morphology 249 and muscle forces were used for both conditions’ models in the absence of significant 250 differences in cartilage and muscle morphology between 1g static and 6g specimens (Figure 251 1A-C, H, H’). The FE meshes were developed and modelled for jaw opening and closing 252 movements as previously published (E.A. Lawrence et al., 2018). Two different versions of the 253 model were created, one for the nanoscale properties derived from AFM and the other using the 254 microscale properties measured by nanoindentation. Relative material properties were derived 255 from AFM and nanoindentation experiments (values used are listed in Table 1). For AFM and 256 nanoindentation experiments separately, values were normalised relative to the 1g static 257 hypertrophic chondrocyte Young’s modulus. In both experiments the Young’s modulus of the 258 joint interzones were set at 0.025% of the 1g static hypertrophic chondrocyte Young’s modulus, 259 a markedly lower value than the chondrocyte cells to enable realistic joint movement, and a 260 Poisson’s ratio of 0.495. To reflect the respective experimental Poisson’s ratios, in the 261 nanoindentation experiment hypertrophic and immature chondrocytes Poisson’s ratios were set 262 at 0.495; in the AFM experiment hypertrophic and immature chondrocytes Poisson’s ratios were 263 set at 0.3. All muscle forces were calculated based on cross-sectional area of the anatomical 264 muscles, with forces reduced to 60% of maximal force (to represent jaw respiratory movement). Nanoindentation 170 265 266 Muscle quantifications and calculation of muscle forces 235 236 Muscle forces for each muscle group were calculated using methodology from (Brunt et al., 237 2016). In brief, muscle fibre number and length was quantified manually in Fiji from confocal 238 images of A4.1025 stained zebrafish and the cross-sectional area of the fibers was calculated 239 using the formula: πr2. The radii of the fibres was calculated by taking a measurement across 240 each fibre and dividing it by two. To calculate the cross-sectional area of the whole muscle 241 group, the resulting value was multiplied by the number of muscle fibers. This area was 242 multiplied by the maximal force generated per unit area for larval zebrafish skeletal muscles 243 (40n N/µm2, from (Iorga et al., 2011)) to give the final force value for the muscle. 244 245 Finite Element Analysis. 246 247 Finite Element (FE) models of the lower jaw for 1g static and 6g conditions were created from a 248 confocal image stack of a representative experiment specimen. The same cartilage morphology 249 and muscle forces were used for both conditions’ models in the absence of significant 250 differences in cartilage and muscle morphology between 1g static and 6g specimens (Figure 251 1A-C, H, H’). The FE meshes were developed and modelled for jaw opening and closing 252 movements as previously published (E.A. Lawrence et al., 2018). Two different versions of the 253 model were created, one for the nanoscale properties derived from AFM and the other using the 254 microscale properties measured by nanoindentation. Relative material properties were derived 255 from AFM and nanoindentation experiments (values used are listed in Table 1). For AFM and 256 nanoindentation experiments separately, values were normalised relative to the 1g static 257 hypertrophic chondrocyte Young’s modulus. In both experiments the Young’s modulus of the 258 joint interzones were set at 0.025% of the 1g static hypertrophic chondrocyte Young’s modulus, 259 a markedly lower value than the chondrocyte cells to enable realistic joint movement, and a 260 Poisson’s ratio of 0.495. To reflect the respective experimental Poisson’s ratios, in the 261 nanoindentation experiment hypertrophic and immature chondrocytes Poisson’s ratios were set 262 at 0.495; in the AFM experiment hypertrophic and immature chondrocytes Poisson’s ratios were 263 set at 0.3. Muscle quantifications and calculation of muscle forces 235 Analysis of joint morphology also reveale 296 fish exposed to hypergravity compared to the 1g controls (Figure 1D 297 Given the association of microgravity with muscle loss (Martin, Edge 298 Caiozzo et al., 1994), we stained larvae with the pan-skeletal myosin 299 visualize muscles in the lower jaw (Figure 1I, I’,). From these images 300 Transmission electron microscopy (TEM) was performed on 5dpf larvae fixed in 2.5% 269 glutaraldehyde in 0.1M sodium cacodylate for 1 hour at RT. These samples were embedded in 270 3% agarose before being osmium/uranyl acetate stained, dehydrated and infiltrated with Epon 271 in a Leica EM TP tissue processor using the standard protocol. Prior to sectioning, samples 272 were laterally embedded in 100% Epon and left to harden at 60°C for two days. These blocks 273 were sectioned at a thickness of 70nm on a Leica EM UC7 RT ultramicrotome using a diamond 274 knife [Diatome]. Sections were dried o/n before staining in uranyl acetate for five minutes 275 followed by dH2O washes, five minutes in lead citrate and a final dH2O wash. Sections were 276 imaged on a Tecnai 12 -FEI 120kV BioTwin Spirit Transmission Electron Microscope. 277 278 Analysis of collagen fibre density from TEM images 279 280 To analyse collagen fibre density, TEM images were loaded into Fiji and a region of interest 281 (ROI) of 1μm2 was drawn in a random location containing ECM. The number of collagen fibres 282 in this ROI was counted using the multipoint tool and this process was repeated for five 283 separate regions per image. Four images were analysed from two separate sections per fish, 284 with the sections originating from one fish per gravity condition. 285 286 Results 287 288 Craniofacial cartilage morphology and musculature are unaffected by hypergravity 289 Having confirmed there was no delay in larval growth (Singleman and Holtzman, 2014) following 290 hypergravity exposure (Supplementary Figure 1E), we investigated the effect of increased 291 mechanical loading through hypergravity on developing cartilage, using type II collagen 292 immunostaining, and Alcian blue and Alizarin red double staining to visualize morphology 293 (Supplementary Figure 2A, Figure 1A, A’, H, H’). These analyses did not reveal significant 294 changes to the overall jaw shape, with jaw length and width not significantly changed in 1g static 295 and 6g fish (Figure 1B, C). Muscle quantifications and calculation of muscle forces 235 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Transmission electron microscopy (TEM) was performed on 5dpf lar 269 glutaraldehyde in 0.1M sodium cacodylate for 1 hour at RT. These s 270 3% agarose before being osmium/uranyl acetate stained, dehydrate 271 in a Leica EM TP tissue processor using the standard protocol. Prior 272 were laterally embedded in 100% Epon and left to harden at 60°C fo 273 were sectioned at a thickness of 70nm on a Leica EM UC7 RT ultram 274 knife [Diatome]. Sections were dried o/n before staining in uranyl ace 275 followed by dH2O washes, five minutes in lead citrate and a final dH 276 imaged on a Tecnai 12 -FEI 120kV BioTwin Spirit Transmission Elec 277 278 Analysis of collagen fibre density from TEM images 279 280 To analyse collagen fibre density, TEM images were loaded into Fiji 281 (ROI) of 1μm2 was drawn in a random location containing ECM. The 282 in this ROI was counted using the multipoint tool and this process wa 283 separate regions per image. Four images were analysed from two se 284 with the sections originating from one fish per gravity condition. 285 286 Results 287 288 Craniofacial cartilage morphology and musculature are unaffec 289 Having confirmed there was no delay in larval growth (Singleman an 290 hypergravity exposure (Supplementary Figure 1E), we investigated t 291 mechanical loading through hypergravity on developing cartilage, us 292 immunostaining, and Alcian blue and Alizarin red double staining to 293 (Supplementary Figure 2A, Figure 1A, A’, H, H’). These analyses did 294 changes to the overall jaw shape, with jaw length and width not sign 295 and 6g fish (Figure 1B, C). Muscle quantifications and calculation of muscle forces 235 Analysis of joint morphology also revealed no significant difference in 296 fish exposed to hypergravity compared to the 1g controls (Figure 1D - G). 297 Given the association of microgravity with muscle loss (Martin, Edgerton and Grindeland, 1988; 298 Caiozzo et al., 1994), we stained larvae with the pan-skeletal myosin marker A4.1025 to 299 visualize muscles in the lower jaw (Figure 1I, I’,). From these images we quantified muscle fibre 300 Transmission electron microscopy (TEM) was performed on 5dpf larvae fixed in 2.5% 269 glutaraldehyde in 0.1M sodium cacodylate for 1 hour at RT. These samples were embedded in 270 3% agarose before being osmium/uranyl acetate stained, dehydrated and infiltrated with Epon 271 in a Leica EM TP tissue processor using the standard protocol. Prior to sectioning, samples 272 were laterally embedded in 100% Epon and left to harden at 60°C for two days. These blocks 273 were sectioned at a thickness of 70nm on a Leica EM UC7 RT ultramicrotome using a diamond 274 knife [Diatome]. Sections were dried o/n before staining in uranyl acetate for five minutes 275 followed by dH2O washes, five minutes in lead citrate and a final dH2O wash. Sections were 276 imaged on a Tecnai 12 -FEI 120kV BioTwin Spirit Transmission Electron Microscope. 277 278 Muscle quantifications and calculation of muscle forces 235 Muscle forces for each muscle group were calculated using methodology from (Brunt et al., 237 2016). In brief, muscle fibre number and length was quantified manually in Fiji from confocal 238 images of A4.1025 stained zebrafish and the cross-sectional area of the fibers was calculated 239 using the formula: πr2. The radii of the fibres was calculated by taking a measurement across 240 each fibre and dividing it by two. To calculate the cross-sectional area of the whole muscle 241 group, the resulting value was multiplied by the number of muscle fibers. This area was 242 multiplied by the maximal force generated per unit area for larval zebrafish skeletal muscles 243 (40n N/µm2, from (Iorga et al., 2011)) to give the final force value for the muscle. 244 245 247 Finite Element (FE) models of the lower jaw for 1g static and 6g conditions were created from a 248 confocal image stack of a representative experiment specimen. The same cartilage morphology 249 and muscle forces were used for both conditions’ models in the absence of significant 250 differences in cartilage and muscle morphology between 1g static and 6g specimens (Figure 251 1A-C, H, H’). The FE meshes were developed and modelled for jaw opening and closing 252 movements as previously published (E.A. Lawrence et al., 2018). Two different versions of the 253 model were created, one for the nanoscale properties derived from AFM and the other using the 254 microscale properties measured by nanoindentation. Relative material properties were derived 255 from AFM and nanoindentation experiments (values used are listed in Table 1). For AFM and 256 nanoindentation experiments separately, values were normalised relative to the 1g static 257 hypertrophic chondrocyte Young’s modulus. In both experiments the Young’s modulus of the 258 joint interzones were set at 0.025% of the 1g static hypertrophic chondrocyte Young’s modulus, 259 a markedly lower value than the chondrocyte cells to enable realistic joint movement, and a 260 Poisson’s ratio of 0.495. To reflect the respective experimental Poisson’s ratios, in the 261 nanoindentation experiment hypertrophic and immature chondrocytes Poisson’s ratios were set 262 at 0.495; in the AFM experiment hypertrophic and immature chondrocytes Poisson’s ratios were 263 set at 0.3. All muscle forces were calculated based on cross-sectional area of the anatomical 264 muscles, with forces reduced to 60% of maximal force (to represent jaw respiratory movement). 265 266 Transmission Electron Microscopy 267 . Craniofacial cartilage morphology and musculature are unaffected by hypergravity 289 Having confirmed there was no delay in larval growth (Singleman and Holtzman, 2014) following 290 hypergravity exposure (Supplementary Figure 1E), we investigated the effect of increased 291 mechanical loading through hypergravity on developing cartilage, using type II collagen 292 immunostaining, and Alcian blue and Alizarin red double staining to visualize morphology 293 (Supplementary Figure 2A, Figure 1A, A’, H, H’). These analyses did not reveal significant 294 changes to the overall jaw shape, with jaw length and width not significantly changed in 1g static 295 and 6g fish (Figure 1B, C). Analysis of joint morphology also revealed no significant difference in 296 fish exposed to hypergravity compared to the 1g controls (Figure 1D - G). 297 Given the association of microgravity with muscle loss (Martin, Edgerton and Grindeland, 1988; 298 Caiozzo et al., 1994), we stained larvae with the pan-skeletal myosin marker A4.1025 to 299 visualize muscles in the lower jaw (Figure 1I, I’,). From these images we quantified muscle fibre 300 Given the association of microgravity with muscle loss (Martin, Edgerton and Grindeland, 1988; 298 Caiozzo et al., 1994), we stained larvae with the pan-skeletal myosin marker A4.1025 to 299 visualize muscles in the lower jaw (Figure 1I, I’,). From these images we quantified muscle fibre 300 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint number, length and force, with no significant differences in craniofacial muscle seen between 301 zebrafish incubated in normal gravity and at 6g between 3-5 dpf (Figure 1J, K, L). 302 303 Material properties are altered in the lower jaw 304 305 Changes to mechanical loading have been observed to change skeletal stiffness at both at the 306 nano and micro scales (Turko et al., 2017). Craniofacial cartilage morphology and musculature are unaffected by hypergravity 289 This led us to investigate the relative material 307 properties of lower jaw cartilage using AFM (Melbourne et al., 2018; Nigmatullin et al., 2018, 308 2019; Swift et al., 2018; Terry et al., 2019; Gubała et al., 2020). Measurements were taken from 309 areas of immature chondrocytes and hypertrophic chondrocytes (location of measurements 310 shown by schematics in Figure 1M, N). In both instances, a positive correlation between the 311 magnitude of gravitational exposure and the measured YM was seen. Fish from 3g and 6g had 312 a significantly higher YM than 1g static or 1g spin fish (Figure 1M,N)), with 6g showing a 313 significant increase in YM compared to 3g fish. This trend represents a stiffening of the cartilage 314 following hypergravity exposure during development. 315 316 Within complex materials, different structures can have a greater influence on stiffness at 317 different length scales. Having used AFM to measure YM at the nano-scale we employed 318 nanoindentation to investigate material properties of lower jaw cartilage at the micro-scale. 319 Measurements from nanoindentation show 6g fish had a significantly higher YM (Ehertz) in 320 hypertrophic chondrocytes when compared to 1g static fish. This pattern of increased stiffness 321 was not seen for immature chondrocytes (Figure 1O). 322 323 Exposure to hypergravity affects chondrocyte maturation and behaviour 324 325 The impact of hypergravity at a cellular level was evaluated by measuring chondrocyte 326 morphology from Alcian blue stained lower jaw sections (Figure 2A - C). Chondrocyte area was 327 significantly reduced in immature and hypertrophic regions in 6g fish, with immature cells at the 328 joint and Meckel’s symphysis showing the largest area reduction (Figure 2B). Immature 329 chondrocytes also showed a significant decrease in circularity (Figure 2C). The regions of most 330 change to cell morphology were co-localised to muscle attachment sites in the mid-Meckel’s 331 cartilage and at the jaw joint. This suggests that short exposure to hypergravity alters 332 chondrocyte behaviour, causing cartilage and resulting bones to develop abnormally if 333 number, length and force, with no significant differences in craniofacial muscle seen between 301 zebrafish incubated in normal gravity and at 6g between 3-5 dpf (Figure 1J, K, L). 302 303 Material properties are altered in the lower jaw 304 305 Changes to mechanical loading have been observed to change skeletal stiffness at both at the 306 nano and micro scales (Turko et al., 2017). Craniofacial cartilage morphology and musculature are unaffected by hypergravity 289 This led us to investigate the relative material 307 properties of lower jaw cartilage using AFM (Melbourne et al., 2018; Nigmatullin et al., 2018, 308 2019; Swift et al., 2018; Terry et al., 2019; Gubała et al., 2020). Measurements were taken from 309 areas of immature chondrocytes and hypertrophic chondrocytes (location of measurements 310 shown by schematics in Figure 1M, N). In both instances, a positive correlation between the 311 magnitude of gravitational exposure and the measured YM was seen. Fish from 3g and 6g had 312 a significantly higher YM than 1g static or 1g spin fish (Figure 1M,N)), with 6g showing a 313 significant increase in YM compared to 3g fish. This trend represents a stiffening of the cartilage 314 following hypergravity exposure during development. 315 316 Within complex materials, different structures can have a greater influence on stiffness at 317 different length scales. Having used AFM to measure YM at the nano-scale we employed 318 nanoindentation to investigate material properties of lower jaw cartilage at the micro-scale. 319 Measurements from nanoindentation show 6g fish had a significantly higher YM (Ehertz) in 320 hypertrophic chondrocytes when compared to 1g static fish. This pattern of increased stiffness 321 was not seen for immature chondrocytes (Figure 1O). 322 323 Exposure to hypergravity affects chondrocyte maturation and behaviour 324 325 The impact of hypergravity at a cellular level was evaluated by measuring chondrocyte 326 morphology from Alcian blue stained lower jaw sections (Figure 2A - C). Chondrocyte area was 327 significantly reduced in immature and hypertrophic regions in 6g fish, with immature cells at the 328 joint and Meckel’s symphysis showing the largest area reduction (Figure 2B). Immature 329 chondrocytes also showed a significant decrease in circularity (Figure 2C). The regions of most 330 change to cell morphology were co-localised to muscle attachment sites in the mid-Meckel’s 331 cartilage and at the jaw joint. This suggests that short exposure to hypergravity alters 332 chondrocyte behaviour, causing cartilage and resulting bones to develop abnormally if 333 number, length and force, with no significant differences in craniofacial muscle seen between 301 zebrafish incubated in normal gravity and at 6g between 3-5 dpf (Figure 1J, K, L). 302 Exposure to hypergravity affects chondrocyte maturation and behaviour 24 344 345 The effect of hypergravity exposure on other ECM components was examined using Safranin O 346 Masson’s trichrome, and picrosirius red staining on sections of the lower jaw including the joint 347 and Meckel’s cartilage. Proteoglycan content and mineralisation of the cartilage ECM was 348 visualized with Safranin O/ Fast Green staining which marks the cartilage red, according to the 349 amount of proteoglycan present, and bone in green (Figure 2D’). The intensity of this stain was 350 measured, revealing fish from the 6g condition had a significantly lower staining intensity in 351 regions of immature and hypertrophic chondrocytes than 1g static fish (Figure 2E), 352 corresponding to a reduction in proteoglycan content in the cartilage following hypergravity 353 exposure. This reduction was more pronounced in regions of ECM surrounding immature 354 chondrocytes (Figure 2E). No areas of mineralisation were seen in the stained sections so no 355 information on bone formation could be gathered from this technique. 356 357 Masson’s trichrome staining was used to test whether hypergravity impacted collagen content in 358 chondrocyte ECM (Figure 2D’’). This stain shows collagen in blue, and measurements of 359 staining intensity showed no significant change to collagen content in the ECM surrounding 360 immature or hypertrophic chondrocytes in 6g fish compared to the ECM in 1g static fish (Figure 361 2F). Alongside measuring total collagen in the ECM through Masson’s trichrome, picrosirius red 362 staining was used to assess the balance of type I and type III collagen fibers in the ECM. Under 363 polarized light, ECM surrounding immature and hypertrophic chondrocytes appeared red/orange 364 (Figure 2D’’’), indicating a predominance of type I collagen fibres over type III (which would give 365 green birefringence). This was unchanged in zebrafish from the 6g condition (Figure 2D’’’). 366 367 Hypergravity causes changes to collagen fibre packing in the ECM 368 Exposure to hypergravity affects chondrocyte maturation and behaviour 24 356 357 Masson’s trichrome staining was used to test whether hypergravity impacted collagen content in 358 335 Histological staining reveals subtle changes to the ECM surrounding chondrocytes in 336 areas of altered cell morphology 337 338 As increased mechanical loading is associated with higher glycosaminoglycan (GAG) synthesis 339 and decreased extracellular matrix (ECM) secretion (Schröder et al., 2019), we performed 340 wholemount Alcian blue and Alizarin red double staining (Figure 1H, H’) and Alcian blue, H & E 341 on sections to visualize GAGs throughout the lower jaw (Figure 2D). From brightfield images, 342 we observed no changes to mineralisation or GAGs at 5dpf (Figure 1H, H’, 2D), suggesting 343 secretion of this ECM component is unaffected by altered loading. 344 345 The effect of hypergravity exposure on other ECM components was examined using Safranin O, 346 Masson’s trichrome, and picrosirius red staining on sections of the lower jaw including the joint 347 and Meckel’s cartilage. Proteoglycan content and mineralisation of the cartilage ECM was 348 visualized with Safranin O/ Fast Green staining which marks the cartilage red, according to the 349 amount of proteoglycan present, and bone in green (Figure 2D’). The intensity of this stain was 350 measured, revealing fish from the 6g condition had a significantly lower staining intensity in 351 regions of immature and hypertrophic chondrocytes than 1g static fish (Figure 2E), 352 corresponding to a reduction in proteoglycan content in the cartilage following hypergravity 353 exposure. This reduction was more pronounced in regions of ECM surrounding immature 354 chondrocytes (Figure 2E). No areas of mineralisation were seen in the stained sections so no 355 information on bone formation could be gathered from this technique. 356 357 335 Histological staining reveals subtle changes to the ECM surrounding chondrocytes in 336 areas of altered cell morphology 337 338 As increased mechanical loading is associated with higher glycosaminoglycan (GAG) synthesis 339 and decreased extracellular matrix (ECM) secretion (Schröder et al., 2019), we performed 340 wholemount Alcian blue and Alizarin red double staining (Figure 1H, H’) and Alcian blue, H & E 341 on sections to visualize GAGs throughout the lower jaw (Figure 2D). From brightfield images, 342 we observed no changes to mineralisation or GAGs at 5dpf (Figure 1H, H’, 2D), suggesting 343 secretion of this ECM component is unaffected by altered loading. Exposure to hypergravity affects chondrocyte maturation and behaviour 24 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint 335 Histological staining reveals subtle changes to the ECM surrounding chondrocytes in 336 areas of altered cell morphology 337 338 As increased mechanical loading is associated with higher glycosaminoglycan (GAG) synthesis 339 and decreased extracellular matrix (ECM) secretion (Schröder et al., 2019), we performed 340 wholemount Alcian blue and Alizarin red double staining (Figure 1H, H’) and Alcian blue, H & E 341 on sections to visualize GAGs throughout the lower jaw (Figure 2D). From brightfield images, 342 we observed no changes to mineralisation or GAGs at 5dpf (Figure 1H, H’, 2D), suggesting 343 secretion of this ECM component is unaffected by altered loading. 344 345 The effect of hypergravity exposure on other ECM components was examined using Safranin O, 346 Masson’s trichrome, and picrosirius red staining on sections of the lower jaw including the joint 347 and Meckel’s cartilage. Proteoglycan content and mineralisation of the cartilage ECM was 348 visualized with Safranin O/ Fast Green staining which marks the cartilage red, according to the 349 amount of proteoglycan present, and bone in green (Figure 2D’). The intensity of this stain was 350 measured, revealing fish from the 6g condition had a significantly lower staining intensity in 351 regions of immature and hypertrophic chondrocytes than 1g static fish (Figure 2E), 352 corresponding to a reduction in proteoglycan content in the cartilage following hypergravity 353 exposure. This reduction was more pronounced in regions of ECM surrounding immature 354 chondrocytes (Figure 2E). No areas of mineralisation were seen in the stained sections so no 355 information on bone formation could be gathered from this technique. Hypergravity causes changes to collagen fibre packing in the ECM 368 Both methods for obtaining material properties of the 392 cartilage show similar change in strain pattern distribution following hypergravity exposure (from 393 1g static to 6g). Similarly, the pattern of von Mises stress is more evenly distributed throughout 394 the jaw in the 1g static condition, whereas in the 6g condition stress is localised in regions 395 already experiencing high stress, specifically the regions surrounding the joints (Supplementary 396 Figure 3). This pattern is true for both opening and closing movements. The altered patterns of 397 strain observed, in which the largest differences are close to the joint could provide an 398 explanation for the subtle changes to cell maturation, which were more pronounced at the joint, 399 and to changes observed to matrix packing. 400 401 369 To further explore how the cartilage ECM is affected by hypergravity, TEM was carried out on 370 sections of ear cartilage, which are comparable to regions of hypertrophic chondrocytes in the 371 lower jaw where the most significant change to material properties was seen. From the 372 micrographs, subtle changes to the collagen fibre packing were observed (Figure 2G), with 373 fibres appearing closer together in 6g fish. This observation was strengthened by quantificatio 374 of fibre density, which revealed a trend for increased fibre density in 6g fish (Figure 2H). 375 376 Taken together with histology data, this suggests that hypergravity induces macromolecular 377 changes in the cartilage which give rise to slight changes at the tissue level. If the larvae had 378 been maintained in hypergravity for longer, it is likely that these changes would have become 379 more pronounced, leading to more severe changes to the tissue. 380 381 Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 exposure 383 384 To assess whether cell and matrix changes could be correlated with altered strain distribution 385 the lower jaw, FE models were generated. As no changes in lower jaw morphology or 386 musculature were observed (Figure 1A-C, H, H’), the same volumetric model and muscle force 387 were used for both gravity conditions. Different normalised material properties from AFM (Figu 388 3A, A’) and nanoindentation (Figure 3B, B’) were applied to these models (Supplementary Tab 389 1). Hypergravity causes changes to collagen fibre packing in the ECM 368 Hypergravity causes changes to collagen fibre packing in the ECM 368 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint 369 To further explore how the cartilage ECM is affected by hypergravity, TEM was carried out on 370 sections of ear cartilage, which are comparable to regions of hypertrophic chondrocytes in the 371 lower jaw where the most significant change to material properties was seen. From the 372 micrographs, subtle changes to the collagen fibre packing were observed (Figure 2G), with 373 fibres appearing closer together in 6g fish. This observation was strengthened by quantification 374 of fibre density, which revealed a trend for increased fibre density in 6g fish (Figure 2H). 375 376 Taken together with histology data, this suggests that hypergravity induces macromolecular 377 changes in the cartilage which give rise to slight changes at the tissue level. If the larvae had 378 been maintained in hypergravity for longer, it is likely that these changes would have become 379 more pronounced, leading to more severe changes to the tissue. 380 381 Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 exposure 383 384 To assess whether cell and matrix changes could be correlated with altered strain distribution in 385 the lower jaw, FE models were generated. As no changes in lower jaw morphology or 386 musculature were observed (Figure 1A-C, H, H’), the same volumetric model and muscle forces 387 were used for both gravity conditions. Different normalised material properties from AFM (Figure 388 3A, A’) and nanoindentation (Figure 3B, B’) were applied to these models (Supplementary Table 389 1). From these models, it can be seen that maximum principal strain is more localised to the 390 joint regions in 6g fish with stiffer cartilage, whereas in 1g fish strain is distributed over a larger 391 area of cartilage elements (Figure 3A-B’). Hypergravity causes changes to collagen fibre packing in the ECM 368 From these models, it can be seen that maximum principal strain is more localised to the 390 joint regions in 6g fish with stiffer cartilage, whereas in 1g fish strain is distributed over a larger 391 area of cartilage elements (Figure 3A-B’). Both methods for obtaining material properties of the 392 cartilage show similar change in strain pattern distribution following hypergravity exposure (fro 393 1g static to 6g). Similarly, the pattern of von Mises stress is more evenly distributed throughou 394 the jaw in the 1g static condition, whereas in the 6g condition stress is localised in regions 395 already experiencing high stress, specifically the regions surrounding the joints (Supplementar 396 Figure 3). This pattern is true for both opening and closing movements. The altered patterns o 397 strain observed, in which the largest differences are close to the joint could provide an 398 explanation for the subtle changes to cell maturation, which were more pronounced at the join 399 and to changes observed to matrix packing. 400 401 Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 402 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Discussion 403 404 The impact of microgravity on the musculoskeletal system has been well studied, with exposure 405 to below-Earth gravity linked to muscle loss and decreased bone density. In comparison, 406 relatively little is known about how short-term hypergravity exposure affects the musculoskeletal 407 system. Here, we show that in zebrafish embryos gross cartilage and muscle morphology is 408 unchanged, but cartilage material properties and its resulting biomechanical performance is 409 affected by short-term exposure to hypergravity during development. We also show that 410 hypergravity leads to altered chondrocyte maturation and subtle changes to the surrounding 411 ECM, which may lead to more dramatic changes to the cartilage over time. 412 413 Gravity is important for cartilage health as it provides a loading force essential for cartilage 414 homeostasis and prevention of degenerative diseases such as OA (Penninx et al., 2001; Bader, 415 Salter and Chowdhury, 2011; Musumeci, Szychlinska and Mobasheri, 2015; Mellor et al., 2017). 416 Although cartilage morphology and skeletal muscle mass has been shown to be affected by 417 altered loading conditions (Vanwanseele et al., 2003; Hinterwimmer et al., 2004; Liphardt et al., 418 2009; Gao et al., 2018), our data suggest that two days of hypergravity exposure is not sufficient 419 to cause gross morphological changes in larval zebrafish. One explanation for this is the length 420 of exposure being insufficient to induce musculoskeletal remodeling, as previous studies have 421 found that longer exposure to non-Earth gravity leads to more severe musculoskeletal 422 transformations (A LeBlanc et al., 2000; Demontis et al., 2017). Another explanation for the lack 423 of morphological alterations is that the level of hypergravity was not high enough to induce 424 changes. However, it has previously been shown by Aceto et. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 al (Aceto et al., 2015) that 425 exposure to 3g between 5 and 9 dpf is sufficient to induce skeletal changes in zebrafish. This 426 indicates that the age at which the zebrafish are exposed to hypergravity is also crucial (Franz- 427 Odendaal and Edsall, 2018). Thus, we hypothesise that extending the amount of time larvae 428 spend in hypergravity to include later time points key in musculoskeletal development would 429 lead to more dramatic changes. 430 431 The hypothesis that more severe cartilage abnormalities would be seen following a lengthier 432 Discussion 403 404 The impact of microgravity on the musculoskeletal system has been well studied, with exposure 405 to below-Earth gravity linked to muscle loss and decreased bone density. In comparison, 406 relatively little is known about how short-term hypergravity exposure affects the musculoskeletal 407 system. Here, we show that in zebrafish embryos gross cartilage and muscle morphology is 408 unchanged, but cartilage material properties and its resulting biomechanical performance is 409 affected by short-term exposure to hypergravity during development. We also show that 410 hypergravity leads to altered chondrocyte maturation and subtle changes to the surrounding 411 ECM, which may lead to more dramatic changes to the cartilage over time. 412 413 Gravity is important for cartilage health as it provides a loading force essential for cartilage 414 homeostasis and prevention of degenerative diseases such as OA (Penninx et al., 2001; Bader, 415 Salter and Chowdhury, 2011; Musumeci, Szychlinska and Mobasheri, 2015; Mellor et al., 2017). 416 Although cartilage morphology and skeletal muscle mass has been shown to be affected by 417 altered loading conditions (Vanwanseele et al., 2003; Hinterwimmer et al., 2004; Liphardt et al., 418 2009; Gao et al., 2018), our data suggest that two days of hypergravity exposure is not sufficient 419 to cause gross morphological changes in larval zebrafish. One explanation for this is the length 420 of exposure being insufficient to induce musculoskeletal remodeling, as previous studies have 421 found that longer exposure to non-Earth gravity leads to more severe musculoskeletal 422 transformations (A LeBlanc et al., 2000; Demontis et al., 2017). Another explanation for the lack 423 of morphological alterations is that the level of hypergravity was not high enough to induce 424 changes. However, it has previously been shown by Aceto et. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 al (Aceto et al., 2015) that 425 exposure to 3g between 5 and 9 dpf is sufficient to induce skeletal changes in zebrafish. This 426 indicates that the age at which the zebrafish are exposed to hypergravity is also crucial (Franz- 427 Odendaal and Edsall, 2018). Thus, we hypothesise that extending the amount of time larvae 428 spend in hypergravity to include later time points key in musculoskeletal development would 429 lead to more dramatic changes. 430 431 The hypothesis that more severe cartilage abnormalities would be seen following a lengthier 432 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint craniofacial cartilage elements. In these areas of altered strain, we observed abnormal 437 chondrocyte maturation, which over time would likely give rise to altered joint shape and 438 cartilage morphology. In conclusion short-term exposure to hypergravity in early development 439 causes changes to ECM content and organisation in zebrafish which could induce more 440 dramatic structural and morphological changes to the musculoskeletal system over extended 441 periods of exposure. 442 443 Competing interests 444 445 The authors declare no competing interests. 446 447 Funding 448 449 EL and JA received funding from the ESA ‘Spin Your Thesis! 2018’ program. EL is also funded 450 by the Wellcome Trust Dynamic Molecular Cell Biology PhD programme. JG is funded by a PhD 451 studentship from the Anatomical Society. PeakForce AFM was carried out with equipment 452 funded by EPSRC under Grant “Atoms to Applications”; Grant ref. (EP/K035746/1). CLH is 453 funded by Versus Arthritis Senior Fellowship 21937. 454 455 Acknowledgements 456 457 The authors would like to thank Kate Robson-Brown for her invaluable expertise, guidance and 458 backing throughout the project. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 They would also like to express their gratitude to the ESA 459 Education team, particularly Nigel Savage and Evelien Lageweg, and Alan Dowson at the LDC 460 for their support and advice throughout the ‘Spin Your Thesis!’ campaign. Finally, they would 461 like to thank David Labonte and Andrea Attipoe for the use of the Chiaro nanoindentation device 462 and for their helpful assistance. 463 464 References 465 466 Aceto, J. et al. 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It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint 677 Figure 1: Lower jaw morphology and musculature are unchanged following hypergravity 678 exposure, but changes to cartilage material properties are observed. (A, A’) 3D surface 679 renders from confocal image stacks of lower jaw cartilage in ventral orientation from 1g static 680 (A) and 6g (A’) zebrafish at 5dpf (MC = Meckel’s cartilage, PQ = palatoquadrate, C = 681 ceratohyal). Scale bar: 100μm. (B, C) Quantification of lower jaw length (B) and width (C). 682 Location of measurements shown by red (width) and blue (length) line in (A) (n=8 for all, 683 different symbols = individual fish). (D) Close-up image of jaw joint from 1g static 3D render, 684 position in lower jaw shown by white dashed box in (A). Orientation compass: A, anterior; L, 685 lateral; M, medial; P, posterior. (E- G) Quantification of joint neck (E) and joint head (F) width, 686 and joint space (G), location of measurements shown in (D), red line = joint neck, yellow line = 687 joint head, white line = joint space (n=8 for all). (H, H’) Brightfield images of Alcian blue Alizarin 688 red stained lower jaws from 1g static (H) and 6g (H’) conditions. Scale bar = 100μm. (I, I’) 689 Maximum projections of ventral confocal image stacks from 5dpf 1g static (I) and 6g (I’) 690 zebrafish immunostained for myosin (A4.1025) (IA = Intermandibularis anterior, IP = 691 Intermandibularis posterior, IH = Interhyoideus, HH-I = Hyohyoideus inferior). Scale bar = 692 100μm. (J, K) Quantification muscle fibre number (J) and length (K) measured from confocal 693 image stacks. Location of muscle groups shown in (I). (L) Quantification of craniofacial muscle 694 forces. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 (M, N) Relative YM values from AFM for immature (M) and hypertrophic (N) 695 chondrocytes from 1g static, 1g, 3g and 6g zebrafish (n=3 for all). Location of measurements 696 taken shown in schematic above graphs. (O) Material properties determined by nanoindentation 697 in 1g static and 6g zebrafish. Data is mean with s.e.m. (E-G show mean with no s.e.m.), 698 D’Agostino and Pearson test performed for all data, followed by student’s unpaired t-test in B, C, 699 E, F and G. One-way ANOVA performed within muscle groups in J, K and L and in M and N. 700 Mann-Whitney u-test in O. * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001, **** p ≤ 0.0001. 701 702 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint picrosirius red (D’’’) stained ventral sections in 1g static and 6g fish. Asterisk = centre of joint, 709 dotted line in (D’’’) = outline of cartilage element from section. Scale bar = 10μm. MC = Meckel’s 710 cartilage, PQ = palatoquadrate, C = ceratohyal. (E, F) Quantification of Safranin O (E) and 711 Masson’s trichrome (F) staining intensity. (G) Electron micrographs of hypertrophic 712 chondrocytes in 1g static and 6g zebrafish. Dashed areas = higher magnification images 713 displayed in the centre and to the right of the panel, red lines = chondrocyte borders. Scale bars 714 are shown below each image. (H) Quantification of collagen fibre density in the ECM of 1g static 715 and 6g fish (n = 1 for both). All data is mean with s.e.m. D’Agostino and Pearson normality test 716 performed in B, C, E and F: followed by Mann-Whitney u-test in B and C, and student’s 717 unpaired t-test in E and F. * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001, **** p ≤ 0.0001. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 718 719 Figure 3: Altered ECM characteristics could result from altered strain distribution in the 720 lower jaw following hypergravity exposure (A,A’) FE models of maximum principal strain 721 incorporating relative material property values from AFM in 1g static (A) and 6g zebrafish (A’). 722 (B,B’) FE models of maximum principal strain incorporating relative material property values 723 from nanoindentation in 1g static (B) and 6g zebrafish (B’). Black arrowheads = areas of high 724 strain; black asterisks = jaw joints; red asterisks = Meckel’s symphysis. Ventral and lateral views 725 shown for opening step in both gravity conditions. 726 727 Table 1: Actual and relative material property values of immature and hypertrophic 728 chondrocytes from AFM and nanoindentation which were used for FE models. Values represent 729 the mean measurement across samples and the figure number of the corresponding FE model 730 is shown in the right-hand column. 731 732 Table 2: Muscle forces used for FE model generation. Values for 1g static and 6g spin fish 733 represent 60% of the maximum muscle force calculated for each muscle group in Figure 1 L 734 with the exception of the adductor mandibulae. 735 736 picrosirius red (D’’’) stained ventral sections in 1g static and 6g fish. Asterisk = centre of joint, 709 dotted line in (D’’’) = outline of cartilage element from section. Scale bar = 10μm. MC = Meckel’s 710 cartilage, PQ = palatoquadrate, C = ceratohyal. (E, F) Quantification of Safranin O (E) and 711 Masson’s trichrome (F) staining intensity. (G) Electron micrographs of hypertrophic 712 chondrocytes in 1g static and 6g zebrafish. Dashed areas = higher magnification images 713 displayed in the centre and to the right of the panel, red lines = chondrocyte borders. Scale bars 714 are shown below each image. (H) Quantification of collagen fibre density in the ECM of 1g static 715 and 6g fish (n = 1 for both). All data is mean with s.e.m. D’Agostino and Pearson normality test 716 performed in B, C, E and F: followed by Mann-Whitney u-test in B and C, and student’s 717 unpaired t-test in E and F. * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001, **** p ≤ 0.0001. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 718 9 1g static 6g spin MC PQ C MC PQ C A B C E F G IA IP IH HH-I 1g static 6g spin H I I’ MC PQ D A P L M A’ H’ 1g static 6g spin 0 50 150 200 250 300 Width (µm) Width of lower jaw 1g static 6g spin 0 50 300 350 400 450 Length of lower jaw Length (!m) 1g static 6g spin 0 5 30 35 40 45 50 Width (µm) Joint head width 1g static 6g spin 0 2 4 6 8 10 Width of space (µm) Joint space 1g static 6g spin 0 2 20 22 24 26 28 30 32 Width (µm) Joint neck width Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Fish 6 Fish 7 Fish 8 1g static 6g spin 0 2 20 22 24 26 28 30 32 Width (µm) Joint neck width Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Fish 6 Fish 7 Fish 8 a4.1025 Alcian blue Alizarin red 1g static 6g spin Figure 1 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint B C 1g static 6g spin 0 50 150 200 250 300 Width (µm) Width of lower jaw 1g static 6g spin 0 50 300 350 400 450 Length of lower jaw Length (!m) Figure 1 . CC-BY 4.0 International license r a o has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; 46 6g spin MC PQ C A’ available unde eer review) is the author/funder, who ://doi.org/10.1101/2020.05.26.11604 B C 1g static MC PQ C A was not certified by p https doi: bioRxiv preprint A’ A C 1g static 6g spin 0 50 150 200 250 300 Width (µm) Width of lower jaw g y the preprint in perpetuity. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 718 719 Figure 3: Altered ECM characteristics could result from altered strain distribution in the 720 lower jaw following hypergravity exposure (A,A’) FE models of maximum principal strain 721 incorporating relative material property values from AFM in 1g static (A) and 6g zebrafish (A’). 722 (B,B’) FE models of maximum principal strain incorporating relative material property values 723 from nanoindentation in 1g static (B) and 6g zebrafish (B’). Black arrowheads = areas of high 724 strain; black asterisks = jaw joints; red asterisks = Meckel’s symphysis. Ventral and lateral views 725 shown for opening step in both gravity conditions. 726 727 Table 1: Actual and relative material property values of immature and hypertrophic 728 chondrocytes from AFM and nanoindentation which were used for FE models. Values represent 729 the mean measurement across samples and the figure number of the corresponding FE model 730 is shown in the right-hand column. 731 732 Table 2: Muscle forces used for FE model generation. Values for 1g static and 6g spin fish 733 represent 60% of the maximum muscle force calculated for each muscle group in Figure 1 L 734 with the exception of the adductor mandibulae. 735 736 picrosirius red (D’’’) stained ventral sections in 1g static and 6g fish. Asterisk = centre of joint, 709 dotted line in (D’’’) = outline of cartilage element from section. Scale bar = 10μm. MC = Meckel’s 710 cartilage, PQ = palatoquadrate, C = ceratohyal. (E, F) Quantification of Safranin O (E) and 711 Masson’s trichrome (F) staining intensity. (G) Electron micrographs of hypertrophic 712 chondrocytes in 1g static and 6g zebrafish. Dashed areas = higher magnification images 713 displayed in the centre and to the right of the panel, red lines = chondrocyte borders. Scale bars 714 are shown below each image. (H) Quantification of collagen fibre density in the ECM of 1g static 715 and 6g fish (n = 1 for both). All data is mean with s.e.m. D’Agostino and Pearson normality test 716 performed in B, C, E and F: followed by Mann-Whitney u-test in B and C, and student’s 717 unpaired t-test in E and F. * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001, ** p ≤ 0.0001. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 1g static 6g spin 1g static 6g spin 0 10000 20000 30000 Cell area (pixels2) Chondrocyte area Immature chondrocytes Hypertrophic chondrocytes ** B 0.0 0.5 1.0 Cell circularity (A.U.) CC-BY 4.0 Internationa available under a e author/funder, who has granted bioRxiv a lic this version posted Ma ; 1/2020.05.26.116046 Figure 2 Figure 2 Cell area (pixels2) Meckel’s cartilage Jaw joint A B * was not certified by peer review) is the a https://doi.org/10.1101/ doi: bioRxiv preprint C B A Chondrocyte circularity Cell circularity Fish Fish Fish Fish lice 1g static 6g spin 1g static 6g spin 0.0 Ce Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 1g static 6g spin 0 C Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 1g static 6g spin 0.0 Cell ci Immature chondrocytes Hypertrophic chondrocytes Hypertrophic H & E, Alcian Blue * MC MC PQ D E F 6g spin Immature 1g static Safranin O/ Fast Green Masson’s Trichrome MC PQ * MC MC PQ MC * MC * MC PQ MC MC PQ * MC MC PQ * 1g static 6g spin 1g static 6g spin 0 50 100 150 200 Gray value (A.U) Safranin O staining intensity Fish 1 Fish 2 Fish 3 ** Hypertrophic chondrocytes Immature chondrocytes 1g static 6g spin 1g static 6g spin 0 20 120 140 160 180 200 220 Gray value (A.U) Masson's trichrome staining intensity Fish 1 Fish 2 Fish 3 Immature chondrocytes Hypertrophic chondrocytes Hypertrophic Immature Picrosirius red MC MC MC PQ PQ * * MC c n c n 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 ** 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish Fish Fish Fish Fish Immature chondrocytes Hypertrophic chondrocytes ** D’ D’’ D’’’ G 1g static 6g spin 1g static 6g spin 1g static 6g spin 0.0 Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 1g static 6g spin 0 Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 1g static 6g spin 0.0 Cel Immature chondrocytes Hypertrophic chondrocytes Hypertrophic H & E, Alcian Blue * MC MC PQ D E F 6g spin Immature 1g static Safranin O/ Fast Green Masson’s Trichrome MC PQ * MC MC PQ MC * MC * MC PQ MC MC PQ * MC MC PQ * 1g static 6g spin 1g static 6g spin 0 50 100 150 200 Gray value (A.U) Safranin O staining intensity Fish 1 Fish 2 Fish 3 ** Hypertrophic chondrocytes Immature chondrocytes 1g static 6g spin 1g static 6g spin 0 20 120 140 160 180 200 220 Gray value (A.U) Masson's trichrome staining intensity Fish 1 Fish 2 Fish 3 Immature chondrocytes Hypertrophic chondrocytes Hypertrophic Immature Picrosirius red MC MC MC PQ PQ * * MC 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes ** 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes D’ D’’ D’’’ 1g static 6g spin 0 50 100 150 Collagen fibre density No. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 1g static 6g spin 0 10000 20000 30000 Cell area (pixels2) Chondrocyte area Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes Meckel’s cartilage Jaw joint A B C tatic spin tatic spin 0.0 0.5 1.0 Cell circularity (A.U.) Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 ** * Figure 2 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Cell circularity (A.U.) 1g static 6g spin 1g static 6g spin 0 10000 20000 30000 Cell area (pixels2) Chondrocyte area Fish Fish Fish Fish Fish Immature chondrocytes Hypertrophic chondrocytes ** ** Meckel’s cartilage Jaw joint A B C atic 0.0 0.5 1.0 Cell circularity (A.U.) * CC-BY 4.0 International lic available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a licens this version posted May 2 ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes ** Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 C static 6g spin static 6g spin Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 ** Figure 2 . al license cense to display the preprint in perpetuity. It is made The copyright holder for this preprint (which ay 28, 2020. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 It is made e copyright holder for this preprint (which MC PQ C MC PQ C E F G IA IP IH HH-I 1g static 6g spin H I I’ 0 50 100 150 200 250 Craniofacial muscle length Length (!m) IA IP IH HH-I Muscle group MC PQ D A P L M H’ 0.000000 0.000001 0.000002 0.000003 0.000004 0.000005 Muscle group Force (N) Muscle forces IA IP IH HH-I 1g static 6g spin 0 50 1g static 6g spin 0 50 1g static 6g spin 0 5 30 35 40 45 50 Width (µm) Joint head width 1g static 6g spin 0 2 4 6 8 10 Width of space (µm) Joint space 1g static 6g spin 0 2 20 22 24 26 28 30 32 Width (µm) Joint neck width Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Fish 6 Fish 7 Fish 8 1g static 6g spin 0 2 20 22 24 26 28 30 32 Width (µm) Joint neck width Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Fish 6 Fish 7 Fish 8 a4.1025 Alcian blue Alizarin red 1g static 6g spin 0 2 4 6 8 10 Number of muscle of fibres Number of fibres IA IP IH HH-I 1g static 6g spin J K L M O 1g static 6g spin 1g static 6g spin 0 20 40 60 80 100 Material properties of Meckel's cartilage from nanoindentation EHertz (kPa) Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 * Immature chondrocytes Hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change Relative material property change of immature chondrocytes ** N 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Relative Young’s modulus Relative material property change of hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change Relative material property change of hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change Relative material property change of immature chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Relative Young’s modulus Relative material property change of immature chondrocytes E H’ 1g static 6g spin 0 2 20 22 24 26 28 30 32 Width (µm) Joint neck width 6g spin F G 1g static 6g spin 0 5 30 35 40 45 50 Width (µm) Joint head width Width of space (µm) Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Fish 6 Fish 7 Fish 8 0 2 20 22 24 26 28 30 32 Width (µm) G I’ 1g static 6g spin 0 2 4 6 8 10 Width of space (µm) Joint space 1g static 6g spin 0 2 20 22 24 26 28 30 32 Joint neck width Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Fish 6 Fish 7 Fish 8 H MC PQ D A P L M 1g static F G D E Width of space (µm) 0 2 4 6 8 0 Width (µm) Width (µm) IA IP IH HH-I 1g static I H I’ 1g H’ I 6g spin 6 6g spin I’ 1 1g static a4.1025 Alcian blue Alizarin red Alcian blue Alizarin red a4.1025 0.000000 0.000001 0.000002 0.000003 0.000004 0.000005 Muscle group Force (N) Muscle forces IA IP IH HH-I 1g static 6g spin L 0 50 100 150 200 250 Craniofacial muscle length Length (!m) IA IP IH HH-I Muscle group K L 0 2 4 6 8 10 Number of muscle of fibres Number of fibres IA IP IH HH-I J J J K Muscle forces Craniofacial muscle length Number of muscle of fibres Force (N) Muscle group Muscle group Muscle group g p N 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Relative Young’s modulus Relative material property change of hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change Relative material property change of hypertrophic chondrocytes c 1g spin 3g spin 6g spin ive material property change of immature chondrocytes N 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Relative Young’s modulus Relative material property change of hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change Relative material property change of hypertrophic chondrocytes atic 1g spin 3g spin 6g spin ative material property change of immature chondrocytes atic 1g spin 3g spin 6g spin ative material property change of immature chondrocytes ** M Relative material property change of immature chondrocytes Relative material property change of immature chondrocytes Relative material property change of immature chondrocytes N Relative material property change of hypertrophic chondrocytes Relative material property change of hypertrophic chondrocytes O O O 1g static 6g spin 1g static 6g spin 0 20 40 60 80 100 Material properties of Meckel's cartilage from nanoindentation EHertz (kPa) Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 * Immature chondrocytes Hypertrophic chondrocytes N M Material properties of Meckel's cartilage from nanoindentation Relative material property change of immature chondrocytes Relative material property change of immat re chondroc tes Relative material property change of immature chondrocytes Relative material property change of hypertrophic chondrocytes Relative material property change of hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Relative Young’s modulus Relative material property change of hypertrophic chondrocytes ** 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change Relative material property change of hypertrophic chondrocytes 1g static 1g spin 3g spin 6g spin 0 1 2 3 4 5 Fold change tatic spin spin spin 0 1 2 3 4 5 Fold change immature chondrocytes tatic spin spin spin 0 1 2 3 4 5 Relative Young’s modulus y Relative Young’s modulus Fold change 0 1 2 3 4 Fold change Fold change Relative Young’s modulus Hypertrophic chondrocytes Immature chondrocytes G 1g static 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 1g static 6g spin 0 10000 20000 30000 Cell area (pixels2) Chondrocyte area Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes Meckel’s cartilage Jaw joint A B C 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes ** Hypertrophic H & E, Alcian Blue * MC MC PQ D E F 6g spin Immature 1g static Safranin O/ Fast Green Masson’s Trichrome MC PQ * MC MC PQ MC * MC * MC PQ MC MC PQ * MC MC PQ * 1g static 6g spin 1g static 6g spin 0 50 100 150 200 Gray value (A.U) Safranin O staining intensity Fish 1 Fish 2 Fish 3 ** Hypertrophic chondrocytes Immature chondrocytes 1g static 6g spin 1g static 6g spin 0 20 120 140 160 180 200 220 Gray value (A.U) Masson's trichrome staining intensity Fish 1 Fish 2 Fish 3 Immature chondrocytes Hypertrophic chondrocytes Hypertrophic Immature Picrosirius red MC MC MC PQ PQ * * MC 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes ** 1g static 6g spin 1g static 6g spin 0.0 0.5 1.0 Cell circularity (A.U.) Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Immature chondrocytes Hypertrophic chondrocytes ** D’ D’’ D’’’ 100 150 Collagen fibre density gen fibres per μm2 H * Figure 2 . Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 of collagen fibres per μm2 H Hypertrophic H & E, Alcian Blue * MC MC PQ D 6g spin Immature 1g static Safranin O/ Fast Green MC PQ * MC MC PQ MC * MC * MC PQ Hypertrophic Immature 0.0 0.5 1.0 Cell circularity (A.U.) D’ Hypertrophic H & E, Alcian Blue * MC MC PQ D 6g spin Immature 1g static Safranin O/ Fast Green Masson’s Trichrome MC PQ * MC MC PQ MC * MC * MC PQ MC MC PQ * MC MC PQ * Hypertrophic Immature Picrosirius red MC MC MC PQ PQ * * MC 0.0 0.5 1.0 Cell circularity (A.U.) 0.0 0.5 1.0 Cell circularity (A.U.) D’ D’’ D’’’ Hypertrophic H & E, Alcian Blue * MC MC PQ D Immature 1g static 1g s 6g 1g s 6g Immature chondrocytes Hypertrophic chondrocytes E 1g static 6g spin 1g static 6g spin 0 50 100 150 200 Gray value (A.U) Safranin O staining intensity Fish 1 Fish 2 Fish 3 Hypertrophic chondrocytes Immature chondrocytes Ch d t i l it tic pin tic pin Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 ** g s Imma E D 1g Hypertrophic Safranin O staining intensity hondrocyte circularity D’ Safranin O/ Fast Green MC PQ * MC D’ Safranin O/ Fast Green D’ Hypertrophic chondrocytes ic Sa Fa Masson’s Trichrome MC PQ MC MC MC PQ * MC MC PQ * Picrosirius red MC MC MC PQ PQ * * MC 0.0 0.5 1.0 Cell circularity (A.U.) 0.0 D’’ D’’’ F C g static F Masson's trichrome staining intensity Chondrocyte circularity static 6g spin static 6g spin F 1g static 6g spin 1g static 6g spin 0 20 120 140 160 180 200 220 Gray value (A.U) Masson's trichrome staining intensity Fish 1 Fish 2 Fish 3 Immature chondrocytes Hypertrophic chondrocytes Chondrocyte circularity Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 **** 1g static 6g spin 1g static 6g spin Immature chondrocytes Hypertrophic chondrocytes Masson’s Trichrome MC MC PQ * D’’ D’’ Immature chondrocytes Hypertrophic chondrocytes G 1g static 6g spin 1g static 6g spin 1g static 6g spin Immature chondrocytes Hypertrophic chondrocytes 1g static 6g spin 0 50 100 150 Collagen fibre density No. of collagen fibres per μm2 H G g sp H No. of collagen fibres per μm2 1g static 6g spin Max. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 principal strain (opening) Max. principal strain (closing) * * * * * * * * * * * * * * * * * * * * Lateral Lateral Ventral Ventral A A’ FE models with material property values from AFM Max. principal strain (opening) ncipal strain (closing) Lateral Ventral Ventral els with material property values from nanoindentation B B’ (Avg: 75%) E, Max. Principal +0.000e+00 +4.167e-04 +8.333e-04 +1.250e-03 +1.667e-03 +2.083e-03 +2.500e-03 +2.917e-03 +3.333e-03 +3.750e-03 +4.167e-03 +4.583e-03 +5.000e-03 * * * * * * * * * * * * * * * * * (Avg: 75%) E, Max. Principal +0.000e+00 +4.167e-04 +8.333e-04 +1.250e-03 +1.667e-03 +2.083e-03 +2.500e-03 +2.917e-03 +3.333e-03 +3.750e-03 +4.167e-03 +4.583e-03 +5.000e-03 Figure 3 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (wh this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint 1g static 6g spin Max. principal strain (opening) Max. principal strain (closing) * * * * * * * * * * * * * * * * * * * * Lateral Lateral Ventral Ventral A A’ FE models with material property values from AFM (Avg: 75%) E, Max. Principal +0.000e+00 +4.167e-04 +8.333e-04 +1.250e-03 +1.667e-03 +2.083e-03 +2.500e-03 +2.917e-03 +3.333e-03 +3.750e-03 +4.167e-03 +4.583e-03 +5.000e-03 Figure 3 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (wh this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Figure 3 1g static A A’ Max. principal strain (opening) Max. principal strain (closing) * * Lateral Lateral Ventral Ventral A FE models with material property values from AFM was n bioRxiv p * * * . 0 International license d bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whic on posted May 28, 2020. FE models with material property values from AFM Ventral Lateral Ventral Max. prin Lateral FE Max. principal strain (opening) Max. principal strain (closing) Lateral Lateral Ventral Ventral FE models with material property values from nanoindentation B Lateral * * Max. principal strain (opening) Max. Finite Element Analyses reveal altered strain distribution in response to hypergravity 382 principal strain (closing) Lateral Lateral Ventral Ventral FE models with material property values from nanoindentation B B’ (Avg: 75%) E, Max. Principal +0.000e+00 +4.167e-04 +8.333e-04 +1.250e-03 +1.667e-03 +2.083e-03 +2.500e-03 +2.917e-03 +3.333e-03 +3.750e-03 +4.167e-03 +4.583e-03 +5.000e-03 * * * * * * * * * * * * * * * * * * * * * B’ B Ventral Lateral Ventral Lateral ty on Method Hypertrophic chondrocytes Immature chondrocytes Figure no. of corresponding model Actual material property value Relative material property value Actual material property value Relative material property value AFM 7.7 MPa 1 4.2 MPa 0.51968 Figure 3 A Nanoindentation 37.39 kPa 1 54.8 kPa 1.46563 Figure 3 B AFM 31.0 MPa 4.13444 15.2 MPa 1.99503 Figure 3 A’ Nanoindentation 62.9 kPa 1.68227 52.63 kPa 1.407597 Figure 3 B’ . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: oRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 28, 2020. ; https://doi.org/10.1101/2020.05.26.116046 doi: bioRxiv preprint Muscle Muscle forces (N) 1g static 6g spin Intermandibularis anterior 1.37E-06 1.35E-06 Intermandibularis posterior/ Interhyoideus (average of both) 1.86E-06 1.68E-06 Adductor mandibulae 2.57E-06 2.57E-06 Muscle Muscle
https://openalex.org/W3192869203	https://www.nature.com/articles/s41598-021-94948-1.pdf	English	null	Distinct mutations in importin-β family nucleocytoplasmic transport receptors transportin-SR and importin-13 affect specific cargo binding	Scientific reports	2,021	cc-by	16,103	cargo binding Makoto Kimura1,5, Kenichiro Imai2,3,5, Yuriko Morinaka1, Yoshiko Hosono‑Sakuma1, Paul Horton4 & Naoko Imamoto1* Makoto Kimura1,5, Kenichiro Imai2,3,5, Yuriko Morinaka1, Yoshiko Hosono‑Sakuma1, Paul Horton4 & Naoko Imamoto1* Importin-(Imp)β family nucleocytoplasmic transport receptors (NTRs) are supposed to bind to their cargoes through interaction between a confined interface on an NTR and a nuclear localization or export signal (NLS/NES) on a cargo. Although consensus NLS/NES sequence motifs have been defined for cargoes of some NTRs, many experimentally identified cargoes of those NTRs lack those motifs, and consensus NLSs/NESs have been reported for only a few NTRs. Crystal structures of NTR–cargo complexes have exemplified 3D structure-dependent binding of cargoes lacking a consensus NLS/ NES to different sites on an NTR. Since only a limited number of NTR–cargo interactions have been studied, whether most cargoes lacking a consensus NLS/NES bind to the same confined interface or to various sites on an NTR is still unclear. Addressing this issue, we generated four mutants of transportin-(Trn)SR, of which many cargoes lack a consensus NLS, and eight mutants of Imp13, where no consensus NLS has been defined, and we analyzed their binding to as many as 40 cargo candidates that we previously identified by a nuclear import reaction-based method. The cargoes bind differently to the NTR mutants, suggesting that positions on an NTR contribute differently to the binding of respective cargoes. Human cells have approximately 20 species of Impβ family NTRs, which share the task of transporting approxi- mately 35% of expressed proteins. Each of the NTRs has been reported to transport a specific group of cargo proteins or RNAs into or out of the nuclei through the nuclear pores1,2, and recent comprehensive analyses further indicated that predominant protein groups in the cargo cohorts are unique to the respective NTRs3,4. Thus, the specific NTR–cargo interaction is expected to be attributed to contact between a confined site on the NTR and an NLS/NES on the cargo. Several NTR-specific NLSs/NESs have been described as short regions with consensus sequences. The most well-studied NLS is the classical NLS5,6 that binds to Impα, which is a cargo-binding adap- tor exclusively for Impβ7–11. Sequences similar to the Impβ binding (IBB)-domain in Impα act as NLSs that bind directly to Impβ12–14. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ domains in addition to the NLS/NES, and in such structures, cargo regions apart from the canonical NLS/NES also interact with the NTR34,39,41. Even if a consensus NLS/NES sequence for an NTR is evident, many cargoes of the same NTR may not have the consensus sequence, and notably, for many NTRs, no consensus NLS/NES sequences have been reported. Recent extensive cargo or binding partner identifications failed to uncover novel consensus NLS/NES sequences3,4,43–46. Crystal structures of NTRs complexed with cargoes lacking the canonical NLS/NES have also been determined: Impβ47–49, Imp950, Imp1351–53, Kap122p54, CSE155, exportin-(Xpo)456, Xpo557, and Xpot58. In these structures, the NTRs interact with not just a short sequence of the cargo, but rather with multiple sites or a surface arranged conformationally, and respective cargoes bind to different positions on the NTRs. Impβ import cargoes, Snail, PTHrP, and SREBP-2 contact Impβ at multiple distinctive sites, which also differ from the IBB-interface47–49. Imp13 alternatively binds to import cargoes, UBE2I (UBC9) and MAGOH (Mago), by its N- and C-terminal region, respectively51,52, and an export cargo, EIF1A, by a middle region adjacent to the MAGOH-binding region53. In the complex of the Impβ/Imp7 heterodimer and histone H1, fuzzy and nonspecific interactions by disordered regions of the cargo also support complex formation59. Thus, it can be postulated that NTRs recognize cargoes lacking a canonical NLS/NES using different contact sites, as required by various three-dimensional cargo structures. Likewise, cargoes containing an NLS/NES may also contact varying NTR sites by regions other than the NLS/NES. Therefore, to understand the specificity determinant of the NTR–cargo interaction, the binding of NTRs to a number of varying proteins or domains lacking a canonical NLS/NES must be characterized.i A substantial number of cargoes or binding partners of NTRs have been identified in recent large-scale studies3,4,43–46, but we cannot expect immediate structure determination of so many NTR–cargo complexes. Biochemical protein–protein binding assays using mutant proteins can infer amino acid residues involved in the binding. If mutations located at distinct positions of an NTR affect the binding of different cargoes, that suggests that the cargoes bind to the NTR in different configurations. gfi g Based on extrapolation from reported crystal structures, we hypothesized that cargoes lacking a consensus NLS/NES bind differently to the same NTR. Results T SR I TrnSR, Imp13, and their cargoes. Previously, we identified candidate cargoes of the 12 import NTRs by the SILAC-Tp method3. In those experiments, the plasma membranes of cells labeled with stable isotopes were permeabilized, and unlabeled proteins in a nuclear extract were imported into the nuclei of the permeabilized cells by an NTR of interest. After a pair of import reactions with (+) and without (−) the NTR, the nuclear proteins were extracted and analyzed by LC–MS/MS to derive the unlabeled/labeled (imported/endogenous) quantitation ratio of each protein. From these data, the proteins were ranked in order of the Z-score (standard deviation value) of the log [(unlabeled/labeled)+NTR/(unlabeled/labeled)-NTR] ratio. The experiments were trip- licated for each NTR. Proteins ranked in the top 4% (49–66 proteins) by the lowest (3rd) Z-score in triplicate (3rd-Z-4% cargoes) were assessed as reliable candidate cargoes, whereas the top 15% (245–309 proteins) by the 2nd Z-score were assessed as possible candidate cargoes (2nd-Z-15% cargoes). Contrary to expectation, we were unable to discern any novel consensus sequence within the identified cargoes, raising the question of how these proteins bind to the NTRs. Thus, we analyzed the binding of the identified cargoes. We focused on TrnSR and Imp13 because (1) some cargo-binding crystal structures are available, (2) many of the identified TrnSR cargoes lack the canonical NLS, SR-domain, and no consensus NLS has been defined for Imp13 cargoes, suggesting that these NTRs provide interfaces structurally distinct from those for canonical NLS interactions, and (3) TrnSR and Imp13 are one of the closely homologous pairs in the Impβ family, but they share few cargoes3 and therefore are a convenient pair for the prediction of specific cargo-binding sites acquired after evolutionary divergence (see the next section). For cargo specimens, we employed 3rd-Z-4% cargoes. Of the 65 and 66 TrnSR and Imp13 3rd- Z-4% cargoes, 41 and 40 cargoes, respectively, were successfully prepared as a GFP-fusion protein in a bacterial extract and quantified by triplicate Western blotting with an anti-GFP antibody (Supplementary Fig. S1). Here, we refer to the cargoes by their gene names and 3rd-Z-rank order in our previous paper3. Selection of sites to mutate in TrnSR and Imp13. To analyze the effects of NTR mutations on the binding of various cargoes, we needed to design NTR mutants that are highly likely to be defective in binding to some cargoes. www.nature.com/scientificreports/ To test this hypothesis, we generated a series of mutants of two human NTRs, TrnSR, of which many identified cargoes lack the SR-domain, which is regarded as its canoni- cal NLS, and Imp13, for which no consensus NLS has been defined, and analyzed the binding to the previ- ously identified cargoes by bead halo assay (BHA). For that purpose, we used NTR mutants already reported to be deleterious to cargo binding and made new mutants, considering the crystal structures and functional differentiation60,61, as TrnSR and Imp13 are one of the close paralogous pairs within the Impβ family. To examine TrnSR and Imp13 cargoes, we prepared approximately 40 candidate cargoes each that we identified previously by SILAC-Tp—a combination of stable isotope labeling by amino acid in cell culture (SILAC), an in vitro transport system that imports proteins from nuclear extract into the nuclei of permeabilized cells, and LC–MS/MS3. The cargoes showed widely varying spectra of mutant NTR binding, suggesting that the configurations of NTR–cargo interactions are more widely diversified than expected. cargo binding Other known NLSs/NESs that bind directly to Impβ family NTRs are the PY-NLS for Trn1 and Trn215–17, the Leu-rich NES for CRM118–20, the SR-domain for TrnSR21, the IK-NLS for yeast Kap121p22,23, and the indefinite β-like importin binding (BIB)-domain, which binds to several NTRs24. In addition, the RG/ RGG-rich segment for Trn1 and the RSY-rich segment for TrnSR were reported recently25. Crystallographic studies revealed the binding structures of these NLSs/NESs and the Impβ family NTRs: IBB-domain/Impβ26–28, PY-NLS/Trn115,29–33, NES/CRM134–39, SR-domain/TrnSR40,41, and IK-NLS/Kap121p22,23,42. In these structures, the NTRs interact with the NLSs/NESs by a confined interface located on a distinct part of each NTR. Some of these structures include only a short peptide containing the NLS/NES, but other structures involve wider cargo 1Cellular Dynamics Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, Japan. 2Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan. 3Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan. 4Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan City, Taiwan. 5These authors contributed equally: Makoto Kimura and Kenichiro Imai. email: makimura@riken.jp; kenichiro.imai@aist.go.jp; nimamoto@riken.jp \| https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 www.nature.com/scientificreports/ Results T SR I Zebrafish ....EAVLFIMASIA-NVDPENNPTLM.... Fly ....EAALFIMQNVA-NILPDENEVIP.... C.elegans ....ESALFVMSAVVQNLLPESDTNMP.... Imp13 Human ....EALLYGFQSIA-TIDVNYSDVVP.... Mouse ....EALLYGFQSIA-TIDVNYSDVVP.... Frog ....EALLYGFQSIA-TIDVNYSDVVP.... Zebrafish ....EALLYGFQSIA-TIDVNYSDVIP.... Fly ....EACIYSFQSVA-HFGGEEKRQIP.... C.elegans ....ESCIFQWECVA-YLGETDYTAIL.... KLsite n= Site n ( ) log ( ) ( ) + ( ) log ( ) ( ) p q TrnSR Human ....EAVLFIMAAIA-SVDPENNPTLV.... Mouse ....EAVLFIMAAIA-SVDPENNPTLV.... Frog ....EAVLFIMAAIA-SIDPENNPTLV.... Zebrafish ....EAVLFIMASIA-NVDPENNPTLM.... Fly ....EAALFIMQNVA-NILPDENEVIP.... C.elegans ....ESALFVMSAVVQNLLPESDTNMP.... Imp13 Human ....EALLYGFQSIA-TIDVNYSDVVP.... Mouse ....EALLYGFQSIA-TIDVNYSDVVP.... Frog ....EALLYGFQSIA-TIDVNYSDVVP.... Zebrafish ....EALLYGFQSIA-TIDVNYSDVIP.... Fly ....EACIYSFQSVA-HFGGEEKRQIP.... C.elegans ....ESCIFQWECVA-YLGETDYTAIL.... KLsite n= Site n ( ) log ( ) ( ) + ( ) log ( ) ( ) p q b p(i): estimated probability of amino acid i at site n of TrnSR q(i): estimated probability of amino acid i at site n of Imp13 p(i): estimated probability of amino acid i at site n of TrnSR q(i): estimated probability of amino acid i at site n of Imp13 Mutagenesis Bead halo binding assay WT GFP Cargo 1 2 3 4 5 6 7 mt 1 mt 2 mt 3 GST mCherry NTR c Cargo clustering by mutant NTR-binding specificity d Cargo clustering by mutant NTR-binding specificity d d c Cargo clustering by mutant NTR-binding specificity Bead halo binding assay Results T SR I We expected that analyses focusing on a couple of distinct regions on the NTRs would yield sufficient information to illustrate the similarity or variety of the binding configurations and considered that the effects of mutations in close proximity should also be compared by introducing a few mutations into a small region. Thus, we selected the residues to substitute in such a way as to form a couple of separated clusters on the NTR; with priority order: (1) residues in contact with cargoes in crystal structures and whose mutations were defective in cargo binding in pull-down assay, (2) residues in contact with cargoes but not assayed by https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ Bead halo binding assay WT GFP Cargo 1 2 3 4 5 6 7 mt 1 mt 2 mt 3 GST mCherry NTR Cargo clustering by mutant NTR-binding specificity a d c b TrnSR Human ....EAVLFIMAAIA-SVDPENNPTLV.... Mouse ....EAVLFIMAAIA-SVDPENNPTLV.... Frog ....EAVLFIMAAIA-SIDPENNPTLV.... Zebrafish ....EAVLFIMASIA-NVDPENNPTLM.... Fly ....EAALFIMQNVA-NILPDENEVIP.... C.elegans ....ESALFVMSAVVQNLLPESDTNMP.... Imp13 Human ....EALLYGFQSIA-TIDVNYSDVVP.... Mouse ....EALLYGFQSIA-TIDVNYSDVVP.... Frog ....EALLYGFQSIA-TIDVNYSDVVP.... Zebrafish ....EALLYGFQSIA-TIDVNYSDVIP.... Fly ....EACIYSFQSVA-HFGGEEKRQIP.... C.elegans ....ESCIFQWECVA-YLGETDYTAIL.... KLsite n= Site n Evolutionary trace ( ) log ( ) ( ) + ( ) log ( ) ( ) p(i): estimated probability of amino acid i at site n of TrnSR q(i): estimated probability of amino acid i at site n of Imp13 p q Mutagenesis Site on concave surfaces with high KL-value Figure 1. Research workflow. (a) In addition to amino acid residues that contact cargoes in reported crystal structures, residues exposed on the concave surface facing the cavity of the TrnSR or Imp13 structure were considered candidate sites for mutational analysis. (b) Residues conserved across species within either TrnSR or Imp13 orthologs but not between them were identified via an ETA of 78 and 73 metazoan TrnSR and Imp13 sequences, respectively. The sequences were aligned, and the obtained amino acid frequencies of each column a b TrnSR Human ....EAVLFIMAAIA-SVDPENNPTLV.... Mouse ....EAVLFIMAAIA-SVDPENNPTLV.... Frog ....EAVLFIMAAIA-SIDPENNPTLV.... Zebrafish ....EAVLFIMASIA-NVDPENNPTLM.... Fly ....EAALFIMQNVA-NILPDENEVIP.... C.elegans ....ESALFVMSAVVQNLLPESDTNMP.... Imp13 Human ....EALLYGFQSIA-TIDVNYSDVVP.... Mouse ....EALLYGFQSIA-TIDVNYSDVVP.... Frog ....EALLYGFQSIA-TIDVNYSDVVP.... Zebrafish ....EALLYGFQSIA-TIDVNYSDVIP.... Fly ....EACIYSFQSVA-HFGGEEKRQIP.... C.elegans ....ESCIFQWECVA-YLGETDYTAIL.... KLsite n= Site n ( ) log ( ) ( ) + ( ) log ( ) ( ) p(i): estimated probability of amino acid i at site n of TrnSR q(i): estimated probability of amino acid i at site n of Imp13 p q Mutagenesis Site on concave surfaces with high KL-value TrnSR Human ....EAVLFIMAAIA-SVDPENNPTLV.... Mouse ....EAVLFIMAAIA-SVDPENNPTLV.... Frog ....EAVLFIMAAIA-SIDPENNPTLV.... www.nature.com/scientificreports/ Y433 has a high KL value, while the KL values of D426 and E478 are somewhat lower than the top 10% KL value. However, in the D426 and E478 positions, residues charged oppositely between TrnSR and Imp13 are conserved through species (Fig. 2b). Y350 was selected in consideration of its structural position and high KL value. Y350, D426, Y433, and E478 locate nearby one another. Imp13-K802E/R803E was characterized by pull-down assay, and K802/R803 interacts with MAGOH51. R748 was selected because of its structural position near K802/R803 and its high KL value. These residues form three clusters on the concave surface of Imp13: N-terminal cluster, Y34/Y35 and E73; middle, Y350, D426, Y433, and E478; and C-terminal, R748 and K802/R803 (Fig. 2b). Bead halo assay. We analyzed the binding of the 3rd-Z-4% cargoes to TrnSR and Imp13 by BHA62 (Figs. 1c, 3). The TrnSR and Imp13 proteins were expressed in bacteria and purified as GST-mCherry-TrnSR and GST- mCherry-Imp13 red fluorescent fusion proteins, respectively. The GST-mCherry-TrnSR or -Imp13 protein was fixed on glutathione (GSH)-Sepharose beads and mixed with an extract containing one of the GFP-cargo fusion proteins. The beads were observed with a confocal laser scanning microscope, and the fluorescent intensities of mCherry and GFP along the rims of the beads were measured from the confocal images (Fig. 3). In the images, only beads around which regions of interest (ROIs) and background (BG) regions can be set as unbroken circles were selected for measurement (Supplementary Fig. S2), yielding one or two beads as the ROIs for most images. We employed the BG-subtracted GFP/mCherry fluorescent ratio as the index for the binding.l p y yl g We fused mCherry into GST-NTR proteins and used the GFP/mCherry fluorescent ratios as the index to improve quantitation accuracy. Initially, we assessed the deviation of the measured values using GST-mCherry- TrnSR and GFP-SRSF1 as an example (Fig. 4a–d) to evaluate our experimental system. In all BHAs, we selected beads 80–150 µm in diameter for quantitation. In this range, intensities of neither mCherry nor GFP fluorescence correlated with the diameter, although both fluctuated independently of the diameter (Fig. 4a, b). However, mCherry and GFP intensities correlated with each other (R = 0.70, Fig. 4c), representing that GFP-cargo binds to a bead in proportion to the amount of GST-mCherry-NTR on the bead. As seen in some images in this report (e.g., Fig. www.nature.com/scientificreports/ is a distinctive feature of this pair, and we expected that cargo-binding sites evolutionarily differentiated between TrnSR and Imp13 could be predicted by ETA. ETA focuses on amino acid positions conserved in each paralog but not across the paralogs, e.g., positions consistently occupied by one amino acid in TrnSR sequences and another amino acid in Imp13 sequences. Such positions are presumably responsible for the functional differentiation between the paralogs in general60,61 and, in particular, the differential cargo recognition of TrnSR and Imp13. TrnSR and Imp13 are close homologs but do differ consistently in a few positions. To systematically identify such positions, we performed a modified ETA using a symmetric KL value60 (Fig. 1b and see Methods). The KL value and the estimated amino acid probability of each position of human TrnSR and Imp13 are presented in Supplementary Table S1. We considered positions with a high (within the top 10%) KL value as the candidate sites for mutagenesis. g According to these considerations, we generated four TrnSR substitution mutants: TrnSR-E398R, -D409R, -R671E, and -D750R/D751R double mutant (Fig. 2a and Table 1). R671 and D750/D751 are located near each other in the crystal structure, and interact with the SR-domains of cargoes SRSF1 (ASF/SF2) and CPSF640,41. TrnSR-R671E and -D750R/D751R were characterized by pull-down assay41. As expected, the alignment sites of R671 and D751 have high KL values (Table 1 and Supplementary Table S1). We selected E398 and D409, located near each other, because they are exposed toward a cargo, SRSF1, and their alignment sites have high KL values. The small region in the TrnSR structure that includes E398 and D409 and another region that includes R671 and D750/D751 are opposed to each other across the cavity (Fig. 2a). y g We also generated eight Imp13 mutants: Imp13-Y34R/Y35R, -E73R, -Y350R, -D426R, -Y433R, -E478R, -R748E, and -K802E/R803E (Fig. 2b and Table 1). Y433, E478, and K802/R803 correspond to Y447, E493, and K814/K815, respectively, in Drosophila Imp13. Y34/Y35 and E73 are located near each other in the crystal structure. Imp13-Y34R/Y35R was characterized by pull-down assay, and Y34/Y35 and E73 interact with a cargo, UBE2I, in the structure52. However, their KL values were unexpectedly low (Table 1 and Supplementary Table S1). Imp13-D426R and -Y433R were characterized by pull-down assay51,52. D426 contacts UBE2I52, and Y433 and E478 contact a cargo, MAGOH51. Bead halo binding assay Figure 1. Research workflow. (a) In addition to amino acid residues that contact cargoes in reported crystal structures, residues exposed on the concave surface facing the cavity of the TrnSR or Imp13 structure were considered candidate sites for mutational analysis. (b) Residues conserved across species within either TrnSR or Imp13 orthologs but not between them were identified via an ETA of 78 and 73 metazoan TrnSR and Imp13 sequences, respectively. The sequences were aligned, and the obtained amino acid frequencies of each column were used to estimate probabilities for use in computing the symmetric Kullback–Leibler divergence (KL value) for each aligned position (site n). (c) WT and mutagenized (mt) TrnSR and Imp13 (NTR) and their cargoes were prepared as bacterially expressed GST-mCherry-NTR fusion and GFP-cargo fusion proteins, respectively. Binding between the GST-mCherry-NTRs and the GFP-cargo was analyzed in all combinations by BHA. GST- mCherry-NTR was fixed on GSH-Sepharose beads and mixed with a bacterial extract containing the GFP-cargo protein. The beads were then observed with a confocal microscope, and the binding intensity was quantified from the fluorescence of mCherry and GFP in the images. (d) The cargoes were clustered based on their NTR mutant-binding specificity. pull-down, and (3) residues facing towards cargoes in crystal structures and predicted as evolutionarily differ- entiated cargo-binding sites by high symmetric Kullback–Leibler information values (KL values) in a modified evolutionary trace analysis60 (ETA, see the next paragraph) (Fig. 1a, b). The selected regions differed between TrnSR and Imp13 because more cargo-contact sites were found, and more mutants were reported for Imp13 than TrnSR. We substituted charged residues with oppositely charged residues because residues were replaced simi- larly in previously characterized mutants defective in cargo binding41,51,52 (e.g., TrnSR-R671E, -D750R/D751R and Imp13-K802E/R803E). p ) ETA aims to correlate sequence variations with differentiated protein functions60,61. Impβ family NTRs share primary structures, although the amino acid identities between quite a few NTRs are less than 30%, and any pair within the 12 import NTRs shares a certain number of the identified cargoes. In general, homologous NTRs share many cargoes3. However, TrnSR and Imp13, which are so similar as to form one of the closest pairs in the phylogenetic analysis of 12 Impβ family import NTRs, are functionally differentiated to share few cargoes3. This https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ 3), some ROIs contain uneven fluorescence, but in most cases, mCherry and GFP show similar uneven fluorescence patterns, indicating proportional binding of GFP-cargo to GST-mCherry-NTR at any place on a bead. Accordingly, the coefficient of variation (C.V. = S.D./mean), which was 0.22 for GFP (Fig. 4b), was reduced to 0.17 for GFP/mCherry (Fig. 4d). All the GFP/mCherry values in Fig. 4d are within the mean ± 36% range, and a significant reduction (e.g., > 40% reduction) in GFP/mCherry values in the following assays of NTR mutants assuredly indicates reduced binding, albeit not precisely quantitative. Note that in the final assays (Supplemen- tary Table S2a and b), the GFP/mCherry ratios of three images were averaged and used to calculate the normal- ized GFP/mCherry values, and therefore, the measurement errors were reduced. In the negative control assay, intact GFP bound to TrnSR weakly, though not to Imp13 (Supplementary Fig. S3a and e). We applied different thresholds for negative binding to TrnSR (GFP/mCherry without normalization, 0.1) and Imp13 (0.05) to avoid unreliable quantification at a low range (Supplementary Fig. S3b–d).i qi g pp y g Next, we assessed the dose–response of the system using two TrnSR- and four Imp13-specific cargoes (Fig. 4e–j). The amount of extract containing GFP-cargo was varied from zero up to that used in the final assays of NTR mutants. In some cases, the BG-subtracted GFP/mCherry ratio responded to the GFP-cargo dose almost linearly (Fig. 4e, g, i), and reduced binding to NTR mutants would be detected sensitively. In other cases, the BG-subtracted GFP/mCherry ratio approached saturation (Fig. 4f, h, j), and a slight reduction in binding would be challenging to detect. Thus, our assays should reliably detect reductions in binding affinity but perhaps with imperfect sensitivity in some cases. In the final assays of NTR mutants below, GFP-cargoes of equal amount were used when possible (see the legend of Supplementary Table S2). www.nature.com/scientificreports/ Scientific Reports \| (2021) 11:15649 \| https://doi.org/10.1038/s41598-021-94948-1 www.nature.com/scientificreports/ Imp13 top3 conserved residues TrnSR top3 conserved residues Y34 Y(0.985), F(0.005), L(0.001) Y(0.802), F(0.116), S(0.054) Y35 R(0.178), Y(0.128), S(0.125) N(0.215), H(0.207), Q(0.113) E73 E(0.805) ,N(0.062), P(0.046) E(0.314), N(0.111), T(0.069) Y350 Y(0.922), F(0.054), L(0.004) H(0.353), Y(0.107), G(0.095) Imp13 top3 conserved residues TrnSR top3 conserved residues Y433 Y(0.757), S(0.109), D(0.087) D(0.903), S(0.047), A(0.008) E478 E(0.614), D(0.214), Q(0.066) K(0.435), R(0.108), Q(0.091) R748 K(0.492), R(0.206), Q(0.102) S(0.916), A(0.050), T(0.007) K802 K(0.705), R(0.207), D(0.041) Q(0.583), E(0.096), D(0.066) a b Residue TrnSR top3 conserved residues Imp13 top3 conserved residues E398 D(0.550), E(0.368), G(0.035) C(0.441), I(0.198), Y(0.076) D409 D(0.903), S(0.047), A(0.008) Y(0.757), S(0.109), D(0.087) R671 R(0.991), K(0.002), G(0.001) S(0.195), T(0.185), K(0.169) D750 D751 D(0.873), E(0.085), N(0.008) E(0.525), D(0.242), H(0.055) D(0.989), E(0.002), G(0.001) S(0.284), A(0.225), G(0.108) e u d i s e R e u d i s e R MAGOH K802 R803 R748 Y350 D426 E478 Y433 Y34 Y35 E73 D750 D751 R671 E398 D409 SRSF1 TrnSR UBE2I Y34 Y35 E73 Y350 D426 E478 R748 R803 K802 Y433 Imp13 Imp13 top3 conserved residues TrnSR top3 conserved residues Y34 Y(0.985), F(0.005), L(0.001) Y(0.802), F(0.116), S(0.054) Y35 R(0.178), Y(0.128), S(0.125) N(0.215), H(0.207), Q(0.113) E73 E(0.805) ,N(0.062), P(0.046) E(0.314), N(0.111), T(0.069) Y350 D426 Y(0.922), F(0.054), L(0.004) H(0.353), Y(0.107), G(0.095) D(0.828), E(0.053), T(0.051) R(0.323), K(0.281), D(0.100) Imp13 top3 conserved residues TrnSR top3 conserved residues Y433 Y(0.757), S(0.109), D(0.087) D(0.903), S(0.047), A(0.008) E478 E(0.614), D(0.214), Q(0.066) K(0.435), R(0.108), Q(0.091) R748 K(0.492), R(0.206), Q(0.102) S(0.916), A(0.050), T(0.007) K802 R803 K(0.705), R(0.207), D(0.041) Q(0.583), E(0.096), D(0.066) K(0.560), R(0.251), S(0.048) R(0.581), K(0.171), C(0.111) a b Residue TrnSR top3 conserved residues Imp13 top3 conserved residues E398 D(0.550), E(0.368), G(0.035) C(0.441), I(0.198), Y(0.076) D409 D(0.903), S(0.047), A(0.008) Y(0.757), S(0.109), D(0.087) R671 R(0.991), K(0.002), G(0.001) S(0.195), T(0.185), K(0.169) D750 D751 D(0.873), E(0.085), N(0.008) E(0.525), D(0.242), H(0.055) D(0.989), E(0.002), G(0.001) S(0.284), A(0.225), G(0.108) e u d i s e R e u d i s e R MAGOH K802 R803 R748 Y350 D426 E478 Y433 Y34 Y35 E73 D750 D751 R671 E398 D409 SRSF1 TrnSR UBE2I Y34 Y35 E73 Y350 D426 E478 R748 R803 K802 Y433 Imp13 Figure 2. Substituted amino acid residues. (a) Positions of the five residues substituted in TrnSR are indicated on the structure of TrnSR binding to SRSF1 (ASF/SF2, gray). www.nature.com/scientificreports/ The three most conserved amino acids in those positions in the 78 metazoan TrnSR orthologs are shown on the right, with probabilities estimated from their amino acid frequencies. The residues were substituted with oppositely charged amino acids. The D750/D751 dipeptide was substituted in one construct following a previous work41. E398 and D409 are proximal, and R671 and D750/D751 are also proximal. See also Table 1. (b) The ten residues substituted in Imp13 are indicated on the structures of Imp13 binding to UBE2I (UBC9, gray) (left) or MAGOH (Mago, gray) (right). The three most conserved amino acids in those positions in the 73 metazoan Imp13 orthologs are shown with probabilities estimated from their amino acid frequencies. These residues were also substituted with oppositely charged amino acids. Two constructs have dipeptide substitutions at Y34/Y35 and K802/R803 following a previous work52. These residues reside in three separated regions: N-terminal region, Y34/Y35 and E73; middle, Y350, D426, Y433, and E478; and C-terminal, R748 and K802/R803. See also Table 1. a Residue TrnSR top3 conserved residues Imp13 top3 conserved residues E398 D(0.550), E(0.368), G(0.035) C(0.441), I(0.198), Y(0.076) D409 D(0.903), S(0.047), A(0.008) Y(0.757), S(0.109), D(0.087) R671 R(0.991), K(0.002), G(0.001) S(0.195), T(0.185), K(0.169) D750 D751 D(0.873), E(0.085), N(0.008) E(0.525), D(0.242), H(0.055) D(0.989), E(0.002), G(0.001) S(0.284), A(0.225), G(0.108) D750 D751 R671 E398 D409 SRSF1 TrnSR a D750 D751 R671 E398 D409 SRSF1 TrnSR a Residue TrnSR top3 conserved residues Imp13 top3 conserved residues E398 D(0.550), E(0.368), G(0.035) C(0.441), I(0.198), Y(0.076) D409 D(0.903), S(0.047), A(0.008) Y(0.757), S(0.109), D(0.087) R671 R(0.991), K(0.002), G(0.001) S(0.195), T(0.185), K(0.169) D750 D751 D(0.873), E(0.085), N(0.008) E(0.525), D(0.242), H(0.055) D(0.989), E(0.002), G(0.001) S(0.284), A(0.225), G(0.108) b MAGOH K802 R803 R748 Y350 D426 E478 Y433 Y34 Y35 E73 UBE2I Y34 Y35 E73 Y350 D426 E478 R748 R803 K802 Y433 Imp13 MAGOH K802 R803 R748 Y350 D426 E478 Y433 Y34 Y35 E73 803 b K802 R803 UBE2I Y34 Y35 E73 Y350 D426 E478 R748 R803 K802 Y433 Imp13 b Imp13 top3 conserved residues TrnSR top3 conserved residues Y34 Y(0.985), F(0.005), L(0.001) Y(0.802), F(0.116), S(0.054) Y35 R(0.178), Y(0.128), S(0.125) N(0.215), H(0.207), Q(0.113) E73 E(0.805) ,N(0.062), P(0.046) E(0.314), N(0.111), T(0.069) Y350 D426 Y(0.922), F(0.054), L(0.004) H(0.353), Y(0.107), G(0.095) D(0.828), E(0.053), T(0.051) R(0.323), K(0.281), D(0.100) e u d i s e R Figure 2. Substituted amino acid residues. (a) Positions of the five residues substituted in TrnSR are indicated on the structure of TrnSR binding to SRSF1 (ASF/SF2, gray). www.nature.com/scientificreports/ The three most conserved amino acids in those positions in the 78 metazoan TrnSR orthologs are shown on the right, with probabilities estimated from their amino acid frequencies. The residues were substituted with oppositely charged amino acids. The D750/D751 dipeptide was substituted in one construct following a previous work41. E398 and D409 are proximal, and R671 and D750/D751 are also proximal. See also Table 1. (b) The ten residues substituted in Imp13 are indicated on the structures of Imp13 binding to UBE2I (UBC9, gray) (left) or MAGOH (Mago, gray) (right). The three most conserved amino acids in those positions in the 73 metazoan Imp13 orthologs are shown with probabilities estimated from their amino acid frequencies. These residues were also substituted with oppositely charged amino acids. Two constructs have dipeptide substitutions at Y34/Y35 and K802/R803 following a previous work52. These residues reside in three separated regions: N-terminal region, Y34/Y35 and E73; middle, Y350, D426, Y433, and E478; and C-terminal, R748 and K802/R803. See also Table 1. Scientific Reports \| (2021) 11:15649 \| https://doi.org/10.1038/s41598-021-94948-1 www.nature.com/scientificreports/ Table 1. TrnSR and Imp13 mutants used. NTR Amino acid substitution KL value (top 10% > 7.5) Cargo binding analyzed by pull- down assay Cargo contact in crystal structure TrnSR E398R 13.966 D409R 10.496 R671E 8.310 SRSF1, CPSF641 SRSF141, CPSF640 D750R/D751R 2.980/13.051 SRSF1, CPSF641 SRSF141, CPSF640 Imp13 Y34R/Y35R 0.957/3.615 UBE2I52 UBE2I52 E73R 2.626 UBE2I52 Y350R 7.590 D426R 6.809 UBE2I52 UBE2I52 Y433R 10.496 Drosophila Mago-Y1451 (Dros- ophila Imp13-Y447) Drosophila Mago51 (Drosophila Imp13-Y447) E478R 7.014 Drosophila Mago51 (Drosophila Imp13-E493) R748E 12.836 K802E/R803E 8.877/2.293 MAGOH-RBM8A (Mago-Y14)52 Drosophila Mago-Y1451 (Dros- ophila Imp13-K814/K815) Drosophila Mago51 (Drosophila Imp13-K814/K815) Table 1. TrnSR and Imp13 mutants used. Table 1. TrnSR and Imp13 mutants used. GSH GSH GSH GSH Bead GST-mCherry-NTR GFP-cargo Bead GFP/mCherry= ROIGFP–BGGFP ROImCherry–BGmCherry a e i b DIC DIC f c mCherry mCherry g j ROI BG d GFP GFP h k 100 µm Figure 3. Bead halo assay. (a–d) An example of an NTR–cargo pair that does not bind. (e–h) An example of an NTR–cargo pair that does bind. (a) A GST-mCherry-NTR is fixed on GSH-Sepharose beads. If GFP-cargo does not bind to the NTR, it remains in the buffer around the beads. (e) If cargo binds to the NTR, it is concentrated on the bead surface. (b, f) Differential interference contrast (DIC) microscopic images of the beads. (c, g) Confocal microscopic images of mCherry fluorescence. www.nature.com/scientificreports/ The yellow square in (g) indicates the magnified region in (j, k). (d, h) Confocal images of GFP fluorescence. (i) After background subtraction, the GFP/mCherry fluorescence ratio around the beads was used as the index for the binding intensity. ROI and BG were defined as in (j, k). The region mean intensities in 12-bit images were used for the calculation. (j) The ROI was set on the mCherry fluorescent image as ring-shaped regions with a 5 pixel width along the inside of the outlines of beads (yellow double line). The same ROI was applied to the corresponding GFP fluorescent image. (k) The BG was established as larger ring-shaped regions with a 5 pixel width and 5 pixels away outside the ROI. Images in (j, k) are magnifications of the square region in (g). In the actual quantitation, ROIs and BGs were set on original images with dimensions of 1024 × 1024 pixels, of which the field size (317 × 317 µm) was the same as that of the images (b–d and f–h). Scale bar: 100 µm. For more examples of the ROI setting, see Supplementary Fig. S2. c mCherry d GFP b DIC 100 µm GSH GSH Bead GST-mCherry-NTR GFP-cargo a d a c GSH GSH Bead e DIC f f h e g i j k k ROI BG Figure 3. Bead halo assay. (a–d) An example of an NTR–cargo pair that does not bind. (e–h) An example of an NTR–cargo pair that does bind. (a) A GST-mCherry-NTR is fixed on GSH-Sepharose beads. If GFP-cargo does not bind to the NTR, it remains in the buffer around the beads. (e) If cargo binds to the NTR, it is concentrated on the bead surface. (b, f) Differential interference contrast (DIC) microscopic images of the beads. (c, g) Confocal microscopic images of mCherry fluorescence. The yellow square in (g) indicates the magnified region in (j, k). (d, h) Confocal images of GFP fluorescence. (i) After background subtraction, the GFP/mCherry fluorescence ratio around the beads was used as the index for the binding intensity. ROI and BG were defined as in (j, k). The region mean intensities in 12-bit images were used for the calculation. (j) The ROI was set on the mCherry fluorescent image as ring-shaped regions with a 5 pixel width along the inside of the outlines of beads (yellow double line). www.nature.com/scientificreports/ The same ROI was applied to the corresponding GFP fluorescent image. (k) The BG was established as larger ring-shaped regions with a 5 pixel width and 5 pixels away outside the ROI. Images in (j, k) are magnifications of the square region in (g). In the actual quantitation, ROIs and BGs were set on original images with dimensions of 1024 × 1024 pixels, of which the field size (317 × 317 µm) was the same as that of the images (b–d and f–h). Scale bar: 100 µm. For more examples of the ROI setting, see Supplementary Fig. S2. https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ 0 100 200 300 400 500 70 90 110 130 150 C.V.=0.22 R=0.39 GFP Diameter (µm) GFP intensity b 0 200 400 600 800 1000 70 90 110 130 150 mCherry Diameter (µm) mCherry intensity C.V.=0.18 R=0.29 a 0 100 200 300 400 500 600 0 200 400 600 800 1000 GFP vs mCherry mCherry GFP R=0.70 c 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 70 90 110 130 150 GFP/mCherry Diameter (µm) GFP/mCherry C.V.=0.17 R=0.28 d TrnSR + 46. PQBP1 GFP-PQBP1 units GFP/mCherry intensity e TrnSR + 56. RTF2 GFP-RTF2 units GFP/mCherry intensity f Imp13 + 4. MAGOH GFP-MAGOH units GFP/mCherry intensity g Imp13 + 6. CHRAC1 GFP-CHRAC units GFP/mCherry intensity h Imp13 + 54. PPIE GFP-PPIE units GFP/mCherry intensity i Imp13 + 58. UBE2I GFP-UBE2I units GFP/mCherry intensity j Figure 4. Evaluation of the bead halo assay system. (a–d) The fluorescence intensity of GST-mCherry- TrnSR and GFP-SRSF1 as measured for 27 single beads is shown to evaluate the deviation of assay values. (a) BG-subtracted mCherry, (b) BG-subtracted GFP, and (d) BG-subtracted GFP/mCherry values are plotted against the bead diameters. (c) BG-subtracted GFP intensities are plotted against the BG-subtracted mCherry intensities of the same beads. C.V., coefficient of variation of the fluorescent values; R, correlation coefficient between the fluorescent values and the bead diameters (a, b, d) or between the mCherry and GFP intensities (c). (e–j) The dosage of bacterial extract containing GFP-cargo was varied from zero to that used in the final assay to evaluate the dose–response. (e) GST-mCherry-TrnSR and GFP-PQBP1, (f) GST-mCherry-TrnSR and GFP-RTF2, (g) GST-mCherry-Imp13 and GFP-MAGOH, (h) GST-mCherry-Imp13 and GFP-CHRAC1, (i) GST-mCherry-Imp13 and GFP-PPIE, and (j) GST-mCherry-Imp13 and GFP-UBE2I were analyzed by BHA. Numbers are the 3rd-Z-ranks of the cargoes. www.nature.com/scientificreports/ Three images for one GFP-cargo dosage were quantified, and the mean intensities of the GFP without BG subtraction (light green), BG of GFP (black), and BG-subtracted GFP ( ) d d d b h G b d Ch l d h h S d d b h 0 200 400 600 800 1000 70 90 110 130 150 mCherry Diameter (µm) mCherry intensity C.V.=0.18 R=0.29 a 0 100 200 300 400 500 70 90 110 130 150 C.V.=0.22 R=0.39 GFP Diameter (µm) GFP intensity b 0 100 200 300 400 500 600 0 200 400 600 800 1000 GFP vs mCherry mCherry GFP R=0.70 c 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 70 90 110 130 150 GFP/mCherry Diameter (µm) GFP/mCherry C.V.=0.17 R=0.28 d b d a c Diameter (µm) Diameter (µm) Diameter (µm) TrnSR + 56. RTF2 GFP-RTF2 units GFP/mCherry intensity f TrnSR + 46. PQBP1 GFP-PQBP1 units GFP/mCherry intensity e f f g Imp13 + 4. MAGOH GFP-MAGOH units GFP/mCherry intensity g e i Imp13 + 6. CHRAC1 GFP-CHRAC units GFP/mCherry intensity h j Imp13 + 54. PPIE GFP-PPIE units GFP/mCherry intensity i Imp13 + 58. UBE2I GFP-UBE2I units GFP/mCherry intensity j i h j Figure 4. Evaluation of the bead halo assay system. (a–d) The fluorescence intensity of GST-mCherry- TrnSR and GFP-SRSF1 as measured for 27 single beads is shown to evaluate the deviation of assay values. (a) BG-subtracted mCherry, (b) BG-subtracted GFP, and (d) BG-subtracted GFP/mCherry values are plotted against the bead diameters. (c) BG-subtracted GFP intensities are plotted against the BG-subtracted mCherry intensities of the same beads. C.V., coefficient of variation of the fluorescent values; R, correlation coefficient between the fluorescent values and the bead diameters (a, b, d) or between the mCherry and GFP intensities (c). (e–j) The dosage of bacterial extract containing GFP-cargo was varied from zero to that used in the final assay to evaluate the dose–response. (e) GST-mCherry-TrnSR and GFP-PQBP1, (f) GST-mCherry-TrnSR and GFP-RTF2, (g) GST-mCherry-Imp13 and GFP-MAGOH, (h) GST-mCherry-Imp13 and GFP-CHRAC1, (i) GST-mCherry-Imp13 and GFP-PPIE, and (j) GST-mCherry-Imp13 and GFP-UBE2I were analyzed by BHA. Numbers are the 3rd-Z-ranks of the cargoes. Three images for one GFP-cargo dosage were quantified, and the mean intensities of the GFP without BG subtraction (light green), BG of GFP (black), and BG-subtracted GFP (green) divided by the mean BG-subtracted mCherry intensity are plotted with the S.D. indicated by the error bar. www.nature.com/scientificreports/ Since TrnSR-D750R/ D751R showed significantly reduced cargo binding in our BHA, we conclude that our system can detect binding reductions of less than one order of magnitude. Binding of the cargoes to the WT NTRs. Imp13 is a bidirectional NTR66, but the candidate cargoes are most likely to be import cargoes because they were identified by the import reaction-based method SILAC- Tp (see the section, TrnSR, Imp13, and their cargoes). As described in the previous paper3, an export cargo of Imp13, EIF1AX66, ranked relatively high (164th in 1671 proteins) by the 3rd-Z-score. The unintended export of endogenous EIF1AX by Imp13 must have raised the imported/endogenous (unlabeled/labeled) ratio in the experiment. Judging by the absolute levels of imported (unlabeled) and endogenous (labeled) proteins in the LC–MS/MS, EIF1AX, abundant in the nuclei, seemed to be the only export cargo ranked high3.f y p g g NTR–cargo complexes pass through the nuclear pores by diffusion, and the transport direction is regulated at the steps of association and dissociation of the complexes in the nuclei and cytoplasm67. RanGTP, which is rich in the nuclei and converted into RanGDP in the cytoplasm, promotes the association and dissociation of export and import NTR–cargo complexes, respectively. We analyzed the binding of all the prepared cargoes in the presence or absence of a GTP-fixed mutant of Ran, Q69L-RanGTP, by BHA (Supplementary Fig. S6). Most of the candidate cargoes that we could analyze bound to their respective NTRs, (35 out of 41 TrnSR cargoes and 27 out of 40 Imp13 cargos), and in most cases except the Imp13 export cargo EIF1AX, the addition of Q69L-RanGTP decreased the GFP/mCherry values (Supplementary Fig. S6, 3rd-Z-rank of EIF1AX is 164 of Imp13), supporting the functionality of the binding. This result attests to the reliability of the SILAC-Tp method, especially when considering that BHAs can detect only the direct binding of a protein to an NTR, but the SILAC-Tp method can also identify indirect cargoes that bind to an NTR by forming complexes with direct cargoes. Piggybacking nuclear import is prevalent in human cells68, and in the STRING database69, quite a few 3rd-Z-4% cargoes of which direct NTR binding was not detected are expected to interact with other 3rd-Z-4% cargoes that bound directly to the NTR (Supplementary Table S2c). Since many unidentified cargoes may also connect the NTRs and indirect cargoes, the possibility of piggybacking may be high for the NTR-unbound cargoes. www.nature.com/scientificreports/ consistent with those in the final assay (Supplementary Fig. S4a–f right panels). Notably, a significant reduction in cargo binding of an NTR mutant could consistently be observed in the repeated experiments. consistent with those in the final assay (Supplementary Fig. S4a–f right panels). Notably, a significant reduction in cargo binding of an NTR mutant could consistently be observed in the repeated experiments. g g y p p We compared the binding of WT and mutant NTRs to the same cargo by localized surface plasmon reso- nance (LSPR) to estimate the physical significance of the major binding reduction observed for NTR mutants (Supplementary Fig. S5). Further, we prepared NTR and cargo proteins without GFP- and mCherry-fusion to avoid optical interference. A His6-tagged cargo protein was fixed on a Ni2+-charged nitrilotriacetic acid (NTA) sensor chip, and the kinetic association and dissociation of a GST-NTR on the sensor were analyzed. Although we tried several NTR–cargo combinations, we could only set up adequate experimental conditions for the TrnSR–DNAJB1 interaction. In our LSPR experiment, TrnSR-WT bound strongly to DNAJB1 with KD ~ 7.4 nM (Supplementary Fig. S5a), comparable to the highest level of NTR–cargo affinity reported for export NTRs in the presence of RanGTP; CRM1–SNUPN (KD ~ 10 nM)63, Xpot–tRNA (KD ~ 2 nM)64, and Xpo4–hypusinated EIF5A (KD ~ 2 nM)65. Strong cargo binding may be an intrinsic feature of TrnSR because TrnSR-WT generally exhibited much higher unnormalized GFP/mCherry ratios than Imp13-WT (Supplementary Table S2a and b). In the LSPR, TrnSR-D750R/D751R bound to DNAJB1 with KD ~ 28 nM (Supplementary Fig. S5b), indicating an ~ 3.8-fold affinity reduction from the WT. In the BHAs, the BG-subtracted GFP/mCherry ratios of TrnSR-D750R/ D751R were as low as 3–25% of the WT in the reproducibility analysis and the final assay (Supplementary Fig. S5c and d, left panels), and the reductions in the GFP/mCherry ratios seem to be more than can be expected from the KD shift in LSPR. However, the GFP/mCherry ratios of TrnSR-D750R/D751R without BG subtraction were 14–52% of WT (Supplementary Fig. S5c and d, right panels), which was more consistent with the affinity reduction in the LSPR. We set BG regions outside beads to highlight the difference in the GFP/mCherry ratios of the ROIs, but the BG regions contain free GFP-cargoes yielding higher signals than the ROIs without GFP-cargo binding (see Supplementary Fig. S3b) because free GFP-cargoes do not penetrate beads. www.nature.com/scientificreports/ Note that the BG of mCherry is low because the beads were used after washing out the unbound GST- mCherry-NTR, while the BG of GFP rises with the dose of GFP-cargo because it includes the fluorescence of free GFP-cargo that is in equilibrium with bead-bound GFP-cargo. All the NTR used are WT. The fitted curves were drawn by Microsoft Excel. Then, we checked the reproducibility of the quantitation (Supplementary Fig. S4a–f left panels), with particu- lar attention to Imp13 since the Imp13 BHA results were more varied and involved more mutants than those of TrnSR. The binding of two TrnSR- and four Imp13-specific cargoes to the WT and mutant NTRs were analyzed three times. The three mean GFP/mCherry values of most triplicates agreed reasonably well with one another. Although the GFP-cargoes used in this reproducibility analysis were prepared independently of those used in the final assays (see Methods), the GFP/mCherry values of mutant NTRs normalized to that of WT were roughly https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ However, we should note that binding inhibition in BHA by the GFP-fusion to cargoes is possible. Inhibition of binding by the N-terminal fusion of GST-mCherry to the NTR is improbable, albeit not impossible, because UBE2I and RanGTP interact with the N-terminal region of the NTR41,51,52 and GST-mCherry-NTR. In any case, the candi- date cargoes that do not bind to the NTRs in BHA could still be authentic. Cargoes bind to the TrnSR and Imp13 mutants with varying affinities. We comprehensively ana- lyzed the binding of 3rd-Z-4% cargoes to the TrnSR and Imp13 mutants by BHA. Examples of the images and GFP/mCherry ratios are shown in Figs. 5 and 6. The GFP/mCherry ratios of the NTR mutants were normalized by the WT NTR ratio for the same cargo to evaluate the mutational effects on the binding of respective cargoes (Supplementary Table S2a and b). Considering the estimated accuracy of the BHA (Fig. 4), we discretized the normalized GFP/mCherry values into three ranks: not reduced (> 0.60), reduced (≤ 0.60 and > 0.20), and much reduced (≤ 0.20). Most of the TrnSR cargoes that bound to TrnSR-WT showed markedly reduced binding to TrnSR-D750R/ D751R. D750/D751 was reported to interact with the SR-domains as referred above, but only a few of the TrnSR cargoes have an apparent SR-domain, including the recently proposed RSY-rich segment25 (Supplementary Table S2a). Thus, D750/D751 should also be involved in the interaction with cargoes lacking an SR-domain. The other mutants, TrnSR-E396R, -D409R, and -R671E, showed diverse effects depending on the combination of the particular mutation and cargo, mostly reducing binding but in a few cases increasing binding (e.g., PPIL1 binding to TrnSR-D409R). Since respective cargoes are affected by different mutations to varying degrees, the cargoes most likely bind to TrnSR in diverse configurations. Likewise, all of the Imp13 cargoes that bound to Imp13-WT exhibited varying affinities to the eight Imp13 mutants, and thus, the cargoes probably differ in how https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ 100 µm mCherry GFP ROI GFP/mCherry 1.09 0.56 0.58 0.77 0.19 0.66 0.15 0.46 0.15 0.02 mCherry GFP ROI GFP/mCherry Enhanced mCherry GFP ROI GFP/mCherry 100 µm 3.84 1.14 2.89 0.50 0.28 mCherry GFP Enhanced ROI GFP/mCherry 0.35 0.09 0.13 0.37 0.12 mCherry GFP ROI GFP/mCherry 1.02 0.94 0.67 0.26 0.25 mCherry GFP ROI GFP/mCherry 2.55 1.35 1.16 0.49 1.11 100 µm WT TrnSR E398R D409R R671E D750R D751R 18. www.nature.com/scientificreports/ SRSF1 mCherry GFP ROI GFP/mCherry 1.09 0.56 0.58 0.77 0.19 0.66 0.15 0.46 0.15 0.02 249. CPSF6 mCherry GFP ROI GFP/mCherry Enhanced 25. NELF mCherry GFP ROI GFP/mCherry 100 µm 3.84 1.14 2.89 0.50 0.28 1 43. RPS6KA3 mCherry GFP Enhanced ROI GFP/mCherry 0.35 0.09 0.13 0.37 0.12 9. SRSF7 mCherry GFP ROI GFP/mCherry 1.02 0.94 0.67 0.26 0.25 WT TrnSR E398R D409R R671E D750R D751R 2. TRAP1 mCherry GFP ROI GFP/mCherry 2.55 1.35 1.16 0.49 1.11 100 µm Figure 5. Bead halo assay of TrnSR mutants. Examples of the GFP and mCherry fluorescent images used to quantify the binding of six GFP-cargoes (number: 3rd-Z-rank) to the WT and four mutants of GST-mCherry- TrnSR are shown. ROIs used for the quantitation are indicated by white circles. BG-subtracted GFP/mCherry ratios calculated from these image values are also designated in the ROI images. Note that these GFP/mCherry ratios are not normalized by the WT ratio and that they slightly differ from those in Supplementary Tale S2a, which are the means of three images. The GFP images of RPS6KA3 and CPSF6 are enhanced equivalently. Scale bar: 100 µm. mCherry GFP ROI GFP/mCherry 1.09 0.56 0.58 0.77 0.19 mCherry GFP ROI GFP/mCherry 1.02 0.94 0.67 0.26 0.25 mCherry GFP ROI GFP/mCherry 2.55 1.35 1.16 0.49 1.11 100 µm 18. SRSF1 mCherry GFP ROI GFP/mCherry 1.09 0.56 0.58 0.77 0.19 9. SRSF7 mCherry GFP ROI GFP/mCherry 1.02 0.94 0.67 0.26 0.25 WT TrnSR E398R D409R R671E D750R D751R 2. TRAP1 mCherry GFP ROI GFP/mCherry 2.55 1.35 1.16 0.49 1.11 100 µm 100 µm 0.77 0.19 0.66 0.15 0.46 0.15 0.02 mCherry GFP ROI GFP/mCherry Enhanced mCherry GFP ROI GFP/mCherry 100 µm 3.84 1.14 2.89 0.50 0.28 mCherry GFP Enhanced ROI GFP/mCherry 0.35 0.09 0.13 0.37 0.12 0.26 0.25 1.16 0.49 WT TrnSR E398R D409R R671E D750R D751R 0.77 0.19 0.66 0.15 0.46 0.15 0.02 249. CPSF6 mCherry GFP ROI GFP/mCherry Enhanced 25. NELF mCherry GFP ROI GFP/mCherry 100 µm 3.84 1.14 2.89 0.50 0.28 1 43. RPS6KA3 mCherry GFP Enhanced ROI GFP/mCherry 0.35 0.09 0.13 0.37 0.12 0.26 0.25 D750R D751R 1.16 0.49 Figure 5. Bead halo assay of TrnSR mutants. Examples of the GFP and mCherry fluorescent images used to quantify the binding of six GFP-cargoes (number: 3rd-Z-rank) to the WT and four mutants of GST-mCherry- TrnSR are shown. ROIs used for the quantitation are indicated by white circles. www.nature.com/scientificreports/ BG-subtracted GFP/mCherry ratios calculated from these image values are also designated in the ROI images. Note that these GFP/mCherry ratios are not normalized by the WT ratio and that they slightly differ from those in Supplementary Tale S2a, which are the means of three images. The GFP images of RPS6KA3 and CPSF6 are enhanced equivalently. Scale bar: 100 µm. 100 µm 0.66 0.15 0.46 0.15 0.02 mCherry GFP ROI GFP/mCherry Enhanced mCherry GFP ROI GFP/mCherry 100 µm 3.84 1.14 2.89 0.50 0.28 mCherry GFP Enhanced ROI GFP/mCherry 0.35 0.09 0.13 0.37 0.12 WT TrnSR E398R D409R R671E D750R D751R 0.66 0.15 0.46 0.15 0.02 249. CPSF6 mCherry GFP ROI GFP/mCherry Enhanced 25. NELF mCherry GFP ROI GFP/mCherry 100 µm 3.84 1.14 2.89 0.50 0.28 1 43. RPS6KA3 mCherry GFP Enhanced ROI GFP/mCherry 0.35 0.09 0.13 0.37 0.12 l Figure 5. Bead halo assay of TrnSR mutants. Examples of the GFP and mCherry fluorescent images used to quantify the binding of six GFP-cargoes (number: 3rd-Z-rank) to the WT and four mutants of GST-mCherry- TrnSR are shown. ROIs used for the quantitation are indicated by white circles. BG-subtracted GFP/mCherry ratios calculated from these image values are also designated in the ROI images. Note that these GFP/mCherry ratios are not normalized by the WT ratio and that they slightly differ from those in Supplementary Tale S2a, which are the means of three images. The GFP images of RPS6KA3 and CPSF6 are enhanced equivalently. Scale bar: 100 µm. they bind. Examples in Fig. 6 clearly illustrate that respective cargoes are affected by different mutations. Two mutants that reduced the binding most among the eight mutants were Imp13-R741E and -K802E/R803E for MAGOH binding, -Y34R/Y35R and -D426R for UBE2I binding, and -Y433R and -E478R for EIF1AX binding. The involvement of the C-terminal, N-terminal, and middle regions of Imp13 in the binding of MAGOH, UBE2I, and EIF1AX, respectively, agrees with the crystal structures51–53. Although EIF1AX is an export cargo of Imp13, they form a complex without RanGTP in the cytoplasm and, RanGTP enhances binding66 (Supplementary Fig. S6). Thus, we believe that BHA reflects cytoplasmic complex formation. Effects of the NTR mutations do not relate to structural similarity of the cargoes. The cargoes may be classified according to the mutant NTR-binding profiles of the cargoes. We clustered the cargoes using Effects of the NTR mutations do not relate to structural similarity of the cargoes. www.nature.com/scientificreports/ The cargoes may be classified according to the mutant NTR-binding profiles of the cargoes. We clustered the cargoes using https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ WT Imp13 Y34R Y35R E73R Y350R D426R E478R R748E K802E R803E Y433R 4. MAGOH mCherry GFP ROI GFP/mCherry 100 µm 1.01 0.46 0.35 0.62 0.72 0.36 1.05 0.15 0.10 58. UBE2I mCherry GFP ROI GFP/mCherry 0.87 0.19 0.26 0.47 0.62 0.75 0.79 0.68 0.10 164. EIF1AX mCherry GFP ROI GFP/mCherry 1.05 0.45 0.49 0.71 0.26 0.13 0.09 0.23 0.32 WT Imp13 Y34R Y35R E73R Y350R D426R E478R R748E K802E R803E Y433R 4. MAGOH mCherry GFP ROI GFP/mCherry 100 µm 1.01 0.46 0.35 0.62 0.72 0.36 1.05 0.15 0.10 58. UBE2I mCherry GFP ROI GFP/mCherry 0.87 0.19 0.26 0.47 0.62 0.75 0.79 0.68 0.10 164. EIF1AX mCherry GFP ROI GFP/mCherry 1.05 0.45 0.49 0.71 0.26 0.13 0.09 0.23 0.32 Figure 6. Bead halo assay of Imp13 mutants. Examples of the GFP and mCherry fluorescent images used to quantify the binding of three GFP-cargoes (number: 3rd-Z-rank) to the WT and eight mutants of GST- mCherry-Imp13 are shown. ROIs used for the quantitation are specified by white circles. BG-subtracted GFP/ mCherry ratios calculated from these image values are also indicated in the ROI images. Note that the GFP/ mCherry ratios are not normalized by the WT ratio and that they slightly differ from those in Supplementary Tale S2b, which are the mean of three images. Scale bar: 100 µm. Ward’s method to explore this possibility (Figs 1d and 7) We assigned discrete values to the three ranks of the WT Imp13 Y34R Y35R E73R Y350R D426R E478R R748E K802E R803E Y433R 4. MAGOH mCherry GFP ROI GFP/mCherry 100 µm 1.01 0.46 0.35 0.62 0.72 0.36 1.05 0.15 0.10 58. UBE2I mCherry GFP ROI GFP/mCherry 0.87 0.19 0.26 0.47 0.62 0.75 0.79 0.68 0.10 164. EIF1AX mCherry GFP ROI GFP/mCherry 1.05 0.45 0.49 0.71 0.26 0.13 0.09 0.23 0.32 WT Imp13 Y34R Y35R E73R Y350R D426R E478R R748E K802E R803E Y433R 4. MAGOH mCherry GFP ROI GFP/mCherry 100 µm 1.01 0.46 0.35 0.62 0.72 0.36 1.05 0.15 0.10 58. UBE2I mCherry GFP ROI GFP/mCherry 0.87 0.19 0.26 0.47 0.62 0.75 0.79 0.68 0.10 164. EIF1AX mCherry GFP ROI GFP/mCherry 1.05 0.45 0.49 0.71 0.26 0.13 0.09 0.23 0.32 Figure 6. Bead halo assay of Imp13 mutants. www.nature.com/scientificreports/ Examples of the GFP and mCherry fluorescent images used to quantify the binding of three GFP-cargoes (number: 3rd-Z-rank) to the WT and eight mutants of GST- mCherry-Imp13 are shown. ROIs used for the quantitation are specified by white circles. BG-subtracted GFP/ mCherry ratios calculated from these image values are also indicated in the ROI images. Note that the GFP/ mCherry ratios are not normalized by the WT ratio and that they slightly differ from those in Supplementary Tale S2b, which are the mean of three images. Scale bar: 100 µm. D426R 0.72 0.10 D426R 0.72 0.10 Figure 6. Bead halo assay of Imp13 mutants. Examples of the GFP and mCherry fluorescent images used to quantify the binding of three GFP-cargoes (number: 3rd-Z-rank) to the WT and eight mutants of GST- mCherry-Imp13 are shown. ROIs used for the quantitation are specified by white circles. BG-subtracted GFP/ mCherry ratios calculated from these image values are also indicated in the ROI images. Note that the GFP/ mCherry ratios are not normalized by the WT ratio and that they slightly differ from those in Supplementary Tale S2b, which are the mean of three images. Scale bar: 100 µm. Ward’s method to explore this possibility (Figs. 1d and 7). We assigned discrete values to the three ranks of the normalized GFP/mCherry values described in the previous section (Supplementary Table S2a and b) as fol- lows: 1 for > 0.6 (not reduced), 0.5 for ≤ 0.60 and > 0.20 (reduced), and 0 for ≤ 0.20 (much reduced), and used the Euclidean distances of these values as the metric. Both the TrnSR and Imp13 cargoes can be classified into a reasonable number of groups. For example, they can be divided into five (Fig. 7a) or six (b) clusters by setting an arbitrary breakpoint. However, the clusters are not separated discretely; the mutant NTR-binding profiles change gradually through the clusters. Ward’s method to explore this possibility (Figs. 1d and 7). We assigned discrete values to the three ranks of the normalized GFP/mCherry values described in the previous section (Supplementary Table S2a and b) as fol- lows: 1 for > 0.6 (not reduced), 0.5 for ≤ 0.60 and > 0.20 (reduced), and 0 for ≤ 0.20 (much reduced), and used the Euclidean distances of these values as the metric. Both the TrnSR and Imp13 cargoes can be classified into a reasonable number of groups. www.nature.com/scientificreports/ 7) and Pfam71 domain https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ E398R D409R R671E D750R/D751R Disordered region Sequence similarity O60568_PLOD3 P33981_TTK Q9Y305_ACOT9 Q9Y3C6_PPIL1 P61006_RAB8A Structural similarity P14635_CCNB1 Q9GZL7_WDR12 Q9H4H8_FAM83D P29372_MPG Q86U70_LDB1 Q69YN2_CWF19L1 Q9UJU6_DBNL P17844_DDX5 P24941_CDK2 Q9BY42_RTF2 Q8IWS0_PHF6 P51812_RPS6KA3 Q9Y6G9_DYNC1LI1 O60828_PQBP1 Q8N7H5_PAF1 Q9NZ63_C9orf78 Q9NTZ6_RBM12 P25685_DNAJB1 Q96ES7_SGF29 Q05048_CSTF1 O00422_SAP18 Q9UDY4_DNAJB4 Q12931_TRAP1 P62136_PPP1CA O00170_AIP Q16629_SRSF7 * Q01081_U2AF1 * Q13247_SRSF6 * Q16630_CPSF6 * Q07955_SRSF1 * * P18615_NELFE Y34R/Y35R E73R Y350R K802E/K803E D426R Y433R E478R R748E Disordered region Sequence similarity Structural similarity P52657_GTF2A2 Q9NUP9_LIN7C O15511_ARPC5 Q9H0A0_NAT10 Q15942_ZYX P63279_UBE2I Q04695_KRT17 P05787_KRT8 P62913_RPL11 Q96GQ7_DDX27 Q969G3_SMARCE1 Q8WUB8_PHF10 P23511_NFYA Q9UNP9_PPIE O43663_PRC1 Q9NQ88_TIGAR Q9H3K6_BOLA2 Q9NRG0_CHRAC1 P13639_EEF2 Q9NRF9_POLE3 P13798_APEH P11413_G6PD Q9BV38_WDR18 P35520_CBS P61326_MAGOH 4 P 7 X A 1 F I E _ 3 1 8 P57772_EEFSEC Q9BQ67_GRWD1 0.0 0.5 1.0 1.5 Distance 2.0 0.0 1.0 Distance 4.0 2.0 3.0 a b TrnSR Imp13 Binding relative to WT Disordered region >0.6 0.2~0.6 ≤0.2 0.6~0.8 0.4~0.6 0.2~0.4 <0.2 Binding relative to WT Disordered region >0.6 0.2~0.6 ≤0.2 0.6~0.8 0.4~0.6 0.2~0.4 <0.2 E398R D409R R671E D750R/D751R Disordered region Sequence similarity O60568_PLOD3 P33981_TTK Q9Y305_ACOT9 Q9Y3C6_PPIL1 P61006_RAB8A Structural similarity P14635_CCNB1 Q9GZL7_WDR12 Q9H4H8_FAM83D P29372_MPG Q86U70_LDB1 Q69YN2_CWF19L1 Q9UJU6_DBNL P17844_DDX5 P24941_CDK2 Q9BY42_RTF2 Q8IWS0_PHF6 P51812_RPS6KA3 Q9Y6G9_DYNC1LI1 O60828_PQBP1 Q8N7H5_PAF1 Q9NZ63_C9orf78 Q9NTZ6_RBM12 P25685_DNAJB1 Q96ES7_SGF29 Q05048_CSTF1 O00422_SAP18 Q9UDY4_DNAJB4 Q12931_TRAP1 P62136_PPP1CA O00170_AIP Q16629_SRSF7 * * Q01081_U2AF1 * Q13247_SRSF6 * Q16630_CPSF6 * Q07955_SRSF1 * * P18615_NELFE 0.0 0.5 1.0 1.5 Distance 2.0 a TrnSR Binding relative to WT Disordered region >0.6 0.2~0.6 ≤0.2 0.6~0.8 0.4~0.6 0.2~0.4 <0.2 E398R D409R R671E D750R/D751R Disordered region Sequence similarity O60568_PLOD3 P33981_TTK Q9Y305_ACOT9 Q9Y3C6_PPIL1 P61006_RAB8A Structural similarity P14635_CCNB1 Q9GZL7_WDR12 Q9H4H8_FAM83D P29372_MPG Q86U70_LDB1 Q69YN2_CWF19L1 Q9UJU6_DBNL P17844_DDX5 P24941_CDK2 Q9BY42_RTF2 Q8IWS0_PHF6 P51812_RPS6KA3 Q9Y6G9_DYNC1LI1 O60828_PQBP1 Q8N7H5_PAF1 Q9NZ63_C9orf78 Q9NTZ6_RBM12 P25685_DNAJB1 Q96ES7_SGF29 Q05048_CSTF1 O00422_SAP18 Q9UDY4_DNAJB4 Q12931_TRAP1 P62136_PPP1CA O00170_AIP Q16629_SRSF7 * * Q01081_U2AF1 * Q13247_SRSF6 * Q16630_CPSF6 * Q07955_SRSF1 * * P18615_NELFE Y34R/Y35R E73R Y350R K802E/K803E D426R Y433R E478R R748E Disordered region Sequence similarity Structural similarity P52657_GTF2A2 Q9NUP9_LIN7C O15511_ARPC5 Q9H0A0_NAT10 Q15942_ZYX P63279_UBE2I Q04695_KRT17 P05787_KRT8 P62913_RPL11 Q96GQ7_DDX27 Q969G3_SMARCE1 Q8WUB8_PHF10 P23511_NFYA Q9UNP9_PPIE O43663_PRC1 Q9NQ88_TIGAR Q9H3K6_BOLA2 Q9NRG0_CHRAC1 P13639_EEF2 Q9NRF9_POLE3 P13798_APEH P11413_G6PD Q9BV38_WDR18 P35520_CBS P61326_MAGOH 4 P 7 X A 1 F I E _ 3 1 8 P57772_EEFSEC Q9BQ67_GRWD1 0.0 0.0 1.0 Distance 4.0 2.0 3.0 b Imp13 Binding relative to WT Disordered region >0.6 0.2~0.6 ≤0.2 0.6~0.8 0.4~0.6 0.2~0.4 <0.2 Binding relative to WT Disordered region >0.6 0.2~0.6 ≤0.2 0.6~0.8 0.4~0.6 0.2~0.4 <0.2 Figure 7. Cargo clustering by the binding specificity to TrnSR or Imp13 mutants. www.nature.com/scientificreports/ For example, they can be divided into five (Fig. 7a) or six (b) clusters by setting an arbitrary breakpoint. However, the clusters are not separated discretely; the mutant NTR-binding profiles change gradually through the clusters. g y g We examined the sequence and structural similarities of the cargoes to relate the mutant NTR-binding spe- cificities and the primary and tertiary structures of the cargoes. From both determined structures and homology modeled structures (Supplementary Table S3a), we calculated structural similarities using MICAN and template modeling scores (TM-scores) of MICAN70 for the structure assigned regions of all possible pairs of cargoes. MICAN is a structural alignment algorithm for identifying the best structural alignment between a protein pair by disregarding the connectivity between secondary structure elements, and thus, it can find similar secondary structure packing arrangements with different topologies. Some TrnSR cargoes share a structural similarity, but they are separated in cargo clustering by mutant TrnSR-binding intensities (Fig. 7a); for example, SRSF7, SRSF6, CPSF6, SRSF1, NELFE, and RBM12 have similar RNA recognition motif domains, but these proteins are distributed in the dendrogram based on mutant-binding specificity. Fewer Imp13 cargoes share structural similarity, and they have also clustered apart (Fig. 7b); high structural similarity is detected only among α-helical substructures of KRT8, KRT17, and PRC1. The sequence similarity search results (Fig. www.nature.com/scientificreports/ (a) The TrnSR cargoes were hierarchically clustered by the binding intensities to the TrnSR mutants. For each cargo, GFP/mCherry ratios of the TrnSR mutants were normalized to WT, and converted into three values, ≤ 0.2 into 0, > 0.2 and ≤ 0.6 into 0.5, and > 0.6 into 1, as indicated by the magenta scale (Supplementary Table S2a). Ward’s method was used among the cargoes with the Euclidean distances calculated from these discrete values. The hierarchical clustering was performed using R version 3.5.1. The dashed line on the dendrogram is an arbitrary boundary to separate the cargoes into five groups. The blue scale signifies the content rates of disordered regions, where ten or more consecutive residues are predicted to be disordered. Sequence similarity was analyzed by SSEARCH, and homologous proteins (E-value < 10–9) are indicated by the same color. 3D-structural similarity was evaluated by the TM-score using determined or homology modeled structures (Supplementary Table S3a). Proteins with high structural similarity (TM-score > 0.6) are designated by the same solid colors, and those with similarity (0.6 > TM-score > 0.5) are represented by the same frame colors. Red asterisks indicate proteins annotated to have an SR-domain (Arg/Ser-rich domain) in UniProt (https://www.uniprot.org), and blue asterisks indicate an RSY-rich segment25 other than the SR-domain. The proximal mutants of TrnSR are specified by the same color. (b) The Imp13 cargoes were clustered by the binding intensities to the Imp13 mutants. Illustrated similarly to (a). The dashed line arbitrarily separates the cargoes into six groups. O60568_PLO P33981_TT Q9Y305_AC Q9Y3C6_PP P61006_RA P14635_CC Q9GZL7_W Q9H4H8_FA P29372_MP Q86U70_LD Q69YN2_CW Q9UJU6_DB P17844_DDX P24941_CD Q9BY42_RT Q8IWS0_PH P51812_RPS Q9Y6G9_DY O60828_PQ Q8N7H5_PA Q9NZ63_C9 Q9NTZ6_RB P25685_DNA Q96ES7_SG Q05048_CS O00422_SA Q9UDY4_DN Q12931_TRA P62136_PPP O00170_AIP Q16629_SR * * Q01081_U2A * Q13247_SR * Q16630_CP * Q07955_SR * * P18615_NEL Y34R/Y35R E73R Y350R K802E/K803E D426R Y433R E478R R748E Disordered region Sequence similarity Structural similarity P52657_GTF2A2 Q9NUP9_LIN7C O15511_ARPC5 Q9H0A0_NAT10 Q15942_ZYX P63279_UBE2I Q04695_KRT17 P05787_KRT8 P62913_RPL11 Q96GQ7_DDX27 Q969G3_SMARCE1 Q8WUB8_PHF10 P23511_NFYA Q9UNP9_PPIE O43663_PRC1 Q9NQ88_TIGAR Q9H3K6_BOLA2 Q9NRG0_CHRAC1 P13639_EEF2 Q9NRF9_POLE3 P13798_APEH P11413_G6PD Q9BV38_WDR18 P35520_CBS P61326_MAGOH 4 P 7 X A 1 F I E _ 3 1 8 P57772_EEFSEC Q9BQ67_GRWD1 0.0 1.0 Distance 4.0 2.0 3.0 b Imp13 0.2~0.4 <0.2 Binding relative to WT Disordered region >0.6 0.2~0.6 ≤0.2 0.6~0.8 0.4~0.6 0.2~0.4 <0.2 Figure 7. Cargo clustering by the binding specificity to TrnSR or Imp13 mutants. (a) The TrnSR cargoes were hierarchically clustered by the binding intensities to the TrnSR mutants. Discussion In this work, we analyzed the effects of NTR mutations on cargo binding. Biochemical studies with mutant proteins may involve the risk of distant effects of overall structural collapse. However, most binding reductions observed here are plausibly due to local effects of the mutations because, for each mutation, a certain number of cargoes are unaffected, and the unaffected cargoes differ between mutations, even those which are proximal to each other (Fig. 7 and Supplementary Table S2). Furthermore, in the verifiable cases, the mutant NTR-binding cargo profiles agree with the reported structures (Fig. 6). In our mutagenesis, we preferentially selected residues that contact cargoes in crystal structures regardless of their KL values, and then selected the other residues located near cargoes and exhibiting high KL values, i.e., E398 and D409 of TrnSR and Y350 and R748 of Imp13. As the four mutants are defective in binding to some cargoes, the combination of structural and evolutionary considerations seems useful to predict amino acid positions significant for ortholog function. However, ETA by itself is not sufficient to identify all cargo binding-related residues, because mutations at positions with lower KL values (e.g., Y34/Y35 and E73 of Imp13) also affected cargo binding. Further analysis of more mutants should help further delineate the utility and limitations of ETA in this setting. To analyze NTR–cargo binding, we applied BHA, which is advantageous to analyze many samples but less quantitative than other methods such as isothermal titration calorimetry or SPR. We reduced measurement deviation using the red fluorescent GST-mCherry-NTR proteins and utilizing the GFP/mCherry ratios (Fig. 4). Based on this deviation, we set the threshold for a sig- nificant reduction in NTR mutant assays as > 40% reduction from the WT value. Adjusting the concentrations of all the cargoes within their linear ranges of NTR binding is difficult, and some of the cargoes were assayed at near saturation levels (Fig. 4f, h, j). In those cases, a significant reduction in the GFP/mCherry value sufficiently demonstrates reduced affinity to the NTR mutants, but values equal to that of WT do not necessarily indicate a WT level of binding affinity. In the final assays, at least one NTR mutant reduced the GFP/mCherry value for most of the respective cargoes (Fig. 7 and Supplementary Table S2a and b), indicating that the assay condition was sufficient to specify the mutation that most impacted the binding to each cargo. Discussion Since the reduced-binding NTR mutants and the degree of the binding reduction differed among the cargoes (Fig. 7 and Supplementary Table S2a and b), respective cargoes are affected differently by a set of NTR mutations, presumably reflecting NTR–cargo binding configuration diversity. g gi g y Another potential concern about our BHA is that we used bacterially expressed proteins devoid of eukary- otic posttranslational modifications (PTMs). Many PTMs on both cargoes and NTRs affect binding specificity2; for example, hypusinated EIF5A purified from HeLa cells binds to Xpo4–RanGTP complexes ~ 35 times more strongly than unmodified recombinant EIF5A65. Thus, our assay may not precisely mirror the binding specificity in vivo, but our results should still reflect the NTR–cargo binding configuration diversity. Degradation products of GFP-cargo proteins in some bacterial extracts used (Supplementary Fig. S1) should also be noted. If degraded cargo fragments have altered affinities to the NTR, the quantitation values in BHA may be deflected. However, the value order of the NTR mutants in binding to a particular cargo can be assumed not to change, because this kind of fragments can usually be regarded to affect uniformly on the values. g y gf y Most TrnSR cargoes have neither an SR-domain nor an RSY-rich segment expected to act as an NLS, and no Imp13-specific NLS is known. Therefore, our results suggest that cargoes without a canonical NLS must interact with varying contact sites on an NTR; at least, each position on an NTR contributes to the binding of different cargo sets, and the degree of contribution also differs widely. Thus, the NTRs presumably accommodate the diverse structures of NLS-less cargoes, differentially using multiple contact sites. Although our mutants do not cover all possible cargo contact sites, they sufficiently elucidate the basic mechanism of cargo recognition. Assuming that an NTR uses multiple contact sites in various combinations to interact with structurally diverse cargoes, correlations among the cargo-binding profiles of NTR mutants may reflect the formation of an inter- face common to some cargoes. The GFP/mCherry values of Imp13-Y34R/Y35R, -E73R, -Y350R, and -Y433R have high correlation coefficients (Supplementary Table S2d), and the N-terminal region containing these sites may form an interface that interacts with the cargoes at partially common and partially distinct contact sites. The binding of UBE2I may be a good example that uses this interface. The cargoes affected by these mutations are clustered proximally in the analysis in Fig. www.nature.com/scientificreports/ For each cargo, GFP/mCherry ratios of the TrnSR mutants were normalized to WT, and converted into three values, ≤ 0.2 into 0, > 0.2 and ≤ 0.6 into 0.5, and > 0.6 into 1, as indicated by the magenta scale (Supplementary Table S2a). Ward’s method was used among the cargoes with the Euclidean distances calculated from these discrete values. The hierarchical clustering was performed using R version 3.5.1. The dashed line on the dendrogram is an arbitrary boundary to separate the cargoes into five groups. The blue scale signifies the content rates of disordered regions, where ten or more consecutive residues are predicted to be disordered Sequence similarity was analyzed by SSEARCH and homologous proteins (E value<10–9) are indicated https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ annotations (Supplementary Table S3a) overlapped with those of the structural similarity search. The fraction of predicted disordered residues of the cargoes also appears irrelevant to the clustering (Fig. 7). Thus, the specific- ity determination of the NTR–cargo interaction must result from more complicated or fortuitous mechanisms than we expected. annotations (Supplementary Table S3a) overlapped with those of the structural similarity search. The fraction of predicted disordered residues of the cargoes also appears irrelevant to the clustering (Fig. 7). Thus, the specific- ity determination of the NTR–cargo interaction must result from more complicated or fortuitous mechanisms than we expected. Discussion 7b, but we could not find the structural similarity among them. A more advanced type of analysis may be needed to extract protein features useful to predict specific cargoes. Not enough mutants were analyzed from TrnSR or other regions of Imp13 to discuss them in similar detail. Nevertheless, our results support the idea extrapolated from a limited number of crystal structures that NTRs bind to diverse cargoes differently. f TrnSR and Imp13 form one of the closest pairs in the Impβ family phylogram but share fewer cargoes3. Although a high KL value in ETA is not essential for a cargo contact site, the selected positions with high KL values appeared significant for the binding of the specific cargoes. Therefore, TrnSR and Imp13 might have diverged evolutionarily from a common ancestral NTR to adapt to bind specific cargoes. In our phylogenetic profiles of TrnSR and Imp13, TrnSR orthologs are widely conserved in eukaryotic species except for excavates, whereas Imp13 orthologs are found only in unikonts (Supplementary Fig. S7). Thus, the functional divergence of TrnSR and Imp13 is likely to have emerged after the divergence to unikonts. However, many Imp13 cargo genes are found in unikonts and widely conserved in eukaryotic species. Moreover, we could not find any relevance between the phylogenetic profiles of the cargoes and the clustering results based on the mutant NTR-binding specificity. It might be possible that cargoes acquired after the divergence to unikonts were assigned to either Scientific Reports \| (2021) 11:15649 \| https://doi.org/10.1038/s41598-021-94948-1 www.nature.com/scientificreports/ TrnSR or Imp13 and also some of the cargoes already present before the divergence to unikonts were reassigned to Imp13. For this type of reassignment, both cargoes and NTRs changed gradually and coevolutionarily during the functional divergence. Consequently, some cargoes and NTRs may settle down to similar binding configura- tions. There were a few mixed pairs of TrnSR and Imp13 cargoes sharing sequence similarities (p value < 10–15), such as the RNA helicases DDX5 and DDX27 (Supplementary Table S3b). DDX5 and DDX27 are a homologous pair (34% sequence identity and p value = 9.9 × 10–45) and are widely conserved in eukaryotic species. Thus, DDX5 and DDX27 are likely to have been present before the functional divergence of TrnSR and Imp13. Methods D i Detection of amino acid sites involved in functional differentiation between TrnSR and Imp13. Seventy-eight and 72 amino acid sequences of metazoan TrnSR and Imp13 orthologs, respectively, were collected from the Inparanoid database72, and a multiple sequence alignment that included all of them was produced using MAFFT73. The amino acid composition at each alignment site was calculated within the two groups of orthologs, TrnSR and Imp13 (Fig. 1a and Supplementary Table 1). In this calculation, the weighting method for the residue count proposed by Gerstein et al.74 was used, and then the probability of each amino acid was estimated using Dirichlet mixture prior75. The difference between the TrnSR and Imp13 orthologs in the amino acid composition at each alignment site was evaluated by the modified KL value60, which is simply the Kullback–Leibler symmetric divergence defined as follows: 20 i=1 p(i)log p(i) q(i) + 20 i=1 q(i)log q(i) p(i) where p(i) and q(i) are the probability of amino acid i at an alignment site of the TrnSR and Imp13 orthologs, respectively. The KL values were used to predict the sites involved in the functional differentiation between TrnSR and Imp13. Sites at which more than half of the sequences had gaps were skipped. where p(i) and q(i) are the probability of amino acid i at an alignment site of the TrnSR and Imp13 orthologs, respectively. The KL values were used to predict the sites involved in the functional differentiation between TrnSR and Imp13. Sites at which more than half of the sequences had gaps were skipped. Plasmids and proteins. WT and mutant GST-mCherry-TrnSR and -Imp13 proteins were expressed from the pGEX-6p3 vector (GE Healthcare). Human TrnSR and Imp13 cDNAs carried in pGEX-6p3 were mutagen- ized using the KOD plus mutagenesis kit (Toyobo) and a PCR fragment encoding mCherry from the pmCherry- C1 plasmid (Clontech) was inserted between the GST and the TrnSR or Imp13 coding regions. The proteins were expressed in Escherichia coli BL21 and purified on GSH-Sepharose 4B (GE Healthcare) and a Mono Q column (GE Healthcare) as described previously76. GST-TrnSR proteins without mCherry-fusion used in the LSPR experiment were also purified similarly on GSH-Sepharose 4B. p pi y p GFP-cargo fusion proteins were also prepared as described previously76. cDNAs for the cargoes were ampli- fied from a HeLa cDNA library (SuperScript, Life Technology) by PCR and inserted into the pQE80L vector (Qiagen) carrying the GFP gene. Methods D i The protein sequences derived from the cloned DNA sequences are shown in Supplementary Table S2e. The proteins were expressed in E. coli BL21 and extracted by extraction buffer [50 mM Tris–HCl (pH 8.0, 4 °C), 500 mM NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.5 mM PMSF]. After dialysis against transport buffer (TB) [20 mM HEPES–KOH (pH 7.3), 110 mM KOAc, 2 mM MgOAc, 5 mM NaOAc, 0.5 mM EGTA], the extracts were cleared by a high-speed centrifuge. The GFP-cargo proteins in the extracts were analyzed and quantified by triplicate Western blotting with an anti-GFP monoclonal antibody (Roche, 1181446), a horseradish peroxidase-conjugated anti-mouse IgG antibody (BioRad, 170-6516), and Immobilon Western chemiluminescent HRP substrate (Millipore). Chemiluminescence images were acquired and analyzed by a Fusion Solo7S image analyzer (Vilber Lourmat). An extract containing intact GFP was used as the standard. The concentrations of GFP moiety and total protein in the extracts were normalized by adding an E. coli extract without GFP. Because the expression levels varied widely, we normalized the extracts into three conditions (Supplementary Table S2a and b). Extracts containing extensively degraded cargo were not analyzed. GFP-cargo proteins used only in the reproducibility analysis (Supplementary Fig. S4a–f, left panels) were prepared similarly but quantified by a fluorometer. i yl His6-tagged DNAJB1 and CBX1 proteins used in the LSPR experiment were expressed and extracted similarly and then purified on TALON metal-affinity resin (Clontech). Bead halo assay. The purified GST-mCherry-TrnSR or -Imp13 protein was mixed with GSH-Sepharose beads at a ratio of 5 mg protein to 1 mL (bed volume) beads in TB, and after incubation for 20 min with occa- sional mixing, the beads were washed three times with TB. The beads were then mixed with an excessive amount of the normalized extract in EHBN buffer62 (10 mM EDTA, 0.5% 1,6-hexanediol, 10 mg/mL BSA, and 125 mM NaCl). After incubation on ice for 30 min, the beads were observed by a confocal laser scanning microscope FX1200 (Olympus) with a 40 × objective UPlanSApo40X2 (NA 0.95). Images were acquired using FV10-ASW software and 473 and 559 nm laser lines for GFP and mCherry, respectively, with Kalman 3 filter mode. Twelve- bit images with pixel dimensions of 1024 × 1024 (317 × 317 µm) were used for quantitation with the Fiji package of ImageJ software77. ROIs for quantitation were set on the mCherry images. Discussion Those genes might have undergone alterations such as mutations on protein surfaces, gain/loss of domains or disorder regions by some constraints during the functional divergence to adapt to recognition by TrnSR or Imp13. Additionally, concave surfaces of NTRs might have changed following the alterations. Further coevolutionary analysis of the NTRs and the cargoes would be vital in uncovering how the functional divergence responsible for unique cargo recognition emerged. www.nature.com/scientificreports/ outlines were fixed as ROIs, and larger ring-shaped regions with the same width but 5 pixels away outside the outlines were established as BG regions (Fig. 3 and Supplementary Fig. S2). The same ROIs and BG regions were applied to the corresponding GFP images. In the final assay, ~ 55%, ~ 40%, ~ 4.6%, and ~ 0.15% of the images included one, two, three, and four beads as the ROIs, respectively. The mean intensities of these regions in an image were quantified, and the value of GFP/mCherry = (ROIGFP-BGGFP)/(ROImCherry-BGmCherry) was used as the index for NTR–cargo binding. Three images for one NTR–cargo pair were quantified and averaged, and then the GFP/mCherry values of NTR mutants were normalized by dividing by that value of WT NTR binding to the same cargo. Statistical analysis of the results of bead halo assay. The Mann–Whitney U test was employed to determine the statistical significance of the differences in GFP/mCherry values between WT and each mutant NTR. In the reproducibility analysis (Supplementary Fig. S4a–f, left panels), all the GFP/mCherry values in trip- licate were pooled and tested. In the final assay (Supplementary Table S2a and b), three values of each WT and mutant NTR were tested, and the possible minimum p value was 0.05. Localized surface plasmon resonance. LSPR experiments were performed on an OpenSPR instrument (Nicoya) using NTA sensor tips (SEN AU NTA). TB containing 0.05% Tween-20 and excluding EGTA flowed at 20 µL/min as the running buffer, and proteins were injected for 217 s at 600-s intervals into the flow cell. After the sensor was charged with Ni2+ ions, 50 µg/mL His6-DNAJB1 was injected into channel 2, His6-CBX1, which does not bind to TrnSR (Supplementary Table S2a), was injected into channel 1 as a reference, and then varying concentrations of GST-TrnSR (see Supplementary Fig. S5a and b) were injected into channels 1 and 2 to observe the association. Dissociation in the buffer flow was observed continuously. The sensor tip was regenerated by 200 mM imidazole and 10 mM glycine–HCl (pH 1.5) and reused no more than three times. The resonance intensity of channel 2 was corrected by subtracting that of channel 1, and the kinetic curves were fitted to the one-to-one model using TraceDrawer 1.8 (Ridgeview Instruments) to derive kon, koff, and KD. Hierarchical clustering of the cargoes based on the difference in binding to the NTR mutants. www.nature.com/scientificreports/ The cargoes were hierarchically clustered using Ward’s method as implemented in the software R (version 3.5.1)78. Based on the experimentally estimated deviation of the BHA values (Fig. 4a–d), the normalized GFP/mCherry ratios (Supplementary Table S2a and b) were converted into three discrete values: GFP/mCherry ratios ≤ 0.2 into 0, > 0.2 and ≤ 0.6 into 0.5, and > 0.6 into 1. The Euclidean distances between the cargoes calcu- lated from these discrete values were used as the metric. Sequence and structural similarity search and disorder prediction. Sequence similarities among the cargo proteins were computed using SSEARCH 36.3.8d79, and sequences with an E-value < 10–9 were judged as similar. Disordered regions were predicted using DISOPRED 3.180. For 3D-structural comparison, structures were aligned using MICAN70, and the similarity was evaluated by the TM-score of MICAN. Structures with TM- scores > 0.6 were judged as highly similar and > 0.5 as similar. For protein regions whose structures are unknown, templates for structural modeling were selected based on HHpred81 alignment, and the structures were modeled using Homology Model application in Molecular Operating Environment (MOE 2016.08; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7). Evolutionary profiles of TrnSR, Imp13, and their cargoes. Orthologous sequences of TrnSR and Imp13 were searched using HMMER-2.2.0 for glocal alignment82. For the glocal profile, an HMM was gen- erated from a multiple sequence alignment produced using MAFFT73 with manually curated orthologous sequences. Sequence search-based ortholog identification of the cargo proteins was conducted automatically using OrthoMCL 2.0.983 with our proteome data sets in a previous study84. RSY‑rich segment search. The sequences of TrnSR cargoes were scanned with sliding windows of 15–30 amino acids in length, and then the composition of R, S, and Y (RSYcomp), dipeptide composition of RS, SR, SY, and YS (dipepRSYcomp), and SY and YS (dipepSYcomp) were calculated to search for RSY-rich segments25. Frag- ments with RSYcomp > 0.3, dipepRSYcomp > 0.2, and dipepSYcomp > 0 were defined as RSY-rich segments. Received: 11 August 2020; Accepted: 20 July 2021 Methods D i The outlines of the beads were selected using the Analyze Particle command with options: size = 5020–17,700 (diameter = 80–150 µm), circu- larity = 0.80–1.00, and "exclude on edges". Ring-shaped regions with a width of 5 pixels along the inside of the https://doi.org/10.1038/s41598-021-94948-1 Scientific Reports \| (2021) 11:15649 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ E., Gu, H. & Chook, Y. M. Modular organization and combinatorial energetics of prolin signals. 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DNA sequencing was performed by the Support Unit for Bio-Material Analysis, RIKEN BSI Research Resource Center. This work was supported by JSPS KAKENHI Grant Numbers 15K07064 and 18K06235 to M.K., Grant Number 18K11543 to K.I., and Grant Numbers 21H02482, 18H02442, and 15H05929 to N.I. Author contributions M.K. and K.I. designed the research; M.K., Y.M., and Y.H.S. performed the experiments; K.I. performed the bioinformatic analyses. P.H. and N.I. supervised research. M.K. and K.I. wrote the original draft; P.H. and N.I. reviewed and edited the manuscript; all authors approved the manuscript. www.nature.com/scientificreports/ DISOPRED3: precise disordered region predictions with annotated protein-binding activity. Bioinfor matics 31, 857–863. https://doi.org/10.1093/bioinformatics/btu744 (2015). 81. Soding, J. Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960. https://doi.org/10.1093/bioin formatics/bti125 (2005).i 82. Eddy, S. R. 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Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-94948-1. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-94948-1. Correspondence and requests for materials should be addressed to M.K., K.I. or N.I. Correspondence and requests for materials should be addressed to M.K., K.I. or N.I. Correspondence and requests for materials should be addressed to M.K., K.I. or N.I Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W3043062835	https://www.nature.com/articles/s41467-020-17041-7.pdf	English	null	Analysis of compositions of microbiomes with bias correction	Nature communications	2,020	cc-by	14,622	ARTICLE Analysis of compositions of microbiomes with bias correction https://doi.org/10.1038/s41467-020-17041-7 OPEN Huang Lin 1 & Shyamal Das Peddada1✉ 1 Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA 15261, USA. ✉email: shyamal.peddada@nih.gov NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications ARTICLE Clearly, the true absolute abundance of each taxon is 50% more in subject B’s ecosystem as compared with subject A’s. However, they each have the same library size (six each) in their respective samples. Furthermore, sample relative abundance as well as sample absolute abundances are identical in the two samples. If a normalization method is based only on the library size and ignores the sampling fraction, then the two samples would be considered as normalized. Con- sequently, an investigator would falsely conclude that none of the taxa are differentially abundant in the two ecosystems. This erroneous conclusion would be avoided if one recognizes that we have a larger sampling fraction in the sample obtained from A’s ANCOM2, which is based on Aitchison’s methodology, uses relative abundances to infer about absolute abundances. According to an extensive simulation study1, among the available methods for DA analysis, only ANCOM performs well in con- trolling FDR while maintaining high power, as long as the sample size is not too small. One of the deﬁciencies of ANCOM is that it does not provide p value for individual taxon, nor can it provide standard errors or conﬁdence intervals of DA for each taxon, and it can be computationally intensive. The Differential Ranking (DR) methodology6 reformulates the DA analysis as a multinomial regression problem. By imposing the Additive Log-Ratio transformation to relative abundances, the DR methodology accounts for compositionality of microbiome data. As demonstrated in6, the ranks of relative differentials perfectly correlate with ranks of absolute differentials. However, similar to ANCOM, the DR procedure does not provide p values or conﬁdence intervals to declare statistical signiﬁcance. g It is important to distinguish between absolute and relative abundances of taxa in a unit volume of an ecosystem. Change in Absolute abundance Ecosystem A Ecosystem B Relative abundance 10% 17% 17% 33% 13% 22% 30% 22% 13% 23% Fig. 1 The distinction between absolute abundances and relative abundances. As shown in this ﬁgure, all taxa (in different colors and shapes) may be identically abundant in a unit volume of two ecosystems (e.g., a unit volume of gut), except for one differentially abundant taxon (the green variety). Due to this one differentially abundant taxon, the two ecosystems may differ in the relative abundance of all taxa. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 A A number of procedures have been proposed and used in the literature for identifying deferentially abundant taxa between two or more ecosystems. A detailed survey of some of the existing methods and their performance has been discussed in Weiss et al.1. As noted in a list of studies2–6, the observed microbiome data are relative abundances which sum to a constant, hence they are compositional. Standard statistical methods are not appropriate for analyzing compositional data7. Methods such as ANOVA, Kruskal–Wallis test do not appro- priately take into consideration the compositional feature of microbiome data when performing differential abundance (DA) analysis. As demonstrated in literatures1,2, these methods are subject to inﬂated false discovery rates (FDR). Although meta- genomeSeq8 was speciﬁcally developed for microbiome data, it too is subject to inﬂated FDR under the Gaussian mixture model1,2. the absolute abundance of a single taxon can alter the relative abundances of all taxa (Fig. 1). The choice of parameter for sta- tistical analysis is important and needs to be clearly stated. Often researchers are interested in identifying taxa that are different in mean absolute abundance between two or more ecosystems6. Testing hypotheses regarding mean relative abundance is not equivalent to testing hypotheses regarding mean absolute abundance2,6. In addition, note that not all samples have the same sampling fraction, which is deﬁned as the ratio of the expected absolute abundance of a taxon in a random sample (e.g., a stool sample) to its absolute abundance in a unit volume of the eco- system (e.g., a unit volume of gut) where the sample was derived from. Consequently, the observed counts are not comparable between samples. Thus, all DA methodologies require the counts to be properly normalized to account for differences in sampling fractions across samples. Sampling fraction is affected by two components, namely, the microbial load in a unit volume of the ecosystem and the library size of the corresponding sample (e.g., total species abundances sequenced from a subject’s stool sam- ple). Therefore, it is not sufﬁcient to normalize the library size across samples as one needs to take into consideration the dif- ferences in the microbial loads. Consider the toy example in Fig. 2. Suppose the gut of subject A as well as B consist of only two taxa, the red and green varieties. Huang Lin 1 & Shyamal Das Peddada1✉ Differential abundance (DA) analysis of microbiome data continues to be a challenging problem due to the complexity of the data. In this article we deﬁne the notion of “sampling fraction” and demonstrate a major hurdle in performing DA analysis of microbiome data is the bias introduced by differences in the sampling fractions across samples. We introduce a methodology called Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC), which estimates the unknown sampling fractions and corrects the bias induced by their differences among samples. The absolute abundance data are modeled using a linear regression framework. This formulation makes a fundamental advancement in the ﬁeld because, unlike the existing methods, it (a) provides statistically valid test with appropriate p-values, (b) provides conﬁdence intervals for differential abundance of each taxon, (c) controls the False Discovery Rate (FDR), (d) maintains adequate power, and (e) is computationally simple to implement. 1 Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA 15261, USA. ✉email: shyamal.peddada@nih.gov NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications 1 Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA 15261, USA. ✉email: shyamal.peddada@nih.gov 1 NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 The offset term serves as the bias correction, and the linear regression framework in log scale is analogous to log-ratio transformation to deal with the compositionality of microbiome data. The case of zero counts NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Ideally, under the null hypothesis, the test statistic for DA analysis should be (at least approximately) centered at zero (i.e., unbiased). However, for many DA methods, this is not always true for at least one of the following reasons: (1) The test statistic may not be designed for testing hypothesis regarding the actual parameter of interest; (2) Data are not properly normalized; (3) Underlying structure, such as compositionality, is ignored. Motivated by the limitations of existing DA methods, in this paper we propose a methodology called Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) that is aimed to address the problems mentioned above. As in ANCOM and DR, the proposed ANCOM-BC methodology assumes that the observed sample is an unknown fraction of a unit volume of the ecosystem, and the sampling fraction varies from sample to sample. ANCOM-BC accounts for sampling fraction by intro- ducing a sample-speciﬁc offset term in a linear regression fra- mework, that is estimated from the observed data. The offset term serves as the bias correction, and the linear regression framework in log scale is analogous to log-ratio transformation to deal with the compositionality of microbiome data. The case of zero counts bas s o sa p g act o s g ves a bette desc pt o o t e t ut than normalization methods that rely purely on the library sizes. Ideally, under the null hypothesis, the test statistic for DA analysis should be (at least approximately) centered at zero (i.e., unbiased). However, for many DA methods, this is not always true for at least one of the following reasons: (1) The test statistic may not be designed for testing hypothesis regarding the actual parameter of interest; (2) Data are not properly normalized; (3) Underlying structure, such as compositionality, is ignored. Motivated by the limitations of existing DA methods, in this paper we propose a methodology called Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) that is aimed to address the problems mentioned above. As in ANCOM and DR, the proposed ANCOM-BC methodology assumes that the observed sample is an unknown fraction of a unit volume of the ecosystem, and the sampling fraction varies from sample to sample. ANCOM-BC accounts for sampling fraction by intro- ducing a sample-speciﬁc offset term in a linear regression fra- mework, that is estimated from the observed data. ARTICLE A researcher may not only be interested in knowing if the mean relative abundance of a taxon is different between two ecosystems but may also want to know if the absolute abundance of a taxon is different in a unit volume of two ecosystems. Fig. 1 The distinction between absolute abundances and relative abundances. As shown in this ﬁgure, all taxa (in different colors and shapes) may be identically abundant in a unit volume of two ecosystems (e.g., a unit volume of gut), except for one differentially abundant taxon (the green variety). Due to this one differentially abundant taxon, the two ecosystems may differ in the relative abundance of all taxa. A researcher may not only be interested in knowing if the mean relative abundance of a taxon is different between two ecosystems but may also want to know if the absolute abundance of a taxon is different in a unit volume of two ecosystems. Fig. 1 The distinction between absolute abundances and relative abundances. As shown in this ﬁgure, all taxa (in different colors and shapes) may be identically abundant in a unit volume of two ecosystems (e.g., a unit volume of gut), except for one differentially abundant taxon (the green variety). Due to this one differentially abundant taxon, the two ecosystems may differ in the relative abundance of all taxa. A researcher may not only be interested in knowing if the mean relative abundance of a taxon is different between two ecosystems but may also want to know if the absolute abundance of a taxon is different in a unit volume of two ecosystems. NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Ecosystem B Subject A Subject B A unit volume of ecosystem Sample A Sample B Sample Ecosystem A B A B 4 4 12 18 2 2 6 9 Sum 6† 6† 18‡ 27‡ † Library size; ‡ microbial load. Ecosystem A Group 1 Group 2 Fig. 2 The bias introduced by cross-sample variations in sampling fractions. Sampling fraction is deﬁned as the ratio of expected absolute abundance in a sample to the corresponding absolute abundance in the ecosystem, which could be empirically estimated by the ratio of library size to the microbial load. Differences in sampling fractions may introduce bias and increase in false positive as well as false negative rates in differential abundance analysis. In this toy example, the microbial load for subject A in a unit volume of ecosystem (e.g., a unit volume of gut) is 18 (12 red + 6 green), while for subject B is 27 (18 red + 9 green). However, the samples taken from subject A and B have the same library size 6 (4 red + 2 green), the same observed absolute abundance as well as the same relative abundance of red and green taxa. Thus, one may mistakenly conclude that the red and green taxa are not differentially abundant, which is not the case in the two ecosystems. This false negative conclusion is caused by differences in the sampling fractions in the two samples. The sampling fraction in sample A is 3/9 and for B it is 2/9. One can similarly construct examples where a false positive conclusion is arrived at. Thus, a normalization method must account for differences in sampling fractions to avoid such erroneous conclusions. Ecosystem B Sample B Sample A Ecosystem A Sample Ecosystem A B A B 4 4 12 18 2 2 6 9 Sum 6† 6† 18‡ 27‡ † Library size; ‡ microbial load. Fig. 2 The bias introduced by cross-sample variations in sampling fractions. Sampling fraction is deﬁned as the ratio of expected absolute abundance in a sample to the corresponding absolute abundance in the ecosystem, which could be empirically estimated by the ratio of library size to the microbial load. Differences in sampling fractions may introduce bias and increase in false positive as well as false negative rates in differential abundance analysis. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 In this toy example, the microbial load for subject A in a unit volume of ecosystem (e.g., a unit volume of gut) is 18 (12 red + 6 green), while for subject B is 27 (18 red + 9 green). However, the samples taken from subject A and B have the same library size 6 (4 red + 2 green), the same observed absolute abundance as well as the same relative abundance of red and green taxa. Thus, one may mistakenly conclude that the red and green taxa are not differentially abundant, which is not the case in the two ecosystems. This false negative conclusion is caused by differences in the sampling fractions in the two samples. The sampling fraction in sample A is 3/9 and for B it is 2/9. One can similarly construct examples where a false positive conclusion is arrived at. Thus, a normalization method must account for differences in sampling fractions to avoid such erroneous conclusions. Fig. 2 The bias introduced by cross-sample variations in sampling fractions. Sampling fraction is deﬁned as the ratio o is also discussed in “Methods” section. This methodology has some conceptual similarities with DR, but is fundamentally dif- ferent. With ANCOM-BC, one can perform standard statistical tests and construct conﬁdence intervals for DA. Moreover, as demonstrated in benchmark simulation studies, ANCOM-BC (a) controls the FDR very well while maintaining adequate power compared with other popular methods, and (b) it is substantially faster than ANCOM. The CPU time (RStudio, x86_64-apple- darwin15.6.0, and macOS) is 0.28 min vs. 63 min when the number of taxa is 500. The CPU time for ANCOM increases dramatically as the number of taxa increases to 1000. In this case, the CPU times for ANCOM-BC and ANCOM are 0.51 and 211 min, respectively. In addition to results based on synthetic data, we also illustrate ANCOM-BC using the well-known global gut microbiota dataset9. ecosystem than from B’s (3 9 vs 2 9), Thus, normalizing data on the basis of sampling fractions gives a better description of the truth than normalization methods that rely purely on the library sizes. ecosystem than from B’s (3 9 vs 2 9), Thus, normalizing data on the basis of sampling fractions gives a better description of the truth than normalization methods that rely purely on the library sizes. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 We note that all existing methods have larger variances compared with ANCOM-BC, and TSS has the largest variance. Except ANCOM-BC, UQ, and TMM, we see from the plot that circles and triangles are systematically separated, which indicates that ELib-UQ, ELib-TMM, CSS, MED, and TSS do not account for systematic bias due to differences in sampling fractions across groups. (circles and triangles) in Fig. 3 should intermix and not cluster by the group labels. with Cumulative-Sum Scaling (CSS) implemented in metagen- omeSeq8, Median (MED) in DESeq210, Upper Quartile (UQ) and Trimmed Mean of M values (TMM), and Total-Sum Scaling (TSS). In addition, we also considered modiﬁed versions of UQ and TMM implemented in edgeR11. These are obtained by multiplying the normalization factors with the corresponding library size to account for “effective library size”12, and are denoted as ELib-UQ and ELib-TMM (see Supplementary Table 7 for formulas and Supplementary Fig. 11 for workﬂow). From Fig. 3 (and Supplementary Figs. 1, 2) we notice that the samples normalized by ANCOM-BC are nicely intermixed and do not cluster by the group labels. This is not the case with most of the remaining methods where residuals cluster by group labels, thus indicating that they are unable to eliminate the underlying differences in sampling fractions between the two groups. Thus, under the null hypothesis of no difference in the absolute abundance of a taxon in two groups, their test statistics are not centered at zero. This results in inﬂated FDR (see Supplementary Discussion). We also note from Fig. 3 and Supplementary Figs. 1, 2, that not only ANCOM-BC does well in estimating the bias due to differences in sampling fraction, the variability in the estimates of the sampling fractions is very small as seen from the height of the box plot for ANCOM-BC. This is an important observation because it suggests that the variability in the estimator of bias due to sampling fraction is potentially negligible in the test statistic described in “Methods” section. y g We considered a variety of simulation scenarios as follows. The details of the simulation study are presented in the Supplementary Notes. (1) Unbalanced microbial load in two experimental groups and balanced library size for each sample. This results in a large variability in sampling fractions (Fig. 3). y p g ( g ) (2) Unbalanced microbial load in two experimental groups and unbalanced library size for each sample. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 −0.4 −0.2 0.0 0.2 0.4 Residual ANCOM−BC (0.00013) ELib−UQ (0.029) ELib−TMM (0.0023) CSS (0.015) MED (0.012) UQ (0.085) TMM (0.05) TSS (0.18) Group 1 Group 2 Fig. 3 Box plot of residuals between true sampling fraction and its estimate for each sample. In the box plot, the lower and upper hinges correspond to the ﬁrst and third quartiles (the 25th and 75th percentiles). The median is represented by a solid line within the box. The upper whisker extends from the hinge to the largest value (maxima) no further than 1.5 times Interquartile Range (IQR, distance between the ﬁrst and third quartiles) from the hinge, the lower whisker extends from the hinge to the smallest value (minima) at most 1.5 times IQR of the hinge. Data beyond the end of the whiskers are called “outlying” points. N = 30 samples examined over two experimental groups (denoted by circles and triangles) and the data points are overlaid in each box. Text on the upper left corner indicates the color for each method and variances are provided within parenthesis for each method. The variability in sampling fractions is set to be large. An ideal box plot should display a narrow height (i.e., smaller variability) and samples from the two groups should be intermixed and not display any systematic separation. We note that all existing methods have larger variances compared with ANCOM-BC, and TSS has the largest variance. Except ANCOM-BC, UQ, and TMM, we see from the plot that circles and triangles are systematically separated, which indicates that ELib-UQ, ELib-TMM, CSS, MED, and TSS do not account for systematic bias due to differences in sampling fractions across groups. −0.4 −0.2 0.0 0.2 0.4 Residual ANCOM−BC (0.00013) ELib−UQ (0.029) ELib−TMM (0.0023) CSS (0.015) MED (0.012) UQ (0.085) TMM (0.05) TSS (0.18) Group 1 Group 2 Residual Fig. 3 Box plot of residuals between true sampling fraction and its estimate for each sample. In the box plot, the lower and upper hinges correspond to the ﬁrst and third quartiles (the 25th and 75th percentiles). The median is represented by a solid line within the box. Results Normalization. Using simulated data, we illustrate how the existing normalization methods fail to eliminate the bias intro- duced by differences in sampling fractions across samples, whereas the normalization method introduced in ANCOM-BC performs well. Speciﬁcally, we compare our proposed method NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 The upper whisker extends from the hinge to the largest value (maxima) no further than 1.5 times Interquartile Range (IQR, distance between the ﬁrst and third quartiles) from the hinge, the lower whisker extends from the hinge to the smallest value (minima) at most 1.5 times IQR of the hinge. Data beyond the end of the whiskers are called “outlying” points. N = 30 samples examined over two experimental groups (denoted by circles and triangles) and the data points are overlaid in each box. Text on the upper left corner indicates the color for each method and variances are provided within parenthesis for each method. The variability in sampling fractions is set to be large. An ideal box plot should display a narrow height (i.e., smaller variability) and samples from the two groups should be intermixed and not display any systematic separation. We note that all existing methods have larger variances compared with ANCOM-BC, and TSS has the largest variance. Except ANCOM-BC, UQ, and TMM, we see from the plot that circles and triangles are systematically separated, which indicates that ELib-UQ, ELib-TMM, CSS, MED, and TSS do not account for systematic bias due to differences in sampling fractions across groups. Fig. 3 Box plot of residuals between true sampling fraction and its estimate for each sample. In the box plot, the lower and upper hinges correspond to the ﬁrst and third quartiles (the 25th and 75th percentiles). The median is represented by a solid line within the box. The upper whisker extends from the hinge to the largest value (maxima) no further than 1.5 times Interquartile Range (IQR, distance between the ﬁrst and third quartiles) from the hinge, the lower whisker extends from the hinge to the smallest value (minima) at most 1.5 times IQR of the hinge. Data beyond the end of the whiskers are called “outlying” points. N = 30 samples examined over two experimental groups (denoted by circles and triangles) and the data points are overlaid in each box. Text on the upper left corner indicates the color for each method and variances are provided within parenthesis for each method. The variability in sampling fractions is set to be large. An ideal box plot should display a narrow height (i.e., smaller variability) and samples from the two groups should be intermixed and not display any systematic separation. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 The two widely used count-based methods in RNA- Seq literature, edgeR (implemented using ELib-TMM12 by default) and DESeq2, generally exceed the 5% nominal FDR level when there are differences in sampling fractions (Fig. 4a and Supplementary Fig. 3a). For instance, edgeR has FDR as large as 40% (Fig. 4a), meaning that 40% of ﬁndings could be potentially false discoveries. The zero-inﬂated Gaussian mixture model used in metagenomeSeq (ZIG) consistently has the largest FDR when sampling fractions are not constant (Fig. 4a and Supplementary Fig. 3a). In some cases, the FDR could be as much as 70%, which perhaps is partly due to the Gaussian distribution assumption for log abundance data. Although metagenomeSeq using zero- inﬂated Log-Gaussian mixture model successfully controls the FDR under 5% in all simulations, it suffers a severe loss of power (Fig. 4b and Supplementary Figs. 3b, 4b). The power of detecting differentially abundant taxa could be lower than 10%. simulation set-up (Supplementary Fig. 5a, b) compared with the simulation using Poisson-Gamma distribution. Note that DESeq2 and edgeR were designed for Poisson-Gamma distribution, and hence it is not surprising that these methods performed poorly in this new set-up. tests without any normalization can also be problematic when the sampling fractions are different across experimental groups (Fig. 4a). The two widely used count-based methods in RNA- Seq literature, edgeR (implemented using ELib-TMM12 by default) and DESeq2, generally exceed the 5% nominal FDR level when there are differences in sampling fractions (Fig. 4a and Supplementary Fig. 3a). For instance, edgeR has FDR as large as 40% (Fig. 4a), meaning that 40% of ﬁndings could be potentially false discoveries. The zero-inﬂated Gaussian mixture model used in metagenomeSeq (ZIG) consistently has the largest FDR when sampling fractions are not constant (Fig. 4a and Supplementary Fig. 3a). In some cases, the FDR could be as much as 70%, which perhaps is partly due to the Gaussian distribution assumption for log abundance data. Although metagenomeSeq using zero- inﬂated Log-Gaussian mixture model successfully controls the FDR under 5% in all simulations, it suffers a severe loss of power (Fig. 4b and Supplementary Figs. 3b, 4b). The power of detecting differentially abundant taxa could be lower than 10%. Illustration using gut microbiota data. We illustrate ANCOM- BC by analyzing the US, Malawi and Venezuela gut microbiota data9. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 This dataset consists of 11,905 OTUs obtained from sub- jects in the USA (n = 317), Malawi (n = 114), and Venezuela (n = 99). We ﬁrst assessed the performance of different normal- ization methods mentioned above. One heuristic approach to gain insights on the impact of normalization is to examine how well the normalized samples separate from each other according to their phenotypes in a nonmetric multidimensional scaling (NMDS) plot. We provide the results for Malawi and Venezuela populations in Fig. 5. As seen from this ﬁgure, ANCOM-BC appears to perform very well visually in separating samples from the two populations and has the largest between-group sum of squares (BSS). BSS measures how well clusters are separated. Larger the BSS value the better a method is in clustering objects according to group labels. ELib-TMM, CSS, and MED also performed well. Consistent with the bias correction and FDR/Power simulations reported in Figs. 3 and 4, where ELib-UQ, UQ, TMM, and TSS perform poorly in correcting biases and have poor FDR control, they also have poor performances in distinguishing samples based on their nationalities. y Similar to ANCOM, ANCOM-BC not only controls the FDR at the nominal level (5%) but also maintains adequate power in all simulation settings considered here. An important observation to be made regarding all methods, other than ANCOM and ANCOM-BC, is that as the sample size within each group increases, so does the FDR. This is perhaps a consequence of the fact that the test statistics are not centered at the true null parameter but are shifted due to differences in the sampling fraction. Hence asymptotically, these tests fail to control the false positive as well as FDR (see Supplementary Discussion). In addition to the above Poisson-Gamma model, we performed simulations using the real global patterns data13, to get a broader perspective on the performance of the various methods (see Supplementary Notes for simulation details). In this case again, ANCOM and ANCOM-BC controlled the FDR and competed well in terms of power with all other methods. The estimated FDR of DESeq2 and edgeR increased further in this We also report results of pairwise DA analyses at phylum level among the above three countries using ANCOM-BC. It is well- known that the infant gut microbiota evolve with their age14 due to changes in the feeding patterns, diet, and other exposures. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 The solid vertical line is the 5% nominal level of FDR, and the dashed vertical line denotes 5% nominal level plus one standard error (SE). By default, ANCOM-BC implements Bonferroni correction and other DA methods implement BH procedure to adjust for multiple comparisons. Color and the name of the corresponding DA method are shown at the bottom within the graph. Two simulation scenarios are considered: small and unbalanced data (n1 = 20, n2 = 30), as well as large and balanced data (n1 = n2 = 50); number of simulations = 100. Results show that only ANCOM and ANCOM-BC control the FDR under the nominal level (5%) while maintaining power comparable with other methods. Gaussian model version of metagenomeSeq has highly inﬂated FDR, while the log-Gaussian version has substantial loss of power, sometimes well below 5%. Other than ANCOM-BC and ANCOM, as the sample size within each group increases, so does the FDR for all other existing methods. Fig. 4 FDR and power comparisons using synthetic data from Poisson-Gamma distributions. The False Discovery Rate (FDR) and power of various differential abundance (DA) analyses (two-sided) are shown in a and b, respectively. The variability in sampling fractions is set to be large. The Y-axis denotes patterns of proportion of differentially abundant taxa. The solid vertical line is the 5% nominal level of FDR, and the dashed vertical line denotes 5% nominal level plus one standard error (SE). By default, ANCOM-BC implements Bonferroni correction and other DA methods implement BH procedure to adjust for multiple comparisons. Color and the name of the corresponding DA method are shown at the bottom within the graph. Two simulation scenarios are considered: small and unbalanced data (n1 = 20, n2 = 30), as well as large and balanced data (n1 = n2 = 50); number of simulations = 100. Results show that only ANCOM and ANCOM-BC control the FDR under the nominal level (5%) while maintaining power comparable with other methods. Gaussian model version of metagenomeSeq has highly inﬂated FDR, while the log-Gaussian version has substantial loss of power, sometimes well below 5%. Other than ANCOM-BC and ANCOM, as the sample size within each group increases, so does the FDR for all other existing methods. tests without any normalization can also be problematic when the sampling fractions are different across experimental groups (Fig. 4a). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 This results in a moderate variability in sampling fractions (Supplementary Fig. 1). Clearly, as seen in Fig. 4a, b and Supplementary Figs. 3a, b, 4a, b, the normalization of data has a major effect on the FDR and power of various methods. g (3) Balanced microbial load in two experimental groups and balanced library size for each sample. This results in a small variability in sampling fractions (Supplementary Fig. 2). Thus, we simulated data where sampling fraction in Group 1 is systematically different from sampling fraction in Group 2. Consequently, observed absolute abundances in the samples in the two groups were systematically different even though the actual absolute abundances in the ecosystems are same. To evaluate the performance of each normalization method, we introduced a residual measure that estimates the deviation between the estimated sampling fraction and the true sampling fraction (see Supplementary Discussion). For simplicity of exposition, we plotted the centered residuals, by subtracting the group average of the residuals. If a normalization method is effective then it should eliminate the bias due to the differences in the sampling fractions so that samples from the two groups DA analyses. Simulating data from Poisson-Gamma distributions (see Supplementary Notes for simulation settings and Supple- mentary Fig. 12 for workﬂow), we evaluated the performance of various methods in terms of FDR and power. Since there is no hard threshold available for DR to declare whether a taxon is differentially abundant or not, it was not included in this simulation study. y Not surprisingly, standard Wilcoxon rank-sum test applied to relative abundance data leads to highly inﬂated FDR (Fig. 4a and Supplementary Figs. 3a, 4a) in all simulation scenarios. This is primarily because such standard tests ignore the compositional structure of the data, and seen from Fig. 3, TSS does not successfully normalize the data. Simply applying nonparametric NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 ARTICLE n = 20/30 n = 50/50 0.05 0.20 0.40 0.60 0.80 0.05 0.20 0.40 0.60 0.80 0.05 0.15 0.25 FDR a n = 20/30 n = 50/50 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.05 0.15 0.25 Power b ANCOM−BC ANCOM DESeq2 edgeR ZILG ZIG Wilcoxon Wilcoxon + TSS Proportion of differentially abundant taxa Fig. 4 FDR and power comparisons using synthetic data from Poisson-Gamma distributions. The False Discovery Rate (FDR) and power of various differential abundance (DA) analyses (two-sided) are shown in a and b, respectively. The variability in sampling fractions is set to be large. The Y-axis denotes patterns of proportion of differentially abundant taxa. The solid vertical line is the 5% nominal level of FDR, and the dashed vertical line denotes 5% nominal level plus one standard error (SE). By default, ANCOM-BC implements Bonferroni correction and other DA methods implement BH procedure to adjust for multiple comparisons. Color and the name of the corresponding DA method are shown at the bottom within the graph. Two simulation scenarios are considered: small and unbalanced data (n1 = 20, n2 = 30), as well as large and balanced data (n1 = n2 = 50); number of simulations = 100. Results show that only ANCOM and ANCOM-BC control the FDR under the nominal level (5%) while maintaining power comparable with other methods. Gaussian model version of metagenomeSeq has highly inﬂated FDR, while the log-Gaussian version has substantial loss of power, sometimes well below 5%. Other than ANCOM-BC and ANCOM, as the sample size within each group increases, so does the FDR for all other existing methods. n = 20/30 n = 50/50 0.05 0.20 0.40 0.60 0.80 0.05 0.20 0.40 0.60 0.80 0.05 0.15 0.25 FDR a ANCOM−BC ANCOM ZILG ZIG Proportion of differentially abundant taxa n = 20/30 n = 50/50 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.05 0.15 0.25 Power b DESeq2 edgeR b a Fig. 4 FDR and power comparisons using synthetic data from Poisson-Gamma distributions. The False Discovery Rate (FDR) and power of various differential abundance (DA) analyses (two-sided) are shown in a and b, respectively. The variability in sampling fractions is set to be large. The Y-axis denotes patterns of proportion of differentially abundant taxa. NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 BSS = 126 BSS = 114 BSS = 38 BSS = 66 BSS = 112 BSS = 28 BSS = 105 BSS = 86 MED UQ TMM TSS ANCOM−BC ELib−UQ ELib−TMM CSS −0.25 0.00 0.25 0.50 −0.25 0.00 0.25 0.50 −0.25 0.00 0.25 0.50 −0.25 0.00 0.25 0.50 −0.2 0.0 0.2 −0.2 0.0 0.2 First NMDS coordinate Second NMDS coordinate Malawi Venezuela Fig. 5 Non-metric multidimensional scaling (NMDS) visualizations of normalized data. First two NMDS coordinates are used to evaluate the performance of various normalization methods (ANCOM-BC, ELib-UQ, ELib-TMM, CSS, MED, UQ, TMM, and TSS) applied on Malawi and Venezuela samples of the global gut microbiota data at genus level. Samples from Malawi are colored in red while samples from Venezuela are colored in green. Visually, ANCOM-BC, ELib-TMM, MED, and CSS appear to provide best separation between Malawi and Venezuela samples. Quantitatively, in terms of Between-Group Sum of Squares (BSS), standardized by Total Sum of Squares (TSS) so that all methods are comparable, ANCOM-BC has the largest BSS, followed by ELib-TMM, and MED. Rest of the methods perform poorly both visually as well as quantitatively with small BSS values. These ﬁndings appear to be consistent with results of the synthetic data shown in Fig. 3 and Supplementary Figs. 1, 2. First NMDS coordinate Fig. 5 Non-metric multidimensional scaling (NMDS) visualizations of normalized data. First two NMDS coordinates are used to evaluate the performance of various normalization methods (ANCOM-BC, ELib-UQ, ELib-TMM, CSS, MED, UQ, TMM, and TSS) applied on Malawi and Venezuela samples of the global gut microbiota data at genus level. Samples from Malawi are colored in red while samples from Venezuela are colored in green. Visually, ANCOM-BC, ELib-TMM, MED, and CSS appear to provide best separation between Malawi and Venezuela samples. Quantitatively, in terms of Between-Group Sum of Squares (BSS), standardized by Total Sum of Squares (TSS) so that all methods are comparable, ANCOM-BC has the largest BSS, followed by ELib-TMM, and MED. Rest of the methods perform poorly both visually as well as quantitatively with small BSS values. These ﬁndings appear to be consistent with results of the synthetic data shown in Fig. 3 and Supplementary Figs. 1, 2. than the US infants (Fig. 6b and Supplementary Table 3). Similarly, the ratio was signiﬁcantly larger among Venezuela infants than the US infants (Fig. 6b and Supplementary Table 3). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Although the differences of the ratio of Bacteroidetes to Firmicutes between US and non-US adults were not signiﬁcant, the effect sizes showed a similar trend as infants indicating that US adults had smaller ratio of Bacteroidetes to Firmicutes. We did not ﬁnd any signiﬁcant differences between Malawi and Venezuelan infants as well as adults. These results are in line with our ﬁndings that there were no differences in the mean absolute abundances of Firmicutes as well as Bacteroidetes among Malawi and Venezuelan infants as well as adults (Fig. 6a). “adults”). Results of all pairwise comparisons are provided in Fig. 6a and Supplementary Table 1. Note that ANCOM-BC is the ﬁrst method in the literature that can not only identify differentially abundant taxa while controlling the FDR for multiple testing, it also provides 95% simultaneous conﬁdence intervals for the mean DA of each taxon in the two experimental groups. These conﬁdence intervals are adjusted for multiplicity using Bonferroni method. Thus, a researcher can evaluate the effect size associated with each taxon when comparing two experimental groups. This is particularly important in the present climate when researchers are increasingly skeptical about making decisions based on p values (alone)15. p Interestingly, phyla such as Cyanobacteria, Elusimicrobia, Euryarchaeota, and Spirochetes, which are known to be associated with rural environment and hygiene16–19, are sig- niﬁcantly more abundant among Malawi than the US infants and adults. We discover an interesting trend in the absolute abundance of phylum Verrucomicrobia, whose absolute abun- dance is known to increase with antibiotics usage to protect against pathogens and other opportunistic bacteria20. Consistent with the high usage of antibiotics in the western world among infants as well as adults, we discover a signiﬁcant increase in the absolute abundance of Verrucomicrobia in US relative to Malawi adults and infants, and relative to Venezuelan adults (Fig. 6a). Similarly, there is a signiﬁcant increase in its absolute abundance among Venezuelan infants compared with Malawi (Fig. 6a). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Hence, for illustration purposes, we performed a stratiﬁed analysis by considering two age groups, infants below 2 years (labeled as “infants”) and adults between 18 and 40 (labeled as TURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 All effect sizes with adjusted p < 0.05 are indicated, signiﬁcant at 5% level of signiﬁcance; signiﬁcant at 1% level of signiﬁcance; *signiﬁcant at 0.1% level of signiﬁcance. Exact adjusted p values can be found in Supplementary Table 1. Diamonds on top of some bars indicate structural zeros. a Pairwise differential abundance analyses stratiﬁed by age using ANCOM-BC: Infants (age ≤2 years, n = 133), and adults (18 ≤age ≤40, n = 83). Infant samples are colored in red and adult samples are colored in green. Phyla Acidobacteria and Chloroﬂexi are not represented in the plot since they are present only in Venezuela samples. b Pairwise tests using ANCOM-BC for the equality of mean log ratio of Bacteroidetes to Firmicutes stratiﬁed by age. Viewing b and Supplementary Table 2 together, lower BMI seems to be associated with higher levels of the Bacteroidetes to Firmicutes ratio, a result widely acknowledged in the literature. reference taxon is not an issue for ANCOM. As demonstrated mathematically in ANCOM methodology2, as long as two taxa are not differentially abundant between two ecosystems, one can draw inferences about DA using differential relative abundance. In many applications, researchers are interested in drawing inferences regarding DA of taxa in more than two ecosystems. We extended ANCOM-BC to deal with such multigroup situations. Extensive simulations suggest that ANCOM-BC controls FDR while maintaining high power (Supplementary Table 5). ANCOM-BC enjoys several important unique characteristics. First, it is the only method available in the literature that esti- mates the sampling fraction and performs DA analysis by cor- recting bias due to differential sampling fractions across samples. It is the only procedure that provides valid p values and con- ﬁdence intervals for each taxon. Second, unlike ANCOM, it simpliﬁes DA analysis by recasting the problem as a linear regression problem with an offset. The offset is due to the sam- pling fraction. By virtue of linear regression formulation, ANCOM-BC can be applied to a broad collection of study designs, including longitudinal data, repeated measurements design, covariance adjusted analysis, and so on. Using a broad range of simulations studies, we demonstrate that ANCOM-BC, like ANCOM, controls the FDR very well, while almost all other methods investigated in this paper fail. Discussion The DA analysis of microbiome data is a challenging problem5,6, in part due to inaccessibility of data necessary for drawing inferences on DA in two or more ecosystems. An important unobservable parameter that impacts DA analysis is the sampling fraction of a sample drawn from a unit volume of ecosystem. As noted in previous studies5,6, the bias correction due to sampling fraction is a major hurdle. While, ANCOM as well as DR pro- cedures ﬁnd ways to get around the problem from different perspectives, there is room for improvement. Secondly, differ- ential relative abundance analysis of microbiome data is not equivalent to differential absolute abundance analysis of micro- biome data. Often simplex or compositional data analysis-based methods transform the simplex coordinate system to Euclidean space by performing log ratio transformation. However, such methods require the researcher to prespecify the reference taxon and the results may be highly dependent on the choice of the reference taxon6. It is important to reiterate that ANCOM computes log-ratios with respect to all taxa and thus the choice of It is well-documented in the literature that BMI is linked to the ratio of Bacteroidetes to Firmicutes21. In our sample, the US infants, as well as adults, had higher BMI than their counterparts in Malawi; The US infants also had higher BMI than Venezuela infants (Supplementary Table 2). Interestingly the ratio of Bacteroidetes to Firmicutes was larger among Malawi infants NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 ) In summary, the proposed ANCOM-BC methodology (1) explicitly tests hypothesis regarding differential absolute abun- dance of individual taxon and provides valid conﬁdence intervals; (2) provides an approach to correct the bias induced by (unob- servable) differential sampling fractions across samples; (3) takes into account the compositionality of the microbiome data, and (4) does not rely on strong parametric assumptions. With the linear regression framework adopted in ANCOM-BC, it allows researchers to derive p value associated with each taxon as well as conﬁdence interval estimation for differential absolute abun- dance. These are unique to ANCOM-BC, to the best of our knowledge. Last but not the least, because of the regression framework adopted in ANCOM-BC, it can be extended to more general settings involving multigroup comparisons, adjusting covariates as well as applying to longitudinal/repeated measurements data. g p p The ANCOM-BC methodology may not perform well when the sample sizes are very small, such as n = 5 per group. The FDR is not controlled by ANCOM-BC in such cases (Supple- mentary Fig. 6a, b). However, when the sample size increases to 10, our simulation results indicate that ANCOM-BC controls FDR with adequate power (Supplementary Fig 6a, b). We also evaluated the performance of ANCOM-BC when the number of taxa is small, as when researchers perform DA analysis at the phylum or class levels. Even in such instances, ANCOM-BC controls the FDR very well while maintaining high power (Supplementary Table 4). ANCOM-BC performs best in terms of FDR control when the proportion of differentially abundant taxa is not too large (e.g., <75%). Otherwise, it may have slightly elevated FDR (Supplementary Fig. 7a, b). However, none of the other methods control the FDR either, in fact, they have larger FDRs than ANCOM-BC. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 ARTICLE MA–VEN VEN–US MA–US −2 0 2 4 Log fold change (Bacteroidetes–Firmicutes) b * * * * * * *** * * * * * * * * * *** * * * * * * * * * * * * MA–US VEN–US MA–VEN −5 0 5 10 −5 0 5 10 −4 0 4 Verrucomicrobia TM7 Tenericutes Synergistetes Spirochaetes Proteobacteria Lentisphaerae Fusobacteria Firmicutes Euryarchaeota Elusimicrobia Cyanobacteria Bacteroidetes Actinobacteria Log fold change a * MA–VEN VEN–US MA–US −2 0 2 4 Log fold change (Bacteroidetes–Firmicutes) b Infants Adults Fig. 6 Analysis of the global gut microbiota data in phylum level. Data are represented by effect size (log fold change) and 95% conﬁdence interval bars (two-sided; Bonferroni adjusted) derived from the ANCOM-BC model. All effect sizes with adjusted p < 0.05 are indicated, signiﬁcant at 5% level of signiﬁcance; signiﬁcant at 1% level of signiﬁcance; signiﬁcant at 0.1% level of signiﬁcance. Exact adjusted p values can be found in Supplementary Table 1. Diamonds on top of some bars indicate structural zeros. a Pairwise differential abundance analyses stratiﬁed by age using ANCOM-BC: Infants (age ≤2 years, n = 133), and adults (18 ≤age ≤40, n = 83). Infant samples are colored in red and adult samples are colored in green. Phyla Acidobacteria and Chloroﬂexi are not represented in the plot since they are present only in Venezuela samples. b Pairwise tests using ANCOM-BC for the equality of mean log ratio of Bacteroidetes to Firmicutes stratiﬁed by age. Viewing b and Supplementary Table 2 together, lower BMI seems to be associated with higher levels of the Bacteroidetes to Firmicutes ratio, a result widely acknowledged in the literature. * * * * *** * *** * * * * * * * * * *** * * * * * * * * * * * * MA–US VEN–US MA–VEN −5 0 5 10 −5 0 5 10 −4 0 4 Verrucomicrobia TM7 Tenericutes Synergistetes Spirochaetes Proteobacteria Lentisphaerae Fusobacteria Firmicutes Euryarchaeota Elusimicrobia Cyanobacteria Bacteroidetes Actinobacteria Log fold change a b a Fig. 6 Analysis of the global gut microbiota data in phylum level. Data are represented by effect size (log fold change) and 95% conﬁdence interval bars (two-sided; Bonferroni adjusted) derived from the ANCOM-BC model. Methods Notation. The notations described in ANCOM-BC methodology are summarized in Table 1. Notation. The notations described in ANCOM-BC methodology are summarized in Table 1. Data preprocessing. We adopted the methodology of ANCOM-II22 as the pre- processing step to deal with different types of zeros before performing DA analysis. There are instances where some taxa are systematically absent in an ecosystem. For example, there may be taxa present in a soil sample from a desert that might absent in a soil sample from a rain forest. In such cases, the observed zeros are called structural zeros. Let pij denote proportion non-zero samples of the ith taxon in the jth group, and let ^pij ¼ 1 nj Pnj k¼1 IðOijk≠0Þ denote the estimate of pij. In practice, we declare the ith taxon to have structural zeros in the jth group if either of the following is true: 7 NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Table 1 Summary of notations. Table 1 Summary of notations. Notation Description i Taxon index, i = 1, 2, …, m. j Group index, j = 1, 2, …, g. k Sample index, k = 1, 2, …, nj. θija Expected absolute abundance of ith taxon in a unit volume of ecosystem in the jth group. Aijkb Unobserved absolute abundance of ith taxon in a unit volume of ecosystem of kth sample in the jth group. A.jkb Microbial load in a unit volume of ecosystem of kth sample in the jth group. Ajk ¼ Pm i¼1 Aijk. γijkb Unobserved relative abundance of ith taxon in a unit volume of ecosystem of kth sample in the jth group. γijk ¼ Aijk Ajk. Oijkb Observed absolute abundance of ith taxon in a random specimen taken from a unit volume of ecosystem of kth sample in the jth group. O.jkb Library size of a random specimen taken from a unit volume of ecosystem of kth sample in the jth group. Ojk ¼ Pm i¼1 Oijk. rijkb Observed relative abundance of ith taxon in a random specimen taken from a unit volume of ecosystem of kth sample in the jth group. rijk ¼ Oijk Ojk. cjka For kth sample from the jth group, cjk represents the proportion of its ecosystem (unobserved absolute abundance) in a random sample (observed absolute abundance), thus cjk ¼ EðOijkjAijkÞ Aijk . Methods Suppose the researcher is interested in comparing the mean absolute abundance of the taxon in the ecosystems of the two groups. Then under the above assumptions, the model describing the study is given by: g y In a similar fashion, we address the outlier zeros as well as sampling zeros using the methodology developed in ANCOM-II22. way ANOVA model. For simplicity, let us suppose we have two groups, say a treatment and a control group. Let us also suppose that there is only one taxon in our microbiome study and n subjects are assigned to the treatment group and n are assigned to the control group. Suppose the researcher is interested in comparing the mean absolute abundance of the taxon in the ecosystems of the two groups. Then under the above assumptions, the model describing the study is given by: Model assumptions. Assumption 0.1. Hence under the null hypothesis μ1 = μ2, Eðy1: y2:Þ ¼ 0. Thus, in this case the t d d t t t i i t H i thi d l th d l t Hence under the null hypothesis μ1 = μ2, Eðy1: y2:Þ ¼ 0. Thus, in this case the standard t-test is appropriate. Hence in this paper we develop methodology to eliminate the bias introduced by the differential sampling fraction by each sample. To do so, we exploit the fact that we have a large number of taxa on each subject and we borrow information across taxa to estimate this bias, which is the essence of the following methodology. ð2Þ standard t-test is appropriate. Hence in this paper we develop methodology to eliminate the bias introduced by the differential sampling fraction by each sample. To do so, we exploit the fact that we have a large number of taxa on each subject and we borrow information across taxa to estimate this bias, which is the essence of the following methodology. where σ2 b;ij = between-sample variation within group j for the ith taxon. where σ2 b;ij = between-sample variation within group j for the ith taxon. ; j The Assumption 0.2 states that for a given taxon, all subjects within and between groups are independent, where θij is a ﬁxed parameter rather than a random variable. Bias and variance of bias estimation under the null hypothesis: From the above model (equation (4)), for each j, note that EðyijÞ ¼ dj þ μij and EðyjkÞ ¼ djk þ μj, where w represents the arithmetic mean over the suitable index. Using the least squares framework, we therefore estimate μij and djk as follows: Methods We shall refer to this constant as “sampling fraction”. yijkb log ðOijkÞ. μija log ðθijÞ. djka log ðcjkÞ. aParameter. bRandom variable. jk For kth sample from the jth group, cjk represents the proportion of its ecosystem (unobserved absolute abundance) in a random sample (observed absolute abundance), thus cjk ¼ EðOijkjAijkÞ Aijk . We shall refer to this constant as “sampling fraction”. (a) ^pij ¼ 0: (b) ^pij 1:96 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ^pijð1^pijÞ nj q ≤0. with EðϵijkÞ ¼ 0; EðyijkÞ ¼ djk þ μij; VarðyijkÞ ¼ VarðϵijkÞ :¼ σ2 ijk: ð5Þ q If a taxon is considered to be a structural zero in an experimental group then, for that speciﬁc ecosystem, the taxon is not used in further analysis. Thus, suppose there are three ecosystems A, B, and C and suppose taxon X is a structural zero in ecosystems A and B but not in C, then taxon X is declared to be differentially abundant in C relative to A and B and not analyzed further. If taxon Y is a structurally zero in ecosystem A but not in B and C, in that case we declare that taxon Y is differentially abundant in B relative to A as well as differentially abundant in C relative to A. We then compare the absolute abundance of taxon Y between B and C using the methodology described in this section. Taxa identiﬁed to be structural zeros among all experimental groups are ignored from the following analyses. ð5Þ Note that with a slight abuse of notation for simplicity of exposition, the above log-transformation of data is inspired by the Box–Cox family of transformations23 which are routinely used in data analysis. Note that d in the above equation is not exactly log(c) due to Jensenʼs inequality, it simply reﬂects the effect of c An important distinction from standard ANOVA: Before we describe the details of the proposed methodology, we like to draw attention to a fundamental difference between the current formulation of the problem and the standard one- way ANOVA model. For simplicity, let us suppose we have two groups, say a treatment and a control group. Let us also suppose that there is only one taxon in our microbiome study and n subjects are assigned to the treatment group and n are assigned to the control group. NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunicatio Model assumptions. Assumption 0.1. EðOijkjAijkÞ ¼ cjkAijk VarðOijkjAijkÞ ¼ σ2 w;ijk; ð1Þ ð1Þ yjk ¼ djk þ μj þ ϵjk; j ¼ 1; 2; k ¼ 1; 2; ¼ ; n: sample mean absolute abundance of jth group is given by yj: ¼ where σ2 w;ijk = variability between specimens within the kth sample from the jth group. Therefore, σ2 w;ijk characterizes the within-sample variability. Typically, researchers do not obtain more than one specimen at a given time in most microbiome studies. Consequently, variability between specimens within sample is usually not estimated. Eðy1: y2:Þ ¼ ðd1: d2:Þ þ ðμ1 μ2Þ. Under the null hypothesis μ1 = μ2, Eðy1 y2 Þ ¼ d1 d2 ≠0, unless d1 ¼ d2 . Thus because of the differenti Eðy1: y2:Þ ¼ ðd1: d2:Þ þ ðμ1 μ2Þ. Under the null hypothesis μ1 = μ2, According to Assumption 0.1, in expectation the absolute abundance of a taxon in a random sample is in constant proportion to the absolute abundance in the ecosystem of the sample. In other words, the expected relative abundance of each taxon in a random sample is equal to the relative abundance of the taxon in the ecosystem of the sample. sampling fractions, which are sample speciﬁc, the numerator of the standard t-test under the null hypothesis for these microbiome data is non-zero. This introduces bias and hence inﬂates the Type I error. On the other hand, the standard one-way ANOVA model for two groups, which is not applicable for the microbiome data described in this paper, is of the form: y p Assumption 0.2. For each taxon i, Aijk, j = 1, …, g, k = 1, …, nj, are independently distributed with Assumption 0.2. For each taxon i, Aijk, j = 1, …, g, k = 1, …, nj, are independently distributed with umption 0.2. For each taxon i, Aijk, j = 1, …, g, k = 1, …, nj, ar yjk ¼ d þ μj þ ϵjk; j ¼ 1; 2; k ¼ 1; 2; ¼ ; n: EðAijkjθijÞ ¼ θij VarðAijkjθijÞ ¼ σ2 b;ij; Hence under the null hypothesis μ1 = μ2, Eðy1: y2:Þ ¼ 0. Thus, in this case the standard t-test is appropriate. Hence in this paper we develop methodology to Hence under the null hypothesis μ1 = μ2, Eðy1: y2:Þ ¼ 0. Lemma 0.1. 1: ð17Þ The above expression is of the form Consequently, aT 1 x1 þ aT 2 x2 þ aT 3 x3 aT 1 1 þ aT 2 1 þ aT 3 1 αTu αT1 ; ð23Þ ð23Þ ^μij!pμij þ dj; as m; n ! 1: ð18Þ ð18Þ The above observations regarding the convergence of various statistics play a critical role in the following. Since the sampling fraction is constant for all taxa The above observations regarding the convergence of various statistics play a critical role in the following. Since the sampling fraction is constant for all taxa within the subject, we attempt to pool information across taxa when estimating δ. We model the taxa using the following Gaussian mixtures model. For the ith taxon, i = 1, 2, …, m, let Δi ¼ ^μi1 ^μi2. Let C0 denote the set of taxa that are not differentially abundant between the two groups, i.e., C0 = {i ∈(1, 2, …, m): μi1 = μi2}, C1 denote the set of taxa whose mean abundance in group 1 is less than that of group 2, i.e., C1 = {i ∈(1, 2, …, m): μi1 < μi2}, and let C2 denote the set of taxa whose mean abundance in group 1 is greater than that of group 2, i.e., C2 = {i ∈ (1, 2, …, m): μi1 > μi2}, Let πr denote the probability that a taxon belongs to set Cr, r = 0, 1, 2. For simplicity of estimation of parameters, similar to GEE, we shall assume that Δi, i = 1, 2, …, m are independently distributed. Thus, we ignore the underlying correlation structure when estimating δ. This is similar to what is often done in other omics studies. Thus, we model the distribution of Δi by Gaussian where where (1) 1 = (1, …, 1)T, (2) ar ¼ ðar1; ar2; ¼ ; armrÞT :¼ ð 1 ν2 irÞT; i 2 Cr; r ¼ 0; 1; 2, (3) xr ¼ ðxr1; xr2; ¼ ; xrmr ÞT :¼ ðΔi liÞT; i 2 Cr; r ¼ 0; 1; 2. Note that l0 = 0, (4) α ¼ ðα1; α2; ¼ ; αmÞT ðaT 1 ; aT 2 ; aT 3 Þ T, (5) u ¼ ðu1; u2; ¼ ; umÞT ðxT 1 ; xT 2 ; xT 3 ÞT. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 with balanced design, i.e., g = 2 and n1 = n2 = n. Later the methodology is easily extended to unbalanced design and multigroup settings. Suppose we have two ecosystems and for each taxon i, i = 1, 2, …m, we wish to test the hypothesis mixture as follows: mixture as follows: f ðΔiÞ ¼ π0ϕðΔi δ νi0 Þ þ π1ϕðΔi ðδ þ l1Þ νi1 Þ þ π2ϕðΔi ðδ þ l2Þ νi2 Þ; ð19Þ ð19Þ H0 : μi1 ¼ μi2 H1 : μi1≠μi2: H0 : μi1 ¼ μi2 H1 : μi1≠μi2: ð7Þ where ð7Þ (1) ϕ is the normal density function, Under the null hypothesis, Eð^μi1 ^μi2Þ ¼ δ ≠0, and hence biased. The goal of ANCOM-BC is to estimate this bias and accordingly modify the estimator ^μi1 ^μi2 so that the resulting estimator is asymptotically centered at zero under the null hypothesis and hence the test statistic is asymptotically centered at zero. First, we make the following observations. Since EðyijÞ ¼ dj þ μij and ^μij ¼ yij, therefore ^μij is an unbiased estimator of dj þ μij. From (5) and Lyapunov central limit theorem, we have: ϕ (2) δ + l1 and δ + l2 are means for Δi∣C1, and Δi∣C2, respectively. l1 < 0, l2 > 0, (3) νi0, νi1, and νi2 are variances of Δi∣C0, Δi∣C1, and Δi∣C2, respectively. so that the resulting estimator is asymptotically centered at zero under the null hypothesis and hence the test statistic is asymptotically centered at zero. First, we make the following observations. Since EðyijÞ ¼ dj þ μij and ^μij ¼ yij, therefore ^μij is an unbiased estimator of dj þ μij. From (5) and Lyapunov central limit theorem, we have: For computational simplicity, we assume that νi1 > νi0, νi2 > νi0. Thus, without loss of generality for κ1, κ2 > 0, let νi1 = νi0 + κ1 and νi2 = νi0 + κ2. While this f h d bl h For computational simplicity, we assume that νi1 > νi0, νi2 > νi0. Thus, without loss of generality for κ1, κ2 > 0, let νi1 = νi0 + κ1 and νi2 = νi0 + κ2. Lemma 0.1. Thus, Thus, We shall denote the resulting estimator of δ by ^δEM: ^ ^djk ¼ yjk yj!pðdjk þ μjÞ ðdj þ μjÞ ¼ djk dj; as m ! 1: ð13Þ U i ( ) d ( ) l Next we estimate Varð^δEMÞ. Since the likelihood function is not a regular likelihood and hence it is not feasible to derive the Fisher information. Consequently, we take a simpler and a pragmatic approach to derive an approximate estimator of Varð^δEMÞ using Varð^δWLSÞ, which is deﬁned below. Extensive simulation studies suggest that ^δEM and ^δWLS are highly correlated (Supplementary Fig. 9) and it appears to be reasonable to approximate Varð^δEMÞ by Varð^δWLSÞ. j j Using (8) and (13), let ^σ2 ij ¼ 1 n2 X n k¼1 ðyijk ^djk ^μijÞ 2 ð14Þ ^σ2 ij ¼ 1 n2 X n k¼1 ðyijk ^djk ^μijÞ 2 ð14Þ approximate estimator of Varð^δEMÞ using Varð^δWLSÞ, which is deﬁned below. Extensive simulation studies suggest that ^δEM and ^δWLS are highly correlated (Supplementary Fig. 9) and it appears to be reasonable to approximate Varð^δEMÞ by Varð^δWLSÞ. denote the mean residual sum of squares. Then under some mild regularity conditions24, we have the following consistency result denote the mean residual sum of squares. Then under some mild regularity conditions24, we have the following consistency result nð^σ2 ij σ2 ijÞ!p0; as m; n ! 1: ð15Þ j for σij in (8) and appealing to Slutsky’s theorem, we have: Let {Cr} = mr, r = 0, 1, 2, then ð15Þ ^δWLS ¼ P i2C0 Δi ^ν2 i0 þ P i2C1 Δi^l1 ^ν2 i1 þ P i2C2 Δi^l2 ^ν2 i2 P i2C0 1 ^ν2 i0 þ P i2C1 1 ^ν2 i1 þ P i2C2 1 ^ν2 i2 ¼ P i2C0 Δi ν2 i0 þ P i2C1 Δil1 ν2 i1 þ P i2C2 Δil2 ν2 i2 P i2C0 1 ν2 i0 þ P i2C1 1 ν2 i1 þ P i2C2 1 ν2 i2 þ opð1Þ: ð22Þ ^μij ðμij þ djÞ ^σij !dNð0; 1Þ; as m; n ! 1: ð16Þ ð22Þ thermore, based on Assumption 0.3, from (8) and (15) we obtai Furthermore, based on Assumption 0.3, from (8) and (15) we obtain: ^σij!p0; as m; n ! 1: ð17Þ ^σij!p0; as m; n ! NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 p σ2 ijk<σ2 0<1 Pm i≠i0 σii0jk m2 ¼ oð1Þ: ð9Þ Denote 1 = (1, 1, …, 1)T, then we have 0 ≤1TΣ1 ¼ X m i¼1 X m i0¼1 σii0jk ¼ X m i¼1 σ2 ijk þ X m i≠i0 σii0jk ≤mσ2 0 þ X m i≠i0 σii0jk; ð10Þ nce 0 ≤1TΣ1 m2 ≤σ2 0 m þ Pm i≠i0 σii0jk m2 ¼ oð1Þ: ð11Þ σ2 ijk<σ2 0<1 Pm i≠i0 σii0jk m2 ¼ oð1Þ: ð9Þ σ2 ijk<σ2 0<1 Pm i≠i0 σii0jk m2 ¼ oð1Þ: Regression framework. From Assumptions 0.1 and 0.2, we have: Regression framework. From Assumptions 0.1 and 0.2, we have: EðOijkÞ ¼ cjkθij VarðOijkÞ ¼ f ðσ2 w;ijk; σ2 b;ijÞ :¼ σ2 t;ijk: ð EðOijkÞ ¼ cjkθij VarðOijkÞ ¼ f ðσ2 w;ijk; σ2 b;ijÞ :¼ σ2 t;ijk: ð3Þ ^djk ¼ yjk yj; k ¼ 1; ¼ ; nj; j ¼ 1; 2; ¼ g; ^μij ¼ yij ^dj ¼ yij; i ¼ 1; ¼ ; m: ð6Þ ð3Þ ð6Þ Motivated by the above set-up, we introduce the following linear model framework for log-transformed OTU counts data: 4Þ Note that Eð^μijÞ ¼ EðyijÞ ¼ μij þ dj. Thus, for each j = 1, 2, …g, ^μij is a biased estimator and Eð^μi1 ^μi2Þ ¼ ðμi1 μi2Þ þ d1 d2. Denote δ ¼ d1 d2: To begin with, in the following we shall assume there are two experimental groups Þ Note that Eð^μijÞ ¼ EðyijÞ ¼ μij þ dj. Thus, for each j = 1, 2, …g, ^μij is a biased estimator and Eð^μi1 ^μi2Þ ¼ ðμi1 μi2Þ þ d1 d2. Denote δ ¼ d1 d2: To begin with, in the following we shall assume there are two experimental groups ð4Þ yijk ¼ djk þ μij þ ϵijk; 8 8 ARTICLE NATURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 While this assumption is not a requirement for our method, it is reasonable to assume that i bili diff i ll b d i l h h h ll For computational simplicity, we assume that νi1 > νi0, νi2 > νi0. Thus, without loss of generality for κ1, κ2 > 0, let νi1 = νi0 + κ1 and νi2 = νi0 + κ2. While this assumption is not a requirement for our method, it is reasonable to assume that variability among differentially abundant taxa is larger than that among the null taxa. By making this assumption, we speed-up the computation time. loss of generality for κ1, κ2 > 0, let νi1 = νi0 + κ1 and νi2 = νi0 + κ2. While this assumption is not a requirement for our method, it is reasonable to assume that variability among differentially abundant taxa is larger than that among the null taxa. By making this assumption, we speed-up the computation time. ^μij ðμij þ djÞ σi !dNð0; 1Þ as n ! 1; where σ2 ij ¼ Varð^μijÞ ¼ Varðyij:Þ ¼ 1 n2 X n k¼1 σ2 ijk: ð8Þ y g p p p p Assuming samples are independent between experimental groups, we begin by ﬁrst estimating ν2 i0 ¼ Varð^μi1 ^μi2Þ ¼ Varð^μi1Þ þ Varð^μi2Þ. Using the estimator stated in (14), we estimate ν2 i0 consistently as follows: ð8Þ ^ν2 i0 ¼ X 2 j¼1 ^σ2 ij ¼ X 2 j¼1 1 n2 X n k¼1 ðyijk ^djk ^μijÞ 2: ð20Þ ð20Þ Let Σjk denote an m × m covariance matrix of ϵjk ¼ ðϵ1jk; ϵ2jk; ¼ ; ϵmjkÞT, where σii0jk is the ði; i0Þth element of Σjk and σ2 ijk is the ith diagonal element of Σjk. Furthermore, suppose In all future calculations, we plug in ^ν2 i0 for ν2 i0. This is similar in spirit to many statistical procedures involving nuisance parameters. The following lemma is useful in the sequel. pp Assumption 0.3. Lemma 0.1. ∂ ∂θ log f ðxÞ ¼ Ef ðzjxÞ½ ∂ ∂θ log f ðzÞ þ ∂ ∂θ log f ðxjzÞ.25 ð ÞT Let Θ ¼ ðδ; π1; π2; π3; l1; l2; κ1; κ2ÞT denote the set of unknown parameters, then for each taxon the log-likelihood can be reformulated using Lemma 0.1, as follows: Denote 1 = (1, 1, …, 1)T, then we have 0 ≤1TΣ1 ¼ X m i¼1 X m i0¼1 σii0jk ¼ X m i¼1 σ2 ijk þ X m i≠i0 σii0jk ≤mσ2 0 þ X m i≠i0 σii0jk; ð Θ arg maxΘ X m i¼1 X 2 r¼0 pr;i½log Prði 2 CrÞ þ log f ðΔiji 2 CrÞ: ð21Þ hen the E–M algorithm is described as follows: ð21Þ Hence Hence i¼1 r¼0 Then the E–M algorithm is described as follows: Then the E–M algorithm is described as follows: Then the E–M algorithm is described as follows: 0 ≤1TΣ1 m2 ≤σ2 0 m þ Pm i≠i0 σii0jk m2 ¼ oð1Þ: ð11Þ ● E-step: Compute conditional probabilities of the latent variable. Deﬁne pr;i ¼ Prði 2 CrjΔiÞ ¼ πrϕðΔi ðδ þ lr Þ νir Þ P rπrϕðΔi ðδ þ lr Þ νir Þ ; r ¼ 0; 1; 2; i ¼ 1; ¼ ; m, which are con- ditional probabilities representing the probability that an observed value follows each distribution. Note that l0 = 0. ● E-step: Compute conditional probabilities of the latent variable. Deﬁne pr;i ¼ Prði 2 CrjΔiÞ ¼ πrϕðΔi ðδ þ lr Þ νir Þ P rπrϕðΔi ðδ þ lr Þ νir Þ ; r ¼ 0; 1; 2; i ¼ 1; ¼ ; m, which are con- ditional probabilities representing the probability that an observed value follows each distribution. Note that l0 = 0. Thus, for each k = 1, 2, …, n, and for each taxon i = 1, 2, …, m, according to Assumption 0.3, we have: 1 m X m i¼1 ðyijk ðdjk þ μijÞÞ!p0 as m ! 1: 1 m X m i¼1 ðyijk ðdjk þ μijÞÞ!p0 as m ! 1: ð12Þ ● M-step: Maximize the likelihood function with respect to the parameters, given the conditional probabilities. ● M-step: Maximize the likelihood function with respect to the parameters, given the conditional probabilities. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Note that αi ¼ 1 VarðΔi ii ð iÞ i1 i0 1 ð Þ ωii ¼ VarðΔiÞ ¼ ν2 i2 ¼ ν2 i0 þ κ2 ¼ Oð1Þ VarðαTu αT1Þ ¼ Pm i¼1 α2 i ωii ðPm i¼1 αiÞ2 þ Pm i≠i0 αiαi0ωii0 ðPm i¼1 αiÞ2 ¼ 1 Pm i¼1 αi þ Pm i≠i0 αiαi0ωii0 ðPm i¼1 αiÞ2 : ð26Þ Since ν2 i0 ¼ Oðn1Þ, ν2 i1 ¼ Oð1Þ, and ν2 i2 ¼ Oð1Þ, consequently, a1i = O(n), 2i = a3i = O(1), and X m i¼1 αi ¼ 1Ta1 þ 1Ta2 þ 1Ta3 ¼ X i2C0 OðnÞ þ X i2C1 Oð1Þ þ X i2C2 Oð1Þ X m i¼1 αi ¼ 1Ta1 þ 1Ta2 þ 1Ta3 ¼ X i2C0 OðnÞ þ X i2C1 Oð1Þ þ X i2C2 Oð1Þ ¼ Oðm0nÞ þ Oðm1Þ þ Oðm2Þ X m i¼1 αi ¼ 1Ta1 þ 1Ta2 þ 1Ta3 ¼ X i2C0 OðnÞ þ X i2C1 Oð1Þ þ X i2C2 Oð1Þ ¼ Oðm0nÞ þ Oðm1Þ þ Oðm2Þ ¼ Oðm0nÞ ifm0n ≥maxfm1; m2g: ð27 ð27Þ ¼ Oðm0nÞ þ Oðm1Þ þ Oðm2Þ Oð Þ if ≥ f ¼ Oðm0nÞ þ Oðm1Þ þ Oðm2Þ ¼ Oðm0nÞ ifm0n ≥maxfm1; m2g Hypothesis testing for multigroup comparison. In some applications, for a given taxon, researchers are interested in drawing inferences regarding DA in more than two ecosystems. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 1: ð32Þ ( r ) Deﬁne the test statistic for pairwise comparison as: We performed extensive simulations to evaluate the bias and variance of ^δEM ^ ^ ^ We performed extensive simulations to evaluate the bias and variance of ^δEM and that of ^δWLS. The scatter plot (Supplementary Fig. 9) of ^δEM and ^δWLS are Wi;jj0 ¼ ^μ ij ^μ ij0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ^σ2 ij þ ^σ2 ij0 q ; i ¼ 1; ¼ ; m; j ≠j0 2 f1; ¼ ; gg: ð40Þ We performed extensive simulations to evaluate the bias and variance of δEM and that of ^δWLS. The scatter plot (Supplementary Fig. 9) of ^δEM and ^δWLS are almost perfectly linear with correlation coefﬁcient nearly 1 in all cases. This suggests that ^δWLS is a very good approximation for ^δEM. The variance of ^δEM as well as that of ^δWLS are roughly of the order n1m1 0 , as we expected. In addition, they are approximately unbiased (Supplementary Table 6). ð40Þ suggests that ^δWLS is a very good approximation for ^δEM. The variance of ^δEM as well as that of ^δWLS are roughly of the order n1m1 0 , as we expected. In addition, they are approximately unbiased (Supplementary Table 6). For computational simplicity, inspired by the William’s type of test32–35, we reformulate the global test with the following test statistic: Wi ¼ max j≠j02f1; ¼ ;gg jWi;jj0j; i ¼ 1; ¼ ; m: ð41Þ ð41Þ Under null, Wi;jj0!dNð0; 1Þ, thus we can construct the null distribution of Wi by simulations, i.e., for each speciﬁc taxon i, Hypothesis testing for two-group comparison. For taxon i, we test the following hypothesis Hypothesis testing for two-group comparison. For taxon i, we test the following hypothesis (a) Generate WðbÞ i;jj0 Nð0; 1Þ; j ≠j0 2 f1; ¼ ; gg; b ¼ 1; ¼ ; B: (b) Compute WðbÞ i ¼ max j≠j02f1; ¼ ;gg WðbÞ i;jj0: (c) Repeat above steps B times (e.g., B = 1000), we then get the null distribution of Wi by ðWð1Þ i ; ¼ ; WðBÞ i Þ T: Finally, p value is calculated as (a) Generate WðbÞ i;jj0 Nð0; 1Þ; j ≠j0 2 f1; ¼ ; gg; b ¼ 1; ¼ ; B: (b) Compute WðbÞ i ¼ max j≠j02f1; ¼ ;gg WðbÞ i;jj0: (c) Repeat above steps B times (e.g., B = 1000), we then get the null (a) Generate WðbÞ i;jj0 Nð0; 1Þ; j ≠j0 2 f1; ¼ ; gg; b ¼ 1; ¼ ; B: (b) Compute WðbÞ i ¼ max j≠j02f1; ¼ ;gg WðbÞ i;jj0: (c) Repeat above steps B times (e.g., B = 1000), we then get the null of Wi by ðWð1Þ i ; ¼ ; WðBÞ i Þ T: (a) Generate WðbÞ i;jj0 Nð0; 1Þ; j ≠j0 2 f1; ¼ ; gg; b ¼ 1; ¼ ; B: (b) Compute WðbÞ i ¼ max j≠j02f1; ¼ ;gg WðbÞ i;jj0: (c) Repeat above steps B times (e.g., B = 1000), we then get the null distribution of Wi by ðWð1Þ i ; ¼ ; WðBÞ i Þ T: Finally, p value is calculated as H0 : μi1 ¼ μi2 H1 : μi1 ≠μi2 using the following test statistic which is approximately centered at zero under the null hypothesis: of Wi by ðWð1Þ i ; ¼ ; WðBÞ i Þ T: of Wi by ðWð1Þ i ; ¼ ; WðBÞ i Þ T: Finally, p value is calculated as Finally, p value is calculated as Finally, p value is calculated as Wi ¼ ^μi1 ^μi2 ^δEM ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ^σ2 i1 þ ^σ2 i2 q : ð33Þ pi ¼ 1 B X B b¼1 IðWðbÞ i >WiÞ; i ¼ 1; ¼ ; m ð42Þ ð33Þ ð42Þ From Slutsky’s theorem, we have: and the Bonferroni correction is applied to control the FDR. Wi!dNð0; 1Þ; as m; n ! 1: Wi!dNð0; 1Þ; as m; n ! 1: ð34Þ ð34Þ Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 For example, for a given taxon, researchers may want to test whether there exists at least one experimental group that is signiﬁcantly different from others, i.e., to test Using these facts and Assumption 0.4 in (26), we get Using these facts and Assumption 0.4 in (26), we get VarðαTu αT1Þ ¼ Oðm1 0 n1Þ þ Pm i≠i0fn1m1αigfn1m1αi0gωii0 n2m2ðPm i¼1 αiÞ2 ¼ Oðm1 0 n1Þ þ 1 m2 Pm i≠i0fn1αigfn1αi0gωii0 ðPm i¼1 n1m1αiÞ2 ¼ Oðm1 0 n1Þ þ 1 m2 Oð1Þoðm2Þ Oð1Þ ¼ Oðm1 0 n1Þ: H0;i : \j≠j02f1; ¼ ;ggμij ¼ μij0 H1;i : ∪j≠j02f1; ¼ ;ggμij ≠μij0: ð28Þ H1;i : ∪j≠j02f1; ¼ ;ggμij ≠μij0: Similar to the two-group comparison, after getting the initial estimates of ^μij Similar to the two-group comparison, after getting the initial estimates of ^μij and ^djk, setting the reference group r (e.g., r = 1), and obtaining the estimator of the bias term ^δrj through E–M algorithm, the ﬁnal estimator of mean absolute abundance of the ecosystem (in log scale) are obtained by transforming ^μij of (6) into: Thus, under Assumption 0.4 regarding ωii0, the contribution of the covariance terms in the above variance expression is negligible as long as m is very large compared with n, which is usually the case. Hence Varð^δWLSÞ ¼ VarðαTu αT1Þ ¼ Oðm1 0 n1Þ: ð29Þ ^μ ij :¼ ^μir; j ¼ r ^μij þ ^δrj; j ≠r 2 1; ¼ ; g ( : ð36Þ ð29Þ ð36Þ Furthermore, appealing to Cauchy–Schwartz inequality we get Furthermore, appealing to Cauchy–Schwartz inequality we get Thus, based on (18) and the E–M estimator of δrj, as m; minðnj; nj0Þ ! NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 Using the above expressions, we compute the variance as follows: large, then we modify the above test statistic as follows: W i ¼ ^μi1 ^μi2 ^δWLS ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ^σ2 i1 þ ^σ2 i2 þ d Varð^δWLSÞ þ 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ð^σ2 i1 þ ^σ2 i2Þd Varð^δWLSÞ q r : ð35Þ Varð^δWLSÞ ¼ VarðαTu αT1Þ ¼ Pm i¼1 α2 i ωii ðPm i¼1 αiÞ2 þ Pm i≠i0 αiαi0ωii0 ðPm i¼1 αiÞ2 : ð25Þ ð25Þ ð35Þ Recall that (a) for i ∈C0, ωii ¼ VarðΔiÞ ¼ ν2 i0 ¼ Oðn1Þ, (b) for i ∈C1, To control the FDR due to multiple comparisons, we recommend applying the Holm–Bonferroni method26 or Bonferroni27,28 correction rather than the Benjamini–Hochberg (BH) procedure29 to adjust the raw p values as research has showed that it is more appropriate to control the FDR when p values were not accurate30, and the BH procedure controls the FDR provided you have either independence or some special correlation structures such as perhaps positive regression dependence among taxa29,31. In our simulation studies, since the absolute abundances for each taxon are generated independently, we compared the ANCOM-BC results adjusted either by Bonferroni correction (Fig. 4) or BH procedure (Supplementary Fig. 10), it is clearly that the FDR control by Bonferroni correction is more conservative while implementing BH procedure results in FDR around the nominal level (5%). Obviously, ANCOM-BC has larger power when using BH procedure. ( ) 0 ii ð iÞ i0 ð Þ ( ) ωii ¼ VarðΔiÞ ¼ ν2 i1 ¼ ν2 i0 þ κ1 ¼ Oð1Þ, and (c) for i ∈C2, ( ) 0 ii ð iÞ i0 ð Þ ( ) ωii ¼ VarðΔiÞ ¼ ν2 i1 ¼ ν2 i0 þ κ1 ¼ Oð1Þ, and (c) for i ∈C2, ii ð iÞ i1 i0 1 ð Þ ( ) 2 ωii ¼ VarðΔiÞ ¼ ν2 i2 ¼ ν2 i0 þ κ2 ¼ Oð1Þ. Note that αi ¼ 1 VarðΔiÞ ¼ 1 ωii, thus we have: ii ð iÞ i1 i0 1 ð Þ ωii ¼ VarðΔiÞ ¼ ν2 i2 ¼ ν2 i0 þ κ2 ¼ Oð1Þ. Lemma 0.1. For the simplicity of notation, we relabel a and x by α and u, respectively. Denote Cov(x) = Cov(u) by Ω, and let ωii0 denotes the ði; i0Þ element of Ω. As in Assumption 0.3, we make the following assumption Assumption 0.4. Pm i≠i0 ωii0 m2 ¼ oð1Þ: ð24Þ ð24Þ 9 TURE COMMUNICATIONS \| (2020) 11:3514 \| https://doi.org/10.1038/s41467-020-17041-7 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-17041-7 1 Covð^μi1 ^μi2; ^δWLSÞ ≤ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Varð^μi1 ^μi2ÞVarð^δWLSÞ q ≤Oðn1=2ÞOðm1=2 0 n1=2Þ ¼ Oðn1m1=2 0 Þ: ð30Þ ^μ ij ^μ ij0 !p 0 if taxo μij μij0 otherw ( Covð^μi1 ^μi2; ^δWLSÞ ≤ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Varð^μi1 ^μi2ÞVarð^δWLSÞ q ≤Oðn1=2ÞOðm1=2 0 n1=2Þ ¼ Oðn1m1=2 0 Þ: ð30Þ ^μ ij ^μ ij0 !p 0 if taxon i is not differentially abundant between group j and j0; μij μij0 otherwise: ( ð37Þ Covð^μi1 ^μi2; ^δWLSÞ ≤ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Varð^μi1 ^μi2ÞVarð^δWLSÞ q ≤Oðn1=2ÞOðm1=2 0 n1=2Þ ¼ Oðn1m1=2 0 Þ: ð30Þ ^μ ij ^μ ij0 !p 0 if taxon i is not differentially abundant between group j and j0; μij μij0 otherwise: ( ð37 ð37Þ Hence, as long as m0 is large, the contribution made by Varð^δWLSÞ and Covð^μi1 ^μi2; ^δWLSÞ relative to Varð^μi1 ^μi2Þ is negligible. Hence, as long as m0 is large, the contribution made by Varð^δWLSÞ and Covð^μi1 ^μi2; ^δWLSÞ relative to Varð^μi1 ^μi2Þ is negligible. ^ Similarly, the estimator of the sampling fraction is obtained by transforming ^djk of (6) into y of (6) into Neglect the covariance term in (26), let ^Cr denote the estimator of Cr, r = 0, 1, 2 from the E–M algorithm, deﬁne ^d jk :¼ ^drk; j ¼ r ^djk ^δrj; j ≠r 2 1; ¼ ; g ( : ð38Þ ð38Þ d Varð^δWLSÞ ¼ 1 P i2^C0 1 ^ν2 i0 þ P i2^C1 1 ^ν2 i1 þ P i2^C2 1 ^ν2 i2 ; ð31Þ ð31Þ As by (13) and the E–M estimator of δrj As by (13) and the E–M estimator of δrj ^d jk!pdjk dras m; minðnj; nj0Þ ! 1; ð39Þ ð39Þ an estimator of Varð^δWLSÞ under the Assumption 0.4. Then which indicates that we are only able to estimate sampling fractions up to an additive constant (dr). which indicates that we are only able to estimate sampling fractions up to an additive constant (dr). d Varð^δWLSÞ!p 1 Pm i¼1 αi ¼ 1 P i2C0 1 ν2 i0 þ P i2C1 1 ν2 i1 þ P i2C2 1 ν2 i2 ; as m; n ! 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 18. Obregon-Tito, A. J. et al. Subsistence strategies in traditional societies distinguish gut microbiomes. Nat. Commun. 6, 6505 (2015). 19. 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https://openalex.org/W4391515880	https://jazindia.com/index.php/jaz/article/download/3506/3112	English	null	Exploring Herbal Remedies For Skin Cancer: A Comprehensive Review	null	2,024	cc-by	4,434	Abstract Received: 04/12/2023 Revised: 06/01/2024 Accepted: 19/01/2024 CC License CC-BY-NC-SA 4.0 Skin cancer, ranked as the fifth most prevalent cancer, is a rising health threat, emphasizing the need for innovative treatments. Genetic mutations cause skin cell abnormalities leading to various cancers, especially melanoma. Current treatments, such as radiation therapy and chemotherapy, have limitations, necessitating advancements in technology and therapy. Early detection is crucial for effective intervention, and diagnostic methods encompass blood tests, imaging, biopsies, and genetic tests, followed by staging to determine disease severity. This review focuses on herbal remedies from medicinal plants like ursolic acid, genistic acid, luteolin, curcumin, and others, demonstrating anti-cancer properties in inhibiting proliferation and modulating molecular processes. Studies, primarily in vitro and animals, offer insights into their potential for skin cancer prevention. The review synthesizes diverse literature, providing nuanced insights into herbal remedies’ molecular mechanisms for innovative therapeutic approaches. Emphasizing the importance of human trials, particularly for compounds like ursolic acid and genistic acid, is crucial for validating efficacy and safety. Herbal remedies align with WHO recommendations, holding promise for more effective and holistic skin cancer treatments. Ongoing research supports the integration of these compounds into treatment protocols, marking a hopeful frontier in combatting this pervasive disease. Keywords: Skin cancer, Herbal remedies, Curcumin, Ursolic acid, Luteolin CC License CC-BY-NC-SA 4.0 Corresponding author: Piyushkumar Sadhu Corresponding author: Piyushkumar Sadhu Department of Pharmacy, Sumandeep Vidyapeeth Deemed to be University, Piparia, Vadodara, Gujarat, India.; Email I’d – piyush.sadhu@yahoo.in; ORCID I’d: 0000-0001-5725-1861 Received: 04/12/2023 Revised: 06/01/2024 Accepted: 19/01/2024 Exploring Herbal Remedies For Skin Cancer: A Comprehensive Review Exploring Herbal Remedies For Skin Cancer: A Comprehensive Review Piyushkumar Sadhua, Falguni Rathodb, Mamta Kumaria, Niyati Shaha, Chitrali Talelea, Chintan Aundhiaa, Nirmal Shaha Piyushkumar Sadhua, Falguni Rathodb, Mamta Kumaria, Niyati Shaha, Chitrali Talelea, Chintan Aundhiaa, Nirmal Shaha aDepartment of Pharmacy, Sumandeep Vidyapeeth Deemed to be University, Piparia, Vadodara, Gujarat – 391760, India. b Faculty of Nursing, Noble University, Junagadh, Gujarat – 360 001, India. 1. INTRODUCTION Available online at: https://jazindia.com 951 Skin cancer, the fifth most prevalent cancer, is projected to become a leading cause of death, potentially surpassing heart disease. With 18.1 million new cancer cases in 2018 and about 9.6 million related deaths, melanoma is estimated to represent 4% of new cases in women and 6% in men by 2023. Genetic flaws or mutations in skin cell DNA are the root cause of aberrant skin cell proliferation leading to skin cancer [1]. The epidermis, composed of melanocytes and epithelial cells, overlays the dermis housing sweat glands, blood 951 Journal of Advanced Zoology vessels, and hair follicles. Non-melanoma skin cancer (NMSC), comprising basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), typically starts in the epidermis. Around 25% of documented cases show a mole progressing to Multiple Myeloma (MM), often with a high recurrence rate, stressing the importance of early detection and eradication. Effective investment in technology and therapy is crucial due to skin cancer’s significant impact on social and psychological well-being. Current treatments like radiation therapy, chemotherapy, and surgery, while commonly used, have drawbacks and can harm healthy cells. Photodynamic therapy (PDT) and photothermal therapy (PTT) offer potential tumor-ablative treatments, selectively targeting cancer cells [2]. [ ] Diagnosis involves a comprehensive evaluation and specific tests such as blood tests, imaging, biopsies, and genetic tests to pinpoint the ailment accurately. Staging, critical after diagnosis, determines the disease’s extent and severity, influencing treatment decisions and prognosis prediction (refer figure 1 and figure 2). It aids in tailoring treatment plans and providing patients with a clearer understanding of their condition and potential outcomes. Skin cancer is a pervasive disease affecting approximately one in every five individuals during their lifetime, but early identification has significantly lowered mortality rates [3]. Unrestrained growth of normal cells characterizes cancer, with two primary types: non-melanoma skin cancer (NMSC) and melanoma skin cancer [4]. NMSC, notably basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is prevalent in regions like North America, Australia, and New Zealand, with an estimated 1,042,056 new cases globally in 2018, linked to about 6% of all deaths. Ultraviolet (UV) radiation is a primary cause of NMSC, particularly affecting those with lighter skin tones. Genetic mutations in certain gene families, including CYP450, GST, and p53, can also contribute to its development. Various unique forms of NMSC, like verrucous carcinoma and squamous cell carcinoma, result from viral infections [5,6]. 1. INTRODUCTION Basal cell carcinoma (BCC), the most common skin cancer type, often stems from sunlight exposure. Despite a low death rate, it accounts for a substantial number of cases, reaching approximately 4.3 million annually in the US. Caucasians exhibit a notably higher prevalence. BCC manifests as flesh-colored bumps or areas on the skin and is primarily associated with areas exposed to sunlight. Genetic mutations and UV radiation are primary causes [7]. Squamous cell carcinoma (SCC), the second most common skin cancer, displays both benign and metastatic potential. It affects keratinocytes in the upper skin layer, appearing as rough red lumps [8]. UV-induced mutations, including P53 mutations, contribute to its development, with subsequent changes in various genes and signaling pathways. Melanoma, arising from melanocytes, presents a more peculiar yet significant challenge. UV light-triggered genetic changes can lead to uncontrolled melanocyte proliferation and cancer. Approximately 75% of skin cancer-related deaths are attributed to melanoma, emphasizing the importance of early detection. Its ABCD rule—assessing asymmetry, border irregularity, coloration, and diameter that helps diagnosing it. Melanoma subtypes, including superficial, nodular, lentigo maligna, and acral lentiginous, vary in appearance, location, and prognosis. While superficial melanoma is the most common subtype, nodular melanoma tends to grow vertically, delaying suspicion. Lentigo maligna commonly occurs in older individuals and can advance swiftly. Acral lentiginous melanoma is less common, often affecting areas unrelated to sun exposure [9,10]. Advanced cases may have worse prognoses, but ongoing research into immunotherapies and targeted drugs offers promise for improved treatment outcomes. Available online at: https://jazindia.com 952 Figure 1. Different stages of skin cancer Reused from Narayanamurthy V et al. [10] Figure 1. Different stages of skin cancer Reused from Narayanamurthy V et al. [10] Available online at: https://jazindia.com Figure 1. Different stages of skin cancer Reused from Narayanamurthy V et al. [10] 952 Journal of Advanced Zoology Figure 2. Different techniques for the diagnosis of skin cancer. Reused from Narayanamurthy V et al. [10] Figure 2. Different techniques for the diagnosis of skin cancer. Reused from Narayanamurthy 2. HERBAL REMEDIES FOR SKIN CANCER Skin cancer poses a significant challenge, underscoring the urgent need for innovative treatment approaches. Natural compounds derived from medicinal plants have demonstrated promising outcomes in inhibiting skin cancer cell growth and progression in various studies involving cell lines and animal models. The World Health Organization (WHO) has outlined dietary recommendations to mitigate cancer risks, emphasizing the importance of integrating phytochemicals from plants into daily consumption as both preventive and therapeutic agents. Epidemiological research has consistently highlighted the risk-lowering benefits of regular fruit and vegetable intake in the development of cancer. Phytochemicals sourced from medicinal plants, fruits, and vegetables have exhibited substantial roles in both preventing and treating skin cancer by modulating various molecular processes [11]. Detailed insights into these phytochemicals, their origins, and the specific molecular pathways are discussed in the following section. These compounds exhibit diverse functions, including inhibiting angiogenesis, metastasis, proliferation, inducing apoptosis, and halting cell cycle progression. 2.1 Ursolic acid (UA) UA, found abundantly in herbs like thyme, basil, and rosemary, exhibits significant potential as a phytochemical. Its beneficial effects include anti-proliferative, chemo-preventive, antioxidant, and anti- inflammatory properties. In studies, UA triggered cell death in certain cancer cell lines through a cascade involving caspase-3 activation via mitochondria, while also influencing the expression of key proteins like p53 and caspase-3 and reducing Bcl-2 levels. Additionally, it affects cell cycle regulation, influencing the G1 phase and the expression of p21 WAF1, which governs cell cycle progression. UA has been shown to inhibit specific pathways like NF-κB signaling by affecting the phosphorylation of p65 and IκBα, leading to decreased expression of certain enzymes involved in cell proliferation. Notably, it demonstrated an ability to reduce UVB- induced oxidative stress in human lymphocytes by lowering lipid hydroperoxide levels and enhancing antioxidants when applied before UV exposure [12]. While there is ongoing interest in exploring UA’s potential in combating skin cancer, no clinical trials on human skin have been reported thus far. However, a liposome- coating formulation containing UA, tested on three healthy subjects, showed an increase in ceramide content in human skin. Yet, the sample size in this experimental study was limited, and the examination was conducted solely on non-cancerous skin [13]. There remains an urgent necessity for extensive human studies on a larger scale specifically focusing on UA’s effects on skin cancer. Available online at: https://jazindia.com 2.5 Indole-3-carbinol (I3C) ( ) Indole-3-carbinol (I3C) is a compound found abundantly in cruciferous vegetables like broccoli, Brussels sprouts, and cauliflower. Its cancer chemopreventive properties have been highlighted across various cancers such as breast, cervical, gastrointestinal, and lung cancers. Studies indicate that I3C induces cell cycle arrest and promotes apoptosis in UVB-sensitized MM cells by inhibiting Bcl-2 and reducing microphthalmia- associated transcription factor (MITF) expression [20]. Additionally, I3C hampers the proliferation of human MM cells by regulating phosphatase and tensin homolog (PTEN) degradation. In animal models, dietary supplementation with I3C has shown to enhance sensitivity to chemotherapy [21]. However, research on I3C has primarily been limited to cellular and mouse models. These initial findings call for extensive scientific investigation to validate its safety and efficacy before potential applications in clinical settings. 2.2 Genisitic acid (GA) GA, a potent isoflavone sourced from soyabeans, has been utilized in soy-rich diets as a supplement for conditions like cancer, heart diseases, and osteoporosis. This phytoestrogen present in soybeans demonstrates considerable potential in antioxidative, anti-inflammatory, anti-proliferative effects, and inhibition of certain 953 Available online at: https://jazindia.com Journal of Advanced Zoology cancers, including breast, neuroblastoma, and both MM and NMSC cancers. It showcases multiple benefits such as anti-angiogenic effects, suppression of tumor growth and metastasis, cell cycle arrest, and facilitation of caspase-mediated cell death. GEN displays protective actions against UV-induced skin damage and photoaging-triggered skin cancer. It’s been observed to inhibit the formation of pyrimidine dimers caused by UVB radiation and has demonstrated photoprotective properties by interfering with the cell cycle in models. Studies also reveal its ability to mitigate oxidative damage triggered by UVB exposure in the skin of mice [14]. In cancer cell cycle progression, GA targets various key elements like p53, p21, checkpoint kinase, and Chk2 in MM cells. Its impact extends beyond cell cycle regulation to include the inhibition of angiogenesis, as supported by numerous scientific findings. While there’s substantial evidence supporting GEN’s applications in preventing and treating MM and NMSC human skin cancers, further research encompassing in vitro and in vivo studies is crucial [15]. 2.4 Curcumin (CUR) CUR, a potent compound derived from the rhizome of Curcumin longa, exhibits notable anti-inflammatory and antioxidant properties, particularly in conditions like psoriasis. Its impact extends to cancer prevention by modulating various pathways such as 5-lipoxygenase (5-LOX), COX-2, NF-κB, STAT3, phosphorylase kinase, and apoptotic cytokines [18]. As early as 1987, Kuttan et al. demonstrated CUR’s anticancer potential in humans by reducing cancer lesion sizes in over 62 patients. Its efficacy extends to various cancers either as a standalone agent or in synergy with other therapeutic agents. Studies have explored CUR’s protective effects against head and neck SCC, prostate, multiple myeloma, pancreatic, lung, and colorectal cancers. In a melanoma mouse model, CUR showcased its ability to upregulate miRNA-2015-5p expression, crucial in modulating apoptosis and proliferation. Furthermore, in a mouse skin model, CUR displayed anti-inflammatory effects against SRB12-p9 skin cancer cells by orally suppressing skin SCC growth and downregulating the pS6 biomarker. Additionally, CUR effectively inhibited proliferation in RB12-p9 cells at specific doses, suggesting its potential efficacy against skin cancer [19]. The safety and efficacy of CUR have been extensively studied in various clinical trials, positioning it as a robust compound for the development of medicines targeting skin cancer. Its administration through different routes, including oral and topical application, has demonstrated promising potential in mouse skin models. 2.3 Luteolin Luteolin, found in various foods like carrots, celery, olives, and peppers, exhibits significant potential as a phytochemical. It demonstrates promising anti-inflammatory, antioxidant, and anti-cancer properties, capable of inhibiting angiogenesis, promoting caspase-mediated cell death, and sensitizing cells to anticancer treatments across a wide spectrum of cancers [16]. Research indicates that luteolin encourages melanogenesis while diminishing the aggressive tendencies of skin cancer cells by influencing β3 integrin and focal adhesion kinase (FAK) signaling pathways. Moreover, luteolin induces apoptosis and halts the growth of skin cancer cells by adjusting the expression of key proteins like Bax and Bcl-2, along with attenuating ERK1/2 signaling [17]. While previous studies have highlighted the potential of this compound in cancer therapeutics, further investigations via in vitro and in vivo studies, as well as human clinical trials, are necessary to gain deeper insights into its efficacy and drug bioavailability. Available online at: https://jazindia.com 2.6 Resveratrol (RV) ( ) Resveratrol (RV) is a stilbene polyphenol commonly found in mulberries, peanuts, and grapes. When topically applied, it exhibits robust inhibitory potential across the three different stages of carcinogenesis in murine models. It is known for its strong anti-cancer effects, RV possesses anti-proliferative, anti-inflammatory, and antioxidant properties. It acts as a potent scavenger for reactive oxygen species (ROS) and has demonstrated 954 Available online at: https://jazindia.com Journal of Advanced Zoology the ability to reduce ROS levels in human skin fibroblast cells in vivo. Despite its antioxidant properties, RV counteracts anti-inflammatory actions by impeding the effects of COX-1 and COX-2, mainly through inhibition of NF-κB expression and suppression of p38 MAPK and ERK. RV shows promise in combination with other phyto-compounds by suppressing tumorigenesis and reducing epidermal hyperplasia while decreasing the expression of specific proteins and enzymes. It also exhibits potential as an adjuvant with other chemotherapeutic agents in treating MM with distant metastatic disease, decreasing skin cancer cell viability, and enhancing the cytotoxic effects of certain drugs. RV’s effectiveness in sensitizing skin cancer cells to drugs like dacarbazine and influencing the expression of Akt/PKB proteins in MM cells showcases its potential as a chemotherapeutic agent [22,23]. However, RV administered orally shows poor bioavailability due to rapid clearance by the liver and intestines, limiting its concentration in the human body. Consequently, topical application of RV appears highly promising for both chemoprevention and chemotherapeutics. Clinical trials involving RV-containing creams have shown promising improvements in skin elasticity, hydration, and luminosity without adverse effects in non-cancerous individuals. Although these trials involved small sample sizes and focused on non-cancerous skin, initial results suggest potential efficacy [24]. However, comprehensive clinical trials exploring the efficacy and safety of RV in preventing and treating MM and NMSC are necessary. 2.8 [6] - Gingerol [6]-Gingerol, a potent phenolic compound extracted from the root of Zingiber officinale, was first studied by Park et al. in 1998. Topical administration showed potential in inhibiting skin papilloma formation. It also demonstrated anti-inflammatory properties by reducing epidermal ornithine decarboxylase activity, inhibiting COX-2, and suppressing NF-κB through modulation of p38 mitogen-activated protein kinase (MAPK) activity. Additionally, [6]-gingerol decreased intracellular ROS levels induced by UV radiation and activated caspases- 3, 8, and 9, influencing antioxidant activity. Other strategies involve [6]-gingerol activating AP-1 DNA binding activity and modulating proteins like p53, Bax, Bcl-2, and surviving [28,29]. No human trials have been published so far, but ongoing investigations aim to incorporate [6]-gingerol into solid nanoparticles for topical administration to enhance safety, efficacy, and stability. Developing such a delivery system for [6]-gingerol could offer a more convenient and stable option for further human clinical studies. 2.7 Capsaicin p There is conflicting scientific evidence surrounding capsaicin’s role as a carcinogenic agent or its potential for both chemoprevention and chemotherapy. While Hwang et al. showed in a mouse model that topical capsaicin use stimulated skin cancer by activating tyrosine kinase EGFR and COX-2, other studies found contrasting results. Some researchers observed no significant increase in skin cancer growth compared to controls and even noted a significant inhibition of papilloma formation in mice, suggesting capsaicin’s potential in inhibiting skin cancer. Capsaicin has displayed strong chemopreventive and chemotherapeutic properties by influencing cell cycle arrest, apoptosis induction, and inhibition of cancer cell proliferation. It antagonizes the expression of NF-κB, AP-1, STAT3, and COX-2, contributing to its therapeutic effects. Additionally, capsaicin induces caspase-mediated cell death in human cutaneous SCC cell lines and exhibits anti-mitogenic activity on metastatic MM cells by down-regulating phosphatidylinositol 3-kinase (PI3-K) expression. Its synergistic effects in inducing caspase-mediated cell death in MM cell lines when combined with HA14-1 further demonstrate its potential [25,26]. Researchers are conducting further studies and epidemiological investigations to ascertain capsaicin’s role in cancer therapeutics. However, while the topical application of capsaicin in treating skin cancer lacks comprehensive studies, experiences from capsaicin’s use in other areas suggest potential drawbacks. One review noted that one in three patients experienced higher rates of side effects like stinging, erythema, and burning compared to a placebo when administered capsaicin topically. These adverse reactions could limit its application in skin cancer chemoprevention and chemotherapy [27]. Therefore, exploring novel drug delivery systems, designs, and formulations involving capsaicin with other agents that have fewer side effects might offer a new avenue for skin cancer treatment. 3. CONCLUSION The exploration of herbal remedies for skin cancer presents a promising frontier in the quest for effective treatments. The utilization of bioactive compounds derived from medicinal plants unveils a potential avenue for inhibiting the development and progression of skin cancer cells. This avenue aligns with the World Health Organization’s dietary recommendations, advocating for the integration of phytochemicals from natural sources into daily consumption for their chemopreventive and chemotherapeutic properties. Epidemiological studies consistently underscore the significance of regular fruit and vegetable intake in lowering the risk of cancer development, further emphasizing the potential of plant-derived compounds in this context. These phytochemicals, found in various natural sources, play pivotal roles in regulating molecular processes fundamental to skin cancer, offering a nuanced and multi-faceted approach to combating this disease. Their ability to influence angiogenesis, metastasis, proliferation, apoptosis, and cell cycle arrest positions these herbal remedies as potential candidates for targeted and comprehensive treatment strategies. As research continues to unravel the specifics of these compounds and their mechanisms of action, the integration of herbal remedies into skin cancer treatment protocols holds promise for more effective and holistic approaches to combating this pervasive disease. 2.9 Epigallocatechin-3-Gallate (EGCG) Available online at: https://jazindia.com 955 p g ( ) EGCG, a potent phytocompound derived from Camellia sinensis, is extensively studied for its potential in both chemoprevention and chemotherapy, showcasing anti-inflammatory, anti-proliferative, and antioxidant properties within green tea phenols (GTP). Pioneering research by Katiyar et al. revealed GTP’s ability to inhibit COX and lipoxygenase activity, reducing skin cancer load by diminishing epidermal hyperplasia and edema [30]. Topical application of EGCG showed a significant reduction in UV radiation-induced ROS products while inhibiting MAPK signaling cascades associated with these radiations. EGCG’s anti- 955 Journal of Advanced Zoology proliferative functions involve modulation of NF-κB signaling cascades, inhibition of tumor-induced activator protein (AP-1), angiogenesis, and recruitment of T cells [31]. Moreover, studies by Nihal et al. highlighted EGCG’s capacity to sensitize MM cells to interferon-induced growth inhibition, reduce cancer cell proliferation, and induce caspase-mediated cell death. The synergistic effect of EGCG with interferon displayed higher effectiveness than their individual applications. EGCG also downregulates inflammation, decreasing interleukin (IL)-1β secretion and NF-κB activity, resulting in reduced cancer cell growth. Recent research demonstrated EGCG’s inhibition of MM cell invasion by reducing tumor necrosis factor (TNF) receptor- associated factor 6 (TRAF6) function [32]. Human trials have showcased EGCG’s strong therapeutic potential for skin cancer in various administration methods. While mice orally administered with GTP or through injection inhibited or reversed UV-induced skin papillomas, another study noted tumor inhibition in mice solely through topical application of EGCG. Topical GTP administration in humans provided protection against UV radiation-induced erythema. However, a single-blind randomized clinical trial on 50 individuals showed that orally administered GTP with vitamin C did not significantly reduce skin leukocyte infiltration and erythema compared to the placebo group [33]. These findings imply that topical use of EGCG holds higher potential than oral application for skin cancer chemoprevention and chemotherapy, warranting further research. Available online at: https://jazindia.com References 1. Sena JS, Girão RJ, Carvalho SM, Tavares RM, Fonseca FL, Silva PB, Barbosa MC. Occupational skin cancer: Systematic review. Rev Assoc Med Bras (1992). 2016;62(3):280-6. doi: 10.1590/1806- 9282.62.03.280. 2. Ng CY, Yen H, Hsiao HY, Su SC. Phytochemicals in Skin Cancer Prevention and Treatme Review. Int J Mol Sci. 2018;19(4):941. doi: 10.3390/ijms19040941. n H, Hsiao HY, Su SC. Phytochemicals in Skin Cancer Prevention and Treatment: An Updated t J Mol Sci. 2018;19(4):941. doi: 10.3390/ijms19040941. 3. Rey-Barroso L, Pena-Gutierrez S, Yanez C, Burgos-Fernández FJ, Vilaseca M, Royo S. Optical Technologies for the Improvement of Skin Cancer Diagnosis: A Review. Sensors (Basel). 2021;21(1):252. doi: 10.3390/s21010252. 3. Rey-Barroso L, Pena-Gutierrez S, Yanez C, Burgos-Fernández FJ, Vilaseca M, Royo S. Optical Technologies for the Improvement of Skin Cancer Diagnosis: A Review. Sensors (Basel). 2021;21(1):252. doi: 10.3390/s21010252. 4. Fuchs E. Skin Stem Cells in Silence, Action, and Cancer. Stem Cell Reports. 2018;10(5):1432-1438. doi: 10.1016/j.stemcr.2018.04.008. 4. Fuchs E. Skin Stem Cells in Silence, Action, and Cancer. Stem Cell Reports. 2018;10(5):1432-1438. doi: 10.1016/j.stemcr.2018.04.008. 5. Didona D, Paolino G, Bottoni U, Cantisani C. Non Melanoma Skin Cancer Pathogenesis Overview. Biomedicines. 2018;6(1):6. doi: 10.3390/biomedicines6010006. 5. Didona D, Paolino G, Bottoni U, Cantisani C. Non Melanoma Skin Cancer Pathogenesis Overview. Biomedicines. 2018;6(1):6. doi: 10.3390/biomedicines6010006. 6. Sample A, He YY. Mechanisms and prevention of UV-induced melanoma. Photodermatol Photoimmunol Photomed. 2018;34(1):13-24. doi: 10.1111/phpp.12329. Á 7. Álvarez-Salafranca M, Ara M, Zaballos P. Dermoscopy in Basal Cell Carcinoma: An Up Actas Dermosifiliogr (Engl Ed). 2021;112(4):330-338. doi: 10.1016/j.ad.2020.11.011. 8. Thompson AK, Kelley BF, Prokop LJ, Murad MH, Baum CL. Risk Factors for Cutaneous Squamous Cell Carcinoma Recurrence, Metastasis, and Disease-Specific Death: A Systematic Review and Meta-analysis. JAMA Dermatol. 2016;152(4):419-28. doi: 10.1001/jamadermatol.2015.4994. ; ( ) j 9. Ke Y, Wang XJ. TGFβ Signaling in Photoaging and UV-Induced Skin Cancer. J Invest Dermatol. 2021;141(4S):1104-1110. doi: 10.1016/j.jid.2020.11.007. Available online at: https://jazindia.com 956 Journal of Advanced Zoology 10. Narayanamurthy V, Padmapriya P, Noorasafrin A, Pooja B, Hema K, Nithyakalyani K, Samsuri F. Skin cancer detection using non-invasive techniques. RSC advances. 2018;8(49):28095-130. 11. Li JY, Kampp JT. Review of Common Alternative Herbal "Remedies" for Skin Cancer. Dermatol Surg. 2019;45(1):58-67. doi: 10.1097/DSS.0000000000001622. 12. Lee YH, Wang E, Kumar N, Glickman RD. Ursolic acid differentially modulates apoptosis in skin melanoma and retinal pigment epithelial cells exposed to UV–VIS broadband radiation. Apoptosis. 2014;19:816-28. 13. Lee YH, Sun Y, Glickman RD. References Ursolic Acid-Regulated Energy Metabolism-Reliever or Propeller of Ultraviolet-Induced Oxidative Stress and DNA Damage? Proteomes. 2014;2(3):399-425. doi: 10.3390/proteomes2030399. p 14. Truchuelo M, Cerdá P, Fernández LF. Chemical Peeling: A Useful Tool in the Office. Actas Dermosifiliogr. 2017;108(4):315-322. English, Spanish. doi: 10.1016/j.ad.2016.09.014. 15. Rkein AM, Ozog DM. Photodynamic therapy. Dermatol Clin. 2014;32(3):415-25, x. doi: 10.1016/j.det.2014.03.009. 16. Juszczak AM, Wöelfle U, Končić MZ, Tomczyk M. Skin cancer, including related pathways and therapy and the role of luteolin derivatives as potential therapeutics. Medicinal Research Reviews. 2022;42(4):1423- 62. 17. Yao X, Jiang W, Yu D, Yan Z. Luteolin inhibits proliferation and induces apoptosis of human melanoma cells in vivo and in vitro by suppressing MMP-2 and MMP-9 through the PI3K/AKT pathway. Food & function. 2019;10(2):703-12. 18. Mirzaei H, Naseri G, Rezaee R, Mohammadi M, Banikazemi Z, Mirzaei HR, Salehi H, Peyvandi M, Pawelek JM, Sahebkar A. Curcumin: A new candidate for melanoma therapy? Int J Cancer. 2016;139(8):1683-95. doi: 10.1002/ijc.30224. ( ) j 19. Jose A, Labala S, Ninave KM, Gade SK, Venuganti VV. Effective skin cancer treatment by topical co- delivery of curcumin and STAT3 siRNA using cationic liposomes. AAPS PharmSciTech. 2018;19:166-75. 20. Kim SY, Kim DS, Jeong YM, Moon SI, Kwon SB, Park KC. Indole-3-carbinol and ultraviolet B induce apoptosis of human melanoma cells via down-regulation of MITF. Die Pharmazie-An International Journal of Pharmaceutical Sciences. 2011;66(12):982-7. ( ) 21. Karimabad MN, Mahmoodi M, Jafarzadeh A, Darekordi A, Hajizadeh MR, Hassanshahi G. Molecular targets, anti-cancer properties and potency of synthetic indole-3-carbinol derivatives. Mini reviews in medicinal chemistry. 2019;19(7):540-54. y ; ( ) 22. Elshaer M, Chen Y, Wang XJ, Tang X. Resveratrol: An overview of its anti-cancer mechanisms. Life sciences. 2018;207:340-9. 23. Aziz SW, Aziz MH. Protective molecular mechanisms of resveratrol in UVR‐induced Skin carcinogenesis. Photodermatology, Photoimmunology & Photomedicine. 2018;34(1):35-41. 24. Chow HH, Garland LL, Heckman-Stoddard BM, Hsu CH, Butler VD, Cordova CA, Chew WM, Cornelison TL. A pilot clinical study of resveratrol in postmenopausal women with high body mass index: effects on systemic sex steroid hormones. J Transl Med. 2014;12:223. doi: 10.1186/s12967-014-0223-0. 25. Georgescu SR, Sârbu MI, Matei C, Ilie MA, Caruntu C, Constantin C, Neagu M, Tampa M. Capsaicin: Friend or Foe in Skin Cancer and Other Related Malignancies? Nutrients. 2017;9(12):1365. doi: 10.3390/nu9121365. 26. Chu H, Li M, Wang X. Capsaicin induces apoptosis and autophagy in human melanoma cells. Oncology letters. 2019;17(6):4827-34. 27. Derry S, Rice AS, Cole P, Tan T, Moore RA. Available online at: https://jazindia.com 33. Fujiki H, Sueoka E, Rawangkan A, Suganuma M. Human cancer stem cells are a target for cancer prevention using (-)-epigallocatechin gallate. J Cancer Res Clin Oncol. 2017 ;143(12):2401-2412. doi: 10.1007/s00432-017-2515-2. multicentre, randomized, crossover, double-blind, placebo-controlled clinical trial. Orphanet J Rare Dis. 2016;11:31. doi: 10.1186/s13023-016-0411-5. multicentre, randomized, crossover, double-blind, placebo-controlled clinical trial. Orphanet J Rare Dis. 2016;11:31. doi: 10.1186/s13023-016-0411-5. 33. Fujiki H, Sueoka E, Rawangkan A, Suganuma M. Human cancer stem cells are a target for cancer References Topical capsaicin (high concentration) for chronic neuropathic pain in adults. Cochrane Database Syst Rev. 2017;1(1):CD007393. doi: 10.1002/14651858.CD007393. 28. Oyagbemi AA, Saba AB, Azeez OI. Molecular targets of [6]‐gingerol: Its potential roles in cancer chemoprevention. Biofactors. 2010;36(3):169-78. p ( ) 29. Kapoor V, Aggarwal S, Das SN. 6‐Gingerol mediates its anti tumor activities in human oral and cervical cancer cell lines through apoptosis and cell cycle arrest. 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https://openalex.org/W2042744344	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0025790&type=printable	English	null	The Atypical Stimulant and Nootropic Modafinil Interacts with the Dopamine Transporter in a Different Manner than Classical Cocaine-Like Inhibitors	PloS one	2,011	cc-by	13,064	Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: maarten.reith@nyumc.org Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan Received July 6, 2011; Accepted September 11, 2011; Published October 17, 2011 Copyright: 2011 Schmitt, Reith. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. mitt, Reith. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: Project supported by United States National Institutes of Health (NIH) Grants DA013261 and DA019676. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: maarten.reith@nyumc.org Competing Interests: The authors have declared that no competing interests exist. * E-mail: maarten.reith@nyumc.org The Atypical Stimulant and Nootropic Modafinil Interacts with the Dopamine Transporter in a Different Manner than Classical Cocaine-Like Inhibitors Kyle C. Schmitt1, Maarten E. A. Reith1,2* 1 Department of Pharmacology, New York University School of Medicine, New York, New York, United States of America, 2 Department of Psychiatry, New York University School of Medicine, New York, New York, United States of America y 1 Department of Pharmacology, New York University School of Medicine, New York, New York, United States of America, 2 Department of Psychiatry, New York University School of Medicine, New York, New York, United States of America York University School of Medicine, New York, New York, United States of America, 2 Department of Psychiatry, New York University York, United States of America October 2011 \| Volume 6 \| Issue 10 \| e25790 Citation: Schmitt KC, Reith MEA (2011) The Atypical Stimulant and Nootropic Modafinil Interacts with the Dopamine Transporter in a Different Manner than Classical Cocaine-Like Inhibitors. PLoS ONE 6(10): e25790. doi:10.1371/journal.pone.0025790 Abstract Modafinil is a mild psychostimulant with pro-cognitive and antidepressant effects. Unlike many conventional stimulants, modafinil has little appreciable potential for abuse, making it a promising therapeutic agent for cocaine addiction. The chief molecular target of modafinil is the dopamine transporter (DAT); however, the mechanistic details underlying modafinil’s unique effects remain unknown. Recent studies suggest that the conformational effects of a given DAT ligand influence the magnitude of the ligand’s reinforcing properties. For example, the atypical DAT inhibitors benztropine and GBR12909 do not share cocaine’s notorious addictive liability, despite having greater binding affinity. Here, we show that the binding mechanism of modafinil is different than cocaine and similar to other atypical inhibitors. We previously established two mutations (W84L and D313N) that increase the likelihood that the DAT will adopt an outward-facing conformational state— these mutations increase the affinity of cocaine-like inhibitors considerably, but have little or opposite effect on atypical inhibitor binding. Thus, a compound’s WT/mutant affinity ratio can indicate whether the compound preferentially interacts with a more outward- or inward-facing conformational state. Modafinil displayed affinity ratios similar to those of benztropine, GBR12909 and bupropion (which lack cocaine-like effects in humans), but far different than those of cocaine, b-CFT or methylphenidate. Whereas treatment with zinc (known to stabilize an outward-facing transporter state) increased the affinity of cocaine and methylphenidate two-fold, it had little or no effect on the binding of modafinil, benztropine, bupropion or GBR12909. Additionally, computational modeling of inhibitor binding indicated that while b-CFT and methylphenidate stabilize an ‘‘open-to-out’’ conformation, binding of either modafinil or bupropion gives rise to a more closed conformation. Our findings highlight a mechanistic difference between modafinil and cocaine-like stimulants and further demonstrate that the conformational effects of a given DAT inhibitor influence its phenomenological effects. Citation: Schmitt KC, Reith MEA (2011) The Atypical Stimulant and Nootropic Modafinil Interacts with the Dopamine Transporter in a Different Manner than Classical Cocaine-Like Inhibitors. PLoS ONE 6(10): e25790. doi:10.1371/journal.pone.0025790 Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan Received July 6, 2011; Accepted September 11, 2011; Published October 17, 2011 Copyright: 2011 Schmitt, Reith. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Project supported by United States National Institutes of Health (NIH) Grants DA013261 and DA019676. Introduction (2009) recently performed a ‘‘receptorome’’ screen, examining the interaction of modafinil with a large array of different neuronal receptor and transporter proteins in vitro [18]. Of the included receptor proteins, the neuronal dopamine transporter (DAT) was the sole target at which modafinil displayed relevant binding (that is, the only protein for which it possessed a Ki value lower than the threshold of 10 mM). However, the addictive stimulants cocaine and methylphenidate also principally target the DAT. What makes modafinil different? One enigmatic aspect of DAT pharmacology is the disparate reinforcing efficacy of various transporter ligands. A particular DAT-inhibiting molecule may have dramatic, mild or even a complete lack of behaviorally rewarding effects, regardless of absolute binding affinity [19,20]. In this sense, the DAT appears to behave somewhat like a classically defined receptor, in that interaction with chemically distinctive ligands can elicit different behavioral effects in vivo. Recently, different chemical classes of ligands have been shown to stabilize the transporter protein in distinct conformational states upon binding; moreover, interaction with a specific conformation has been posited to affect the ‘‘addictiveness’’ of a given ligand [21]. It is important to note that rate of onset has also been shown to affect the addictiveness of DAT ligands—compounds with a rapid onset of action tend to exhibit greater reinforcing efficacy than those with a slower onset rate [22–25]. Compared to cocaine, modafinil has a slower onset of action [26]; hence, it is possible that this characteristic also contributes to its low addictive liability. resolution crystallographic structures of a related transporter protein—a leucine transporter from the bacterium Aquifex aeolicus (LeuT)—bound to a variety of substrate-like and inhibitor-like ligands [27–29] provided a groundbreaking template for in silico molecular modeling of DAT ligand-binding dynamics [30,31]. LeuT is a prokaryotic member of the neurotransmitter/sodium symporter (NSS) family of proteins, which also includes the eukaryotic transporters for serotonin, noradrenaline and dopa- mine (SERT, NET and DAT, respectively). The crystal structures, combined with a plethora of additional investigations of LeuT binding kinetics [32,33] and single-molecule dynamics [34,35] suggest an alternating access translocation cycle with at least three dominant low-energy conformational states (depicted in Fig. 1). The substrate interaction pocket at the center of the 12 transmembrane domain (TM) transporter protein (referred to as the ‘S1’ or primary substrate site) can be occluded from solution by both intra- and extracellular gating networks. Introduction modafinil has shown considerable promise as a therapeutic in the treatment of addiction to cocaine, one of the most frequently- used recreational drugs and likely the most addictive, based upon the percentage of both initial and regular users that transition into severe addicts [12,13]. Modafinil attenuates craving for cocaine during drug withdrawal and has also been shown to decrease self- administration of smoked cocaine base (crack) in habitual crack users [14,15]. Importantly, a recent study of modafinil self- administration in human cocaine addicts demonstrated that modafinil was not administered more frequently than placebo, nor did it occasion cocaine-like subjective effects [16]. Modafinil (2-(benzhydrylsulfinyl)acetamide) is a mild psycho- stimulant-like agent that increases wakefulness, improves attention and enhances performance in a variety of cognitive tasks [1–3]. Modafinil has been shown to exert antidepressive effects [4] and like other stimulants is an effective adjuvant for those experiencing only marginal improvement with serotonergic compounds [5,6]. Classical psychostimulants, such as dextroamphetamine and methylphenidate exhibit dose-dependent biphasic effects on cognition—enhancing performance, learning and memory con- solidation at moderate doses, but impairing cognitive function when used at high doses [7–9]. From a phenomenological perspective, modafinil has nootropic (pro-cognitive) effects similar to those of low-dose classical psychostimulants. However, compared to typical stimulants, modafinil possess a far more subtle and benign pharmacological profile [10]. Modafinil appears to lack many of the undesirable side effects of other stimulants, most notably: cardiovascular strain, sympathomimetic peripheral stimulation and significant addictive liability [11]. As such, The pharmacodynamic mechanism of modafinil is rather poorly understood and a wide-ranging variety of neurochemical systems have been previously implicated in its activity (for review, see e.g. [17]). One of the most prominent unresolved questions regarding modafinil’s mechanism of action is: why does it lack the notable addictive potential of classical stimulants, such as cocaine? An understanding of why modafinil has a far lower abuse liability than prototypical stimulants may facilitate the design of novel and improved stimulant therapeutics for ADHD, cognitive enhance- PLoS ONE \| www.plosone.org October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 1 October 2011 \| Volume 6 \| Issue 10 \| e25790 Modafinil Is an Atypical DAT Inhibitor ment, depression and cocaine addiction. In order to address this question, however, one must first possess insight into the protein target(s) of modafinil in the brain. Zolkowska et al. Introduction These gates are formed by a small number of critical residue side-chains (highly- conserved throughout the NSS family), via networks of ionic, p- cation and hydrogen-bonding interactions [36]. Disruption and reformation of these interaction networks—mediated by the binding of ions and substrate or other ligands [34]—likely underlies the alternating access mechanism, allowing transition between terminal ‘‘open-to-out’’ (outward-facing) and ‘‘open-to- in’’ (inward-facing) conformations, with a dually occluded intermediate. Further studies with LeuT have revealed the presence an additional substrate-binding domain (dubbed the ‘S2’ site) located in the extracellular vestibule of the transporter, 11–13 A˚ above the central S1 site. This vestibular site appears to bind a variety of different ligands, including a second molecule of the substrate leucine [32], alkylglucoside detergents [37] and a The specific molecular mechanism underlying the DAT’s substrate translocation cycle is not known. However, high- Figure 1. Cartoon representation of the DAT alternating access conformational cycle. (A) A fully outward-facing conformation with an open extracellular gating network (open-to-out) is established by binding of Na+ at the S1 site and is therefore the predominant state in the presence of high extracellular Na+ levels and absence of substrate. (B) Following Na+ binding, substrate interaction with S1 site residues triggers closure of the extracellular gate, establishing an occluded (closed-to-out) intermediate conformation. (C) Putative interaction of a second molecule of substrate with the vestibular S2 site helps facilitate opening of the intracellular gating network, giving rise to a fully inward-facing (open-to-in) conformation capable of releasing S1-bound substrate and ions into the cytoplasm. doi:10.1371/journal.pone.0025790.g001 Figure 1. Cartoon representation of the DAT alternating access conformational cycle. (A) A fully outward-facing conformation with an open extracellular gating network (open-to-out) is established by binding of Na+ at the S1 site and is therefore the predominant state in the presence of high extracellular Na+ levels and absence of substrate. (B) Following Na+ binding, substrate interaction with S1 site residues triggers closure of the extracellular gate, establishing an occluded (closed-to-out) intermediate conformation. (C) Putative interaction of a second molecule of substrate with the vestibular S2 site helps facilitate opening of the intracellular gating network, giving rise to a fully inward-facing (open-to-in) conformation capable of releasing S1-bound substrate and ions into the cytoplasm. doi:10.1371/journal.pone.0025790.g001 October 2011 \| Volume 6 \| Issue 10 \| e25790 October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 2 Modafinil Is an Atypical DAT Inhibitor wells. Homology modeling and flexible docking gy g g The DAT protein homology model was generated in a manner similar to the procedure detailed in Schmitt et al. (2010) [47]. The crystal structure of LeuT bound to the ligands leucine and the tricyclic antidepressant (TCA) desipramine ([28]; PDB Index 2QJU) was used as the structural template, employing the NSS- family protein amino acid sequence alignment proposed by Beuming et al. (2006) [48]. Since the sequence of LeuT is shorter than that of the DAT, parts of the intracellular termini were excluded from the model (N-terminal residues M1-V55 and residues K589-V620 on the C-terminus). In addition, all water and b-octylglucoside molecules and the ligands present in the template LeuT crystal were not included in the DAT model. The sodium ions were initially placed in the DAT model based upon their location in LeuT, but were allowed to move freely during energy minimization, docking and optimization rounds. The DAT chloride ion was initially placed at the position corresponding to E290 in the LeuT structure (in the DAT, this residue is S357—the negative charge provided by glutamate renders LeuT Cl2- insensitive) [49,50]. Introduction Binding reactions were initiated by addition of 50 mL cell suspension to buffer containing radioligand and varying concen- trations of test ligand, for a final per-well reaction volume of 200 mL. Cells were incubated with 2–4 nM [3H]CFT (85.9 Ci/ mmol) and test compounds for 15 min at 21uC. Nonspecific binding was determined using 1 mM non-radiolabeled b-CFT. Binding was terminated by vacuum filtration onto a filtermat (Wallac A) and washing with 0.9% ice-cold saline using a Tomtec automatic 96-pin cell harvester (Tomtec, Orange, CT, USA). Tritium accumulation was quantified using a Microbeta 1405 liquid scintillation counter (Perkin-Elmer, Boston, MA, USA). variety of antidepressant compounds, both tricyclics [28,38] and SSRIs like fluoxetine and sertraline [39]. Interestingly, whereas tricyclics and other inhibitors that bind at the S2 site stabilize LeuT in an occluded state, binding of the competitive inhibitor tryptophan (which binds at the S1 site, displacing leucine itself) stabilizes an open-to-out conformational state [29]. Mutagenesis and cysteine-accessibility studies suggest that cocaine and structural analogues preferentially stabilize the DAT in the open-to-out conformation [40,41]. In contrast, atypical inhibitors—compounds that potently inhibit the DAT, yet do not share cocaine’s abuse potential (such as benztropine, GBR12909 and bupropion)—stabilize a ‘‘closed-to-out’’ conformation; that is, either an occluded or inward-facing state [21,42]. Here, we present evidence that modafinil displays atypical-like binding characteristics—stabilizing the DAT in a different conformation than cocaine-like compounds. We have previously characterized two DAT mutations (W84L and D313N) that disrupt the transition between outward- and inward-facing states, increasing the likelihood that the transporter will adopt an outward-facing conformation [43]. These mutations considerably increase the affinity of cocaine-like inhibitors as measured by inhibition of [3H]CFT binding, but have negligible or opposing effects on the affinity of atypical inhibitors [42,44]. Thus, a given DAT ligand’s affinity ratio at mutant versus WT transporters can offer insight into whether the ligand preferentially interacts with the outward- or the inward-facing conformational state. We employed these mutants, as well as conformation-biasing ionic conditions [45], to investigate the binding mechanism of modafinil at the DAT. Additionally, we performed in silico induced-fit docking of the atypical inhibitors modafinil and bupropion and the cocaine-like inhibitors b-CFT and methylphenidate, in order to probe possible structural differences in DAT interaction between the two classes of compounds. Data analysis and statistics Kinetic parameters, such as the equilibrium dissociation constant of radioligand binding (KD), were determined by respective competition analysis with non-radiolabeled b-CFT, using Kell RADLIG (Biosoft, Cambridge, UK). For each tested DAT ligand, the IC50 for inhibition of [3H]CFT binding was calculated with Origin 7.5. IC50 values for the DAT ligands were converted into relative inhibition constants (Ki) using the Cheng- Prusoff equation [46]. Materials and Methods Generation of cell lines stably expressing WT and mutant DATs Generation of cell lines stably expressing WT and mutant DATs In this work, we used Human Embryonic Kidney cells (HEK293) stably expressing WT human DAT, or the human DAT mutants W84L or D313N. HEK cells were obtained from ATCC (ATCC CRL 1573) as previously described; transfected cell lines were prepared by us for studies previously reported [43,44]. Human DAT mutant plasmids were generated using site- directed mutagenesis as previously outlined [43]. Mutations were screened by PCR and restriction enzyme mapping. The cells were stably transfected with the various DAT plasmids using Lipofecta- mine (Invitrogen, Carlsbad, CA, USA) and were maintained with ,250 mM geneticin (G418). Homology modeling was performed using the MODELLER algorithm and the resultant lowest energy structure was imported into the Molecular Operating Environment (MOE) program suite (Version 2009.10; Chemical Computing Group, Montreal, CA). The Protonate3D function in MOE was used to calculate residue protonation states and assign hydrogen atom coordinates; partial charges were assigned according to the AMBER99 forcefield. In order to refine residue stereochemistry and relieve any steric clashes in the protein prior to ligand docking, the DAT model was subjected to several rounds of energy minimization, employing the AMBER99 forcefield and the generalized Born (GB/VI) implicit solvation model [51]. During the first round, protein backbone atoms were dynamic and the model was minimized until hitting a convergence gradient of 0.05 kcal mol21 A˚ 21. Subsequent mini- mization rounds focused on optimizing side chain geometry of particular residues, hence backbone atoms were tethered and a more stringent convergence value (0.001 kcal mol21 A˚ 21) was employed. Analysis of the final DAT model with PROCHECK [52] indicated that 98.9% of the residues fell within either the ‘most favored’ or ‘additionally allowed’ Ramachandran plot region (86.8% most favored); only four residues (0.9%) fell within the ‘generously allowed’ region and only one residue (Q373; 0.2%) PLoS ONE \| www.plosone.org [3H]CFT binding inhibition assays For binding assays, suspensions of intact HEK-hDAT were prepared according to the method outlined previously [42,44]. Cell slurry was incubated for 1 hr at 21uC and centrifuged; the supernatant was discarded and the subsequent pellet was washed and gently resuspended in 6 mL KRH buffer solution in preparation for assay. Modified Krebs/Ringer/HEPES (KRH) buffer containing 1 mM ascorbic acid and 0.1 mM tropolone was used. In the ‘sodium free’ binding conditions, buffer NaCl was isotonically replaced with N-methyl-D-glucamine chloride (NMDG-Cl). For zinc-modulated binding conditions, 10 mM Zn2+ was added to the assay buffer before the addition of the unlabeled test ligand and [3H]CFT. Assays were conducted in 96- well plates at 21uC, with all determinations performed in triplicate PLoS ONE \| www.plosone.org October 2011 \| Volume 6 \| Issue 10 \| e25790 3 Modafinil Is an Atypical DAT Inhibitor was deemed to be in the ‘disallowed’ region (for further discussion of model stereochemical quality, see [47]). was set at 0.01 kcal mol21 A˚ 21. Final ray-traced models depicted in figures were rendered with PyMOL 1.4 (Schro¨dinger LLC, New York, NY, USA). All MOE simulations were performed on a standard quad-core 664 computer running Windows 7. Ligand binding sites in the DAT model were identified with the Site Finder tool implemented in MOE—after manual elimination of sites lying directly on the exterior, cytoplasmic or extracellular faces of the protein, two binding pockets (approximately overlapping with the S1 and S2 sites of LeuT) were identified. Dummy atoms were placed at the centroids of alpha spheres defining these two sites to assist in ligand docking. For docking, ligand structures were imported into MOE, protonated, assigned partial charges and energy minimized (,0.001 kcal mol21 A˚ 21) using the MMFF94x forcefield with GB/VI implicit solvation. In the preliminary docking process, ligand bond length and DAT protein atoms are held constant and various ligand orientations and conformational rotomers are systematically positioned in the active site such that no steric clashes between ligand and residue side-chains occur. The top 50 non-duplicate docked poses (London dG scoring method) were output to a MOE database and manually sorted into two population clusters, representative of binding at either the central S1 site (below the R85-D476 gating interaction) or the vestibular S2 site (above the R85-D476 gate). Examples of S1-localized highly-populated ‘‘metapose’’ clusters are shown in the Supporting Information (Fig. S1). [3H]CFT binding inhibition assays An energetically favorable (top-scoring) pose from each population was chosen as a representative for ligand-adaptive geometric optimization; however, poses that did not display any strong molecular interactions (e.g. hydrogen bonds, cation-p and aromatic p-stacking interactions) with specific residues within their binding pocket were not considered. Binding and mutant affinity-shift profile of modafinil and DAT inhibitors Modafinil and other compounds—representing different chem- ical classes of DAT ligands (Fig. 2)—were assayed for their ability to inhibit [3H]CFT binding to WT or mutant DATs expressed in whole HEK293 cells. The binding affinities (Ki values) of the tested compounds and the observed WT/mutant affinity ratios are listed in Table 1. Modafinil’s binding affinity at WT transporters was relatively low (Ki = 2.1 mM); compared to the other reference ligands, modafinil was anywhere from 6- to 100-fold weaker (Table 1). The micromolar level affinity is consistent with prior literature reports of modafinil radioligand binding at the DAT and likely underlies the comparatively high effective dose of modafinil (200–600 mg) in humans [18,53]. At the W84L mutant, modafinil showed a significant decrease in affinity (an increase in Ki value to 3.8 mM; p,0.05) compared with the WT transporter, resulting in a WT/W84L Ki ratio of 0.56 (Table 1). This mutant affinity-shift was strikingly similar to that observed with the atypical ligands benztropine, GBR12909 and bupropion (for each of these ligands, the WT/W84L Ki ratio was approximately 0.5). In contrast, the classical DAT inhibitors cocaine, b-CFT and methylphenidate all showed significantly increased binding affinity (decreased Ki value) at the W84L mutant: the tropane compounds both gave 3.5-fold improvements, whereas methylphenidate displayed a more modest 2-fold gain. At the D313N mutant, modafinil showed little change in affinity compared with WT (having a WT/D313N Ki ratio of 0.95), behaving similarly to bupropion and GBR12909—which gave WT/D313N Ki ratios of 0.90 and 1.05, respectively—but not to any of the cocaine-like ligands (Table 1). Representative poses were then refined by further minimization of the protein/ligand. In refinement rounds, protein backbone atoms were weakly tethered (1 kcal mol21 A˚ 21 force constant) and the side-chain and ligand atoms completely unconstrained to allow for flexible ‘‘ligand adaptive’’ docking—for the last minimization round, the backbone tethering constant was increased to 10 kcal mol21 A˚ 21 and the convergence gradient Figure 2. Chemical structures of modafinil and other tested DAT inhibitor ligands. Atypical inhibitors (top row) exhibited preferential interaction with a more inward-facing transporter conformation, whereas cocaine-like inhibitors (bottom row) preferentially bound to the outward- facing DAT conformation. While modafinil has a chiral sulfoxide moiety, the enantiomers possess little difference in pharmacodynamic activity (hence, only the racemate was tested). doi:10.1371/journal.pone.0025790.g002 Figure 2. Chemical structures of modafinil and other tested DAT inhibitor ligands. Effects of ionic conformational manipulation on modafinil and DAT inhibitor binding demonstrated to increase the relative number of inward-facing DATs [56]. Replacement of buffer sodium resulted in a decrease of affinity (increase in Ki value) for all of the tested DAT inhibitors (compare Ki values of WT transporter in Table 1 to those of the Na+-Free condition listed in Table 2). However, amongst the inhibitors, modafinil and GBR12909 were least impacted by sodium depletion, displaying 1.4- and 1.8-fold increases in respective Ki values. Various endogenous ionic species are known affect the conformational equilibrium of the DAT and other NSS-family proteins. For example, recent biophysical studies with LeuT have demonstrated that binding of Na+ to the substrate-free (apo) form of the transporter induces a conformational shift toward the open- to-out state, increasing accessibility of the extracellular vestibule [34] and constricting residues near the intracellular gating network [35,54]. The sodium gradient present under normal physiological conditions (high extracellular Na+ concentration and low intra- cellular Na+ concentration) therefore gives rise to a population of transporters that are predominantly outward-facing, primed to bind ligands approaching from the extracellular milieu [55]. In the absence of significant sodium levels, the transporter effectively shifts between outward and inward-facing conformations [35]. Hence, changing the ionic conditions by removing extracellular sodium (without grossly altering intracellular ionic components) would be expected to increase the preponderance of a ‘‘closed-to- out’’ state amongst the overall population of transporters. Applying this logic to the DAT, we performed intact-cell binding assays with buffer Na+ isotonically substituted for the inert and membrane-impermeant cation NMDG+ (yielding a functionally 0 mM concentration of extracellular Na+ without significantly affecting intracellular ionic conditions), a treatment previously Zinc is another important endogenous modulator of the DAT; in vivo, it forms organometallic coordinations with three residues at the top of the extracellular vestibule of the transporter (H193, H375 and E396). By loosely ‘‘grasping’’ these three residues on the external protein face, zinc likely impedes the transition between outward- and inward-facing conformations, biasing the equilibri- um in favor of the outward-facing state [41]. Effects of exogenously-applied Zn2+ are observable experimentally at micromolar concentrations: Zn2+ increases the binding of b-CFT and cocaine [44,45] and can partially overcome the effects of DAT mutations exerting an inward-facing conformational bias (the opposite of the W84L or D313N mutations), such as the Y335A [41], D345N [57] and W267L [58] mutants. We thus used Zn2+ to investigate the conformational preference of modafinil and the other DAT ligands. Binding and mutant affinity-shift profile of modafinil and DAT inhibitors Data for inhibitors other than modafinil included from [42] for reference. doi:10.1371/journal.pone.0025790.t001 rmed using intact stably-transfected HEK293 cells; values are means 6 SEM for 3–6 experiments, each performed in triplicate. sus wild-type binding affinity (p,0.05; t test, two-tailed). Data for inhibitors other than modafinil included from [42] for reference. 0025790 t001 Binding and mutant affinity-shift profile of modafinil and DAT inhibitors Atypical inhibitors (top row) exhibited preferential interaction with a more inward-facing transporter conformation, whereas cocaine-like inhibitors (bottom row) preferentially bound to the outward- facing DAT conformation. While modafinil has a chiral sulfoxide moiety, the enantiomers possess little difference in pharmacodynamic activity (hence, only the racemate was tested). doi:10.1371/journal.pone.0025790.g002 Figure 2. Chemical structures of modafinil and other tested DAT inhibitor ligands. Atypical inhibitors (top row) exhibited preferential interaction with a more inward-facing transporter conformation, whereas cocaine-like inhibitors (bottom row) preferentially bound to the outward- facing DAT conformation. While modafinil has a chiral sulfoxide moiety, the enantiomers possess little difference in pharmacodynamic activity (hence, only the racemate was tested). doi:10.1371/journal.pone.0025790.g002 Figure 2. Chemical structures of modafinil and other tested DAT inhibitor ligands. Atypical inhibitors (top row) exhibited preferential interaction with a more inward-facing transporter conformation, whereas cocaine-like inhibitors (bottom row) preferentially bound to the outward- facing DAT conformation. While modafinil has a chiral sulfoxide moiety, the enantiomers possess little difference in pharmacodynamic activity (hence, only the racemate was tested). doi:10.1371/journal.pone.0025790.g002 PLoS ONE \| www.plosone.org October 2011 \| Volume 6 \| Issue 10 \| e25790 October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 4 Modafinil Is an Atypical DAT Inhibitor Table 1. Potencies of modafinil and other DAT inhibitors, assessed by displacement of intact-cell [3H]CFT binding to WT or mutant hDAT. Compound Whole-Cell hDAT Binding Ki (nM) Ki [WT]/Ki [Mutant] Ratio WT W84L D313N WT/W84L WT/D313N b-CFT 15.462.1 4.4460.69* 6.1460.29* 3.47 2.51 (2)-cocaine 163.661.20 46.764.52* 51.565.06* 3.50 3.18 (6)-methylphenidate 21.263.7 11.161.6* 11.460.38* 1.91 1.86 benztropine 75.367.4 189.566.82* 181.4630.3* 0.40 0.42 (6)-bupropion 319.5624.9 745.9614.0* 353.9616.7 0.43 0.90 GBR12909 53.2619.7 108610.7* 50.661.2 0.49 1.05 (6)-modafinil 21436215 38166266* 22556229 0.56 0.95 Binding assays were performed using intact stably-transfected HEK293 cells; values are means 6 SEM for 3–6 experiments, each performed in triplicate. Significant difference versus wild-type binding affinity (p,0.05; t test, two-tailed). Data for inhibitors other than modafinil included from [42] for reference. doi:10.1371/journal.pone.0025790.t001 Table 1. Potencies of modafinil and other DAT inhibitors, assessed by displacement of intact-cell [3H]CFT binding to WT or mutant hDAT. Table 1. Potencies of modafinil and other DAT inhibitors, assessed by displacement of intact-cell [3H]CFT binding to WT or mutant hDAT. Binding assays were performed using intact stably-transfected HEK293 cells; values are means 6 SEM for 3–6 experiments, each performed in triplicate. Significant difference versus wild-type binding affinity (p,0.05; t test, two-tailed). Adaptive docking of modafinil and other inhibitors in an hDAT model ( g ) The cocaine-like inhibitors b-CFT and d-methylphenidate also yielded highly populated pose clusters when docked in the S1 and S2 sites (a representative pose cluster for CFT docked at the S1 site is shown in Fig. S1B). At the S1 site, the tropane amine of CFT engaged in hydrogen bonding with D79, with the N-methyl group oriented downward towards F76 and neighboring residues in TMs 1 and 6 (Fig. 5A). The tropane ethylene bridge was directed upward toward the extracellular gate, likely blocking the aromatic side chain of F320 from establishing an interaction with the cationic nitrogen. In addition, the 3b-fluorophenyl ring of CFT participated in p-p stacking aromatic interaction with the side- chain of F326 and the 2b-carbomethoxy moiety formed a hydrogen bond with S422 of TM8 (Figs. 5A and 6A). Many of the interactions and binding pocket residues found for CFT were consonant with those reported in prior molecular simulations of phenyltropane binding at the S1 site (e.g. [31]). In the S2 site, CFT was oriented perpendicular to the plane of the membrane, with the charged tropane amine directed towards the top of the extracellular vestibule (Fig. 5B). Residues from extracellular loop 4 (D385, G386 and P387) helped to shield CFT from the extracellular space, with the backbone of D385 forming a hydrogen bond with the tropane nitrogen (Fig. 6B). The 2b- carbomethyoxy moiety was situated directly adjacent to the side- chains of R85, F155 and D476, but did not disrupt the interaction between R85 and D476. In contrast to the other DAT inhibitors docked in the S2 site, the aromatic portion of CFT dipped below the R85-D476 extracellular gate (Fig. 5B), enabling a p-p stacking interaction between the S1-localized residue Y156 and the 3b- fluorophenyl substituent (Fig. 6B). This binding orientation is relatively consistent with other computational studies modeling cocaine and phenyltropane binding in the extracellular vestibule (S2 site) of the dopamine and noradrenaline transporters in the presence of respective substrates bound at S1 [67,68]. In an attempt to gain structural insight into the differential interactions of cocaine and modafinil with the DAT, we employed a homology model of the human DAT and docked (R)-modafinil, as well as (S)-bupropion, (d)-methylphenidate and b-CFT with a flexible ligand-adaptive docking procedure. Specific enantiomers of the various DAT inhibitors were used in order to simplify the docking protocol. Effects of ionic conformational manipulation on modafinil and DAT inhibitor binding By increasing the population of outward- facing DATs and (at least partially) reversing the effect of extracellular Na+ depletion, zinc can highlight compounds that Table 2. [3H]CFT binding potency of modafinil and other DAT ligands in the absence of extracellular Na+ and the effect of Zn2+ on binding affinity. Compound Whole-Cell WT hDAT Binding Ki (nM) Zn2+ Effect Ratio Na+-Free (0 mM) Buffer Na+-Free+10 mM Zn2+ Ki [0 mM]/Ki [10 mM] (2)-cocaine 415.3641.1 229.2627.7* 1.81 (6)-methylphenidate 252.9624.1 98.9268.94* 2.56 benztropine 231.1617.0 209.9618.1 1.10 (6)-bupropion 709.3665.3 737.1655.4 0.96 GBR12909 95.9968.74 126.8615.6 0.76 (6)-modafinil 29636161 34706261 0.85 Assays were performed in Na+-free conditions (buffer sodium was isotonically replaced with the impermeant cation NMDG+) in the presence and absence of 10 micromolar zinc; values are means 6 SEM for 3–7 experiments, each performed in triplicate. Significant difference versus Na+-Free affinity value (p,0.05; t test, two-tailed). doi:10.1371/journal.pone.0025790.t002 Table 2. [3H]CFT binding potency of modafinil and other DAT ligands in the absence of extracellular Na+ and the effect of Zn2+ on binding affinity. otency of modafinil and other DAT ligands in the absence of extracellular Na+ and the effect of Zn2+ on Assays were performed in Na+-free conditions (buffer sodium was isotonically replaced with the impermeant cation NMDG+) in the presence and absence of 10 micromolar zinc; values are means 6 SEM for 3–7 experiments, each performed in triplicate. Significant difference versus Na+-Free affinity value (p,0.05; t test, two-tailed). doi:10.1371/journal.pone.0025790.t002 October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 5 Modafinil Is an Atypical DAT Inhibitor selectively bind to an outward-facing state. Under sodium-free buffer conditions, the addition of 10 mM Zn2+ significantly increased the binding affinity (decreased the Ki value) of cocaine and methylphenidate at WT transporters (Table 2). For inhibition of [3H]CFT binding by cold b-CFT, the presence of Zn2+ under sodium-free conditions increased the Bmax value of labeled [3H]CFT by a factor of four, from 125615.8 fmole/well to 502678 fmole/well. The calculated absolute Kd values for the sodium-free and +10 mM Zn2+ conditions were not significantly different: 49.3269.69 and 57.0866.67, respectively. This zinc- mediated effect—alteration in the Bmax, but not the Kd kinetic parameter—has been demonstrated before in both Na+-free [58] and physionormal Na+ (130 mM) buffers [45,59]. It is likely that the particular kinetic effects of micromolar Zn2+-levels depend on the specific assay protocol and nonlinear curve-fitting algorithm used. Effects of ionic conformational manipulation on modafinil and DAT inhibitor binding Addition of Zn2+, however, had little impact on the atypical DAT inhibitors overall (the ratio of Ki values obtained in the absence and presence of zinc was close to unity for each compound; Table 2). This finding suggests that unlike b-CFT, cocaine or methylphenidate, the interaction of modafinil (like GBR12909, benztropine and bupropion) with the DAT is far less dependent on the transporter assuming an open-to-out conforma- tional state. examples of pose clusters from which potential candidates were selected). Following docking at the S1 site, modafinil was oriented horizontally (parallel to the plane of the membrane), with the diphenyl ring system facing V152, G153 and Y156 of TM3 and the sulfinylacetamide chain surrounded by F76, A77, D79 of TM1 and F320, S321 and L322 of TM6 (Fig. 3A). In this pose, few strong molecular interactions between modafinil and the DAT were observed, save for hydrogen bonds formed between modafinil’s terminal amide nitrogen and residues F76, A77 and D79 (Fig. 4A). At the S2 site, modafinil was positioned just above the extracellular vestibule gating residues R85, F320 and D476 (Fig. 3B); one phenyl ring formed a cation-p interaction with R85 and the protonated amide displayed a combination of hydrogen bonding with D476 and a cation-p interaction with the aromatic side chain of F320 (Fig. 4B). Bupropion docked at a slightly lower position in S1 (Fig. 3C), but like modafinil, the aromatic portion of the molecule was oriented parallel to V152 and enveloped by residues of TM3, whereas the amine nitrogen and bulky tert-butyl group were oriented towards D79, F320 and other adjacent residues of TMs 1 and 6 (Fig. 4C). In the S2 site, while bupropion was positioned marginally higher than modafinil in the extracel- lular vestibule (Fig. 3D), its strongest molecular interactions—a cation-p interaction with R85 and a hydrogen bond between the amine and D476—were similar (Fig. 4D). Adaptive docking of modafinil and other inhibitors in an hDAT model Selected binding pocket residues are labeled and rendered as sticks; bound ligand molecules (also shown as sticks) are highlighted using gray- colored carbon atoms. The distance between the carboxylate oxygen atom of D79 and the ring hydroxyl moiety of Y156 is displayed in the lower right of each panel (in yellow). (A, B) (R)-modafinil docked at the S1 and S2 sites, respectively—at the S1 site (A), modafinil primarily interacts with D79 and adjacent TM1 residues, whereas at the S2 site (B), it mainly interacts with residues that form the extracellular gating network. (C, D) (S)- bupropion docked at both the S1 (C) and S2 sites (D). Note that for each of the DAT/inhibitor models, the bound inhibitor molecule does not disrupt the D79-Y156 hydrogen bond (i.e. the interatomic distance remains less than 3.5 A˚ following adaptive docking procedures). doi:10.1371/journal.pone.0025790.g003 Figure 3. Final energy-minimized poses of atypical inhibitors docked at the DAT primary (S1) and vestibular (S2) substrate binding sites. Selected binding pocket residues are labeled and rendered as sticks; bound ligand molecules (also shown as sticks) are highlighted using gray- colored carbon atoms. The distance between the carboxylate oxygen atom of D79 and the ring hydroxyl moiety of Y156 is displayed in the lower right of each panel (in yellow). (A, B) (R)-modafinil docked at the S1 and S2 sites, respectively—at the S1 site (A), modafinil primarily interacts with D79 and adjacent TM1 residues, whereas at the S2 site (B), it mainly interacts with residues that form the extracellular gating network. (C, D) (S)- bupropion docked at both the S1 (C) and S2 sites (D). Note that for each of the DAT/inhibitor models, the bound inhibitor molecule does not disrupt the D79-Y156 hydrogen bond (i.e. the interatomic distance remains less than 3.5 A˚ following adaptive docking procedures). doi:10.1371/journal.pone.0025790.g003 greatest difference in the binding models of the two inhibitors involved F320: for methylphenidate, the charged piperidine amine group formed both a cation-p interaction with the aromatic side- chain of F320 and a hydrogen bond with the backbone. However, at the S2 site, methylphenidate exhibited an interaction pattern and binding orientation more akin to that of modafinil—forming a cation-p interaction between the ligand aromatic ring and R85, with the protonated ligand amine anchored by a combination of hydrogen bonding with D476 and a cation-p interaction with the aromatic side chain of F320 (Figs. 5D and 6D). Adaptive docking of modafinil and other inhibitors in an hDAT model The (S)-enantiomer of bupropion was selected based upon the stereoselective dopaminergic activity of its primary metabolite (S,S)-hydroxybupropion [60] and the comparatively greater isomeric potency of other (S)-cathinones [61,62]. Dex- methylphenidate (the threo-(R,R)-isomer of methylphenidate) has been extensively shown to be wholly responsible for the DAT- mediated physiological effects of the racemate [63,64] and was therefore selected for modeling. The stereochemistry of modafinil differs from other DAT ligands, as modafinil’s stereocenter is not the typical asymmetric carbon atom, but a sulfinyl moiety (Fig. 2). Unlike other DAT ligands, which generally possess significant enantioselectivity, (R)- and (S)-modafinil show only mild differences in DAT affinity, with the (R)-enantiomer having marginally greater affinity [65]. In humans, racemic modafinil and (R)- modafinil are active at similar doses, but the (R)-isomer has a more stable pharmacokinetic profile [66] and was recently released to the market as an enantiopure drug (armodafinil); hence, it was selected as the more ‘‘active’’ isomer for docking. b-CFT was chosen over cocaine for its structural rigidity, as flexibility imparted by cocaine’s benzoyloxy moiety prevented the docking procedure from converging upon particularly consistent pose clusters. The hDAT model was based upon the structure of LeuT co-crystallized with its substrate leucine, as well as the tricyclic antidepressant desipramine [28]. We previously employed this DAT model in docking of substrates and bivalent substrate-like inhibitors [47]. Two ligand-binding pockets identified in the hDAT model were used for docking—roughly corresponding with the S1 and S2 sites of LeuT—and each inhibitor was docked in both sites. A single candidate was selected from a cluster of top- scoring poses and used as the initial input for further energy minimization of the protein/ligand complex (see Fig. S1 for Despite adopting a slightly different orientation, we found that d-methylphenidate shared many of the same interactions and binding pocket residues with b-CFT when docked at the S1 site (Fig. 5C). In particular, the methyl ester moiety of methylpheni- date engaged in hydrogen bonding with the side-chain of S422 and the cationic amine formed a bond with D79 (Fig. 6C). The PLoS October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 6 Modafinil Is an Atypical DAT Inhibitor Figure 3. Final energy-minimized poses of atypical inhibitors docked at the DAT primary (S1) and vestibular (S2) substrate binding sites. PLoS ONE \| www.plosone.org Discussion a preserved hydrogen bond and a closed extracellular gating network. Similarly, the atypical inhibitor bupropion gave respec- tive interatomic distances of 2.34 A˚ and 2.37 A˚ when docked at the S1 and S2 sites (Fig. 3C–D). In accordance with the findings of Beuming et al. (2008), docking of b-CFT at the S1 site resulted in a D79-Y156 distance of 4.85 A˚ , indicative of an open extracellular gate (Fig. 5A). Interestingly, at the S2 site, extension of CFT’s 3b- fluorophenyl moiety downward into the S1 site permitted an aromatic stacking interaction with Y156, pushing the tyrosine ring aside and expanding the D79-Y156 distance to 4.94 A˚ (Fig. 5B). In addition, the classical inhibitor d-methylphenidate also disrupted the D79-Y156 hydrogen bond, yielding S1- and S2-bound distances of 4.12 A˚ and 3.57 A˚ , respectively (Fig. 5C–D). This suggests that cocaine-like phenyltropane inhibitors and methyl- phenidate are capable of inducing an open-to-out transporter conformation upon binding at either the S2 or S1 site. a preserved hydrogen bond and a closed extracellular gating network. Similarly, the atypical inhibitor bupropion gave respec- tive interatomic distances of 2.34 A˚ and 2.37 A˚ when docked at the S1 and S2 sites (Fig. 3C–D). In accordance with the findings of Beuming et al. (2008), docking of b-CFT at the S1 site resulted in a D79-Y156 distance of 4.85 A˚ , indicative of an open extracellular gate (Fig. 5A). Interestingly, at the S2 site, extension of CFT’s 3b- fluorophenyl moiety downward into the S1 site permitted an aromatic stacking interaction with Y156, pushing the tyrosine ring aside and expanding the D79-Y156 distance to 4.94 A˚ (Fig. 5B). In addition, the classical inhibitor d-methylphenidate also disrupted the D79-Y156 hydrogen bond, yielding S1- and S2-bound distances of 4.12 A˚ and 3.57 A˚ , respectively (Fig. 5C–D). This suggests that cocaine-like phenyltropane inhibitors and methyl- phenidate are capable of inducing an open-to-out transporter conformation upon binding at either the S2 or S1 site. The stimulant and nootropic compound modafinil was initially assumed not to possess a dopaminergic mechanism of action, due to its structural dissimilarity to other DAT ligands and its relatively low micromolar-level affinity for the DAT [71]. However, recent broad-spectrum receptor screening assays have identified the DAT as the only protein target displaying significant (,10 mM) affinity for modafinil (although Madras et al. Adaptive docking of modafinil and other inhibitors in an hDAT model moiety of Y156 helps to close the extracellular gate, protecting the S1-bound substrate from infiltration by water from the extracel- lular space [31]. Hence, the presence of a D79-Y156 hydrogen bond is associated with a ‘‘closed-to-out’’ transporter state. In their molecular dynamics simulations, Beuming et al. (2008) showed that an interatomic distance of less than 3.5 A˚ (indicative of an intact hydrogen bond) was maintained between the oxygen atoms of D79 and Y156 during binding of DAT substrates (dopamine, amphetamine and MDMA) in the S1 site. In contrast, binding of the classical inhibitors b-CFT and cocaine yielded D79-Y156 distances greater than the 3.5 A˚ maximum for hydrogen bonding (<5.5 A˚ and <7.5 A˚ , respectively), signifying an open vestibular gate in each case. Binding of the atypical inhibitor benztropine, however, resulted in a preserved D79-Y156 hydrogen bond (i.e. an interatomic distance less than 3.5 A˚ ), suggesting that—unlike cocaine—binding of benztropine at the S1 site does not prevent closure of the gate. Our in silico modeling data are also consistent with the idea that modafinil interacts with the DAT in a different manner than cocaine-like inhibitors. In a recent study combining molecular simulation and site-directed mutagenesis, Beuming et al. (2008) showed that the presence or absence of a hydrogen bond between D79 and Y156 in a given DAT/ligand complex can provide an indication of the conformational bias engendered by the ligand [31]. The highly conserved TM3 tyrosine residue Y156 interacts with the substrate dopamine as it binds at the S1 site and also participates in the vestibular gating network—consisting of R85, F320 and D476—that partitions the S1 and S2 sites [69,70]. When dopamine is bound at the S1 site, a hydrogen bond formed between the side chain oxygen atoms of D79 and the hydroxyl In an effort to expand upon this finding, we measured the terminal D79-Y156 distance for each of the modeled DAT inhibitors when bound at either the S1 or the S2 site (Figs. 3 and 4, distance values are indicated in yellow at the bottom of each panel). Modafinil docked at the S1 and S2 sites yielded respective D79-Y156 distances of 2.29 A˚ and 2.25 A˚ (Fig. 3A–B), suggesting PLoS ONE \| www.plosone.org 7 October 2011 \| Volume 6 \| Issue 10 \| e25790 Modafinil Is an Atypical DAT Inhibitor Figure 4. Molecular interaction diagrams of docked atypical inhibitors. Adaptive docking of modafinil and other inhibitors in an hDAT model For each panel, the interaction map depicts DAT residues located within 4.5 A˚ of the bound inhibitor molecule (hydrophobic residues are colored green and polar residues are purple). The most significant (non van der Waals) DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond (green), main-chain hydrogen bond (blue), cation-p bond ({+) or aromatic p-stacking ({{). (A, B) Residue interaction maps for modafinil bound at the S1 (A) and S2 sites (B). (C, D) Interaction maps for bupropion bound at the S1 (C) and S2 sites (D), respectively. For both of the atypical inhibitors, binding at the S1 site (panels A and C) gives rise to few strong interactions with the DAT—only their protonated nitrogen atoms form hydrogen bonds—suggesting that recognition of these relatively modest inhibitors (Ki.100 nM) is influenced more by molecular shape and steric bulk than by specific polar interactions. doi:10.1371/journal.pone.0025790.g004 Figure 4. Molecular interaction diagrams of docked atypical inhibitors. For each panel, the interaction map depicts DAT residues located within 4.5 A˚ of the bound inhibitor molecule (hydrophobic residues are colored green and polar residues are purple). The most significant (non van der Waals) DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond (green), main-chain hydrogen bond (blue), cation-p bond ({+) or aromatic p-stacking ({{). (A, B) Residue interaction maps for modafinil bound at the S1 (A) and S2 sites (B). (C, D) Interaction maps for bupropion bound at the S1 (C) and S2 sites (D), respectively. For both of the atypical inhibitors, binding at the S1 site (panels A and C) gives rise to few strong interactions with the DAT—only their protonated nitrogen atoms form hydrogen bonds—suggesting that recognition of these relatively modest inhibitors (Ki.100 nM) is influenced more by molecular shape and steric bulk than by specific polar interactions. doi:10.1371/journal.pone.0025790.g004 Discussion doi:10.1371/journal.pone.0025790.g005 proclaim that modafinil may have significant abuse liability, akin to that of traditional cocaine-like DAT inhibitors (e.g. [72]). In addition, while certain behavioral studies in animals have shown that modafinil is not self-administered via the IV route and does not induce place preference [73,74], others have found that high doses of modafinil fully substitute for cocaine in drug discrimina- tion tests [75,76] and that modafinil occasions conditioned place preference and cocaine-like locomotor sensitization in mice [77]. Clinical and preclinical studies, however, suggest that modafinil neither elicits stimulant-like subjective effects nor encourages self- administration in frequent cocaine users [16], unlike the classical dopamine uptake inhibitor methylphenidate [78]. And while it is widely accepted that interaction with the DAT underlies cocaine’s strong addictive potential, extensive research has shown that a number of atypical DAT inhibitors—such as benztropine, GBR12909 and bupropion—have limited reinforcing effects in humans [19,79,80], despite fully substituting for cocaine in animal drug discrimination protocols (e.g. [76,81]). Moreover, numerous animal studies have shown that exceptionally potent and selective DAT inhibitors derived from benztropine or GBR12909 incom- pletely substitute for cocaine in drug discrimination tests and also decrease cocaine self-administration [82–85]. As benztropine is also a potent antagonist at muscarinic M1 and histamine H1 receptors, some have argued that activity at these targets (as opposed to the DAT itself) underlies benztropine’s low addictive liability. However, antihistaminergic and antimuscarinic com- pounds do not attenuate the reinforcing effects of cocaine [86,87]. Additionally, benztropine analogues with lower affinity for the M1 muscarinic receptor than benztropine itself do not exhibit cocaine- like effects [87], making it unlikely that these non-DAT side effects are responsible for the behavioral profile of benztropine and its derivatives. It has also been argued that a slow onset of action (compared to cocaine) is responsible for the non-classical behavioral effects of various benztropine-derived atypical DAT ligands [88,89]. However, a recent study by Li et al. (2011) found that a number of N-substituted benztropine analogues possessing rapid onset rates did not induce cocaine-like place preference, suggesting that a slow onset rate is not required for atypical-like behavioral effects [20]. proclaim that modafinil may have significant abuse liability, akin to that of traditional cocaine-like DAT inhibitors (e.g. [72]). PLoS ONE \| www.plosone.org October 2011 \| Volume 6 \| Issue 10 \| e25790 Discussion (2006) showed that modafinil also inhibits noradrenaline uptake by the NET, albeit with an IC50 value of <36 mM) [53]. This is consistent with our finding that modafinil inhibits [3H]CFT binding to human DAT with relatively low affinity (Ki = 2.1 mM). Despite its modest affinity, recent findings that modafinil occupies brain DATs in humans at clinically-relevant doses—and, like any DAT inhibitor, causes an increase in extraneuronal dopamine—have prompted some to PLoS ONE \| www.plosone.o October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 8 Modafinil Is an Atypical DAT Inhibitor Figure 5. Final energy-minimized poses of cocaine-like inhibitors docked at the DAT S1 and S2 sites. Selected binding pocket residues are labeled and rendered as sticks; bound ligand molecules are highlighted using gray-colored carbon atoms. The distances between the oxygen atoms of D79 and Y156 are displayed in the lower right of each panel (in yellow). (A, B) b-CFT docked at the S1 (A) and S2 sites (B); binding of b-CFT at either site disrupts the hydrogen bond between and D79 and Y156 (interatomic distance .3.5 A˚), indicating that it promotes an open-to-out conformational state. (C, D) Dexmethylphenidate docked at the respective S1 (C) and S2 sites (D)—similar to CFT, methylphenidate disrupts the D79- Y156 hydrogen bond upon binding at the S1 site (however, at the S2 site, the D79-Y156 interatomic distance is roughly <3.6 A˚, hence the effect of methylphenidate on the integrity of the hydrogen bond is less conclusive). doi:10.1371/journal.pone.0025790.g005 Figure 5. Final energy-minimized poses of cocaine-like inhibitors docked at the DAT S1 and S2 sites. Selected binding pocket residues are labeled and rendered as sticks; bound ligand molecules are highlighted using gray-colored carbon atoms. The distances between the oxygen atoms of D79 and Y156 are displayed in the lower right of each panel (in yellow). (A, B) b-CFT docked at the S1 (A) and S2 sites (B); binding of b-CFT at either site disrupts the hydrogen bond between and D79 and Y156 (interatomic distance .3.5 A˚), indicating that it promotes an open-to-out conformational state. (C, D) Dexmethylphenidate docked at the respective S1 (C) and S2 sites (D)—similar to CFT, methylphenidate disrupts the D79- Y156 hydrogen bond upon binding at the S1 site (however, at the S2 site, the D79-Y156 interatomic distance is roughly <3.6 A˚, hence the effect of methylphenidate on the integrity of the hydrogen bond is less conclusive). Discussion At the S2 site, the interaction pattern of methylphenidate is similar to that of modafinil (compare Figure 6D with Figure 4B). doi:10.1371/journal.pone.0025790.g006 Figure 6. Molecular interaction diagrams of cocaine-like inhibitors docked at the S1 and S2 sites. For each panel, the interaction map depicts DAT residues located within 4.5 A˚ of the bound inhibitor. As described for Figure 4, the residues are colored based upon their chemical nature and the most significant DAT/inhibitor interactions are labeled with dotted lines and a symbol depicting the chemistry of the interaction formed. (A, B) Residue interaction maps for b-CFT bound at the S1 (A) and S2 sites (B). (C, D) Interaction maps for dexmethylphenidate bound at the S1 (C) and S2 sites (D), respectively. At the S2 site, the interaction pattern of methylphenidate is similar to that of modafinil (compare Figure 6D with Figure 4B). doi:10.1371/journal.pone.0025790.g006 g doi:10.1371/journal.pone.0025790.g006 doi:10.1371/journal.pone.0025790.g006 conformational states. Experimentally, this idea is supported by the finding that cocaine and benztropine differentially affect the vulnerability of extracellular-facing DAT cysteine residues towards reaction with impermeant sulfhydryl reducing reagents, indicating that these inhibitors stabilize different conformations [40]. In addition, binding of cocaine-like compounds has been shown to protect DAT transmembrane arginine residues from covalent reaction with phenylglyoxal, whereas benztropine-like compounds failed to affect phenylglyoxal reactivity, further hinting at specific conformational effects that vary depending upon the structure of the bound inhibitor [90]. In prior site-directed mutagenesis studies, we identified two DAT mutants (W84L and D313N) that bias the conformational equilibrium of the transporter towards the open-to-out (outward-facing) state [43]. By impeding the transition from open-to-out to occluded and inward-facing conformations, the W84L and D313N mutants enhance the binding affinity of cocaine-like DAT ligands, which bind to and stabilize the outward-facing state. However, the mutations display either unchanged or decreased affinity for atypical inhibitors—as well as DAT substrates (such as dextroamphetamine) and certain bivalent substrate-like ligands (see [47])—allowing them to be used as tools to determine whether or not a particular ligand possesses a cocaine-like mechanism of action. In a previous structure-activity relationship (SAR) investigation of a variety of structurally unique DAT inhibitors, we used these two transporter mutants to show that the presence of a diphenylmethoxy moiety was sufficient (but not necessary) to engender a given DAT inhibitor molecule with an atypical binding profile [42]. Discussion In addition, while certain behavioral studies in animals have shown that modafinil is not self-administered via the IV route and does not induce place preference [73,74], others have found that high doses of modafinil fully substitute for cocaine in drug discrimina- tion tests [75,76] and that modafinil occasions conditioned place preference and cocaine-like locomotor sensitization in mice [77]. p [ ] Clinical and preclinical studies, however, suggest that modafinil neither elicits stimulant-like subjective effects nor encourages self- administration in frequent cocaine users [16], unlike the classical dopamine uptake inhibitor methylphenidate [78]. And while it is widely accepted that interaction with the DAT underlies cocaine’s strong addictive potential, extensive research has shown that a number of atypical DAT inhibitors—such as benztropine, GBR12909 and bupropion—have limited reinforcing effects in humans [19,79,80], despite fully substituting for cocaine in animal drug discrimination protocols (e.g. [76,81]). Moreover, numerous animal studies have shown that exceptionally potent and selective DAT inhibitors derived from benztropine or GBR12909 incom- pletely substitute for cocaine in drug discrimination tests and also decrease cocaine self-administration [82–85]. As benztropine is also a potent antagonist at muscarinic M1 and histamine H1 receptors, some have argued that activity at these targets (as Hence, it appears that addictiveness is not a property shared by all DAT-inhibiting compounds, but instead may be contingent upon a specific sort of molecular interaction with the DAT protein. In this study, we compared the nature of modafinil’s molecular interaction with the dopamine transporter to that of characterized cocaine-like and atypical uptake inhibitors, employ- ing a combination of biochemical and computational techniques. There is ample evidence that different classes of DAT inhibitors preferentially bind to (or induce upon binding) distinct transporter PLoS ONE \| www.plosone.org 9 October 2011 \| Volume 6 \| Issue 10 \| e25790 Modafinil Is an Atypical DAT Inhibitor Figure 6. Molecular interaction diagrams of cocaine-like inhibitors docked at the S1 and S2 sites. For each panel, the interaction map depicts DAT residues located within 4.5 A˚ of the bound inhibitor. As described for Figure 4, the residues are colored based upon their chemical nature and the most significant DAT/inhibitor interactions are labeled with dotted lines and a symbol depicting the chemistry of the interaction formed. (A, B) Residue interaction maps for b-CFT bound at the S1 (A) and S2 sites (B). (C, D) Interaction maps for dexmethylphenidate bound at the S1 (C) and S2 sites (D), respectively. Discussion Binding of the benztropine analogue JHW007, a potent DAT inhibitor that elicits neither self- administration nor place preference in behavioral reinforcement tests, has also been found to be largely insensitive to extracellular sodium levels [80]. Under these sodium-depleted conditions, ‘‘rescue’’ of the outward-facing transporter state by addition of 10 mM Zn2+—which interacts with the DAT above the vestibular S2 site and promotes conformational reorientation from inward- to outward-facing states—dramatically increased the binding of cocaine, b-CFT and methylphenidate, but had no effect on binding of modafinil or the other atypicals (benztropine, bupropion and GBR12909). exact binding location of uptake inhibitors in NSS proteins has been intensely debated, particularly following the discovery of tricyclic binding at the S2 site in the bacterial NSS family member LeuT. Our docking models, however, suggest that cocaine-like and atypical inhibitors can exert differential conformational effects in the transporter protein upon binding at either site. Interestingly, the D79-Y156 hydrogen bond is also preserved in models of DAT substrate binding [31,47]. This raises the possibility that, despite not being translocated across the membrane, atypical inhibitors like modafinil interact with the DAT in substrate-like manner. It has been recently proposed that stabilization of an occluded or inward-facing conformational state, similar to that induced (transiently) during substrate translocation, underlies the ‘co- caine-antagonist’ properties of benztropine and other atypical inhibitors [84]. The rationale being that having a significant percentage of DATs stabilized in a substrate-like closed confor- mation will prevent cocaine from interacting with the transporter. This idea is in fact consistent with the preclinical literature, which suggests that substrates (such as dextroamphetamine) and atypical DAT inhibitors (such as modafinil and the benztropines) are more effective as treatments for cocaine addiction than methylpheni- date, which preferentially interacts with the same transporter conformation as cocaine [91]. In order to provide a structural context for the binding and mutagenesis results, we also performed computational studies of inhibitor interaction with a DAT molecular model. Docking models of b-CFT and dexmethylphenidate demonstrated that these inhibitors promote an outward-facing conformation by breaking a critical D79-Y156 hydrogen bond. By breaking this interaction, cocaine-like inhibitors appear to impede closure of the extracellular gating network and therefore prevent the transporter from transitioning from the open-to-out state to the occluded state. Discussion This particular functional group is a structural feature common to benztropine, GBR12909 and their respective 3a-diarylmethoxytropane and 1,4-dialkylpiperazine derivatives investigated as therapeutics for cocaine addiction [19]. The fact that modafinil possesses a similar diphenylmethyl structural moiety—albeit with a sulfinyl functionality in place of the diphenylmethoxy ether oxygen atom—was a motivation for investigating its potential conformation-specific interaction with the DAT. conformational states. Experimentally, this idea is supported by the finding that cocaine and benztropine differentially affect the vulnerability of extracellular-facing DAT cysteine residues towards reaction with impermeant sulfhydryl reducing reagents, indicating that these inhibitors stabilize different conformations [40]. In addition, binding of cocaine-like compounds has been shown to protect DAT transmembrane arginine residues from covalent reaction with phenylglyoxal, whereas benztropine-like compounds failed to affect phenylglyoxal reactivity, further hinting at specific conformational effects that vary depending upon the structure of the bound inhibitor [90]. In prior site-directed mutagenesis studies, we identified two DAT mutants (W84L and D313N) that bias the conformational equilibrium of the transporter towards the open-to-out (outward-facing) state [43]. By impeding the transition from open-to-out to occluded and inward-facing conformations, the W84L and D313N mutants enhance the binding affinity of cocaine-like DAT ligands, which bind to and stabilize the outward-facing state. However, the mutations display either unchanged or decreased affinity for atypical inhibitors—as well as DAT substrates (such as dextroamphetamine) and certain bivalent substrate-like ligands (see [47])—allowing them to be used The data obtained with our outward-biasing DAT mutants are consistent with the idea that modafinil exhibits an interaction mode akin to that of the diphenylmethoxy-based inhibitors benztropine and GBR12909, but different than that of cocaine and methylphenidate. That is, like other atypical DAT inhibitors, modafinil preferentially interacts with a ‘‘closed-to-out’’ transport- PLoS ONE \| www.plosone.org October 2011 \| Volume 6 \| Issue 10 \| e25790 October 2011 \| Volume 6 \| Issue 10 \| e25790 PLoS ONE \| www.plosone.org 10 Modafinil Is an Atypical DAT Inhibitor er conformation. This conclusion is further supported by the binding assays we performed under conformation-biasing ionic conditions, as well as our computational modeling data. Amongst the DAT inhibitors tested, the binding affinity of modafinil was the least impacted by replacement of extracellular sodium with the inert cation NMDG, a treatment known to shift the dynamic equilibrium of the transporter from a predominately open-to-out state to a more inward-facing one. Supporting Information Figure S1 Representative clusters of docking poses (‘‘metaposes’’) showing potential ligand binding geom- etries. 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PLoS ONE \| www.plosone.org October 2011 \| Volume 6 \| Issue 10 \| e25790 12 October 2011 \| Volume 6 \| Issue 10 \| e25790 October 2011 \| Volume 6 \| Issue 10 \| e25790 October 2011 \| Volume 6 \| Issue 10 \| e25790 Modafinil Is an Atypical DAT Inhibitor Desai RI, Kopajtic TA, French D, Newman AH, Katz JL (2005) Relationship between in vivo occupancy at the dopamine transporter and behavioral effects of cocaine, GBR 12909 [1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpro- pyl)piperazine], and benztropine analogs. J Pharmacol Exp Ther 315: 397–404. 81. Tella SR, Ladenheim B, Cadet JL (1997) Differential regulation of dopamine transporter after chronic self-administration of bupropion and nomifensine. J Pharmacol Exp Ther 281: 508–513. 90. Volz TJ, Kim M, Schenk JO (2004) Covalent and noncovalent chemical modifications of arginine residues decrease dopamine transporter activity. Synapse 52: 272–282. 82. 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https://openalex.org/W2118246348	https://europepmc.org/articles/pmc2945326?pdf=render	English	null	Left ventricular noncompaction and myocardial fibrosis: a case report	International archives of medicine	2,010	cc-by	3,094	© 2010 Kalavakunta et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Pertinent positives in her review of systems included decreased appetite, paroxysmal nocturnal dyspnea, orthop- nea, lower extremity swelling and intermittent chest pain. The patient denied fever, chills, or cough. Her past medi- cal history was significant for type 2 diabetes, asthma, and osteoarthritis. Medications included theophylline, predni- sone, furosemide (Lasix), fluticasone & salmeterol (Advair) and albuterol. She quit smoking 20 years ago and denied alcohol or intravenous drug abuse. Family history was negative for coronary artery disease at an early age. The physical examination was significant for tachycardia, raised jugular venous pressure, lower extremity edema and expiratory wheezes upon chest examination. Noncompaction of the ventricular myocardium, also called left ventricular noncompaction (LVNC), is a rare congenital abnormality seen in only 0.05% of adults [1]. It is characterized by spongy myocardium and results from arrest of the compaction of the loosely interwoven meshwork of myocardial fibers during endomyocardial morphogenesis between 5-8 weeks of fetal life. With the advent of new diagnostic imaging techniques, more cases of LVNC are being detected. Early diagnosis is crucial due to associated high morbidity and mortality. Abstract Background: Left ventricular noncompaction (LVNC) is a rare congenital abnormality. It is currently classified as a genetic cardiomyopathy and results from early arrest of endomyocardial morphogenesis. The pathophysiology of left ventricular dysfunction, which becomes apparent beyond the 4th decade of life, is unclear. Case report: We report a case of 60-year-old woman who presented with shortness of breath and showed noncompacted endocardium on echocardiography. Cardiac catheterization and viral studies were unremarkable. Histology revealed endomyocardial fibrosis without disarray. She was subsequently diagnosed with LVNC and treated with medications. Discussion: Cardiologists and other physicians should be aware of LVNC due to its high likelihood of misdiagnosis and associated high complication rates. Early diagnosis, intervention and screening among family members can decrease the morbidity and mortality associated with LVNC. Jagadeesh K Kalavakunta1, Hemasri Tokala1, Aparna Gosavi2, Vishal Gupta1* Jagadeesh K Kalavakunta1, Hemasri Tokala1, Aparna Gosavi2, Vishal Gupta1* Kalavakunta et al. International Archives of Medicine 2010, 3:20 http://www.intarchmed.com/content/3/1/20 Kalavakunta et al. International Archives of Medicine 2010, 3:20 http://www.intarchmed.com/content/3/1/20 Open Access * Correspondence: vishalgupta@borgess.com 1Department of Internal Medicine: Division of Cardiology, Michigan State University/Kalamazoo Center for Medical Studies/Borgess Medical Center, Kalamazoo, MI, USA Full list of author information is available at the end of the article Case Report Laboratory tests revealed elevated brain natriuretic peptide at 1020 pg/ml (normal <100 pg/ml), and nega- tive cardiac enzymes with troponin levels consistently below 0.01 ng/ml (normal 0.00-0.03 ng/ml). Electrocar- diogram revealed sinus tachycardia, left atrial enlarge- ment, poor R wave progression and nonspecific ST-T wave changes in all leads specifically T wave inversion in the lateral leads (Figure 1). Chest x-ray showed cardi- omegaly with pulmonary vascular congestion. Pulmon- ary embolism was ruled out by spiral computer tomography (CT) scan. A 2D echocardiogram with albu- min echo contrast showed left ventricular (LV) ejection fraction of 25-30% with moderate to severe global A 60-year-old Caucasian woman with a frequent history of asthma, presented to the hospital with several weeks of progressively worsening shortness of breath. She provided a history of intermittent chest pain which, at one time, was relieved with nitroglycerin and morphine, given in the emergency department. As the patient continued having increasing shortness of breath despite adjustments in her asthma medications, she was admitted for further workup. * Correspondence: vishalgupta@borgess.com 1Department of Internal Medicine: Division of Cardiology, Michigan State University/Kalamazoo Center for Medical Studies/Borgess Medical Center, Kalamazoo, MI, USA Full list of author information is available at the end of the article Kalavakunta et al. International Archives of Medicine 2010, 3:20 http://www.intarchmed.com/content/3/1/20 Page 2 of 4 Figure 1 A 12-lead electrocardiogram showing sinus tachycardia, left atrial enlargement, poor R wave progression and nonspecific ST-T wave changes and T wave inversion in the lateral leads. Figure 1 A 12-lead electrocardiogram showing sinus tachycardia, left atrial enlargement, poor R wave progression and nonspecific ST-T wave changes and T wave inversion in the lateral leads. electrocardiogram showing sinus tachycardia, left atrial enlargement, poor R wave progression and nonspecific and T wave inversion in the lateral leads. hypokinesis, further workup was done to rule out other causes of cardiomyopathy. Viral cultures were negative for enteric cytopathic human orphan [ECHO] and cox- sackie viruses. To further elucidate the cause of the car- diomyopathy, LV endomyocardial biopsy was performed. Histology showed myocardial fibrosis suggestive of car- diomyopathy, possibly secondary to LVNC (Figure 3). hypokinesis of the left ventricle and moderately enlarged left atrium. It also showed a normal sized ventricle with multiple trabeculation in the mid LV cavity and apex suggesting either an apical form of hypertrophic cardio- myopathy or LVNC (Figure 2). She underwent cardiac catheterization which revealed normal coronary arteries. Discussion Left ventricular noncompaction is a rare cause of cardio- myopathy, and patients present with systolic dysfunction commonly of the left, and sometimes of the right ventri- cle. The incidence of LVNC in clinical practice is low because it is an under-recognized phenomenon and most cases are diagnosed as idiopathic cardiomyopathy. Age of onset and degree of clinical symptoms depend on the extent of the noncompacted cardiac segments [2]. Clinical manifestation of LVNC ranges from absence of symptoms to disabling congestive heart failure, arrhythmias, such as atrial fibrillation, ventricular tachyarrhythmias, and sudden cardiac death, and throm- boembolic events [20]. LVNC association with neuro- muscular disorders is commonly seen with Barth syndrome and mitochondrial disorders [21]. With its varied presentation and genetic association, patients diagnosed with LVNC warrant genetic counseling, DNA diagnostics, and cardiological family screening to make an early diagnosis and prevent complications [22]. The exact pathophysiology of the ventricular dysfunc- tion is not known. Jenni, et al, suggested subendocardial hypoperfusion and microcirculatory dysfunction in development of ventricular dysfunction and arrhythmo- genesis [3]. Echocardiography is the reference standard for diagnosing LVNC, although it can be well defined by CT scan, positron emission tomography and magnetic resonance imaging [4-8]. Echocardiogram shows a two- layered hypokinetic myocardium with thin, compacted epicardium, thick, noncompacted endocardium, and a noncompaction (NC) to compaction (C) ratio >2, and blood flowing directly from the ventricular cavity in to the deep intertrabecular recess [2,9]. In this case the NC/C was >2 with multiple trabeculations and it could be classified into the prominent trabeculation morpholo- gical group of LVNC [10]. Treatment is symptomatic, including consideration of heart transplantation. Medical treatment depends mainly upon the clinical presentation. Cardiac resynchroniza- tion therapy (CRT) or placement of an implantable car- dioverter-defibrillators (ICD) should be considered when the LV ejection fraction ≤35% and prior to heart trans- plantation. In one study, the largest cohort of LVNC patients showed the effectiveness of ICD for secondary or primary prevention of sudden cardiac death, and improvement of New York Heart Association (NYHA) class in patients with LV ejection fraction ≤35% or LVNC with CRT [23]. There are no concrete guidelines for anticoagulation therapy as a primary prevention of thromboembolism among LVNC patients. Many authors believe that it should be considered among LVNC patients with atrial fibrillation, severe LV dysfunction, or a history of thromboembolism. Case Report In view of the normal coronaries and severe global Figure 2 Transthoracic echocardiogram (A, B, C, D) four chamber view with albumin contrast showing numerous trabeculations (white arrow) in the left ventricular apex, along with deep intertrabecular recesses. (RA- right atrium, LA- Left atrium, RV-right ventricle, LV- left ventricle). Figure 3 Endomyocardial biopsy of the left ventricle (hematoxylin and eosin stain) showing the myocardial fibrosis (100×, 400×), along with the cardiac myocytes. Figure 2 Transthoracic echocardiogram (A, B, C, D) four chamber view with albumin contrast showing numerous trabeculations (white arrow) in the left ventricular apex, along with deep intertrabecular recesses. (RA- right atrium, LA- Left atrium, RV-right ventricle, LV- left ventricle). Figure 3 Endomyocardial biopsy of the left ventricle (hematoxylin and eosin stain) showing the myocardial fibrosis (100×, 400×), along with the cardiac myocytes. Page 3 of 4 Kalavakunta et al. International Archives of Medicine 2010, 3:20 http://www.intarchmed.com/content/3/1/20 disarray. Absence of disarray in the histology excluded hypertrophic cardiomyopathy in this case. Discussion In our case, implantation of an ICD, warfarin anticoagulation was offered to the patient but she refused. g g LVNC is genetically heterogeneous, with predomi- nance of autosomal dominant inheritance, and is cur- rently classified under cardiomyopathies as a genetic disease [11]. LVNC leading to myocardial fibrosis has not been well defined in the literature [12]. The etiology of the fibrosis is unclear, but it explains the LV dysfunc- tion associated with LVNC. Recent studies have reported a significant prevalence of mitochondrial myo- pathy and genetic mutations in patients with LVNC. Myopathies with cardiac involvement have also been implicated in LVNC, including Barth syndrome, Emery- Dreifuss muscular dystrophy, and myotubular myopathy [13,14]. Genetic mutations, especially the alpha-dystro- brevin gene, Cypher/ZASP and gene G4.5 of the Xq28 chromosome region and loss of the cardiac-specific gene CSX have been well described in the literature as causes of LVNC [15-17]. These genetic mutations and myopa- thies may eventually lead to fibrosis of the myocardium causing severe LV dysfunction. This process may take several years, thus explaining why heart failure in patients with LVNC usually occurs beyond the 4th dec- ade of life. LVNC is misdiagnosed or missed in a major- ity of the cases due to its varied presentation and is not associated with any specific histological finding [18]. In a small case series, Burke, et al, found that LVNC was associated with endocardial fibroelastosis and anasto- mosing or polypoid endocardial trabeculations [19]. In our case, biopsy showed myocardial fibrosis without any Prognosis, in general, is poor and improves signifi- cantly with heart transplantation. A larger case series of 34 patients with long-term follow-up of 44 months, showed mortality of 35%, and with sudden cardiac death accounting for 50% of fatalities [2]. In the same study, 24% of patients had thromboembolic events and 41% had ventricular tachyarrhythmias. A recent similar study showed lower mortality and fewer complications [24]. This variation is secondary to the inclusion of milder/ preclinical cases in the later study, along with advance- ment in the echocardiographic technology that facilitate earlier diagnosis. Heart transplantation is considered only when medical therapy fails to control the progres- sion of heart failure. Our patient responded well to treatment with angiotensin-converting enzyme inhibitors and beta blockers. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: y y 10. 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Author details 1 f ut o deta s 1Department of Internal Medicine: Division of Cardiology, Michigan State University/Kalamazoo Center for Medical Studies/Borgess Medical Center, Kalamazoo, MI, USA. 2Department of Internal Medicine: Division of Cardiology, University of Missouri-Columbia, MO, USA. 17. Pauli RM, Scheib-Wixted S, Cripe L, Izumo S, Sekhon GS: Ventricular noncompaction and distal chromosome 5q deletion. Am J Med Genet 1999, 85:419-423. Conclusion Cardiologists and other physicians should be aware of LVNC due to its high likelihood of misdiagnosis and consequent high complication rates. Early diagnosis and Page 4 of 4 Kalavakunta et al. International Archives of Medicine 2010, 3:20 http://www.intarchmed.com/content/3/1/20 Kalavakunta et al. International Archives of Medicine 2010, 3:20 http://www.intarchmed.com/content/3/1/20 12. Finsterer J, Stollberger C, Feichtinger H: Histological appearance of left ventricular hypertrabeculation/noncompaction. 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Murphy RT, Thaman R, Blanes JG, Ward D, Sevdalis E, Papra E, Kiotsekolglou A, Tome MT, Pellerin D, McKenna WJ, Elliott PM: Natural history and familial characteristics of isolated left ventricular non- compaction. Eur Heart J 2005, 26:187-192. y 4. Conces DJ Jr, Ryan T, Tarver RD: Noncompaction of ventricular myocardium: CT appearance. Am J Roentgenol 1991, 156:717-718. 5. Daimon Y, Watanabe S, Takeda S, Hijikata Y, Komuro I: Two-layered appearance of noncompaction of the ventricular myocardium on magnetic resonance imaging. Circ J 2002, 66:619-621. doi:10.1186/1755-7682-3-20 Cite this article as: Kalavakunta et al.: Left ventricular noncompaction and myocardial fibrosis: a case report. International Archives of Medicine 2010 3:20. doi:10.1186/1755-7682-3-20 Cite this article as: Kalavakunta et al.: Left ventricular noncompaction and myocardial fibrosis: a case report. International Archives of Medicine 2010 3:20. appearance of noncompaction of the ventricular myocardium on magnetic resonance imaging. Circ J 2002, 66:619-621. 6. Senior R: Left ventricular contrast echocardiography: role for evaluation of function and structure. Echocardiography 2002, 19:615-620. 7. Hany TF, Jenni R, Debatin JF: MR appearance of isolated noncompaction of the left ventricle. J Magn Reson Imaging 1997, 7:437-438. g g g 6. Authors’ contributions 18. Weiford BC, Subbarao VD, Mulhern KM: Noncompaction of the ventricular myocardium. Circulation 2004, 109:2965-2971. All the four authors contributed to the conception, design, analysis and preparation of the manuscript. All authors read and approved the final manuscript. 19. Burke A, Mont E, Kutys R, Virmani R: Left ventricular noncompaction: a pathological study of 14 cases. Hum Pathol 2005, 36:403-411. 20. Agmon Y, Connolly HM, Olson LJ, Khandheria BK, Seward JB: Competing interests h h d l h 20. Agmon Y, Connolly HM, Olson LJ, Khandheria BK, Seward JB: Noncompaction of the ventricular myocardium. J Am Soc Echocardiogr 1999, 12:859-863. Competing interests The authors declare that they have no competing interests. Received: 15 February 2010 Accepted: 15 September 2010 Published: 15 September 2010 References Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ, Seidman CE, Young JB: Contemporary definitions and classification of the cardiomyopathies: an American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 2006, 113:1807-1816.
https://openalex.org/W2773370922	https://hal-insu.archives-ouvertes.fr/insu-03463397/file/essd-9-993-2017.pdf	English	null	Meteorological and snow distribution data in the Izas Experimental Catchment (Spanish Pyrenees) from 2011 to 2017	Earth system science data	2,017	cc-by	9,644	Meteorological and snow distribution data in the Izas Experimental Catchment (Spanish Pyrenees) from 2011 to 2017 Jesús Revuelto, Cesar Azorin-Molina, Esteban Alonso-González, Alba Sanmiguel-Vallelado, Francisco Navarro-Serrano, Ibai Rico, Juan Ignacio López-Moreno Meteorological and snow distribution data in the Izas Experimental Catchment (Spanish Pyrenees) from 2011 to 2017 Jesús Revuelto, Cesar Azorin-Molina, Esteban Alonso-González, Alba Sanmiguel-Vallelado, Francisco Navarro-Serrano, Ibai Rico, Juan Ignacio López-Moreno To cite this version: Jesús Revuelto, Cesar Azorin-Molina, Esteban Alonso-González, Alba Sanmiguel-Vallelado, Francisco Navarro-Serrano, et al.. Meteorological and snow distribution data in the Izas Experimental Catch- ment (Spanish Pyrenees) from 2011 to 2017. Earth System Science Data, 2017, 9 (2), pp.993-1005. 10.5194/essd-9-993-2017. insu-03463397 Distributed under a Creative Commons Attribution 4.0 International License Meteorological and snow distribution data in the Izas Experimental Catchment (Spanish Pyrenees) from 2011 to 2017 Jesús Revuelto1,2, Cesar Azorin-Molina1,3, Esteban Alonso-González1, Alba Sanmiguel-Vallelado1, Francisco Navarro-Serrano1, Ibai Rico1,4, and Juan Ignacio López-Moreno1 1Pyrenean Institute of Ecology, CSIC, Zaragoza, Spain 2Météo-France – CNRS, CNRM (UMR3589), Centre d’Etudes de la Neige, Grenoble, France 3Regional Climate Group, Department of Earth Sciences, University of Gothenburg, Gothenburg, Sweden 4University of the Basque Country. Department of Geography, Prehistory and Archaeology, Vitoria, Spain Correspondence: Jesús Revuelto (jesus.revuelto@meteo.fr) Received: 20 May 2017 – Discussion started: 1 June 2017 Revised: September 2017 – Accepted: 30 October 2017 – Published: 13 December 2017 Jesús Revuelto1,2, Cesar Azorin-Molina1,3, Esteban Alonso-González1, Alba Sanmiguel-Vallelado1, Francisco Navarro-Serrano1, Ibai Rico1,4, and Juan Ignacio López-Moreno1 1Pyrenean Institute of Ecology, CSIC, Zaragoza, Spain 2Météo-France – CNRS, CNRM (UMR3589), Centre d’Etudes de la Neige, Grenoble, France 3Regional Climate Group, Department of Earth Sciences, University of Gothenburg, Gothenburg, Sweden 4University of the Basque Country. Department of Geography, Prehistory and Archaeology, Vitoria, Spain Jesús Revuelto1,2, Cesar Azorin-Molina1,3, Esteban Alonso-González1, Alba Sanmiguel-Vallelado1, Francisco Navarro-Serrano1, Ibai Rico1,4, and Juan Ignacio López-Moreno1 1Pyrenean Institute of Ecology, CSIC, Zaragoza, Spain 2Météo-France – CNRS, CNRM (UMR3589), Centre d’Etudes de la Neige, Grenoble, France 3Regional Climate Group, Department of Earth Sciences, University of Gothenburg, Gothenburg, Sweden 4University of the Basque Country. Department of Geography, Prehistory and Archaeology, Vitoria, Spain Correspondence: Jesús Revuelto (jesus.revuelto@meteo.fr) Received: 20 May 2017 – Discussion started: 1 June 2017 Revised: September 2017 – Accepted: 30 October 2017 – Published: 13 December 2017 Abstract. This work describes the snow and meteorological data set available for the Izas Experimental Catch- ment in the Central Spanish Pyrenees, from the 2011 to 2017 snow seasons. The experimental site is located on the southern side of the Pyrenees between 2000 and 2300 m above sea level, covering an area of 55 ha. The site is a good example of a subalpine environment in which the evolution of snow accumulation and melt are of major importance in many mountain processes. The climatic data set consists of (i) continuous meteorological variables acquired from an automatic weather station (AWS), (ii) detailed information on snow depth distribution collected with a terrestrial laser scanner (TLS, lidar technology) for certain dates across the snow season (between three and six TLS surveys per snow season) and (iii) time-lapse images showing the evolution of the snow-covered area (SCA). HAL Id: insu-03463397 https://insu.hal.science/insu-03463397v1 Submitted on 2 Dec 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Earth Syst. Sci. Data, 9, 993–1005, 2017 https://doi.org/10.5194/essd-9-993-2017 © Author(s) 2017. This work is distributed under the Creative Commons Attribution 3.0 License. Meteorological and snow distribution data in the Izas Experimental Catchment (Spanish Pyrenees) from 2011 to 2017 The meteorological variables acquired at the AWS are precipitation, air temperature, incoming and reﬂected solar radiation, infrared surface temperature, relative humidity, wind speed and direction, atmospheric air pressure, surface temperature (snow or soil surface), and soil temperature; all were taken at 10 min intervals. Snow depth distribution was measured during 23 ﬁeld campaigns using a TLS, and daily information on the SCA was also retrieved from time-lapse photography. The data set (https://doi.org/10.5281/zenodo.848277) is valuable since it provides high-spatial-resolution information on the snow depth and snow cover, which is par- ticularly useful when combined with meteorological variables to simulate snow energy and mass balance. This information has already been analyzed in various scientiﬁc studies on snow pack dynamics and its interaction with the local climatology or topographical characteristics. However, the database generated has great potential for understanding other environmental processes from a hydrometeorological or ecological perspective in which snow dynamics play a determinant role. Published by Copernicus Publications. 2.1 The Pyrenees The Pyrenees lie on the northeastern border of the Iberian Peninsula (Fig. 1) and form an orographic barrier be- tween the north and south faces. Due to this, progres- sively higher aridity is found toward the south as the mountain range blocks humid air masses from the At- lantic (López-Moreno and Vicente-Serrano, 2007; Vicente- Serrano, 2005). Thus, the natural barrier directly inﬂuences precipitation, leading to areas above 2000 m a.s.l. receiving about 2000 mm year−1, increasing to 2500 mm year−1 in the highest divides of the mountain range and rapidly decreasing to 600–800 mm year−1 in low-elevation areas on the south- ern side (García-Ruiz, et al., 2001). Another distinct feature of the Pyrenees is their location between two water masses with contrasting conditions, i.e., the Atlantic Ocean is on the west side, while the Mediter- ranean Sea lies in the east. This position between both water masses causes a climatic transition from oceanic to mediter- ranean conditions in the east. During autumn, fronts ap- proaching from the Atlantic bring the highest monthly aver- ages of precipitation in the western observatories, with their total contribution accounting for 40 % of total annual precip- itation in this area (Creus-Novau, 1983). Conversely, spring and summer storms mostly affect the eastern areas of the Pyrenees, promoted by the development of zones in which sea breezes and local winds converge to initiate deep moist convection along the eastern fringe of the Iberian Mediter- ranean area (Azorin-Molina et al., 2015). Therefore, Pyre- nean observatories in the east record a large number of con- vective events, i.e up to 32 % of total annual precipitation in eastern valleys, but dropping below 16 % of annual pre- cipitation in western valleys (Cuadrat et al., 2007). In early winter, the arrival of fronts from the northwest and west are the most frequent, leading to the highest snow accumula- tion found in the western Pyrenees (Navarro-Serrano and López-Moreno, 2017). The Azores high, which usually af- fects the Iberian Peninsula at certain times in the winter, gives rise to relatively long periods with no snow accumula- tion in this season. Subsequently, in spring, snow accumula- tion is associated with southwesterly advections, which lead to heavy snow accumulations in the western Pyrenees (Re- vuelto et al., 2012). Snow remains for long periods above 1600 m a.s.l., between November and April (López-Moreno and Nogués-Bravo, 2006). 2 Study area characteristics and climatology p ) The Pyrenees are a midlatitude mountain range, with sig- niﬁcant snowfalls in the high-elevation areas throughout the year. During the spring, Pyrenean river discharges depend on the snowmelt timing, with approximately 40 % of spring runoff being directly attributable to snow (López-Moreno and García-Ruiz, 2004). Thus, snow accumulation has a heavy inﬂuence on Pyrenean headwaters. This dependence is mostly due to the generally continuous snow cover from November to April above 2000 m above sea level (a.s.l.) (Alvera and Garcia-Ruiz, 2000; García-Ruiz et al., 1986; López-Moreno et al., 2002) and, therefore, the study of the snowpack at high elevations in the Pyrenees is crucial for understanding and managing mountain river discharges (López-Moreno, 2005), especially in the scenario of global climate change (García-Ruiz et al., 2011). However, contin- uous snow observations above 2000 m a.s.l. are scarce in this mountain range since most only have information from 1600 to 2000 m a.s.l. and those that are available only cover short time spans. Therefore, well-established study areas at high elevations with continuous measurements of meteorological variables and snowpack distribution are required in the Pyre- nees. 1 Introduction et al., 2009) are directly controlled by the evolution of snow cover over time. Conversely, snowmelt dynamics are also of major importance from a hydrological perspective since one- sixth of the Earth’s total population depends on the water storage in mountain river headwaters (Barnett et al., 2005). In downstream areas exposed to extreme climatic conditions, the snowmelt runoff from mountain areas becomes a key ele- ment (Viviroli et al., 2007), especially in zones affected by Snowpack distribution and its temporal evolution have a marked inﬂuence on many mountain processes. These in- clude erosion rates and sediment transport (Colbeck et al., 1979; Lana-Renault et al., 2011) and geomorphological and glaciological processes (López-Moreno et al., 2017; Ser- rano et al., 2001). Phenological cycles (Liston, 1999; Wipf J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 994 water shortages. This is the case of semiarid regions, like the Mediterranean area, which are characterized by an ir- regular climate with long drought periods (Vicente-Serrano, 2006), and therefore are highly dependent on water stored in mountain areas, such as the Pyrenees (López-Moreno, 2005; López-Moreno et al., 2008). the SCA derived from time-lapse images; Sect. 5 concludes with information for downloading the database; and ﬁnally Sect. 6 summarizes all information available and the poten- tial application of the database. www.earth-syst-sci-data.net/9/993/2017/ 2.1 The Pyrenees The right map (c) shows the topographic characteristics of the catchment and the location of the TLS scanning positions (Scan stations), the meteorological station and the ﬁeld of view of the time-lapse camera (continuous lines from Scan station 1). thermal amplitude observed in the western Pyrenees is be- cause of the proximity of the ocean (Cuadrat et al., 2007). As a general tendency in the Central Pyrenees, the annual 0 ◦C isotherms lie between 2700 and 2900 m a.s.l. (del Barrio et al., 1990; Chueca, 1993). Spain–France border (Fig. 1). The Izas Experimental Catch- ment exempliﬁes the general characteristics of subalpine ar- eas of the Pyrenees. In this environment, snowpack dynam- ics are of major importance throughout the year. Thus, the atmosphere–snowpack interactions observed at this experi- mental site will enable a better understanding of many pro- cesses in subalpine areas. ) Additionally the Pyrenees exhibit a high interannual vari- ability in air temperature and precipitation, which makes the annual snow accumulation very uncertain (López-Moreno, 2005). This variability is inﬂuenced by the interannual vari- ability in atmospheric circulation, with a decrease in snow accumulation weather types being identiﬁed under positive North Atlantic Oscillation (NAO) phases (López-Moreno and Vicente-Serrano, 2007). As observed with precipitation, snow accumulation correlates to Atlantic–Mediterranean proximity and distance from the main divide of the moun- tain range (Revuelto et al., 2012), and it is strongly depen- dent on the ﬂuctuations of the 0 ◦C isotherm during winter and spring. This high climatic variability is also the cause of large interannual variability in total snow accumulation and its temporal distribution across the snow season (López- Moreno, 2005). The mean annual precipitation is 2000 mm, and snow ac- counts for approximately 50 % of total precipitation (Ander- ton et al., 2004). For an average of 130 days each year the mean daily air temperature is below 0 ◦C, with a mean an- nual air temperature of 3 ◦C (del Barrio et al., 1997). Snow covers a high percentage of the catchment from Novem- ber to the end of May (López-Moreno et al., 2010). Lithol- ogy shows limestones and sandstones of the Cretaceous pe- riod, and limestones of the Paleocene, much more resistant to erosion. The zonal vegetation type corresponds to a high mountain steppe, mainly covered by bunch grasses, namely Festuca eskia, Nardus stricta, Trifolium alpinum, Plantago alpine and Carex sempervirens. 2.1 The Pyrenees This paper presents the recently acquired data set of mete- orological and snowpack variables obtained from a small ex- perimental catchment on the southern face of the Pyrenees. Although meteorological and hydrological data are avail- able from previous years (some variables have been mea- sured since the late 1980s; Alvera and Garcia-Ruiz, 2000), we present data from the 2011/12 to 2016/17 snow sea- sons, as data series provide higher quality and continuity, and they also match in situ observations of snow depth and snow cover. The data set consists of (i) continuous meteoro- logical variables acquired from an automatic weather station (AWS), (ii) detailed information on snow depth distribution collected with a terrestrial laser scanner (TLS, lidar technol- ogy) for certain dates across the snow season (between two and six TLS surveys per snow season) and (iii) time-lapse im- ages showing the snow-covered area (SCA) evolution. Some years of this data set have already been used to study the topographic control on snow depth distribution (Revuelto et al., 2014b), the spatial variability in snowpack at different distances (López-Moreno et al., 2012) or to investigate how detailed snowpack simulation could be improved by includ- ing snow distribution information (Revuelto et al., 2016a, b). The paper is structured as follows: Sect. 2 describes the study area characteristics; Sect. 3 presents meteorological data acquired from the AWS with a general description of the observed climatology; Sect. 4 describes the distributed measurements on snow depth distribution from the TLS and Similar to precipitation, air temperature is inﬂuenced by the Atlantic–Mediterranean transitions, but elevation plays a major role in its distribution. For instance, the lower annual Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 995 Figure 1. The Izas Experimental Catchment study site. Panel (a) shows the location of the study site. Panel (b) shows an overview of the catchment with marginal snow presence. The right map (c) shows the topographic characteristics of the catchment and the location of the TLS scanning positions (Scan stations), the meteorological station and the ﬁeld of view of the time-lapse camera (continuous lines from Scan station 1). Figure 1. The Izas Experimental Catchment study site. Panel (a) shows the location of the study site. Panel (b) shows an overview of the catchment with marginal snow presence. 2.1 The Pyrenees Rocky outcrops dominate the upper and steeper slopes (less than 15 % of the study area). There are no trees present in the study area. The catch- ment is predominantly east-facing, with some areas also fac- ing north or south. The mean slope of the catchment is 16◦ (López-Moreno et al., 2012), with the topographic charac- teristics displaying the typical high spatial heterogeneity of subalpine areas, with ﬂat concave and convex areas. www.earth-syst-sci-data.net/9/993/2017/ 3.1 Wind speed and direction The AWS is equipped with a Young wind monitor – Alpine model (Young Company, 2010), placed at the highest point of the meteorological tower (8 m above the ground). The Pyre- nees are commonly affected by strong westerly to northerly winds as shown in the wind roses displayed in Fig. 3. With the exception of south winds that mainly occur during the melt period, westerly to northerly winds dominate. Addition- ally, the most frequently moderate to strong winds come from the northwest. J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees Figure 2. Pictures of the experimental site equipment. (a) AWS sensors. 1A: Young wind sensor; 2A: radiation shield with HMP 155 humidity and temperature probe; 3A: BP1 air pressure recorder; 4A: IR100 infrared remote temperature sensor; 5A: CMA6 Kipp & Zonen albedometer; 6A: SR50A range sensor; 7A: Geonor T-200B with wind shield; 8A: CR3000 data logger and modem; 9A: solar panel and battery; 10A: Campbell Scientiﬁc 107 ground temperature probes. (b) RIEGL LPM-321 TLS mounted on the tripod during an acquisition campaign. The upper-right part shows one of the 12 ﬁxed reﬂective targets ﬁxed on the terrain. (c) Campbell CC640 camera mounted in the metal structure with 1C: digital camera inside the enclosure house; 2C: modem; 3C: protection glass of the digital camera; and 4C: frontal view of the camera and its structure. Figure 2. Pictures of the experimental site equipment. (a) AWS sensors. 1A: Young wind sensor; 2A: radiation shield with HMP 155 humidity and temperature probe; 3A: BP1 air pressure recorder; 4A: IR100 infrared remote temperature sensor; 5A: CMA6 Kipp & Zonen albedometer; 6A: SR50A range sensor; 7A: Geonor T-200B with wind shield; 8A: CR3000 data logger and modem; 9A: solar panel and battery; 10A: Campbell Scientiﬁc 107 ground temperature probes. (b) RIEGL LPM-321 TLS mounted on the tripod during an acquisition campaign. The upper-right part shows one of the 12 ﬁxed reﬂective targets ﬁxed on the terrain. (c) Campbell CC640 camera mounted in the metal structure with 1C: digital camera inside the enclosure house; 2C: modem; 3C: protection glass of the digital camera; and 4C: frontal view of the camera and its structure. 3 Meteorological data Since the main application of the data collected by the AWS is to assess the evolution of snow cover in the study area, in the following subsections we focus our analyses on the accumulation and melt periods, i.e., accumulation (Jan- uary, February and March; JFM) and melt (April, May and June; AMJ). Annual values observed during a whole snow season are also presented for each subsection. The study site is equipped with an AWS located in the lower elevation of the catchment (42◦44′33.65′′ N, 0◦25′8.83′′ W, 2113 m a.s.l.; Fig. 1), located in a ﬂat open area with sparse vegetation (mountain pastures). The AWS measures wind speed and direction, atmospheric air temperature, relative hu- midity and air pressure, soil temperature for 0, 5, 10, and 20 cm, temperature of the surface close to the AWS (snow or soil, depending on whether snow is present or not), global and reﬂected solar irradiance, snow depth, and precipitation (the precipitation gauge is located at 15 m from the AWS tower) (see Fig. 2). Information on the main atmospheric variables has been recorded since the end of 2011 (AWS in- stalled in November 2011). Therefore, data availability cov- ers ﬁve complete snow seasons. Since the station is located in the lower elevation of the catchment and despite air tem- perature lapse rate with elevation, the AWS records serve to describe the evolution of atmospheric variables occurring at the Izas Experimental Catchment. 2.2 The Izas Experimental Catchment The Izas Experimental Catchment (42◦44′ N, 0◦25′ W) has a surface area of 33 ha, but snow depth information cov- ers a total of 55 ha, with elevations ranging between 2075 and 2325 m a.s.l. This area is close to the main divide of the Pyrenees in the headwaters of the Gállego River, near the Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 996 J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 997 J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 997 Figure 3. Wind roses showing the frequency (%) of wind speed and direction observed in the AWS for accumulation (a) and melt (b) snow seasons. Figure 3. Wind roses showing the frequency (%) of wind speed and direction observed in the AWS for accumulation (a) and melt (b) snow seasons. Table 1. Mean and standard deviation of air temperature for the ﬁve snow seasons for the annual, accumulation and melt periods. Also shown are maximum and minimum air temperatures for each period of the snow seasons. Air temperature (◦C) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Annual 5.13 ± 7.73 3.50 ± 6.88 4.17 ± 6.11 5.26 ± 7.02 5.08 ± 6.69 Nan Mean Accumulation −1.15 ± 5.69 −2.78 ± 4.57 −1.71 ± 3.44 −1.65 ± 4.87 −1.66 ± 3.69 −0.56 ± 4.20 Melting 5.80 ± 6.60 2.79 ± 4.79 5.51 ± 4.07 7.23 ± 4.86 4.45 ± 5.12 7.58 ± 6.00 Annual 25.87 20.85 21.42 24.07 24.23 Nan Max Accumulation 7.89 10.69 10.20 10.98 11.62 11.39 Melting 18.29 17.13 18.32 23.07 19.26 22.51 Annual −18.51 −15.26 −11.35 −15.24 −11.78 Nan Min Accumulation −18.51 −15.26 −11.35 −15.24 −11.78 −14.97 Melting −9.33 −9.04 −3.71 −4.76 −8.20 −8.33 Nan: no data observed during the period. Nan: no data observed during the period. with 67% during the accumulation period and 66% during the melt. Similarly, atmospheric air pressure has a mean an- nual value of 791 mbar, with 787 mbar for the accumulation period and 792 mbar for the melt. Over the six snow seasons analyzed, the mean annual air temperature ranged between 5.26 ◦C (2014/15) and 3.51 ◦C (2012/13), with an average value of 4.59 ◦C. The mean air temperature in the accumulation period ranged from −2.78 ◦C (2012/13) to −0.56 ◦C (2016/17), with an average value of −1.59 ◦C for the whole study period. Finally, the melt period returned a mean value of 5.56 ◦C ranging from 2.79 ◦C (2012/13) to 7.58 ◦C (2016/17). Table 1 shows that the 2012/13 snow season was the coldest in the study period. Figure 4 depicts the temporal evolution of air temperature and other variables observed in the AWS from 2011 to 2016. Thus, this ﬁgure shows the control points for air temperature on the ground and the surface temperature. www.earth-syst-sci-data.net/9/993/2017/ 3.2 Air temperature, relative humidity and atmospheric air pressure The data acquisition system consists of a Campbell Scien- tiﬁc CR3000 data logger that samples each instrument and stores data at 10 min time intervals. All data are transmit- ted via modem to the Pyrenean Institute of Ecology where automatic quality-control checks are applied to remove out- liers. Data gaps are rare for almost all variables and, there- fore, instead of gap ﬁlling with interpolation methods, only measured data are available. However, some variables had long data gaps and certain periods have been discarded from further analysis. This is the case of precipitation for the ﬁrst three snow seasons, which were useless because of the length of data gaps. Air temperature and relative humidity were measured with the HMP155 Vaisala sensor (Vaisala Company, 2012), and atmospheric air pressure was recorded with the BP1 sen- sor from ADCON Telemetry (ADCON Telemetry Company, 2015). The HMP 155 humidity and temperature probe was placed inside a standard radiation shield at 3.2 m from the ground in order to prevent the snowpack from eventually covering the sensors. Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 999 Table 3. Mean and standard deviation of atmospheric air pressure for the ﬁve snow seasons for the annual, accumulation and melt periods. Atmospheric air pressure (mbar) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Annual 794.5 ± 5.9 790.7 ± 7.7 791.3 ± 6.5 792.4 ± 6.9 791.8 ± 7.1 Nan Accumulation 790.9 ± 7.2 784.7 ± 8.3 786.4 ± 6.9 789.7 ± 9.3 786.8 ± 7.9 788.5 ± 5.5 Melting 797.1 ± 3.6 790.9 ± 6.6 791.8 ± 4.6 794.2 ± 4.4 788.9 ± 5.4 791.5 ± 5.0 Nan: no data observed during the period. Table 3. Mean and standard deviation of atmospheric air pressure for the ﬁve snow seasons for the annual, accumulation and melt periods. on of atmospheric air pressure for the ﬁve snow seasons for the annual, accumulation and melt periods. Table 3. Mean and standard deviation of atmospheric air pressure for the ﬁve snow seasons for the annual, accumulation and melt periods. Nan: no data observed during the period. Table 4. Mean and standard deviation ground temperature for different depths for the ﬁve snow seasons for the annual, accumulation and melt periods. Mean and standard deviation ground temperature for different depths for the ﬁve snow seasons for the annual, accumulation and ds. Ground temperatures (◦C) Depth 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 (cm) Annual 0 Nan 4.60 ± 6.71 5.13 ± 6.45 5.98 ± 7.02 4.24 ± 6.02 Nan 5 Nan 4.35 ± 5.67 5.61 ± 6.52 6.06 ± 5.52 4.66 ± 5.12 Nan 10 Nan 4.38 ± 5.19 5.07 ± 5.46 5.99 ± 6.09 4.55 ± 4.87 Nan 20 Nan 4.26 ± 4.66 5.01 ± 4.62 5.08 ± 3.26 4.51 ± 3.88 Nan Acc. 3.5 Global and reﬂected solar irradiance The AWS also obtains information on the global and re- ﬂected solar irradiance with a CMA 6 Kipp & Zonen albedometer (Kipp & Zonen B. V., 2016) placed at 3.4 m height. Figure 4 shows the daily evolution of the values recorded and how these are interrelated, with the reﬂected ra- diation increasing at the same time as the incident. The aver- age values of these variables are presented in Table 6. For the whole period, the average values of the incident radiation are 207.97 W m−2 day, taking complete snow seasons into ac- count, 164.73 m−2 day for accumulation and 280.95 m−2 day J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 0 Nan 0,22 ± 0,05 0.03 ± 0.04 −0.26 ± 0.87 −0.66 ± 1.13 Nan 5 Nan 0,69 ± 0.12 0.11 ± 0.08 −0.39 ± 0.54 0.99 ± 0.10 Nan 10 Nan 1.10 ± 0.16 0.31 ± 0.18 −0.27 ± 0.23 0.98 ± 0.11 Nan 20 Nan 1.34 ± 0.19 0.94 ± 0.06 0.39 ± 0.08 1.57 ± 0.17 Nan Melting 0 Nan 1.21 ± 3.49 5.53 ± 6.41 7.87 ± 6.41 4.57 ± 5.46 Nan 5 Nan 1.04 ± 2.45 5.19 ± 6.08 7.03 ± 5.71 4.43 ± 5.09 Nan 10 Nan 1.06 ± 1.78 4.15 ± 4.68 6.46 ± 5.32 4.15 ± 4.79 Nan 20 Nan 1.04 ± 1.36 3.46 ± 3.49 5.35 ± 4.15 3.50 ± 3.47 Nan Nan: no data observed during the period. Table 4. Mean and standard deviation ground temperature for different depths for the ﬁve snow season melt periods. Nan: no data observed during the period. The infrared remote sensor shows the tendency of the snow surface to cool faster than soil. During winter and spring, while snow is present on the ground, the differences between air and surface temperature are more marked, with surface temperatures always observed to be lower (see the occurrence of snow below the AWS when lower surface tem- peratures are observed in Fig. 4). This plainly exempliﬁes the higher energy irradiance of snow when compared to snow- free soils. perature probes were damaged by cows. The average values during the period with information for the 0, 5, 10 and 20 cm depths are, respectively, 5.26 ± 6.22, 4.97, 4.93 ± 6.17 and 4.89 ± 4.56 ◦C. The temporal evolution of air and ground temperatures de- picts the impact of the snowpack on ground energy dynam- ics. The snowpack shelters the ground from the high tem- poral variability in air temperature. Therefore, the daily vari- ability in ground temperatures is signiﬁcantly lower. Further- more, the different ground temperatures tend to reach 0 ◦C while snow covers the ground, i.e., the typical soil–snow in- terface temperature. 3.3 Ground temperature On 22 November 2012 four Campbell Scientiﬁc 107 tem- perature probes (Campbell Scientiﬁc Ltd, 2012) were in- stalled in the AWS to measure ground temperature at dif- ferent depths. One sensor was located in the atmosphere– ground interface (slightly buried, 0 cm depth), while the other three were placed at depths of 5, 10 and 20 cm. Ta- ble 4 and Fig. 4 show the average values of ground tem- peratures and the temporal evolution of ground temperature. Data are lacking from August 2016 onwards because tem- The relative air humidity and the atmospheric air pressure are shown in Tables 2 and 3, respectively. The mean annual value of the relative humidity for the ﬁve seasons is 65%, www.earth-syst-sci-data.net/9/993/2017/ Earth Syst. Sci. Data, 9, 993–1005, 2017 998 J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees l i f l i l i bl f 2011 2016 b i d l evolution of meteorological variables from 2011 to 2016. From top to bottom are air temperature and surface temperature ); ground temperature for the four depths; global (Incident) and reﬂected solar irradiance; punctual snow depth (SD); and precipitation (Pcp) (sum of solid and liquid). Figure 4. Temporal evolution of meteorological variables from 2011 to 2016. From top to bottom are air temperature and surface temperature (from the IR sensor); ground temperature for the four depths; global (Incident) and reﬂected solar irradiance; punctual snow depth (SD); and daily accumulated precipitation (Pcp) (sum of solid and liquid). Table 2. Mean and standard deviation of relative humidity for the ﬁve snow seasons for the annual, accumulation and melt periods. Relative air humidity (%) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Annual 59.9 ± 18.9 70.1 ± 17.1 68.8 ± 17.3 64.8 ± 19.2 65.9 ± 18.5 Nan Accumulation 67.1 ± 18.1 70.5 ± 19.3 72.7 ± 15.8 62.8 ± 22.2 71.3 ± 18.3 61.0 ± 20.8 Melting 57.1 ± 15.2 74.4 ± 14.5 68.7 ± 15.9 63.9 ± 15.8 69.9 ± 14.1 62.9 ± 15.65 Nan: no data observed during the period. dard deviation of relative humidity for the ﬁve snow seasons for the annual, accumulation and melt periods. ble 2. Mean and standard deviation of relative humidity for the ﬁve snow seasons for the annual, accumulatio www.earth-syst-sci-data.net/9/993/2017/ Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ Table 5. Mean surface temperature from the infrared sensor for the ﬁve snow seasons for the annual, accumulation and melt periods. SD (cm) 14.74 ± 14.60 145.61 ± 52.3 148.54 ± 41.60 55.90 ± 36.50 81.42 ± 31.67 114.44 ± 73.80 Pcp (mm) Nan Nan Nan 454.35 147.22 82.38 SD ( ) 1 60 ± 1 57 131 42 ± 64 64 51 57 ± 64 95 12 70 ± 22 24 64 30 ± 59 85 25 14 ± 32 22 Table 7. Accumulated precipitation (liquid and solid) for snow seasons with observations available. Average snow depth values for accumu- lation and melt periods for the ﬁve snow seasons. Accumulated total precipitation (mm) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Ann Pcp (mm) Nan Nan Nan 1572 411 Nan Table 7. Accumulated precipitation (liquid and solid) for snow seasons with observations available. Average snow depth values for accumu- lation and melt periods for the ﬁve snow seasons. A l t d t t l i it ti ( ) Table 7. Accumulated precipitation (liquid and solid) for snow seasons with observations available. Average snow depth values for accumu- lation and melt periods for the ﬁve snow seasons. Table 7. Accumulated precipitation (liquid and solid) for snow seasons with observations available. Average snow depth values for accumu- ation and melt periods for the ﬁve snow seasons. Accumulated total precipitation (mm) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Ann Pcp (mm) Nan Nan Nan 1572 411 Nan Acc. SD (cm) 14.74 ± 14.60 145.61 ± 52.3 148.54 ± 41.60 55.90 ± 36.50 81.42 ± 31.67 114.44 ± 73.80 Pcp (mm) Nan Nan Nan 454.35 147.22 82.38 Mlt. SD (cm) 1.60 ± 1.57 131.42 ± 64.64 51.57 ± 64.95 12.70 ± 22.24 64.30 ± 59.85 25.14 ± 32.22 Pcp (mm) Nan Nan Nan 249.61 121.05 162.51 Nan: no data observed during the period. Nan: no data observed during the period. surface and the sensor (the sensor is placed 2.64 m from the ground, and the snow depth is obtained by subtracting this value from the observed distance). This sensor worked un- interruptedly during the study period and provided a good record of the snow depth evolution in the Izas Experimen- tal Catchment. Therefore, the information on the snow depth can be used as a reference for other observations of snow- pack evolution. The average values for the whole study pe- riod are 93.4 cm for the accumulation period and 47.8 cm for the melt period (Table 7 shows the seasonal values). Table 5. Mean surface temperature from the infrared sensor for the ﬁve snow seasons for the annual, accumulation and melt periods. The tem- poral evolution of the snow depth is shown in Fig. 4. for all melt periods. Similarly, the reﬂected radiation av- erage values are 83.67 m−2 day for entire snow seasons, 109.69 m−2 day for the accumulation and 117.06 m−2 day for melt periods. Similar to ground and surface temperatures, the radia- tion reﬂected is heavily inﬂuenced by the presence of snow. When snow covers the ground, the sensor shows higher val- ues of reﬂected radiation in comparison with snow-free peri- ods (Fig. 4). 3.4 Surface temperature Together with the installation of the ground temperature sen- sors, an IR100 infrared remote temperature sensor (Campbell Scientiﬁc Ltd, 2015) was also set up to measure surface tem- perature of near-target ground or snow. Table 5 shows the average land surface temperatures. The mean annual surface temperature is 2.56 ◦C, with a mean value of −4.58 ◦C during the accumulation period and 3.94 ◦C during the melt period. www.earth-syst-sci-data.net/9/993/2017/ Earth Syst. Sci. Data, 9, 993–1005, 2017 1000 Table 5. Mean surface temperature from the infrared sensor for the ﬁve snow seasons for the annual, accumulation and melt periods. emperature from the infrared sensor for the ﬁve snow seasons for the annual, accumulation and melt periods. Table 5. Mean surface temperature from the infrared sensor for the ﬁve snow seasons for the annual, accumulation and melt periods. Table 5. Mean surface temperature from the infrared sensor for the ﬁve snow seasons for the annual, accumulation and melt periods. Surface temperature (◦C) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Annual Nan 1.29 ± 7.83 2.44 ± 7.06 3.26 ± 8.14 3.26 ± 7.71 Nan Accumulation Nan −5.38 ± 3.58 −4.18 ± 2.65 −5.36 ± 3.61 −4.32 ± 2.99 −3.68 ± 3.58 Melting Nan −0.09 ± 3.44 3.75 ± 5.16 5.95 ± 6.02 3.47 ± 5.96 6.64 ± 6.67 Nan: no data observed during the period. n global and reﬂected radiation for the ﬁve snow seasons for the annual, accumulation and melt periods. Table 6. Mean global and reﬂected radiation for the ﬁve snow seasons for the annual, accumulation and melt periods. Radiation (W m−2 day) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Annual Global 219.48 ± 110.60 205.36 ± 114.50 196.64 ± 110.49 207.63 ± 116.50 211.03 ± 113.95 Nan Reﬂected 82.87 ± 49.60 96.20 ± 64.92 79.35 ± 52.78 76.34 ± 64.90 83.61 ± 53.76 Nan Acc. Global 181.09 ± 68.18 154.83 ± 67.30 150.04 ± 84.02 166.97 ± 65.80 152.83 ± 83.18 182.64 ± 85.36 Reﬂected 99.14 ± 40.34 117.04 ± 44.35 108.50 ± 47.43 114.24 ± 44.35 108.94 ± 48.59 110.29 ± 41.13 Melting Global 245.37 ± 120.56 289.59 ± 114.10 283.33 ± 102.80 287.65 ± 117.15 278.71 ± 114.37 301.07 ± 107.57 Reﬂected 103.11 ± 67.15 169.56 ± 60.28 114.83 ± 61.10 90.51 ± 60.28 120.06 ± 67.30 104.28 ± 66.7 Nan: no data observed during the period. Table 6. Mean global and reﬂected radiation for the ﬁve snow seasons for the annual, accumulation a Table 7. Accumulated precipitation (liquid and solid) for snow seasons with observations available. Average snow depth values for accumu- lation and melt periods for the ﬁve snow seasons. Accumulated total precipitation (mm) 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17 Ann Pcp (mm) Nan Nan Nan 1572 411 Nan Acc. 4.1 TLS acquisitions of snow depth distribution During the ﬁve snow seasons presented here, three to six TLS surveys were carried out each year in the Izas Experimental Catchment. TLSs are devices using lidar technology, a re- mote sensing method to obtain the distance between a target area and the device. During a TLS data acquisition, the de- vice measures the distance of some hundreds of thousands of points within the area deﬁned by the operator, creating a cloud of data points representing the topography of the target surface. The device used in this study is a long-range TLS (RIEGL LPM-321 (Fig. 2), RIEGL Laser Measurements, 2010). The technical characteristics of this model include (i) light pulses of 905 nm wavelength (near infrared), appro- priate for acquiring data from snow cover (Prokop, 2008); (ii) a minimum angular step width of 0.018◦; (iii) a laser beam divergence of 0.046◦; and (iv) a maximum working distance of 6000 m. In order to reduce topographic shad- owing (note that terrain topography limits the line of sight of the TLS), two scanning positions (Scan station in Fig. 1) were established within the study site (Fig. 1). Also ﬁxed on the terrain, were 12 reﬂected targets (Fig. 2). The location of these targets was acquired on each TLS acquisition date since this information is used in the post-processing phase for comparing the point clouds acquired on different dates. The protocol for obtaining the information in the ﬁeld and the methodology for generating the snow depth distribution maps for the different TLS survey dates is fully explained in Revuelto et al. (2014a). The method is mainly based on cal- culating the elevation difference between the point clouds ob- tained on different dates with and without snow cover across the study area. The ﬁnal products are snow depth distribu- tion maps with a grid size of 1 m × 1 m, with a mean abso- lute error of 0.07 m in the snow depth values (Revuelto et al., 2014a). for dates on which the snow cover had already completely melted over wide areas of the catchment. Table 8 presents the average snow depth and the maximum snow depth value observed for each TLS acquisition. This table also shows the coefﬁcient of variation on each snow distribution map and the fraction of the SCA. 3.6 Snow depth and precipitation In addition, Fig. 4 shows the precipitation values for the period with data in the precipitation gauge (from the end of July 2014). The sensor installed is a Geonor T-200B with a wind shield (Geonor A/S, 2010), which continuously weighs the accumulated precipitation (liquid and solid). The height The AWS is also equipped with a Campbell SR50A sonic ranging sensor (Campbell Scientiﬁc Ltd, 2011). For the sake of simplicity we will refer to it as a snow depth sensor since it is used for measuring the changing distance between the Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 1001 Table 8. Observed mean and maximum snow depth values, snow- covered area (SCA, % of the total area covered by the TLS), and coefﬁcient of variation for the observed snow distribution on the TLS survey dates. of the gauge oriﬁce is 3.25 m (2.5 m metal pedestal plus the height of the T-200B inlet). The precipitation accumulated over a certain period was calculated by subtracting ﬁnal and initial weighted values. Table 7 includes the accumulated precipitation for the whole snow year and also during the ac- cumulation and melt periods. Snow Date Mean SD Max SD SCA CV season (m) (m) (%) 2011/12 22 Feb 0.46 5.53 67.2 1.35 2 Apr 0.17 3.86 33.5 2.23 17 Apr 0.56 5.34 94.1 1.07 2 May 0.90 6.11 98.8 0.74 14 May 0.21 4.47 30.9 1.90 24 May 0.09 4.32 18.9 1.29 2012/13 17 Feb 2.91 10.89 98.8 0.63 3 Apr 3.19 11.20 100 0.56 25 Apr 2.42 10.10 96.3 0.76 6 Jun 1.98 9.64 86.4 0.86 12 Jun 1.69 8.90 77.1 0.90 20 Jun 0.76 7.97 67.0 1.35 2013/14 3 Feb 2.16 10.20 96.0 0.59 22 Feb 2.56 10.47 98.6 0.57 9 Apr 2.54 9.72 89.0 0.65 5 May 1.67 9.02 75.2 0.87 2014/15 6 Nov 0.22 2.78 85.0 0.81 26 Jan 0.74 4.88 89.3 0.85 6 Mar 2.13 11.55 94.0 0.69 12 May 0.67 7.75 56.0 1.21 4 Feb 0.82 6.20 91.1 0.63 2015/16 25 Apr 1.86 10.82 97.0 0.50 26 May 1.16 7.81 74.8 0.70 2016/17 20 Jan 1.26 6.33 93 0.72 8 May 0.77 7.25 57.2 0.81 4.1 TLS acquisitions of snow depth distribution The values ob- tained depict the heavy accumulation of snow in some areas of the catchment, while the average snow depth is lower. www.earth-syst-sci-data.net/9/993/2017/ 4.2 Snow-covered area from time-lapse photographs Figure 5 shows the snow depth maps obtained for the 2012/13 snow season. The information for this snow season is presented because six TLS surveys were completed. Fur- thermore, the accumulated snow depths were signiﬁcant and thus provide an interesting example of snowpack evolution over time. These maps show the high spatial variability in the snowpack within the study area, with marked changes in the snow depth distribution within short distances. It was also observed how high-accumulation areas had large accumula- tions during the whole snow season, with a thick snowpack The Izas Experimental Catchment is also equipped with a Campbell CC640 digital camera (Campbell Scientiﬁc Ltd, 2010). This camera was mounted on a solid metal struc- ture set into the ground with concrete (Fig. 2), which en- sured a constant position to obtain consistent information. The digital camera has a resolution of 640 × 480 pixels with a focal length of 6–12 mm. The ﬁeld of view of the pho- tographs obtained with the camera mounted on the metal structure covers approximately 30 ha (Fig. 1), which repre- www.earth-syst-sci-data.net/9/993/2017/ Earth Syst. Sci. Data, 9, 993–1005, 2017 1002 J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees Figure 5. Snow depth distribution maps obtained for the six TLS acquisition dates of the 2012/13 snow season. Figure 6. Example of a sequence of four photographs for the 2012/13 snow season, showing the evolution of the snow cover. J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 1002 Figure 5. Snow depth distribution maps obtained for the six TLS acquisition dates of the 2012/13 snow season. gure 5. Snow depth distribution maps obtained for the six TLS acquisition dates of the 2012/13 snow season. Figure 5. Snow depth distribution maps obtained for the six TLS acquisition dates of the 2012/13 sno Figure 6. Example of a sequence of four photographs for the 2012/13 snow season, showing the evolution of the snow cover. sequence of four photographs for the 2012/13 snow season, showing the evolution of the snow cover. Figure 6. Example of a sequence of four photographs for the 2012/13 snow season, showing the evolution o sents about 52 % of the total surface covered by the TLS. J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 1003 Figure 7. Number of days with snow present for each pixel for 2011/12 and 2012/13 snow seasons. Figure 7. Number of days with snow present for each pixel for 2011/12 and 2012/13 snow seasons. Additionally, great effort has been made on ﬁeld data ac- quisition with TLS over the last ﬁve snow seasons and is ongoing. The data set described here is novel in the Pyre- nees because, for the ﬁrst time, it represents high-spatial- resolution information on the snowpack distribution and its evolution in time, as well as making continuous information available on meteorological variables. The high quality of the information obtained has already been exploited for differ- ent studies on the understanding of snowpack dynamics and the improvement of simulation approaches to snowpack evo- lution in mountain areas (López-Moreno et al., 2012, 2014, Revuelto et al., 2014b, 2016a, 2016b). However, many sci- entiﬁc questions still go unanswered, such as the long-term inﬂuence of topography on snow dynamics, and the spatial distribution of snow during precipitation and strong wind events. Also, the high interannual variability in snow accu- mulation in the Pyrenees has serious consequences for water management, especially in the Mediterranean area (García- Ruiz et al., 2011). Thus, it is very important to continue ob- taining information on snowpack evolution and the meteoro- logical variables controlling snow dynamics. This informa- tion will allow the scientiﬁc community to better understand processes involved in snow dynamics and make for better adaptation to climate change scenarios. Moreover, offering the possibility of exploiting the information to other ﬁelds provides, as INARCH does, the opportunity of establishing new collaboration networks to push forward the frontiers of science in mountain areas. ages can be binarized to create daily snow presence–absence maps. This information can also be used for other applica- tions, such as to observe the growth timing of plant species. Since the binarized snow presence–absence maps were recorded on almost a daily frequency (note that about 20 % of all photographs from the camera had to be discarded because cloud or snow obscured the camera lens), many parameters can be derived from this information, including the SCA tem- poral evolution, the numbers of days with snow presence or the melt-out date (MOD) on each pixel. Figure 7 shows an example of the number of days with snow presence for the 2011/12 and 2012/13 snow seasons. 5 Data availability The database presented and described in this arti- cle is available for download at Zenodo (Revuelto et al., 2017; https://doi.org/10.5281/zenodo.848277). Meteoro- logical data of the AWS are given in .csv format. The mete- orological data set includes observations at 10 min intervals. The TLS survey snow depth distribution maps are available online (one ﬁle for each TLS acquisition). These ﬁles are in ASCII format in the UTM 30T north coordinate system with a 1 m × 1 m spatial resolution. The DEM of the study area is also provided in same coordinate system. Photographs of cloud-free days from the time-lapse camera are available in the online repository, with the correspondence of pixel ground control points to GPS coordinates. Information on the optics and chip size of the camera is also provided. Ad- ditionally, all available MOD distribution maps (last Julian day with snow presence on each pixel) are included in the database. Competing interests. The authors declare that they have no con- ﬂict of interest. Special issue statement. This article is part of the special is- sue “Hydrometeorological data from mountain and alpine research catchments”. It is not associated with a conference. www.earth-syst-sci-data.net/9/993/2017/ 4.2 Snow-covered area from time-lapse photographs The camera obtained three pictures per day (time-lapse photogra- phy) at 10:00, 11:00 and 12:00 UTC, ensuring good illumi- nation of the area. Figure 6 contains four photographs from the 2012/13 snow season, showing how the SCA evolved in time. pio (2004) and the speciﬁc features of the methodology ap- plied in the Izas Experimental Catchment are fully described in Revuelto et al. (2016a). The routines applied ﬁrst make a viewing transformation allowing for the optics of the camera and, second, a perspective projection, providing a virtual im- age of the DEM. Therefore, in the second step, the correspon- dence of ground control points with the pixels of the photo- graph must be established. Since this stage is quite sensitive, the coordinates of ground control points were acquired with a differential GPS. With this process, images projected into the DEM had a 3.3 pixel performance in the calibration of the transformation. Finally, the daily series of the projected im- The pictures can be projected into a digital elevation model (DEM) of the study site. Projecting the pictures into the 1 m × 1 m DEM for an entire snow season provides dis- tributed information on the evolution of the SCA in the same reference system as snow depth maps. The approach for pro- jecting the pictures into the DEM is described by Corri- Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees References ADCON Telemetry Company: Adcon BP1 Barometric Pressure Sensor, Copyright © 2017 Adcon Telemetry, OTT Hydromet GmbH, Klosterneuburg, Austria, 2017. Geonor A/S: Geonor T-200B series, All-weather precipitation gauges speciﬁcations, © 2010 Geonor A/S, Oslo, Norway, 2010. 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Campbell Scientiﬁc Ltd: SR50A Sonic Ranging Sensor, User Guide, Copyright © 2007–2011 Campbell Scientiﬁc Ltd, Camp- bell Park, Shepshed, Loughborough, UK, 2011. López-Moreno, J. I. and Nogués-Bravo, D.: Interpolating local snow depth data: an evaluation of methods, Hydrol. Process., 20, 2217–2232, 2006. Campbell Scientiﬁc Ltd: 107 temperature Probe, User Guide, Copy- right © 2003–2012 Campbell Scientiﬁc Ltd, Campbell Park, Shepshed, Loughborough, UK, 2012. López-Moreno, J. I. and Vicente-Serrano, S. 6 Summary The Izas Experimental Catchment is a well-established study area on the south face of the Pyrenees, in which different me- teorological and snow variables are automatically acquired. Earth Syst. Sci. Data, 9, 993–1005, 2017 www.earth-syst-sci-data.net/9/993/2017/ J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees J. Revuelto et al.: Snow and meteorological data in the Central Pyrenees 1004 Acknowledgements. This study was funded by the research projects CGL2014-52599-P “Estudio del manto de nieve en la mon- taña española y su respuesta a la variabilidad y cambio climatico” (Ministry of Economy and Development, MINECO) and CLIMPY “Characterization of the evolution of climate and provision of information for adaptation in the Pyrenees” (FEDER-POCTEFA). The authors are thankful for this unique opportunity to share information through the International Network for Alpine Research Catchment Hydrology (INARCH). Jesús Revuelto is supported by a postdoctoral Fellowship from the AXA research fund 2016 (le Post-Doctorant Jesús Revuelto est bénéﬁciaire d’une bourse postdoctorale du Fonds AXA pour la Recherche). Cesar Azorin- Molina is supported by the Marie Skłodowska-Curie Individual Fellowship (STILLING project – 703733) funded by the European Commission. Acknowledgements. This study was funded by the research projects CGL2014-52599-P “Estudio del manto de nieve en la mon- taña española y su respuesta a la variabilidad y cambio climatico” (Ministry of Economy and Development, MINECO) and CLIMPY “Characterization of the evolution of climate and provision of information for adaptation in the Pyrenees” (FEDER-POCTEFA). The authors are thankful for this unique opportunity to share information through the International Network for Alpine Research Catchment Hydrology (INARCH). Jesús Revuelto is supported by a postdoctoral Fellowship from the AXA research fund 2016 (le Post-Doctorant Jesús Revuelto est bénéﬁciaire d’une bourse postdoctorale du Fonds AXA pour la Recherche). Cesar Azorin- Molina is supported by the Marie Skłodowska-Curie Individual Fellowship (STILLING project – 703733) funded by the European Commission. 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https://openalex.org/W2166596743	https://nutritionandmetabolism.biomedcentral.com/counter/pdf/10.1186/1743-7075-8-78	English	null	Effects of 28 days of resistance exercise while consuming commercially available pre- and post-workout supplements, NO-Shotgun® and NO-Synthesize® on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers in males	Nutrition & metabolism	2,011	cc-by	7,875	Effects of 28 days of resistance exercise while consuming commercially available pre- and post- workout supplements, NO-Shotgun® and NO- Synthesize® on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers in males Effects of 28 days of resistance exercise while consuming commercially available pre- and post- workout supplements, NO-Shotgun® and NO- Synthesize® on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers in males * Correspondence: darryn_willoughby@baylor.edu 1Department of Health, Human Performance, and Recreation, Baylor University, Box 97313, Waco, TX76798, USA Full list of author information is available at the end of the article © 2011 Spillane et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: darryn_willoughby@baylor.edu 1Department of Health, Human Performance, and Recreation, Baylor University, Box 97313, Waco, TX76798, USA Full list of author information is available at the end of the article © 2011 Spillane et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH Open Access Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Spillane et al. Nutrition & Metabolism 2011, 8:78 Abstract Purpose: The effects of 28 days of heavy resistance training while ingesting the pre- and post-workout supplements, NO-Shotgun® and NO-Synthesize® were determined on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers. Methods: Nineteen non-resistance-trained males participated in a resistance training program 4 times/week for 28 days while either ingesting 27 g/day of carbohydrate (CARB) or NO-Shotgun® 30 min pre-exercise and 27 g/day of carbohydrate or NO- Synthesize® 30 min post-exercise (NOSS). Data were analyzed with separate 2 × 2 ANOVA (p < 0.05). Results: Total body mass was increased in both groups (p = 0.001), but not different between groups. Fat mass was unchanged with CARB, but NOSS decreased fat mass (p = 0.026). Both groups increased fat-free mass (p = 0.001); however, the increases were greater with NOSS (p = 0.023). NOSS underwent greater increases in upper-body (p = 0.023) and lower-body (p = 0.035) strength than CARB. Myofibrillar protein significantly increased in both groups (p = 0.041), with NOSS being greater than CARB (p = 0.049). All of the MHC isoforms were significantly increased in both groups; however, NOSS was greater than CARB for MHC 1 (p = 0.013) and MHC 2A (p = 0.046). All of the myogenic regulatory factors were significantly increased in both groups; however, NOSS was greater than CARB for Myo-D (p = 0.038) and MRF-4 (p = 0.001). For the whole blood and serum clinical chemistry markers, all variables remained within normal clinical ranges. Conclusions: Heavy resistance training for 28 days, with NO-Shotgun® and NO-Synthesize® ingested before and after exercise, respectively, significantly improved body composition and increased muscle mass and performance without abnormally impacting any of the clinical chemistry markers. Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 2 of 11 Testing sessions The study included baseline testing at day 0 and a fol- low-up testing session at day 29 in which blood and muscle samples were obtained and where body compo- sition and muscle performance tests were performed. Participants Nineteen apparently healthy, recreationally active, non- resistance trained [no consistent (at least thrice weekly) resistance training for one year prior to the study] males with an average age of 22.8 ± 4.67 yr, height of 179.5 ± 6.38 cm, and total body mass of 79.1 ± 16.13 kg com- pleted the study. Enrollment was open to men of all eth- nicities. All participants passed a mandatory medical screening. Participants with contraindications to exercise as outlined by the American College of Sports Medicine and/or who had consumed any nutritional supplements (excluding multi-vitamins) such creatine monohydrate, nitric oxide-stimulating, hydroxy-beta-methylbutyrate (HMB), various androstenedione derivatives, or pharma- cologic agents such as anabolic steroids three months prior to the study were not allowed to participate. All eligible participants signed a university-approved informed consent document based on the guidelines set forth by the Institutional Review Board for the Protec- tion of Human Subjects of Baylor University. Addition- ally, all experimental procedures involved in this study conformed to the ethical considerations of the Helsinki Code. y As a result, many recent studies have chosen to pro- vide nutrients in close proximity (either before and/or after) to resistance exercise [11-14]. This concept of nutrient timing has been demonstrated in a 10 week- study in which a supplement comprised of protein, creatine, and glucose was given immediately before and after each resistance exercise session or in the morning and evening. Providing the supplement before and after exercise resulted in a greater improvement in muscle strength and mass, Type II muscle fiber cross- sectional area, and contractile protein content [14]. However, more recently it was shown that a protein supplement provided before and after resistance exer- cise for 10 weeks was no more effective at increasing muscle strength and mass compared to when the pro- tein supplement was provided in the morning and eve- ning [7]. As such, there appears to be disagreement in the literature regarding this nutritional timing strategy during resistance training, yet it continues to be con- sidered to be a more effective method of bolstering increases in muscle mass and strength compared to resistance training without pre- and/or post-exercise nutrient provision. Introduction [15], and using the same experimental design in the pre- sent study, we wanted to also provide a nutritional sup- plement post-exercise to compare the effects compared to carbohydrate. Heavy resistance training augments muscle protein synthesis [1-3], thereby resulting in increases in muscle strength and hypertrophy [4-6]. It has been suggested that the ingestion of specific nutrients (e.g., protein, amino acids, carbohydrate, creatine, etc.) [7-10], or a combination of nutrients (i.e., protein+carbohydrate, protein+carbohydrate+creatine, protein+amino acids, etc.) [11-13] within approximately one hour before and/ or after resistance exercise will augment substrate avail- ability that is necessary during exercise and several hours into the recovery period. The ingestion of either protein or creatine before and after resistance exercise for 16 weeks was shown to be more effective in increas- ing muscle strength and satellite cell activation than resistance training without nutrient provision [9]. We have shown that ingesting protein (whey and casein) and amino acids before and after resistance exercise for 10 weeks resulted in significantly greater increases in muscle strength and mass compared to iso-caloric car- bohydrate [13]. Therefore, the purpose of this study was to compare the effects of four weeks of heavy resistance training performed in conjunction with either carbohydrate or NO-Shotgun® before and NO-Synthesize® after each exercise session on muscle strength, body composition, markers of protein synthesis, and clinical safety markers in men. Resistance-training protocol Based on our previous study [15], participants com- pleted a periodized 28-day resistance-training program split into two upper-extremity and two lower-extremity exercise sessions each week. This constituted a total of 16 exercise sessions, with eight upper-body and eight lower-body exercise sessions. Prior to each exercise ses- sion, participants performed a standardized series of stretching exercises. The participants then performed an upper-extremity resistance-training program consisting of nine exercises (bench press, lat pull, shoulder press, seated rows, shoulder shrugs, chest flies, biceps curl, triceps press down, and abdominal curls) twice per week and a program consisting of seven lower-extremity exer- cises (leg press or squat, back extension, step ups, leg curls, leg extension, heel raises, and abdominal crunches). Participants performed three sets of 10 repe- titions at 70 - 80% 1-RM. Rest periods were two min between exercises and sets. The resistance exercise ses- sions were not supervised; however, it was required that each participant completed detailed daily resistance- training logs. Whole blood and serum clinical chemistry analyses o b ood a d u a t y a a y Whole blood was collected and immediately analyzed for standard cell blood counts with percentage differen- tials (hemoglobin, hematocrit, RBC, MCV, MCH, MCHC, RDW, WBC counts, neutrophils, lymphocytes, monocytes, eosinophils, basophils and leukocyte differ- entials) using an automated hematology analyzer (Sysmex XS-1000i, Mundelein, IL). The instrument’s flow system was primed and the background counts checked daily to ensure appropriate RBC and WBC Strength assessment Upper- and lower-body one repetition maximum (1- RM) strength tests were performed using the free weight bench press and angled leg press exercises (Nebula, Ver- sailles, OH), respectively. Initially, an estimated 50% 1- RM was utilized to complete 5 to 10 repetitions. After a two min rest period, a load of 70% of estimated 1-RM was utilized to perform 3 to 5 repetitions. Weight was gradually increased until a 1-RM was reached with each following lift, with a two min rest period in between Recently we conducted a study to determine the effects of an alleged pre-workout supplement and demonstrated that four weeks of heavy resistance train- ing in conjunction with the provision of the nutritional supplement, NO-Shotgun®, 30 min prior to each exer- cise session was more effective at increasing muscle strength and mass and markers indicative of muscle protein synthesis and satellite cell activation when com- pared to carbohydrate [15]. Based on our previous study Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 3 of 11 Synthesize® within 30 min following exercise. Immedi- ately upon waking on non-training days, CARB ingested 27 g of the supplement, whereas NOSS ingested 27 g of NO-Synthesize®. For supplementation compliance, participants completed questionnaires and returned empty containers during the post-study test- ing session on day 29. each successful lift. Test-retest reliability of performing these strength assessments on subjects within our laboratory during the previous year has demonstrated low mean coefficients of variation and high reliability for the bench press (1.7%, intra-class r = 0.92) and leg press (0.72%, intra-class r = 0.93), respectively. Dietary monitoring I d i Total body mass (kg) was determined on a standard dual beam balance scale (Detecto Bridgeview, IL). Per- cent body fat, fat mass, and fat-free mass were deter- mined using DEXA (Hologic Discovery Series W, Waltham, MA). Quality control calibration procedures were performed on a spine phantom (Hologic X-CALI- BER Model DPA/QDR-1 anthropometric spine phan- tom) and a density step calibration phantom prior to each testing session. The DEXA scans were segmented into regions (right & left arm, right & left leg, and trunk). Each of these segments was analyzed for fat mass, lean mass, and bone mass. Based on previous quality control testing in our laboratory from the pre- vious year, the accuracy of the DEXA for body composi- tion assessment is ± 2.3% as assessed by direct comparison with hydrodensitometry and scale weight. Total body water volume was determined by bioelectric impedance analysis (Xitron Technologies Inc., San Diego, CA) using a low energy, high frequency current (500 micro-amps at a frequency of 50 kHz). In order to monitor dietary intake, participants were required to record their food and drink intake for four days prior to each of the two testing sessions at day 0 and day 29. For standardization purposes, participants’ diets were not standardized and subjects were asked not to change their dietary habits during the course of the study. The four-day dietary recalls were evaluated with the Food Processor IV Nutrition Software (ESHA, Salem OR) to determine the average daily macronutrient intake of fat, carbohydrate, and protein for the duration of the study. Venous blood sampling and percutaneous muscle biopsies Venous blood samples were obtained from the antecubi- tal vein into a 10 ml collection tube using a standard vacutainer apparatus. Blood samples were allowed to stand at room temperature for 10 min and then centri- fuged. The serum was removed and frozen at -80°C for later analysis. Percutaneous muscle biopsies (50-70 mg) were obtained from the middle portion of the vastus lateralis muscle of the dominant leg at the midpoint between the patella and the greater trochanter of the femur at a depth between 1 and 2 cm. After sample removal, adipose tissue was trimmed from the muscle specimens, immediately frozen in liquid nitrogen, and stored at -80°C for later analysis. Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Myogenic regulatory factor quantitation Myogenic regulatory factor quantitation The muscle protein expression of the MRFs was assessed through the use of ELISA [15]. Polyclonal anti- bodies specific for Myo-D, myogenin, and MRF-4 were purchased from Santa Cruz Biotech (Santa Cruz, CA). Initially, the antibodies were diluted to 1 μg/ml in coat- ing buffer (Na2CO3, NaHCO3, and ddH2O, pH 9.6) Myosin heavy chain isoform protein quantitation The MHC protein isoform composition within 20 μg muscle homogenates was determined under denaturing conditions using an Experion Pro260 automated electro- phoresis system (Bio-Rad, Hercules, CA) using the prin- ciples of SDS-PAGE and LabChip (Caliper Life Sciences, Hopkinton, MA) technology [13]. The Experion Pro260 analysis kit has a resolution and quantitation of 10-260 kDa proteins while also separating and detecting 2.5- 2,000 ng/μl protein. The Experion Pro260 system com- bines electrophoresis, staining, destaining, imaging, band detection, and basic data analysis into a single, automated step. Gel images were then processed and displayed on a computer monitor and MHC bands identified by migra- tion relative to the molecular weight marker (data not shown). The density of the MHC bands was determined using Experion Imaging software (Bio-Rad, Hercules, CA), expressed in arbitrary density units. Supplementation protocol Participants were assigned to a 28-day supplementation protocol, in a double-blind, placebo-controlled manner. Participants ingested either 54 g/day of maltodextrose (CARB) or 27 g/day of NO-Shotgun® and 27 g/day of NO-Synthesize® (NOSS). For CARB, 27 g were ingested 30 min prior to exercise and 27 g within 30 min following exercise. NOSS ingested 27 g of NO- Shotgun® 30 min prior to exercise and 27 g/day NO- Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 4 of 11 and allowed to incubate at room temperature overnight. Following incubation, the plates were washed (1X phos- phate buffered saline, Tween-20), blocked (10X phos- phate buffered saline, bovine serum albumin, ddH2O), washed, and then incubated with a secondary antibody (IgG conjugated to HRP) diluted to 1 μg/ml in dilution buffer (10X phosphate buffered saline, Tween-20, bovine serum albumin, ddH2O). After washing, a stabilized TMB chromogen was added and the plates were covered and placed in the dark for the last 30-min prior to being stopped with 0.2 M sulphuric acid. The subsequent absorbances, which were directly proportional to the concentration of the MRFs in the samples, were mea- sured at a wavelength of 450 nm. There were no stan- dards used in these ELISAs, thus no standard curve was created. Therefore, the absorbances relative to muscle weight were assessed. The overall intra-assay percent coefficients of variation were 7.12%, 6.47%, and 8.03% for Myo-D, myogenin, and MRF-4, respectively. linearity. Based on the quality control standards from the manufacturer, the coefficients of variation for the Sysmex XS-1000i were 0.82%, 0.84%, 0.026%, 0.75%, and 0.82% for neutrophils, lymphocytes, monocytes, eosino- phils, and basophils, respectively, and fell within the recommended ranges. Serum samples were out-sourced (Quest Diagnostics, Dallas, TX) and assayed for general clinical chemistry markers (total cholesterol, high-density lipoproteins, low-density lipoproteins, triglycerides, albumin, glucose, GGT, LDH, uric acid, BUN, creatinine, BUN/creatinine ratio, calcium, creatine kinase, total protein, total biliru- bin, ALP, ALT, and AST). Based on the methodology employed for analysis, the coefficients of variation for all analyses reported by Quest Diagnostics (Dallas, TX) were no greater than 6%. Serum IGF-1 analysis Serum samples were analyzed in duplicate for IGF-1 (Enzo Life Sciences, Plymouth Meeting, PA) and HGF (R&D Systems, Minneapolis, MN) using an ELISA. For IGF-1, this assay has a sensitivity of 34.20 pg/ml and does not cross-react with IGFBPs 2, 3, and 4, HGF, or insulin. For IGF-1, the subsequent absorbances, which were directly proportional to the concentration of ana- lyte in the sample, were measured at a wavelength of 450 nm using a microplate reader (iMark, Bio-Rad, Her- cules, CA). A set of standards of known concentrations for IGF-1 was utilized to construct a standard curve by plotting the net absorbance values of the standards against the respective peptide concentrations. By apply- ing a four-part parameter curve using data reduction software (Microplate Manager, Bio-Rad, Hercules, CA), the serum IGF-1 concentrations were calculated. The overall intra-assay percent coefficient of variation was 5.3% for IGF-1. Myofibrillar protein content Myofibrillar protein was isolated from the skeletal mus- cle cellular extracts with repeated incubations in 0.1% SDS at 50°C and separated by centrifugation. Myofibril- lar protein content was determined spectrophotometri- cally based on the Bradford method at a wavelength of 595 nm [13,15]. A standard curve was generated (R = 0.99, p = 0.001) using bovine serum albumin (Bio-Rad, Hercules, CA), and myofibrillar protein content was expressed relative to muscle wet-weight. Skeletal muscle cellular extraction Each muscle sample was weighed and approximately 20 mg were homogenized using a commercial cell extrac- tion buffer (Biosource, Camarillo, CA) and a tissue homogenizer. The cell extraction buffer was supplemen- ted with 1 mM phenylmethanesulphonylfluoride (PMSF) and a protease inhibitor cocktail (Sigma Chemical Com- pany, St. Louis, MO) with broad specificity for the inhi- bition of serine, cysteine, and metallo-proteases. Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Muscle strength For muscle strength, both groups underwent significant increases with training; however, NOSS underwent greater increases in upper-body (p = 0.023) and lower- body (p = 0.035) strength compared to CARB (Table 2). Body composition l b d Twenty-two participants began the study; however, three were withdrawn due to reasons unrelated to the study. One participant contracted mononucleosis and another injured his knee and neither were able to exercise for several weeks. The third participant withdrew because he did not have an adequate amount of time in his schedule to remain compliant with the resistance train- ing program. As a result, 19 participants completed the study. The CARB group (n = 9) had an average (±SD) age of 20.00 ± 1.41 yr, height of 179.75 ± 6.22 cm, and total body mass of 81.43 ± 16.46 kg. The NOSS group (n = 10) had an age of 21.20 ± 1.98 yr, height of 178.00 ± 4.88 cm, and total body mass of 81.41 ± 29.39 kg. Total body mass was significantly increased in both groups with training (p = 0.001) with no significant changes occurring in total body water (p = 0.345). Fat mass was unchanged with CARB, but NOSS decreased fat mass (p = 0.026). Both groups increased fat-free mass with training (p = 0.001); however, the increases were greater with NOSS (p = 0.023) (Table 2). Reported side effects from supplements Participants reported by questionnaire at the testing ses- sion on day 29 how well they tolerated the supplemen- tation protocol, in addition to reporting any medical Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 5 of 11 Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 problems and/or symptoms they may have encountered during the study. problems and/or symptoms they may have encountered during the study. All participants appeared to have exhibited 100% com- pliance with the resistance training and supplementation protocol, and were able to complete the required dosing regimen and testing procedures. Over the course of the 28 days, two participants in CARB and three in NOSS reported side effects. For CARB, both participants reported feelings of nausea, one reported a rapid heart rate, and one reported shortness of breath. For NOSS, three participants reported dizziness, two reported feel- ings of nausea, three reported headache, two reported a rapid heart rate, one reported shortness of breath, and one reported nervousness. Dietary analysis, supplement compliance, and reported side effects Serum IGF-1 was significantly increased with training (p = 0.038); however, NOSS and CARB did not differ (p = 0.385) (Table 3). The diet logs were used to analyze the average daily caloric and macronutrient consumption (Table 1). Neither group significantly increased their caloric intake during the course of the study. In addition, no signifi- cant differences existed between groups for total caloric (p = 0.129), protein (p = 0.216), carbohydrate (p = 0.106), and fat intake (p = 0.665). Statistical analysis Data were analyzed with separate 2 (group) × 2 (time) ANOVA with repeated measures on the second factor with SPSS 16.0 software (SPSS inc., Chicago, IL). Signifi- cant differences among groups were identified by a Tukey HSD post-hoc test. A probability level of < 0.05 was adopted throughout. Data are presented as means and standard deviations. Myogenic regulatory factors All of the myogenic regulatory factors were significantly increased in both groups; however, NOSS was greater All of the myogenic regulatory factors were significantly increased in both groups; however, NOSS was greater Table 1 Dietary caloric and macronutrient intake for the CARB and NOSS groups Variable Group Day 0 Day 29 Test (p < .05) Group x Test (p < .05) Total Calories (kcal/kg) .683 .129 NOSS 30.12 ± 9.94 31.61 ± 10.58 CARB 41.81 ± 18.98 35.41 ± 16.16 Protein (g/kg) .763 .216 NOSS 1.17 ± 0.33 1.25 ± 0.42 CARB 1.57 ± 0.77 1.31 ± 0.43 Carbohydrate (g/kg) .932 .106 NOSS 3.64 ± 1.39 4.01 ± 1.41 CARB 5.03 2.82 4.13 2.22 Fat (g/kg) .551 .665 NOSS 1.17 ± 0.41 1.15 ± 0.44 CARB 1.47 ± 0.64 1.27 ± 0.77 D t t d d t d d d i ti Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 6 of 11 Table 2 Body composition and muscle strength variables for the CARB and NOSS groups Variable Group Day 0 Day 29 Test (p < .05) Group x Test (p < .05) Body Mass (kg) .010* .793 NOSS 81.41 ± 16.46 82.64 ± 15.97 CARB 84.41 ± 29.39 85.44 ± 29.32 Body Water (kg) .345 .587 NOSS 43.07 ± 10.33 43.43 ± 9.68 CARB 43.18 ± 6.50 44.12 ± 7.13 Body Fat (%) .026* .014† NOSS < PLC NOSS 17.88 ± 7.67 16.53 ± 7.35 CARB 21.18 ± 9.06 21.26 ± 9.55 Fat Mass (kg) .046* .026† NOSS < PLC NOSS 14.21 ± 8.31 13.40 ± 8.10 CARB 18.64 ± 18.16 18.99 ± 18.64 Fat-Free Mass (kg) .001* .023† NOSS > PLC NOSS 57.80 ± 8.01 59.92 ± 7.57 CARB 56.43 ± 10.33 57.02 ± 9.86 Upper-Body Strength (kg/kg) .016* .023† NOSS > PLAC NOSS 1.03 ±0.15 1.16 ±0.21 CARB 1.07 ±0.25 1.08 ± 0.23 Lower-Body Strength (kg/kg) .001* .035† NOSS > PLAC NOSS 4.04 ± 0.55 4.90 ± 0.64 CARB 4.19 ± 0.58 4.64 ± 0.84 Data are presented as means and standard deviations. * Denotes a significant increase at Day 29 compared to Day 0. † Denotes a significant difference between CARB and NOSS. than CARB for Myo-D (p = 0.038) and MRF-4 (p = 0.001) (Table 3). than CARB for Myo-D (p = 0.038) and MRF-4 (p = 0.001) (Table 3). Myofibrillar protein and MHC isoforms Myofibrillar protein significantly increased in both groups with training (p = 0.041), with NOSS being greater than CARB (p = 0.049). All of the MHC iso- forms were significantly increased in both groups with training; however, NOSS was greater than CARB for MHC 1 (p = 0.013) and MHC 2A (p = 0.046) (Table 3). Our rationale to use carbohydrate as a comparator was based on the premise that there is empirical evi- dence to suggest that carbohydrate supplementation prior to and after resistance exercise results in the maintenance of muscle glycogen [16], in addition to the fact that the insulin response associated with carbohy- drate ingestion up-regulates signal transduction path- ways in muscle which can activate muscle-specific gene expression and protein synthesis [17]. Therefore, many recent studies have provided nutrient provision in close proximity (either before and/or after) to resistance exer- cise and, in so doing, have used carbohydrate as a com- parator based on the premise that carbohydrate provided in conjunction with other nutrients such as protein [17], amino acids [18], and creatine [11] has Myogenic regulatory factors before and after resistance exercise, respectively, and in conjunction with heavy resistance training, is more effective than carbohydrate at increasing fat-free mass, muscle strength and mass, and markers of muscle pro- tein synthesis in untrained males, while having no effect on whole blood and serum clinical safety markers. In regard to the various ingredients contained in both sup- plements, based on previous research it is conceivable that the primary active ingredients are whey protein, creatine, leucine, beta-alanine, and KIC. Serum and whole blood clinical chemistry markers Serum creatinine was significantly increased with train- ing (p = 0.016), but was not different between groups (p = 0.413). In addition, basophils were significantly less at Day 29 for NOSS (p = 0.05). Regarding all other serum and whole blood clinical chemistry markers assessed, there were no significant changes due to training or between groups (p > 0.05), and all variables remained within normal clinical ranges throughout the duration of the study (Tables 4 and 5). Discussion h l The results of the present study indicate that NO-Shot- gun® and NO-Synthesize® supplementation provided Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 7 of 11 Table 3 Serum and muscle markers indicative of muscle protein synthesis for the CARB and NOSS groups Variable Group Day 0 Day 29 Test (p < .05) Group x Test (p < .05) IGF-1 (pg/ml) .038* .385 NOSS 3491.53 ± 597.34 3609.63 ± 497.11 CARB 3018.43 ± 690.91 3339.24 ± 70.94 Myofibrillar Protein (μg/mg) .041* .049† NOSS > PLC NOSS 0.089 ± 0.019 0.115 ± 0.033 CARB 0.087 ± 0.128 0.092 ± 0.022 MHC 1 (arbitrary density units) .001* .013† NOSS > PLC NOSS 1072.93 ± 206.16 1582.37 ± 247.55 CARB 1114.95 ± 448.29 1381.76 ± 423.04 MHC 2A (arbitrary density units) .001* .046† NOSS > PLC NOSS 904.06 ± 500.22 1502.84 ± 412.07 CARB 944.11 ± 458.98 1385.97 ± 310.87 MHC 2X (arbitrary density units) .003* .244 NOSS 878.45 ± 328.28 731.70 ± 266.26 CARB 979.89 ± 226.74 676.78 ± 163.27 Myo-D (Abs/mg) .005* .038† NOSS > PLC NOSS 1.72 ± 0.491 2.03 ± 0.399 CARB 1.65 ± 0.339 1.74 ± 0.462 Myogenin (Abs/mg) .017* .091 NOSS 1.63 ± 0.398 1.85 ± 0.422 CARB 1.57 ± 0.240 1.78 ± 0.405 MRF-4 (Abs/mg) .001* .001† NOSS > PLC NOSS 1.87 ± 0.236 2.25 ± 0.247 CARB 1.83 ± 0.005 1.97 ± 0.003 Data are presented as means and standard deviations. * Denotes a significant increase at Day 29 compared to Day 0. † Denotes a significant difference between CARB and NOSS. been shown to augment the responses to resistance training. and amino acids [13], and whey protein and amino acids [12] produces greater increases in muscle strength. Our results demonstrated that both NOSS and CARB significantly increased total body mass (p = 0.001) with no associated increases in total body water (p = 0.345). Additionally, fat-free mass was increased in both groups (p = 0.001) with NOSS demonstrating significantly greater improvements than CARB (p = 0.023). These findings for NOSS are similar to our previous study [15] as well as a study that observed 12 weeks of heavy resis- tance training and creatine supplementation to induce a greater increase in fat-free mass compared to the carbo- hydrate group [19]. Discussion h l In addition, 10 weeks of heavy resis- tance training and whey protein and amino acid supplementation resulted in greater increases in fat-free mass compared to carbohydrate [13]. Our present results demonstrated that NOSS supple- mentation results in preferential increases in myofibrillar (p = 0.049) protein and Type I (p = 0.013) and IIa MHC (p = 0.046) when compared to carbohydrate. Our results are similar to a study in which creatine supplementation in conjunction with 12 weeks of resistance training resulted in an increase in myofibrillar protein and MHC isoform content when compared to carbohydrate [19]. Additionally, a protein and amino acid supplement ingested in concert with 10 weeks of heavy resistance training induced a greater increase in myofibrillar pro- tein compared to carbohydrate [13]. As with our previous study [15], we observed serum IGF-1 to be increased with heavy resistance training after four weeks; however, there was no difference between groups (p = 0.385). Previous studies have demonstrated heavy resistance training to either increase [20] or have no effect [21] on serum IGF-1. We have previously shown that 10 weeks of heavy resistance training combined with a daily supplement containing Increases in both upper- (p = 0.023) and lower-body (p = 0.035) muscle strength were significantly greater in NOSS compared to CARB. The present data are corroborated by our previous study [15], along with previous other studies which have demonstrated heavy resistance training, when combined with creatine [9,19] whey and casein protein Spillane et al. Data are presented as means and standard deviations. * Denotes a significant increase at Day 29 compared to Day 0. Discussion h l Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 8 of 11 Table 4 Serum clinical chemistry markers for the CARB and NOSS groups Variable Group Day 0 Day 29 Test (p < .05) Group x Test (p < .05) Glucose (mg/dL) .142 .315 NOSS 82.00 ± 4.98 83.00 ± 11.45 CARB 81.80 ± 6.52 86.90 ± 5.56 BUN (mg/dL) .117 .117 NOSS 14.80 ± 3.42 14.80 ± 3.22 CARB 15.33 ± 2.82 12.88 ± 3.85 Creatinine (mg/dL) .016* .413 NOSS 0.896 ± 0.150 0.949 ± 0.145 CARB 0.916 ± 0.087 1.07 ± 0.201 Sodium (mmol/L) .304 .681 NOSS 137.40 ± 2.79 139.72 ± 2.26 CARB 136.67 ± 2.06 137.67 ± 9.08 Potassium (mmol/L) .107 .671 NOSS 4.06 ± 0.177 4.34 ± 0.568 CARB 3.95 ± 0.274 4.12 ± 0.446 Chloride (mmol/L) .665 .561 NOSS 101.70 ± 2.79 103.24 ± 2.85 CARB 101.33 ± 2.64 101.11 ± 8.47 CO2 (mmol/L) .787 .349 NOSS 21.40 ± 1.07 22.40 ± 1.07 CARB 20.11 ± 2.20 19.55 ± 4.90 Calcium (mg/dL) .799 .247 NOSS 9.27 ± 0.577 9.58 ± 0.322 CARB 9.28 ± 0.481 9.07 ± 1.24 Protein (mg/dL) .914 .336 NOSS 6.79 ± 0.645 6.95 ± 0.353 CARB 6.85 ± 0.657 6.65 ± 1.18 Albumin (mg/dL) .653 .244 NOSS 4.41 ± 0.338 4.50 ± 0.205 CARB 4.48 ± 0.341 4.28 ± 0.695 Globulin (mg/dL) .622 .622 NOSS 2.38 ± 0.410 2.45 ± 0.310 CARB 2.36 ± 0.393 2.36 ± 0.574 Albumin/Globulin .157 .436 NOSS 1.89 ± 0.237 1.87 ± 0.231 CARB 1.93 ± 0.300 1.86 ± 0.304 Bilirubin (mg/dL) .181 .465 NOSS 0.550 ± 0.295 0.440 ± 0.171 CARB 0.667 ± 0.282 0.633 ± 0.400 ALP (U/L) .066 .816 NOSS 52.30 ± 12.84 56.40 ± 18.36 CARB 58.55 ± 15.56 63.77 ± 21.77 AST (U/L) .982 .403 NOSS 17.80 ± 4.15 17.40 ± 4.67 CARB 17.55 ± 5.07 17.88 ± 4.59 ALT (U/L) .785 .785 NOSS 10.70 ± 4.16 10.70 ± 6.26 CARB 7.66 ± 1.93 8.33 ± 5.63 Data are presented as means and standard deviations. * Denotes a significant increase at Day 29 compared to Day 0. Table 4 Serum clinical chemistry markers for the CARB and NOSS groups Spillane et al. Discussion h l Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 10 of 11 Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 10 of 11 The MRFs (Myo-D, myogenin, MRF-4, myf5) are tran- scription factors that play a role in muscle hypertrophy by binding to E-boxes in the promoter region of various sarcomeric genes [4], thereby up-regulating transcription which can invariably lead to an increase in protein synthesis. It appears that myogenin and MRF-4 specifi- cally up-regulate the expression of genes specific to con- tractile protein [26,27] and fast and slow muscle fiber differentiation [28]. Type I and II muscle fibers have been shown to preferentially accumulate myogenin and Myo-D, respectively [26]. Creatine supplementation in conjunction with resistance training has been shown to increase MyoD, myogenin, and MRF-4 that were corre- lated with increased MHC and myofibrillar protein [29] and myofiber size [30]. In line with our previous studies, the present results demonstrated that both groups underwent significant increases in MRF content [15,29] and all three MHCs [15,19]. However, NOSS underwent even greater increases in Myo-D (p = 0.038) and MRF-4 (p = 0.001) and Type 1 and 2A MHC. It is difficult to conclude which specific ingredient elicited these results; however, based on previous research we can speculate a role for creatine since 12 weeks of supplementation and heavy resistance training resulted in muscle hypertrophy, along with concomitant increases in MHC Type 1, 2A, and 2X protein, and myofibrillar protein [19]. respectively, with neither being different from placebo. Myofibrillar protein was preferentially increased by 70.39% in the NO group in our previous study (15) and 29.21% in our present study. For the MRFs, in our pre- vious study, they were preferentially increased in the NO group by 70.91%, 56.24%, and 71.17% for Myo-D, MRF-4, and myogenin, respectively. While still preferen- tially increased in the NOSS group in our present study, Myo-D, MRF-4, and myogenin increased 18.02%, 20.32%, and 13.49%. NO-Shotgun® and NO-Synthesize® contain a proprie- tary blend of a number of compounds assumed to be effective at increasing muscle strength and mass such as creatine, arginine, glutamine, beta-alanine, keto-iso- caproate (KIC), and leucine, casein and whey protein, branched-chain amino acids, lysine, phenylalanine, threonine, histidine, and methionine. As a result, attempting to isolate which specific ingredient has the greatest impact on our outcome measures is not feasi- ble. Authors’ contributions All researchers involved independently collected, analyzed, and interpreted the results from this study. MS assisted in coordination of the study, data acquisition, in performing the statistical analysis, and drafting the manuscript. NS, SL, TC, MM, and VL participated in the data acquisition. DSW conceived the study, developed the study design, secured the funding for the project, assisted and provided oversight for all data acquisition and statistical analysis, assisted and provided oversight in drafting the manuscript, and served as the faculty mentor for the project. All authors have read and approved the final manuscript. Author details 1 Author details 1Department of Health, Human Performance, and Recreation, Baylor University, Box 97313, Waco, TX76798, USA. 2Institute for Biomedical Studies, Baylor University, Waco, TX87898, USA. 1Department of Health, Human Performance, and Recreation, Baylor University, Box 97313, Waco, TX76798, USA. 2Institute for Biomedical Studies, Baylor University, Waco, TX87898, USA. Discussion h l However, our present results indicate that supple- mentation protocol of providing NO-Shotgun® pre- exercise, NO-Synthesize® post-exercise, and NO-Synthe- size® on non-exercise days for 28 days is more effective than carbohydrate at increasing muscle mass and strength and markers indicative of muscle protein synth- esis, while having no negative impact on the clinical chemistry variables assessed. Furthermore, our present results demonstrate preferential improvements in mus- cle strength and mass and agree with our previous study [15], and suggest that nutrient provision before and after resistance exercise is effective in preferentially aug- menting muscle strength and mass. As with most nutritional supplements, NO-Shotgun® and NO-Synthesize® are comprised of a number of differ- ent compounds with most having no little, if any, clinical safety data available. During the course of the study, we observed no significant changes beyond the normal clini- cal ranges in regard to clinical safety measures in either group. These data indicate that the ingestion of carbohy- drate, NO-Shotgun®, and NO-Synthesize® for a period of 28 days has no detrimental clinical effects with regard to the whole blood and serum variables assessed. Acknowledgements W ld lik h We would like to thank the individuals that participated as subjects in this study. A purpose of the present study was to compare the effects of NO-Shotgun® given pre-exercise and NO- Synthesize® given post-exercise to our previous study (15) in which only NO-Shotgun® was given pre-exercise to determine if additional post-exercise nutrient provi- sion would provide an augmented effect. Our present data showed that total body mass to increase by 1.51% and fat-free mass to increase 3.66% in the NOSS group. This mirrors the results observed in our previous study in which the NO-Shotgun® (NO) group increased 2.59% and fat-free mass 4.75% (15). In our previous study, upper- and lower-body strength increased 8.82% and 18.40%, respectively, with only lower-body being greater than placebo (15). However, our present results show a preferential increase of 12.62% and 21.28%, respectively, for upper- and lower-body strength with both being greater in NOSS. In our previous (15) and present study, serum IGF-1 increased 9.34% and 3.38%, Discussion h l Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Page 9 of 11 Table 5 Whole blood clinical chemistry markers for the CARB and NOSS groups Variable Group Day 0 Day 29 Test (p < .05) Group x Test (p < .05) WBC (106/L) .148 .300 NOSS 6.06 ± 1.32 5.91 ± 1.27 CARB 7.11 ± 1.74 6.28 ± 1.34 RBC (109/L) .243 .755 NOSS 4.99 ± 0.304 4.94 ± 0.257 CARB 4.94 ± 0.312 4.85 ± 0.302 HGB (g/dL) .100 .695 NOSS 15.24 ± 0.815 15.01 ± 0.750 CARB 14.96 ± 0.593 14.60 ± 0.563 HCT (%) .509 .997 NOSS 44.28 ± 2.12 43.92 ± 2.34 CARB 43.44 ± 1.81 43.08 ± 2.03 MCV (fL) .093 .226 NOSS 88.85 ± 3.90 88.90 ± 4.06 CARB 88.05 ± 4.00 88.86 ± 4.05 MCH (pg) .103 .886 NOSS 30.57 ± 1.31 30.42 ± 1.34 CARB 30.32 ± 1.85 30.14 ± 1.62 MCHC (g/dL) .015 .235 NOSS 34.42 ± 0.399 34.22 ± 0.482 CARB 34.45 ± 1.01 33.92 ± 1.03 Platelets (106/L) .243 .861 NOSS 211.24 ± 28.57 221.82 ± 38.27 CARB 228.89 ± 62.43 236.78 ± 77.51 Neutrophils (cells/μl) .178 .111 NOSS 2798.63 ± 1056.71 2868.65 ± 886.18 CARB 4038.93 ± 1448.59 3263.78 ± 767.91 Lymphocytes (cells/μl) .445 .566 NOSS 2512.93 ± 516.94 2344.63 ± 355.10 CARB 2164.57 ± 620.78 2139.84 ± 629.11 Monocytes (cells/μl) .613 .888 NOSS 536.70 ± 121.60 509.40 ± 124.70 CARB 599.11 ± 142.53 583.67 ± 263.01 Eosinophils (cells/μl) .602 .926 NOSS 186.40 ± 113.01 177.10 ± 143.54 CARB 287.33 ± 161.21 274.03 ± 183.27 Basophils (cells/μl) .293 .050† NOSS < PLC NOSS 25.70 ± 10.34 17.40 ± 8.51 CARB 21.88 ± 11.43 24.55 ± 11.10 Data are presented as means and standard deviations. † Denotes a significant difference between CARB and NOSS. Table 5 Whole blood clinical chemistry markers for the CARB and NOSS groups whey/casein protein and free amino acids increased cir- culating IGF-1 levels [13]. Even though serum IGF-1 was increased in the present study, we can conceivably conclude that none of the ingredients contained in the supplements ingested by both groups served as IGF-1 secretagogues. Even so, this outcome may not be germane to the results as hepatically-derived circulating IGF-1 appears to have no direct effect on muscle hyper- trophy [22] compared to skeletal-muscle derived IGF-I which has been shown to increase in response to resis- tance training [23] and induces muscular protein synth- esis [24,25]. doi:10.1186/1743-7075-8-78 / Cite this article as: Spillane et al.: Effects of 28 days of resistance exercise while consuming commercially available pre- and post- workout supplements, NO-Shotgun®® and NO-Synthesize®® on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers in males. Nutrition & Metabolism 2011 8:78. 10. Rankin J, Goldman L, Puglisi M, Nickols-Richardson S, Earthman C, Gwazdauskas F: Effect of post-exercise supplement consumption on adaptations to resistance training. J Am Coll Nutr 2004, 23:322-30. 11. Cribb P, Williams A, Hayes A: A creatine-protein-carbohydrate supplement enhances responses to resistance training. Med Sci Sports Exerc 2007, 39:1960-68. 12. Beck T, Housh T, Johnson G, Coburn J, Malek M, Cramer J: Effects of a drink containing creatine, amino acids, and protein combined with ten weeks of resistance training on body composition, strength, and anaerobic performance. J Strength Cond Res 2007, 21:100-04. 13. Willoughby D, Stout J, Wilborn C: Effects of resistance training and protein plus amino acid supplementation on muscle anabolism, mass, and strength. Amino Acids 2007, 32:467-77. 14. Cribb P, Hayes A: Effects of supplement timing and resistance exercise on skeletal muscle hypertrophy. Med Sci Sports Exerc 2006, 38:1918-25. 15. Shelmadine B, Cooke M, Buford T, Hudson G, Redd L, Leutholtz B, Willoughby D: Effects of 28 days of resistance exercise and consuming a commercially available pre-workout supplement, NO-Shotgun, on body composition, muscle strength and mass, markers of satellite cell activation, and clinical safety markers in males. J Int Soc Sports Nutr 2009, 5:6-16. 16. Haff G, Lehmkuhl J, McCoy B, Stone H: Carbohydrate supplementation and resistance training. J Strength Cond Res 2003, 17:187-96. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 17. Competing interests This study was supported by an independent research grant from VPX (Davie, FL) awarded to Baylor University. DSW has previously served as a paid consultant for VPX; however, he was not serving in this capacity during the time in which this study was being conducted, and has no financial interests concerning the outcome of the investigation. The authors declare that they have no competing interests. Page 11 of 11 Spillane et al. Nutrition & Metabolism 2011, 8:78 http://www.nutritionandmetabolism.com/content/8/1/78 Received: 22 September 2011 Accepted: 3 November 2011 Published: 3 November 2011 Received: 22 September 2011 Accepted: 3 November 2011 Published: 3 November 2011 trained young and older men and women. Am J Physiol Endocrinol Metab 2006, 291:E937-46. 21. McCall G, Byrnes W, Fleck S, Dickinson A, Kraemer W: Acute and chronic hormonal responses to resistance training designed to promote muscle hypertrophy. Can J Appl Physiol 1999, 24:96-107. References Buckingham M, Houzelstein D, Lyons G, Ontell M, Ott M, Sassoon D: Expression of muscle genes in the mouse embryo. 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Burke D, Chilibeck P, Davidson K, Candow D, Farthing J, Smith-Palmer T: The effect of whey protein supplementation with and without creatine monohydrate combined with resistance training on lean tissue mass and muscle strength. Int J Sport Nutr Exerc Metab 2001, 11:349-64. 30. Hespel P, Op’t Eijnde B, Van Leemputte M, Urso B, Greenhaff P, Labarque V, Dymarkowski S, Van Hecke P, Richter E: Oral creatine supplementation faciltates the rehabilitation of disuse atrophy and alters the expression of muscle myogenic factors in humans. J Physiol 2001, 536:625-35. faciltates the rehabilitation of disuse atrophy and alters the expression of muscle myogenic factors in humans. J Physiol 2001, 536:625-35. 9. Olsen S, Aagard P, Kadi F, Tufekovic G, Verney J, Olesen J, Suetta C, Kjaer M: Creatine supplementatin augments the increase in satellite cell and myonuclei number in human skeletal muscle induced by strength training. J Physiol 2006, 573:525-34. References 1. 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Florini J, Ewton D, Coolican S: Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 1996, 17:481-517. 25. Yang Y, Creer A, Jemiolo B, Trappe S: Time course of myogenic and metabolic gene expression in response to acute exercise in human skeletal muscle. J Appl Physiol 2005, 98:1745-1752. 4. Bergstrom D, Penn B, Strand A, Perry R, Rudnicki M, Tapscott S: Promoter- specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol Cell 2002, 9:587-600. 4. Bergstrom D, Penn B, Strand A, Perry R, Rudnicki M, Tapscott S: Promoter- specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol Cell 2002, 9:587-600. y 26. Mak K, Kong Y, Konieczny S: The MRF4 activation domain is required to induce muscle-specific gene expression. Mol Cell Biol 1992, 12:4224-46. 5. Bickel S, Slade J, Mahoney E, Haddad F, Dudley G, Adams G: Time course of molecular responses of human skeletal muscle to acute bouts of resistance exercise. J Appl Physiol 2005, 98:482-88. 5. Bickel S, Slade J, Mahoney E, Haddad F, Dudley G, Adams G: Time course of molecular responses of human skeletal muscle to acute bouts of resistance exercise. J Appl Physiol 2005, 98:482-88. 27. Yutzey K, Rhodes S, Konieczny S: Differential transactivation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4. Mol Cell Biol 1990, 10:3934-44. 6. doi:10.1186/1743-7075-8-78 Beelen M, Tieland M, Gijsen A, Vandereyt H, Kies A, Kuipers H, Saris W, Koopman R, van Loon L: Coingestion of carbohydrate and protein hydrolysate stimulates muscle protein synthesis during exercise in young men, with no further increase during subsequent overnight recovery. J Nutr 2008, 138:2198-204. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 18. Vieillevoye S, Poortmans J, Duchateau J, Carpentier A: Effects of a combined essential amino acids/carbohydrate supplementation on muscle mass, architecture and maximal strength following heavy-load training. Eur J Appl Physiol 2010, 110:479-88. 19. Willoughby D, Rosene J: Effects of oral creatine and resistance training on myosin heavy chain expression. Med Sci Sports Exerc 2001, 33:1674-81 20. Petrella J, Kim J, Cross J, Kosek D, Bamman M: Efficacy of myonuclear addition may explain differential myofiber growth among resistance- 20. Petrella J, Kim J, Cross J, Kosek D, Bamman M: Efficacy of myonuclear addition may explain differential myofiber growth among resistance-
https://openalex.org/W2945203438	https://europepmc.org/articles/pmc6567366?pdf=render	English	null	A Miniaturized Multiband Antenna Array for Robust Navigation in Aerial Applications	Sensors	2,019	cc-by	9,899	Received: 11 April 2019; Accepted: 14 May 2019; Published: 16 May 2019 Abstract: Satellite navigation is more and more important in a plethora of very diﬀerent application ﬁelds, ranging from bank transactions to shipping, from autonomous driving to aerial applications, such as avionics as well as unmanned aerial vehicles (UAVs). Due to the increasing dependency on satellite navigation, the need for robust systems able to counteract unintentional or intentional interferences is growing. When considering interference-robust designs; however, the complexity increases. Top performance is obtained through the use of multi-antenna receivers capable of performing spatial nulling in the direction of the interference signals. In particular, mobile applications (aeronautics, UAVs, automotive) have a substantial interest in robust navigation, but they also have the strongest constraints on the weight and available places for installation, with the use of bigger and heavier systems posing a substantial problem. In order to overcome this limitation, the present work shows a miniaturized ﬁve element (4+1) antenna array, which operates at the L1/E1 band (with array capability), as well as at the L5/E5 band (as a single antenna). The proposed antenna array is able to ﬁt into a 3.5-inch footprint, i.e., is compliant with the most widespread footprints for single antennas. Moreover, it is capable of multiband operation and meets the requirements of dual-frequency multi-constellation (DFMC) systems. Thanks to its extreme miniaturization and its compliance with current airborne single antenna footprints, the presented antenna array is suitable for easy integration in future aerial platforms, while enabling robustness and enhancing interference mitigation techniques using multi-antenna processing. Keywords: GNSS; antenna; antenna array; robustness; interference; UAV; jamming Keywords: GNSS; antenna; antenna array; robustness; interference; UAV; jamming sensors sensors Article A Miniaturized Multiband Antenna Array for Robust Navigation in Aerial Applications A Miniaturized Multiband Antenna Array for Robust Navigation in Aerial Applications Stefano Caizzone * , Georg Buchner, Mihaela-Simona Circiu , Manuel Cuntz, Wahid Elmarissi and Emilio Pérez Marcos Institute of Communications and Navigation, German Aerospace Center (DLR), 82234 Wessling, Germany; georg.buchner@dlr.de (G.B.); mihaela-simona.circiu@dlr.de (M.-S.C.); manuel.cuntz@dlr.de (M.C.); wahid.elmarissi@dlr.de (W.E.); emilio.perezmarcos@dlr.de (E.P.M.) * Correspondence: stefano.caizzone@dlr.de Stefano Caizzone * , Georg Buchner, Mihaela-Simona Circiu , Manuel Cuntz, Wahid Elmarissi and Emilio Pérez Marcos Stefano Caizzone , Georg Buchner, Mihaela-Simona Circiu , Manuel Cuntz, Wahid Elmarissi and Emilio Pérez Marcos Institute of Communications and Navigation, German Aerospace Center (DLR), 82234 Wessling, Germany; georg.buchner@dlr.de (G.B.); mihaela-simona.circiu@dlr.de (M.-S.C.); manuel.cuntz@dlr.de (M.C.); wahid.elmarissi@dlr.de (W.E.); emilio.perezmarcos@dlr.de (E.P.M.) * Correspondence: stefano.caizzone@dlr.de Wahid Elmarissi and Emilio Pérez Marcos Institute of Communications and Navigation, German Aerospace Center (DLR), 82234 Wessling, Germany; georg.buchner@dlr.de (G.B.); mihaela-simona.circiu@dlr.de (M.-S.C.); manuel.cuntz@dlr.de (M.C.); wahid.elmarissi@dlr.de (W.E.); emilio.perezmarcos@dlr.de (E.P.M.) * Correspondence: stefano.caizzone@dlr.de Institute of Communications and Navigation, German Aerospace Center (DLR), 82234 Wessling, Germany; georg.buchner@dlr.de (G.B.); mihaela-simona.circiu@dlr.de (M.-S.C.); manuel.cuntz@dlr.de (M.C.); wahid.elmarissi@dlr.de (W.E.); emilio.perezmarcos@dlr.de (E.P.M.) * Correspondence: stefano.caizzone@dlr.de 1. Introduction The use of satellite navigation is nowadays very widespread and embraces almost all ﬁelds of modern life [1]. Beyond being an incredible booster for location-based services, such ubiquitous use of satellite navigation also poses serious risks, due to the increasing dependency of safety-critical systems with respect to global navigation satellite systems (GNSS) [2]. Threats due to unintentional as well as intentional interferences can cause enormous damage [3], both in terms of costs and lives. Countermeasures are currently being deployed worldwide. Multi-antenna receivers could till now prove the best performance. They are able to place spatial nulls in the direction of the interference signals and therefore can limit their eﬀect on the position solution [4,5]. Such systems are capable of suppressing interferences by orders of magnitude stronger than the navigation signals; their drawback, however, is usually their complexity in terms of size, weight, and power consumption. Such limitation becomes a crucial point for mobile applications, such as airborne ones. Sensors 2019, 19, 2258; doi:10.3390/s19102258 Sensors 2019, 19, 2258 2 of 12 In order to overcome it, diﬀerent groups have lately been developing miniaturized antennas and receivers [6–9]; however, the antenna size was still bigger than commercial single antennas (which usually ﬁt into a footprint of 3.5 inches) and therefore their use in civilian airborne applications to date has been limited. The present work shows a miniaturized ﬁve element (4+1) antenna array, with four antennas operating at the L1/E1 band and a single antenna receiving L5/E5a signals. Such an array is able to ﬁt into a 3.5-inches footprint, and is therefore suitable for use in safety-of-life airborne applications, thanks to both its reduced dimensions and to the dual frequency capability which is needed, for example, for ionospheric corrections. The concept of the proposed antenna array will be shown first with measurements in anechoic chambers validating the simulation results. GNSS measurements performed with the antenna will also show its usability in the satellite navigation context. Moreover, its capability to enable interference suppression will be verified with the help of simulations with typical algorithms for interference mitigation. Finally, the installed performance of the antenna on top of a commercial aircraft and an octocopter will be analyzed through precise electromagnetic simulations, showing its capability to perform as good as current single antenna systems when used in reference mode (i.e., in the absence of interferences). 2. Antenna Array 3 of 12 3 of 12 Sensors 2019, 19, 2258 Sensors 2019, 19, x FOR Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 2. Simulated realized gain for the central antenna element: the solid line is the RHCP, while the dotted line is the Left Hand Circular Polarization (LHCP). Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 2. Simulated realized gain for the central antenna element: the solid line is the RHCP, whil the dotted line is the Left Hand Circular Polarization (LHCP). The antenna array has been manufactured (Figure 4) and tested both in a semi-anechoic Figure 2. Simulated realized gain for the central antenna element: the solid line is the RHCP, while the dotted line is the Left Hand Circular Polarization (LHCP). The antenna array has been manufactured (Figure 4) and tested both in a semi-anechoic near Fi 1 T i ( ) d id i (b ) f h Figure 1. Top view (top) and side view (bottom) of the antenna arr Figure 1 Top view (top) and side view (bottom) of the antenna array Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 1. Top view (top) and side view (bottom) of the antenna array. Fi 1 T i (t ) d id i (b tt ) f th t Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 1. Top view (top) and side view (bottom) of the antenna array. Figure 2. Simulated realized gain for the central antenna element: the solid line is the RHCP, while Figure 2. Simulated realized gain for the central antenna element: the solid line is the RHCP, while the dotted line is the Left Hand Circular Polarization (LHCP). Figure 2. Simulated realized gain for the central antenna element: the solid line is the RHCP, while the dotted line is the Left Hand Circular Polarization (LHCP). the dotted line is the Left Hand Circular Polarization (LHCP). 2. Antenna Array The antenna array has been manufactured (Figure 4) and tested both in a semi-anechoic near field chamber (Satimo Starlab) as well as in the bigger Compact Test Range far-field chamber, both available at DLR. Two different ground planes have been used, having a flat zone with a diameter of 40 cm and 122 cm, respectively; both of them have rolled edges to minimize diffraction effects from the edges of the ground plane. The bigger ground plane was manufactured following the specifications of DO-373 [11]: such a ground plane, however, is heavy and needs anechoic chambers with large quiet zones (as the Compact Test Range (CTR) chamber available on the DLR premises) to be measured. For the sake of comparison, a smaller ground plane (with a 40 cm flat zone diameter) The antenna array has been manufactured (Figure 4) and tested both in a semi-anechoic near field chamber (Satimo Starlab) as well as in the bigger Compact Test Range far-field chamber, both available at DLR. Two different ground planes have been used, having a flat zone with a diameter of 40 cm and 122 cm, respectively; both of them have rolled edges to minimize diffraction effects from the edges of the ground plane. The bigger ground plane was manufactured following the specifications of DO-373 [11]: such a ground plane, however, is heavy and needs anechoic chambers with large quiet zones (as the Compact Test Range (CTR) chamber available on the DLR premises) to be measured. For the sake of comparison, a smaller ground plane (with a 40 cm flat zone diameter) The antenna array has been manufactured (Figure 4) and tested both in a semi-anechoic near ﬁeld chamber (Satimo Starlab) as well as in the bigger Compact Test Range far-ﬁeld chamber, both available at DLR. Two diﬀerent ground planes have been used, having a ﬂat zone with a diameter of 40 cm and 122 cm, respectively; both of them have rolled edges to minimize diﬀraction eﬀects from the edges of the ground plane. The bigger ground plane was manufactured following the speciﬁcations of DO-373 [11]: such a ground plane, however, is heavy and needs anechoic chambers with large quiet zones (as the Compact Test Range (CTR) chamber available on the DLR premises) to be measured. For the sake of comparison, a smaller ground plane (with a 40 cm ﬂat zone diameter) was also manufactured. 2. Antenna Array The basic requirement for the antenna design is the standard 3.5 inches (~90 mm) footprint with four screws for installation. Due to the very limited space available, strong miniaturization of the single antennas, as well as an extremely reduced mutual distance between the elements, is required (Figure 1). In order to cope with the contrasting requirements of miniaturization and bandwidth, a dielectric resonator antenna technology has been chosen. Each antenna has two feeding pins that excite linear polarizations. Such pins are then connected to broadband hybrid circuits for the generation of Right Hand Circular Polarization (RHCP). The antenna design has been recently patented [10]. The antenna has been simulated using Ansys High Frequency Electromagnetic Field Simulation Software (HFSS). In the simulations, the antenna is surrounded by air. The antenna has been simulated using Ansys High Frequency Electromagnetic Field Simulation Software (HFSS). In the simulations, the antenna is surrounded by air. The simulated results for the antenna in terms of realized gain are shown in Figures 2 and 3, both for the central antenna (operating at L5/E5) and for one of the lateral antennas (covering the L1/E1 band). The simulated results for the antenna in terms of realized gain are shown in Figures 2 and 3, both for the central antenna (operating at L5/E5) and for one of the lateral antennas (covering the L1/E1 band). Moreover, the antenna can operate in an “array mode” when no interference is detected. In that case, the antenna outputs will be combined constructively in order to obtain a smoother pattern with higher gain levels. We will refer to it as “Mode 1”. Critical antenna design performance metrics like good matching and low mutual coupling between the antennas (with the maximum mutual S-parameter being ~−12 dBic at the L1/E1 central frequency) are met. RHCP gain at zenith is about 2.5 dBic for the central antenna at the L5/E5a central frequency, while at the L1/E1 band, it is about 2.6 dBic for Mode 1. The lateral antennas, when considered singularly, have an RHCP gain at the boresight of about −3.5 dBic at the L1/E1 central frequency (smaller than the central antenna due to coupling eﬀects, causing pattern distortion with maximum gain not being at the boresight anymore, as shown later) The axial ratio at the boresight of the central frequency is 1.6 dBic for the central antenna and 2.2 dBic for the lateral antennas, respectively. 2. Antenna Array In this case, the ground plane ﬁt into the smaller anechoic chambers such as the Starlab. 4 of 12 such as Sensors 2019, 19, 2258 was also manufact th St l b the Starlab. Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. Fi Si l d li d i f f h l l l ( d) d M d 1 (bl ) h Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. g g ( ) ( ) solid line is the RHCP, while the dotted line is the LHCP. Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-field chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, d i th l t ti t i th C t T t R t th DLR f iliti Figure 3. Simulated realized gain for one of the lateral antenna elements (red) and Mode 1 (blue): the solid line is the RHCP, while the dotted line is the LHCP. solid line is the RHCP, while the dotted line is the LHCP. Figure 4. 2. Antenna Array Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-field chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, during the electromagnetic measurement in the Compact Test Range at the DLR facilities. Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-ﬁeld chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, during the electromagnetic measurement in the Compact Test Range at the DLR facilities. Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-field chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-field chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, during the electromagnetic measurement in the Compact Test Range at the DLR facilities. Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-ﬁeld chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, during the electromagnetic measurement in the Compact Test Range at the DLR facilities. Figure 4. 2. Antenna Array Small diﬀerences between the measurements on the two ground planes appear mostly on the Moreover, the frequency trends appear similar on both ground planes. Small differences between the measurements on the two ground planes appear mostly on the Moreover, the frequency trends appear similar on both ground planes. Small differences between the measurements on the two ground planes appear mostly on the mall diﬀerences between the measurements on the two ground planes appear mostly on the components and when looking at the pattern cuts, as for instance in Figure 9. mall differences between the measurements on the two ground planes appear mostly on the omponents and when looking at the pattern cuts, as for instance in Figure 9. all differences between the measurements on the two ground planes appear mostly on the omponents and when looking at the pattern cuts, as for instance in Figure 9. Small diﬀerences between the measurements on the two ground planes appear mostly on LHCP components and when looking at the pattern cuts, as for instance in Figure 9. Small differences between the measurements on the two ground planes appear mostly on HCP components and when looking at the pattern cuts, as for instance in Figure 9. Small differences between the measurements on the two ground planes appear mostly on HCP components and when looking at the pattern cuts, as for instance in Figure 9. LHCP components and when looking at the pattern cuts, as for instance in Figure 9. LHCP components and when looking at the pattern cuts, as for instance in Figure 9. LHCP components and when looking at the pattern cuts, as for instance in Figure 9. They are due to a reﬂection happening on the surface of the big ground plane. This causes more ripples in the pattern at high elevations (Figure 9), which are however not due to the antenna intrinsic characteristics but only to the overlapping of the reﬂected waves with the ones originating by the antenna itself (The use of the small ground plane has indeed been suggested recently by the authors to standardize bodies as a more valid approach for the characterization of the antenna in a standalone conﬁguration, i.e., without the speciﬁc aeronautic platform on which it will be mounted). They are due to a reflection happening on the surface of the big ground plane. 2. Antenna Array This causes more ripples in the pattern at high elevations (Figure 9), which are however not due to the antenna intrinsic characteristics but only to the overlapping of the reflected waves with the ones originating by the antenna itself (The use of the small ground plane has indeed been suggested recently by the authors to standardize bodies as a more valid approach for the characterization of the antenna in a standalone configuration, i.e., without the specific aeronautic platform on which it will be mounted) They are due to a reflection happening on the surface of the big ground plane. This causes more ripples in the pattern at high elevations (Figure 9), which are however not due to the antenna intrinsic characteristics but only to the overlapping of the reflected waves with the ones originating by the antenna itself (The use of the small ground plane has indeed been suggested recently by the authors to standardize bodies as a more valid approach for the characterization of the antenna in a standalone configuration, i.e., without the specific aeronautic platform on which it will be mounted) Figure 5. Gain as measured in the Starlab near-field semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-field semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-ﬁeld semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-field semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-field semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-ﬁeld semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-field semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. 2. Antenna Array Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-field chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-field chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, during the electromagnetic measurement in the Compact Test Range at the DLR facilities. Figure 4. Top left: manufactured DLR array (on the top left) compared with a commercial avionic single antenna with the same 3.5-inch footprint; top right: antenna array placed on the small rolled edges ground plane during the electromagnetic measurement in the semi-anechoic near-ﬁeld chamber at the DLR facilities; down: antenna array, placed on the big rolled edges ground plane, during the electromagnetic measurement in the Compact Test Range at the DLR facilities. Sensors 2019, 19, 2258 S 2019 19 FOR Sensors 2019 19 x FOR P 5 of 12 5 of 12 of 12 Results from the measurements in both chambers / on both ground planes are shown in Figures 5–8. The measured results are in good agreement with the simulated ones, shown in Figures 2 and 3. Moreover, the frequency trends appear similar on both ground planes. Results from the measurements in both chambers / on both ground planes are shown in Figure 5–8. The measured results are in good agreement with the simulated ones, shown in Figure 2 and Figure 3. Moreover, the frequency trends appear similar on both ground planes. Results from the measurements in both chambers / on both ground planes are shown in Figure 5–8. The measured results are in good agreement with the simulated ones, shown in Figure 2 and Figure 3. Moreover, the frequency trends appear similar on both ground planes. Moreover, the frequency trends appear similar on both ground planes. Moreover, the frequency trends appear similar on both ground planes. Moreover, the frequency trends appear similar on both ground planes. Moreover, the frequency trends appear similar on both ground planes. 2. Antenna Array Gain as measured in the Starlab near-ﬁeld semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-field semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-field semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-ﬁeld semi-anechoic chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 5. Gain as measured in the Starlab near-field semi-anechoic chamber for the central antenna: he solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-field semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. , Figure 6. Gain as measured in the Starlab near-field semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-ﬁeld semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-field semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-field semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 6. Gain as measured in the Starlab near-ﬁeld semi-anechoic chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. nsors 2019, 19, 2258 6 of 1 Sensors 2019, 19, x FOR PEER REVIEW 6 of Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. ensors 2019, 19, x FOR PEER REVIEW 6 of Figure 7. 2. Antenna Array Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. RHCP, while the dotted line is the LHCP. Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reflection from the ground plane itself. Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reflection from the ground plane itself. Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reflection from the ground plane itself. Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reﬂection from the ground plane itself. , Fi 9 P tt t f th i f l t l t (bl ) RHCP ( d) LHCP (S lid Li ) bi Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane Ripples in the solid lines are due to reflection Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big d l ( d ) ll d l l h l d l d ﬂ Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reflection from the ground plane itself Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. 2. Antenna Array Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. nsors 2019, 19, x FOR PEER REVIEW 6 of 1 Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. 6 of 12 6 of 12 6 of 12 6 of 12 Sensors 2019, 19, 2258 S 2019 19 FO Sensors 2019, 19, x FOR Sensors 2019, 19, x FOR ors 2019, 19, 2258 6 of sors 2019, 19, x FOR PEER REVIEW 6 o Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. ors 2019, 19, x FOR PEER REVIEW 6 of Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. ors 2019, 19, x FOR PEER REVIEW 6 of Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. ensors 2019, 19, 2258 6 of Sensors 2019, 19, x FOR PEER REVIEW 6 o Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP Figure 7. 2. Antenna Array Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP ensors 2019, 19, x FOR PEER REVIEW 6 of Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. ensors 2019, 19, x FOR PEER REVIEW 6 of Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. Figure 7. Gain as measured in the CTR chamber for the central antenna: the solid line is the RHCP, while the dotted line is the LHCP. while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP while the dotted line is the LHCP Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Fi 8 G i d i h CTR h b f f h l l h lid li i h Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP while the dotted line is the LHCP Figure 8. Gain as measured in the CTR chamber for one of the lateral antennas: the solid line is the RHCP, while the dotted line is the LHCP. Figure 8. 2. Antenna Array (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reflection from the ground plane itself. Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reflection from the ground plane itself. Figure 9. Pattern cut of the gain of one lateral antenna: (blue) RHCP; (red) LHCP. (Solid Line) on big ground plane; (Dotted Line) on small ground plane. Ripples in the solid lines are due to reﬂection from the ground plane itself. 7 of 12 12 ies, i.e., lement Sensors 2019, 19, 2258 S 2019 19 FOR P Figure 10 sho at 1575 MHz for t Figure 10 shows the sky plots of the single antenna element gain at their central frequencies, i.e., at 1575 MHz for the lateral antennas and at 1175 MHz for the central one. While the center element has a uniform coverage, as expected from a single antenna, the patterns of the lateral antennas are distorted due to the close vicinity of each element with other antennas resonating at the same frequency. Though such an eﬀect does not allow for optimal coverage by each antenna, it ensures that all sky sectors are well covered by at least one antenna. By combining the lateral antenna outputs for Mode 1 operation, moreover, a smooth and uniform coverage throughout the upper hemisphere is achieved, as shown in Figure 11. Figure 10 shows the sky plots of the single antenna element gain at their central frequencies, i.e., at 1575 MHz for the lateral antennas and at 1175 MHz for the central one. While the center element has a uniform coverage, as expected from a single antenna, the patterns of the lateral antennas are distorted due to the close vicinity of each element with other antennas resonating at the same frequency. Though such an effect does not allow for optimal coverage by each antenna, it ensures that all sky sectors are well covered by at least one antenna. By combining the lateral antenna outputs for Mode 1 operation, moreover, a smooth and uniform coverage throughout the upper hemisphere is achieved, as shown in Figure 11. 2. Antenna Array has a uniform coverage, as expected from a single antenna, the patterns of the lateral antennas are distorted due to the close vicinity of each element with other antennas resonating at the same frequency. Though such an effect does not allow for optimal coverage by each antenna, it ensures that all sky sectors are well covered by at least one antenna. By combining the lateral antenna outputs for Mode 1 operation, moreover, a smooth and uniform coverage throughout the upper hemisphere is achieved, as shown in Figure 11. Figure 10. Sky plots of the embedded antenna RHCP gain in dBic, as measured in the anechoic chamber Figure 10. Sky plots of the embedded antenna RHCP gain in dBic, as measured in the anechoic chamber. Figure 10. Sky plots of the embedded antenna RHCP gain in dBic, as measured in the anechoic chamber. Figure 10. Sky plots of the embedded antenna RHCP gain in dBic, as measured in the anechoic h b Figure 10. Sky plots of the embedded antenna RHCP gain in dBic, as measured in the anechoic chamber. chamber. a e Figure 11. Sky plot of the Mode 1 RHCP gain in dBic, as measured in the anechoic chamber. Figure 11. Sky plot of the Mode 1 RHCP gain in dBic, as measured in the anechoic chamber. Figure 11. Sky plot of the Mode 1 RHCP gain in dBic, as measured in the anechoic chamber. Figure 11. Sky plot of the Mode 1 RHCP gain in dBic, as measured in the anechoic chamber. Figure 11. Sky plot of the Mode 1 RHCP gain in dBic, as measured in the anechoic chamber. Figure 11. Sky plot of the Mode 1 RHCP gain in dBic, as measured in the anechoic chamber. Figure 13. Sky plots of the recorded C/N0 L5/E5a (right). L5/E5a (right). 4. Interference Suppression Capability 4. Interference Suppression Capability In order to show that, despite the extreme miniaturization, the proposed antenna array can properly suppress interferences and hence add robustness with respect to single antenna designs, simulations of the antenna array in conjunction with interference suppression algorithms have been performed. The simulations take into consideration the patterns of each of the antenna elements in the array and several Continuous Wave (CW) interferences. Figure 14 shows the resulting digitally formed antenna-array gain patterns after the interference has been mitigated. On the left side of 4. Interference Suppression Capability In order to show that, despite the extreme miniaturization, the proposed antenna array can properly suppress interferences and hence add robustness with respect to single antenna designs, simulations of the antenna array in conjunction with interference suppression algorithms have been performed. The simulations take into consideration the patterns of each of the antenna elements in the array and several Continuous Wave (CW) interferences. Figure 14 shows the resulting digitally formed antenna-array gain patterns after the interference has been mitigated. On the left side of Figure 8, a single CW interference was simulated, while on the right side, three CW interferences In order to show that, despite the extreme miniaturization, the proposed antenna array can properly suppress interferences and hence add robustness with respect to single antenna designs, simulations of the antenna array in conjunction with interference suppression algorithms have been performed. The simulations take into consideration the patterns of each of the antenna elements in the array and several Continuous Wave (CW) interferences. Figure 14 shows the resulting digitally formed antenna-array gain patterns after the interference has been mitigated. On the left side of Figure 8, a single CW interference was simulated, while on the right side, three CW interferences (impinging the antenna-array from diﬀerent directions) were simulated. y g p g Figure 8, a single CW interference was simulated, while on the right side, three CW interferences (impinging the antenna-array from different directions) were simulated. Good interference suppression can be observed for both cases, with the coverage of the g , g , g , (impinging the antenna-array from different directions) were simulated. Good interference suppression can be observed for both cases, with the coverage of the remaining sky plot areas becoming, as expected, worse as the number of interferers increases. 3 GNSS Measurement of the Standalone Antenna 3. GNSS Measurement of the Standalone Antenna 3. GNSS Measurement of the Standalone Antenna span of 9 h have been collected: the sky plot of the recorded C/N0 values can be seen in Figure 13, both for L1/E1 and L5/E5 bands; good reception from the whole sky plot is visible, with C/N0 values of up to 47 dB-Hz at both bands recorded for medium to high elevations. both for L1/E1 and L5/E5 bands; good reception from the whole sky plot is visible, with C/N0 values of up to 47 dB-Hz at both bands recorded for medium to high elevations. Figure 12. DLR antenna on a small ground plane and connected to a Javad receiver for GNSS field tests. Figure 12. DLR antenna on a small ground plane and connected to a Javad receiver for GNSS ﬁeld tests. Figure 12. DLR antenna on a small ground plane and connected to a Javad receiver for GNSS field tests. Figure 12. DLR antenna on a small ground plane and connected to a Javad receiver for GNSS field tests. Figure 12. DLR antenna on a small ground plane and connected to a Javad receiver for GNSS ﬁeld tests. Figure 13 Sky plots of the recorded C/N0 values over the different satellite passes for L1/E1 (left) and Figure 13. Sky plots of the recorded C/N0 values over the different satellite passes for L1/E1 (left) and L5/E5 ( i ht) Figure 13. Sky plots of the recorded C/N0 values over the diﬀerent satellite passes for L1/E1 (left) and L5/E5a (right). Figure 13. Sky plots of the recorded C/N0 values over the different satellite passes for L1/E1 (left) and Figure 13. Sky plots of the recorded C/N0 values over the diﬀerent satellite passes for L1/E1 (left) and L5/E5a (right). 3 GNSS Measurement of the Standalone Antenna 3. GNSS Measurement of the Standalone Antenna 3. GNSS Measurement of the Standalone Antenna 3. GNSS Measurement of the Standalone Antenna The proposed antenna has been tested also from a GNSS point of view. For such scope, the antenna alone (i.e., not mounted on any aerial vehicle, but only placed on the small ground plane) was connected to a Javad Delta receiver and placed in an open field on the DLR premises (Figure 12) to minimize multipath effects. GNSS observables and C/N0 values on L1 and L5 signals over a time The proposed antenna has been tested also from a GNSS point of view. For such scope, the antenna alone (i.e., not mounted on any aerial vehicle, but only placed on the small ground plane) was connected to a Javad Delta receiver and placed in an open field on the DLR premises (Figure 12) to minimize multipath effects. GNSS observables and C/N0 values on L1 and L5 signals over a time The proposed antenna has been tested also from a GNSS point of view. For such scope, the antenna alone (i.e., not mounted on any aerial vehicle, but only placed on the small ground plane) was connected to a Javad Delta receiver and placed in an open ﬁeld on the DLR premises (Figure 12) to minimize multipath eﬀects. GNSS observables and C/N0 values on L1 and L5 signals over a time span of 9 h have been collected: the sky plot of the recorded C/N0 values can be seen in Figure 13, both for L1/E1 and 8 of 12 8 of 12 13 Sensors 2019, 19, 2258 Sensors 2019, 19, x FOR L5/E5 bands; good reception from the whole sky plot is visible, with C/N0 values of up to 47 dB-Hz at both bands recorded for medium to high elevations. span of 9 h have been collected: the sky plot of the recorded C/N0 values can be seen in Figure 13, both for L1/E1 and L5/E5 bands; good reception from the whole sky plot is visible, with C/N0 values of up to 47 dB-Hz at both bands recorded for medium to high elevations. both for L1/E1 and L5/E5 bands; good reception from the whole sky plot is visible, with C/N0 values of up to 47 dB-Hz at both bands recorded for medium to high elevations. L5/E5 bands; good reception from the whole sky plot is visible, with C/N0 values of up to 47 dB-Hz at both bands recorded for medium to high elevations. 5 Installed Performance Analysis 5. Installed Performance Analysis In order to verify the perform l tf i t ll d f i For the aircraft, a simplified model of an Airbus A320 (as the DLR one shown in Figure 15) has been considered For the drone a commercial DJI S1000 drone is being considered (Figure 15 right) Two examples have been investigated: an aircraft installation and a drone installation. For the aircraft, a simpliﬁed model of an Airbus A320 (as the DLR one shown in Figure 15) has been considered. For the drone, a commercial DJI S1000 drone is being considered (Figure 15 right). one commonly used in the GNSS community. The relationship of the coordinate systems (GNSS vs. e.m. simulations) is shown in Figure 16.) Good behavior in the case of the aircraft can be observed (Figure 17), with a broad beamwidth enabling optimal coverage of the sky plot in case of no interference The results for the drone (Figure considered. For the drone, a commercial DJI S1000 drone is being considered (Figure 15 right). (please be aware of the coordinate system being used in this section, which is different from the one commonly used in the GNSS community. The relationship of the coordinate systems (GNSS vs. e m simulations) is shown in Figure 16 ) (please be aware of the coordinate system being used in this section, which is diﬀerent from the one commonly used in the GNSS community. The relationship of the coordinate systems (GNSS vs. e.m. simulations) is shown in Figure 16.) enabling optimal coverage of the sky plot in case of no interference. The results for the drone (Figure 18) also show good coverage of the upper hemisphere, with more waviness due to strong scattering from the many metallic parts in the close vicinity of the antenna. Also more backradiation due to the limited ground plane/absence of flush mounting can be observed. ood behavior in the case of the aircraft can be observed (Figure 17), with a broad beamwidth abling optimal coverage of the sky plot in case of no interference. The results for the drone (Figure ) also show good coverage of the upper hemisphere, with more waviness due to strong scattering om the many metallic parts in the close vicinity of the antenna. Also more backradiation due to the mited ground plane/absence of flush mounting can be observed. Figure 15. Left: DLR Advanced Technology Research Aircraft (“ATRA”): Airbus A320; right: DJI S1000 drone cad model. Figure 15. 5 Installed Performance Analysis 5. Installed Performance Analysis In order to verify the perform l tf i t ll d f i y The extreme compactness of the antenna array, as well as its multiband capability, make it particularly suitable for mobile applications, such as airborne ones, where place and weight constraints play a major role The extreme compactness of the antenna array, as well as its multiband capability, make it particularly suitable for mobile applications, such as airborne ones, where place and weight constraints play a major role. platforms, installed performance simulations have been performed, where the measured antenna characteristics have been integrated with the platform CAD model. In particular, the currents of the antenna array in Mode 1, as measured in the semi-anechoic chamber and reconstructed on a box, have been used as field sources in an electromagnetic simulator once placed in the installed position constraints play a major role. In order to verify the performance of the antenna array once mounted on different airborne platforms, installed performance simulations have been performed, where the measured antenna characteristics have been integrated with the platform CAD model. In particular, the currents of the antenna array in Mode 1, as measured in the semi-anechoic chamber and reconstructed on a box, have been used as field sources in an electromagnetic simulator, once placed in the installed position on the platform In order to verify the performance of the antenna array once mounted on diﬀerent airborne platforms, installed performance simulations have been performed, where the measured antenna characteristics have been integrated with the platform CAD model. In particular, the currents of the antenna array in Mode 1, as measured in the semi-anechoic chamber and reconstructed on a box, have been used as ﬁeld sources in an electromagnetic simulator, once placed in the installed position on the platform. have been used as field sources in an electromagnetic simulator, once placed in the installed position on the platform Two examples have been investigated: an aircraft installation and a drone installation. For the aircraft, a simplified model of an Airbus A320 (as the DLR one shown in Figure 15) has been considered. For the drone, a commercial DJI S1000 drone is being considered (Figure 15 right). (please be aware of the coordinate system being used in this section, which is different from the one commonly used in the GNSS community The relationship of the coordinate systems (GNSS vs on the platform Two examples have been investigated: an aircraft installation and a drone installation. Figure 13. Sky plots of the recorded C/N0 L5/E5a (right). L5/E5a (right). 4. Interference Suppression Capability More Good interference suppression can be observed for both cases, with the coverage of the remaining sky plot areas becoming, as expected, worse as the number of interferers increases. More details on the use of interference suppression algorithms with miniaturized antenna arrays can be found in [12]. 9 of 12 Sensors 2019, 19, 2258 ors 2019, 19, x FOR PEER REVIEW Figure 14 Sky plots of the array gain after interferer(s). Interference Direction of Arriva right: three interferers. nstalled Performance Analysis applying weights for nulling in the direction of l (DOA) marked with a red rhomb. Left: one interfe Figure 14 Sky plots of the array gain after applying weights for nulling in the direction of the interferer(s). Interference Direction of Arrival (DOA) marked with a red rhomb. Left: one interferer; right: three interferers. Figure 14. Sky plots of the array gain after applying weights for nulling in the direction of the interferer(s). Interference Direction of Arrival (DOA) marked with a red rhomb. Left: one interferer; right: three interferers. Figure 14 Sky plots of the array gain after applying weights for nulling in the direction of the interferer(s). Interference Direction of Arrival (DOA) marked with a red rhomb. Left: one interferer; right: three interferers. Installed Performance Analysis The extreme compactness of the antenna array, as well as its multiband capability, make articularly suitable for mobile applications, such as airborne ones, where place and weig onstraints play a major role. Figure 14 Sky plots of the array gain after applying weights for nulling in the direction of the interferer(s). Interference Direction of Arrival (DOA) marked with a red rhomb. Left: one interferer; right: three interferers. Figure 14. Sky plots of the array gain after applying weights for nulling in the direction of the interferer(s). Interference Direction of Arrival (DOA) marked with a red rhomb. Left: one interferer; right: three interferers. The extreme compactness of the antenna array, as well as its multiband capability, mak icularly suitable for mobile applications, such as airborne ones, where place and we straints play a major role. 5 Installed Performance Analysis 5. Installed Performance Analysis In order to verify the perform l tf i t ll d f i Left: DLR Advanced Technology Research Aircraft (“ATRA”): Airbus A320; right: DJI S1000 drone cad model. Figure 15. Left: DLR Advanced Technology Research Aircraft (“ATRA”): Airbus A320; right: DJI S1000 drone cad model. Figure 15. Left: DLR Advanced Technology Research Aircraft (“ATRA”): Airbus A320; right: DJI S1000 drone cad model. 10 of 12 Sensors 2019, 19, 2258 Figure 16. Coordinate Systems for both the GNSS results (elevation and azimuth) and e.m. simulations (phi and theta). Figure 16. Coordinate Systems for both the GNSS results (elevation and azimuth) and e.m. simulations (phi and theta). sors 2019, 19, x FOR PEER REVIEW 10 of 1 Figure 16. Coordinate Systems for both the GNSS results (elevation and azimuth) and e.m. simulations (phi and theta). Figure 16. Coordinate Systems for both the GNSS results (elevation and azimuth) and e.m. simulations (phi and theta). Good behavior in the case of the aircraft can be observed (Figure 17), with a broad beamwidth enabling optimal coverage of the sky plot in case of no interference. The results for the drone (Figure 18) also show good coverage of the upper hemisphere, with more waviness due to strong scattering from the many metallic parts in the close vicinity of the antenna. Also more backradiation due to the limited ground plane/absence of ﬂush mounting can be observed. Figure 16. Coordinate Systems for both the GNSS results (elevation and azimuth) and e.m. simulations (phi and theta). Figure 17. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on an aircraft: (top) 3D view; (bottom) 2D view. Figure 17. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on an aircraft: (top) 3D view; (bottom) 2D view. Figure 17. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on an aircraft: (top) 3D view; (bottom) 2D view. Figure 17. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on an aircraft: (top) 3D view; (bottom) 2D view Figure 17. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on an aircraft: (top) 3D view; (bottom) 2D view. 11 of 12 of 12 Sensors 2019, 19, 2258 Sensors 2019, 19, x FOR , , Figure 18. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on a drone: (top) 3D view; (bottom) 2D view. Figure 18. 5 Installed Performance Analysis 5. Installed Performance Analysis In order to verify the perform l tf i t ll d f i RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on a drone: (top) 3D view; (bottom) 2D view. Figure 18. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on a drone: (top) 3D view; (bottom) 2D view. Figure 18. RHCP radiation pattern of the DLR antenna array in Mode 1 once mounted on a drone: (top) 3D view; (bottom) 2D view. 5. Conclusion 6. Conclusions In this work, a new and miniaturized five-element antenna array (with four L1/E1 elements and one L5/E5 element) fitting into a 3.5-inch footprint has been shown. Both electromagnetic and GNSS tests have validated the antenna suitability for use in mobile applications. It has been demonstrated how, in an interference-free scenario, the antenna array is capable of ensuring good satellite signal reception at the foreseen bands in almost the whole upper hemisphere. Moreover, it has been shown through simulations how the antenna array properly enables interference suppression/mitigation techniques based on digital array processing. Finally, the installed performance of the array once mounted on an aircraft or a drone has been analyzed, confirming its suitability for airborne In this work, a new and miniaturized ﬁve-element antenna array (with four L1/E1 elements and one L5/E5 element) ﬁtting into a 3.5-inch footprint has been shown. Both electromagnetic and GNSS tests have validated the antenna suitability for use in mobile applications. It has been demonstrated how, in an interference-free scenario, the antenna array is capable of ensuring good satellite signal reception at the foreseen bands in almost the whole upper hemisphere. Moreover, it has been shown through simulations how the antenna array properly enables interference suppression/mitigation techniques based on digital array processing. Finally, the installed performance of the array once mounted on an aircraft or a drone has been analyzed, conﬁrming its suitability for airborne applications. applications. Funding: The APC was funded by M.C. Author Contributions: Conceptualization—S C and M C ; Methodology—S C ; software S C and E P M ; Author Contributions: Conceptualization—S.C. and M.C..; Methodology—S.C.; software, S.C. and E.P.M.; Validation—S.C., W.E., G.B. and M.S-.C..; Writing and original draft preparation—S.C..; Writing, review, and editing—all; Funding acquisition—M.C. p Validation—S.C., W.E., G.B. and M.S-.C.. Funding: The APC was funded by M.C. p Validation—S.C., W.E., G.B. and M.S-.C.. Funding: The APC was funded by M.C. editing—all; Funding acquisition—M.C. Conﬂicts of Interest: The authors declare no conﬂict of interest. 978-1-60807-810-3. 3 Gü th C A f fi d t N i ti 2013 9 159 177 3. Günther, C. A survey of spooﬁng and counter-measures. Navigation 2013, 9, 159–177. 3. Günther, C. A survey of spoofing and counter-measures Navigation 2013, 9, 159–177. 4. Cuntz, M.; Konovaltsev, A.; Meurer, M. Concepts, Development and Validation of Multi-Antenna GNSS R i f R ili N i i P IEEE 2016 104 1288 1301 y p g g 4. Cuntz, M.; Konovaltsev, A.; Meurer, M. Concepts, Development and Validation of Multi-Antenna GNSS Receivers for Resilient Navigation. Proc. IEEE 2016, 104, 1288–1301. [CrossRef] Germany, 2017. 2. Dovis, F. GNSS Interference, Threats, and Countermeasures; Artech House: Boston, MA, USA, 2015; ISBN 2. Dovis, F. GNSS Interference, Threats, and Countermeasures; Artech House: Boston, MA, USA, 2015; ISBN 978-1-60807-810-3. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). R f References References 1. Teunissen, P.T.J.; Montenbruck, O. Handbook of Global Navigation Satellite Systems; Springer: Berlin, 1. Teunissen, P.T.J.; Montenbruck, O. Handbook of Global Navigation Satellite Systems; Springer: Berlin, Germany, 2017. Germany, 2017. 2. Dovis, F. GNSS Interference, Threats, and Countermeasures; Artech House: Boston, MA, USA, 2015; ISBN 2. Dovis, F. GNSS Interference, Threats, and Countermeasures; Artech House: Boston, MA, USA, 2015; ISBN 978-1-60807-810-3. 3. Günther, C. A survey of spoofing and counter-measures Navigation 2013, 9, 159–177. 4. Cuntz, M.; Konovaltsev, A.; Meurer, M. Concepts, Development and Validation of Multi-Antenna GNSS R i f R ili t N i ti P IEEE 2016 104 1288 1301 4. Cuntz, M.; Konovaltsev, A.; Meurer, M. Concepts, Development and Validation of Multi-Antenna GNSS Receivers for Resilient Navigation. Proc. IEEE 2016, 104, 1288–1301. [CrossRef] Receivers for Resilient Navigation. Proc. IEEE 2016, 104, 1288–1301 5. Vagle, N.; Broumandan, A.; Lachapelle, G. Analysis of Multi-Antenna GNSS Receiver Performance under Jamming Attacks. Sensors 2016, 16, 1937. [CrossRef] [PubMed] 12 of 12 Sensors 2019, 19, 2258 12 of 12 6. Basta, N.; Dreher, A.; Caizzone, S.; Sgammini, M.; Antreich, F.; Kappen, G.; Irteza, S.; Stephan, R.; Hein, M.A.; Richter, A.; et al. System Concept of a Compact Multi-Antenna GNSS Receiver. In Proceedings of the 2012 The 7th German Microwave Conference, Ilmenau, Germany, 12–14 March 2012; pp. 1–4. 7. Caizzone, S. Miniaturized E5a/E1 Antenna Array for Robust GNSS Navigation. IEEE Antennas Wir Lett. 2017, 16, 485–488. [CrossRef] 8. JKasemodel, J.A.; Chen, C.C.; Gupta, I.J.; Volakis, J.L. Miniature Continuous Coverage Antenna Array for GNSS Receivers. IEEE Antennas Wirel. Prop. Lett. 2008, 7, 592–595. [CrossRef] 9. Volakis, J.L.; Brien, A.J.O.; Chen, C.C. Small and Adaptive Antennas and Arrays for GNSS Applications. Proc. IEEE 2016, 104, 1221–1232. [CrossRef] 10. Caizzone, S.; Elmarissi, W.; Buchner, G.; Cuntz, M. Controlled Radiation Pattern Antenne. German Patent Pending 2018. 11. DO-373-MOPS for GNSS Airborne Active Antenna Equipment for the L1/E1 and L5/E5a Frequency Bands; RTCA Inc.: Washington, DC, USA, 2018. 12. Pérez Marcos, E.; Caizzone, S.; Cuntz, M.; Konovaltsev, A.; Meurer, M. STAP Performance and Antenna Miniaturization in Multi Antenna GNSS Receivers. In Proceedings of the ION GNSS+ 2019, Miami, FL, USA, 16–20 September 2019. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3034395599	https://edoc.unibas.ch/77447/1/20200615130809_5ee7569930ba5.pdf	English	null	Positional Isomerism in the N^N Ligand: How Much Difference Does a Methyl Group Make in [Cu(P^P)(N^N)]+ Complexes?	Molecules/Molecules online/Molecules annual	2,020	cc-by	11,370	Positional Isomerism in the NˆN Ligand: How Much Diﬀerence Does a Methyl Group Make in [Cu(PˆP)(NˆN)]+ Complexes? Fabian Brunner , Alessandro Prescimone , Edwin C. Constable and Catherine E. Housecroft * Department of Chemistry, University of Basel, BPR 1096, Mattenstrasse 24a, CH-4058 Basel, Switzerland; fabian.brunner@unibas.ch (F.B.); alessandro.prescimone@unibas.ch (A.P.); edwin.constable@unibas.ch (E.C.C.) * Correspondence: catherine.housecroft@unibas.ch; Tel.: +41-61-207-1008 Academic Editor: Barbara Modec Received: 29 May 2020; Accepted: 12 June 2020; Published: 15 June 2020 Received: 29 May 2020; Accepted: 12 June 2020; Published: 15 June 2020 Abstract: The synthesis and structural characterization of 5,6′-dimethyl-2,2′-bipyridine (5,6′-Me2bpy) are reported, along with the preparations and characterizations of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] (POP = bis(2-(diphenylphosphanyl)phenyl)ether, xantphos = 4,5- bis(diphenylphosphanyl)-9,9-dimethyl-9H-xanthene). Single-crystal X-ray structure determinations of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] conﬁrmed distorted tetrahedral copper(I) coordination environments with the 5-methylpyridine ring of 5,6′-Me2bpy directed towards the (C6H4)2O unit of POP or the xanthene unit of xantphos. In the xantphos case, this preference may be attributed to C–H . . . π interactions involving both the 6-CH unit and the 5-methyl substituent in the 5-methylpyridine ring and the arene rings of the xanthene unit. 1H NMR spectroscopic data indicate that this ligand orientation is also preferred in solution. In solution and the solid state, [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] are yellow emitters, and, for powdered samples, photoluminescence quantum yields (PLQYs) are 12 and 11%, respectively, and excited-state lifetimes are 5 and 6 µs, respectively. These values are lower than PLQY and τ values for [Cu(POP)(6,6′-Me2bpy)][PF6] and [Cu(xantphos)(6,6′-Me2bpy)][PF6], and the investigation points to the 6,6′-dimethyl substitution pattern in the bpy ligand being critical for enhancement of the PLQY. Keywords: copper(I); bisphosphane; 2,2′-bipyridine; X-ray crystallography; photoluminescence; heteroleptic coordination compounds molecules molecules Molecules 2020, 25, 2760; doi:10.3390/molecules25122760 molecules molecules 1. Introduction The structures of xantphos (4,5-bis(diphenylphosphanyl)-9,9-dimethyl-9H-xanthene, IUPAC PIN (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane)) and POP (bis(2-(diphenylphosphanyl)phenyl)ether, IUPAC PIN oxydi(2,1-phenylene)]bis(diphenylphosphane)). The labelling scheme is used for the NMR spectroscopic assignments; the PPh2 phenyl rings are labelled D. Scheme 1. The structures of xantphos (4,5-bis(diphenylphosphanyl)-9,9-dimethyl-9H-xanthene, IUPAC PIN (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane)) and POP (bis(2- (diphenylphosphanyl)phenyl)ether, IUPAC PIN oxydi(2,1-phenylene)]bis(diphenylphosphane)). The labelling scheme is used for the NMR spectroscopic assignments; the PPh2 phenyl rings are labelled D. Scheme 1. The structures of xantphos (4,5-bis(diphenylphosphanyl)-9,9-dimethyl-9H-xanthene, IUPAC PIN (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane)) and POP (bis(2-(diphenylphosphanyl)phenyl)ether, IUPAC PIN oxydi(2,1-phenylene)]bis(diphenylphosphane)). The labelling scheme is used for the NMR spectroscopic assignments; the PPh2 phenyl rings are labelled D. Since the potential for copper(I) iTMCs as light-emitting components in LECs was first demonstrated [11,12], xantphos and POP have been combined with many structurally and electronically diverse diimine ligands [5]. Some of the highest PLQY values and the best device electroluminescence (EL) performances have been achieved using bpy ligands containing simple substituents, including 6-alkyl and 6,6'-dialkyl groups [13,14], 6-alkyloxy and 6-alkylthio substituents [15], 6,6'-bis(alkyloxy) groups [16] and 6,6'-dihalo substituents [17]. Methyl substituents, in particular, lead to high PLQY values [13,14] and LEC performances. For example, a maximum luminance of 53 cd m−2 was observed in a LEC containing [Cu(POP)(6,6′-Me2bpy)][PF6] in the emissive layer [13]. Considering photoluminescence data for powdered samples, there is a significant improvement in quantum yield on going from [Cu(POP)(bpy)][PF6] (3% [17]), to [Cu(POP)(6-Mebpy)][PF6] (9.5% [13]) or [Cu(POP)(6,6′-Me2bpy)][PF6] (43.2% [13]), and from [Cu(xantphos)(bpy)][PF6] (1.7% [17]) to [Cu(xantphos)(6-Mebpy)][PF6] (34% [14]) or [Cu(xantphos)(6,6′-Me2bpy)][PF6] (37% [14]) (6-Mebpy = 6-methyl-2,2'-bipyridine, 6,6'-Me2bpy = 6,6'-dimethyl-2,2'-bipyridine). These data indicate that the introduction of at least one methyl group into the 6-position of bpy is beneficial for enhanced emission. In the case of the POP complex, there is a further enhancement when a second methyl group is introduced, but this is less pronounced in the case of the xantphos-containing analogue. Much of the effort in ligand design has emphasized electronic tuning through substituents attached at the 4- and 6-positions of the pyridine rings. In contrast, substitution at the 5-position has a similar inductive but a slightly different resonance electronic effect (6-Me, σ –0.17, σI –0.04, σR –0.04; 5-Me, σ –0.07, σI – 0.04, σR –0.14) [18,19] but significantly changes the steric demands of the ligand. We were interested in comparing complexes containing the N^N ligand 5,6'-dimethyl-2,2'-bipyridine (5,6'-Me2bpy) with those containing 6-Mebpy or (isomeric) 6,6'-Me2bpy. 1. Introduction The development of solid-state lighting technologies has revolutionized modern domestic and commercial lighting, primarily through the development of devices which are cheaper to manufacture and operate [1]. Although light-emitting diodes (LEDs) and organic light-emitting diodes (OLEDs) lead the market, light-emitting electrochemical cells (LECs) oﬀer a promising alternative [2,3]. LECs employ ionic transition metal compounds (iTMCs) as the light-emitting materials, the most commonly encountered of which are cyclometallated iridium(III) complexes [2,4] and heteroleptic [Cu(PˆP)(NˆN)]+ complexes [5] in which PˆP is a wide bite-angle bisphosphane [6], such as xantphos and POP (Scheme 1), and NˆN is typically a 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen) chelating ligand. This last class of compound follows from the seminal work of McMillin and coworkers, who observed that [Cu(PˆP)(NˆN)]+ complexes exhibit low-lying metal-to-ligand charge transfer (MLCT) excited states [7,8]. More recently, it has been demonstrated that many [Cu(PˆP)(NˆN)]+ complexes exhibit thermally activated delayed ﬂuorescence (TADF) [9,10], and this has increased interest in this family of copper(I) emitters. Triplet and singlet excited states are statistically present in a 3:1 ratio, and, in TADF, Molecules 2020, 25, 2760; doi:10.3390/molecules25122760 www.mdpi.com/journal/molecules 2 of 15 ed that [9 10] Molecules 2020, 25, 2760 to-ligand charge tra there is a fast intersystem crossing from the lowest-lying singlet excited state (S1) to the triplet excited state (T1). The T1 state is long-lived, with a relatively slow phosphorescence. If the energy gap between the S1 and T1 states is small, thermal repopulation of the S1 state via a reverse intersystem crossing can occur, leading to enhanced ﬂuorescence from the S1 state and an increased photoluminescence quantum yield (PLQY). and this has increased interest in this family of copper(I) emitters. Triplet and singlet excited states are statistically present in a 3:1 ratio, and, in TADF, there is a fast intersystem crossing from the lowest-lying singlet excited state (S1) to the triplet excited state (T1). The T1 state is long-lived, with a relatively slow phosphorescence. If the energy gap between the S1 and T1 states is small, thermal repopulation of the S1 state via a reverse intersystem crossing can occur, leading to enhanced fluorescence from the S1 state and an increased photoluminescence quantum yield (PLQY). Scheme 1. The structures of xantphos (4,5-bis(diphenylphosphanyl)-9,9-dimethyl-9H-xanthene, IUPAC PIN (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane)) and POP (bis(2- (diphenylphosphanyl)phenyl)ether, IUPAC PIN oxydi(2,1-phenylene)]bis(diphenylphosphane)). The labelling scheme is used for the NMR spectroscopic assignments; the PPh2 phenyl rings are labelled D. Scheme 1. 1. Introduction Here we describe the synthesis, characterization, and photophysical and electrochemical properties of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'-Me2bpy)][PF6] and assess the effects of introducing the second methyl group into the 5- rather than the 6-position. Since the potential for copper(I) iTMCs as light-emitting components in LECs was ﬁrst demonstrated [11,12], xantphos and POP have been combined with many structurally and electronically diverse diimine ligands [5]. Some of the highest PLQY values and the best device electroluminescence (EL) performances have been achieved using bpy ligands containing simple substituents, including 6-alkyl and 6,6′-dialkyl groups [13,14], 6-alkyloxy and 6-alkylthio substituents [15], 6,6′-bis(alkyloxy) groups [16] and 6,6′-dihalo substituents [17]. Methyl substituents, in particular, lead to high PLQY values [13,14] and LEC performances. For example, a maximum luminance of 53 cd m−2 was observed in a LEC containing [Cu(POP)(6,6′-Me2bpy)][PF6] in the emissive layer [13]. Considering photoluminescence data for powdered samples, there is a signiﬁcant improvement in quantum yield on going from [Cu(POP)(bpy)][PF6] (3% [17]), to [Cu(POP)(6-Mebpy)][PF6] (9.5% [13]) or [Cu(POP)(6,6′-Me2bpy)][PF6] (43.2% [13]), and from [Cu(xantphos)(bpy)][PF6] (1.7% [17]) to [Cu(xantphos)(6-Mebpy)][PF6] (34% [14]) or [Cu(xantphos)(6,6′-Me2bpy)][PF6] (37% [14]) (6-Mebpy = 6-methyl-2,2′-bipyridine, 6,6′-Me2bpy = 6,6′-dimethyl-2,2′-bipyridine). These data indicate that the introduction of at least one methyl group into the 6-position of bpy is beneﬁcial for enhanced emission. In the case of the POP complex, there is a further enhancement when a second methyl group is introduced, but this is less pronounced in the case of the xantphos-containing analogue. Much of the eﬀort in ligand design has emphasized electronic tuning through substituents attached at the 4- and 6-positions of the pyridine rings. In contrast, substitution at the 5-position has a similar inductive but a slightly diﬀerent resonance electronic eﬀect (6-Me, σ –0.17, σI –0.04, σR –0.04; 5-Me, σ –0.07, σI –0.04, σR –0.14) [18,19] but signiﬁcantly changes the steric demands of the ligand. We were interested in comparing complexes containing the NˆN ligand 5,6′-dimethyl-2,2′-bipyridine (5,6′-Me2bpy) with those containing 6-Mebpy or (isomeric) 6,6′-Me2bpy. Here we describe the synthesis, characterization, and photophysical and electrochemical properties of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] and assess the eﬀects of introducing the second methyl group into the 5- rather than the 6-position. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy Single crystals of 5,6'-Me2bpy were obtained by diffusion of cyclohexane into an ethyl ac ynthesis of 5,6'-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 ° e 2. Synthesis of 5,6'-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 °C, 2h 5-methyl-2,2'-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 °C, 60 h. Ring labelling is for scopic assignments. Scheme 2. Synthesis of 5,6′-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 ◦C, 2 h; (ii) 2-bromo-5-methyl-2,2′-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 ◦C, 60 h. Ring labelling is for NMR spectroscopic assignments. romo-5-methyl-2,2'-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 °C, 60 h. Ring labelling is for NMR pectroscopic assignments. ingle crystals of 5 6'-Me2bpy were obtained by diffusion of cyclohexane into an ethyl a me 2. Synthesis of 5,6'-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 °C, 2h; -5-methyl-2,2'-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 °C, 60 h. Ring labelling is for oscopic assignments. Scheme 2. Synthesis of 5,6′-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 ◦C, 2 h; (ii) 2-bromo-5-methyl-2,2′-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 ◦C, 60 h. Ring labelling is for NMR spectroscopic assignments. bromo-5-methyl-2,2'-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 °C, 60 h. Ring labelling is for NMR spectroscopic assignments. Single crystals of 5,6'-Me2bpy were obtained by diffusion of cyclohexane into an ethyl ac ngle crystals of 5,6'-Me2bpy were obtained by diffusion of cyclohexane into an ethyl n of the compound. 5,6'-Me2bpy crystallizes in the monoclinic space group P21/n re is shown in Figure 1. The compound adopts the expected trans-conformation and th n the least-squares planes through the two pyridine rings is 16.5°. Bond lengths and xceptional, and selected bond lengths are listed in the caption to Figure 1. The molecul rosymmetric pairs (Figure 2a, pairs of molecules A/B, B/A', and A'/B'). The first inte s face-to-faceπ-stacking of pyridine rings between molecules A/B and A'/B'. The d n the planes of the pyridine rings containing N2 and N2i (symmetry code i = 1 – x, 1 – Å, and the intercentroid distance is 3.73 Å. For the second pairing (B/A' in Figure 2a) nteraction is important with a CMe…centroid distance of 3.51 Å. The shortest C es are 3.68 and 3.69 Å, which are similar to the sum of the van der Waals radii for CMe a [22]. We consider the CMe…Cpy rather than the HMe…Cpy distances because the H atom group are in calculated positions. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy When the lattice is viewed along the a-axis, we obs ly into zigzag chains (Figure 2b). The latter features a close interaction between the 5 d h 6 b i d idi ( ) i f dj l l Th l C Single crystals of 5,6′-Me2bpy were obtained by diﬀusion of cyclohexane into an ethyl acetate solution of the compound. 5,6′-Me2bpy crystallizes in the monoclinic space group P21/n and the structure is shown in Figure 1. The compound adopts the expected trans-conformation and the angle between the least-squares planes through the two pyridine rings is 16.5◦. Bond lengths and angles are unexceptional, and selected bond lengths are listed in the caption to Figure 1. The molecules pack in centrosymmetric pairs (Figure 2a, pairs of molecules A/B, B/A’, and A’/B’). The ﬁrst interaction involves face-to-face π-stacking of pyridine rings between molecules A/B and A’/B’. The distance between the planes of the pyridine rings containing N2 and N2i (symmetry code i = 1 – x, 1 – y, 1 – z) is 3.46 Å, and the intercentroid distance is 3.73 Å. For the second pairing (B/A’ in Figure 2a), a CMe–H . . . π interaction is important with a CMe . . . centroid distance of 3.51 Å. The shortest CMe . . . Cpy distances are 3.68 and 3.69 Å, which are similar to the sum of the van der Waals radii for CMe and Csp2 (3.70 Å) [22]. We consider the CMe . . . Cpy rather than the HMe . . . Cpy distances because the H atoms of the methyl group are in calculated positions. When the lattice is viewed along the a-axis, we observe an assembly into zigzag chains (Figure 2b). The latter features a close interaction between the 5-methyl substituent and the 6-substituted pyridine (py) ring of an adjacent molecule. The closest CMe . . . Cpy separation is 3.79 Å, which is slightly longer than the sum of the CMe and Csp2 van der Waals radii [22]. The relationship between the zigzag chains and the centrosymmetric pairs of molecules can be appreciated by comparing Figure 2a,b. olution of the compound. 5,6'-Me2bpy crystallizes in the monoclinic space group P21/n and th tructure is shown in Figure 1. The compound adopts the expected trans-conformation and the ang etween the least-squares planes through the two pyridine rings is 16.5°. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy Bond lengths and angle re unexceptional, and selected bond lengths are listed in the caption to Figure 1. The molecules pac n centrosymmetric pairs (Figure 2a, pairs of molecules A/B, B/A', and A'/B'). The first interactio nvolves face-to-faceπ-stacking of pyridine rings between molecules A/B and A'/B'. The distanc etween the planes of the pyridine rings containing N2 and N2i (symmetry code i = 1 – x, 1 – y, 1 – s 3.46 Å, and the intercentroid distance is 3.73 Å. For the second pairing (B/A' in Figure 2a), a CM H…π interaction is important with a CMe…centroid distance of 3.51 Å. The shortest CMe…C istances are 3.68 and 3.69 Å, which are similar to the sum of the van der Waals radii for CMe and Cs 3.70 Å) [22]. We consider the CMe…Cpy rather than the HMe…Cpy distances because the H atoms of th methyl group are in calculated positions. When the lattice is viewed along the a-axis, we observe a ssembly into zigzag chains (Figure 2b). The latter features a close interaction between the 5-meth ubstituent and the 6-substituted pyridine (py) ring of an adjacent molecule. The closest CMe…C eparation is 3.79 Å, which is slightly longer than the sum of the CMe and Csp2 van der Waals rad 22]. The relationship between the zigzag chains and the centrosymmetric pairs of molecules can b ppreciated by comparing Figure 2a,b. py (py) g j n is 3.79 Å, which is slightly longer than the sum of the CMe and Csp2 van der W elationship between the zigzag chains and the centrosymmetric pairs of molecu ed by comparing Figure 2a,b. Figure 1. ORTEP representation of the structure of 5,6'-Me2bpy with ellipsoids plotted at a 40% probability level. Selected bond lengths: N1–C1 = 1.343(2), N1–C6 = 1.347(2), N2–C7 = 1.341(2), N2– C12 = 1.338(2), C1–C2 = 1.503(2), C10–C11 = 1.504(2) Å. Figure 1. ORTEP representation of the structure of 5,6′-Me2bpy with ellipsoids plotted at a 40% probability level. Selected bond lengths: N1–C1 = 1.343(2), N1–C6 = 1.347(2), N2–C7 = 1.341(2), N2–C12 = 1.338(2), C1–C2 = 1.503(2), C10–C11 = 1.504(2) Å. Figure 1. ORTEP representation of the structure of 5,6'-Me2bpy with ellipsoids plotted at a 40% probability level. Selected bond lengths: N1–C1 = 1.343(2), N1–C6 = 1.347(2), N2–C7 = 1.341(2), N2– C12 = 1.338(2), C1–C2 = 1.503(2), C10–C11 = 1.504(2) Å. Figure 1. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy y f py The ligand 5,6'-Me2bpy has previously been prepared in 66% overall yield using a Stille coupling [20,21] but we preferred to develop a tin-free approach. The palladium-catalyzed cross-coupling The ligand 5,6′-Me2bpy has previously been prepared in 66% overall yield using a Stille coupling [20,21] but we preferred to develop a tin-free approach. The palladium-catalyzed cross-coupling shown in Scheme 2 (in which the intermediate is made in situ and was not isolated) yielded 5,6′-Me2bpy, after work up, in 48% yield. Although the yield is lower than those reported 3 of 15 ) yield d for th ded 5,6 the Still Molecules 2020, 25, 2760 in Scheme 2 (in w y after work up i hown in Scheme 2 Me2bpy after work u for the Stille route [20,21], we consider that the procedure described here is advantageous in that no intermediate organometal reagent needs to be isolated. The 1H NMR spectrum (assigned using COSY and NOESY methods) agreed with that published [20], and the 13C{1H} NMR spectrum (not previously reported) was assigned using HMQC and HMBC methods. 20,21], we consider that the procedure described here is advantageous in that no interm metal reagent needs to be isolated. The 1H NMR spectrum (assigned using COSY and N ds) agreed with that published [20], and the 13C{1H} NMR spectrum (not previously re signed using HMQC and HMBC methods. p g rganometal reagent needs to be isolated. The 1H NMR spectrum (assigned using COSY and NOES methods) agreed with that published [20], and the 13C{1H} NMR spectrum (not previously reported was assigned using HMQC and HMBC methods. me 2. Synthesis of 5,6'-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 °C, 2h; -5-methyl-2,2'-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 °C, 60 h. Ring labelling is for oscopic assignments. Scheme 2. Synthesis of 5,6′-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 ◦C, 2 h; (ii) 2-bromo-5-methyl-2,2′-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 ◦C, 60 h. Ring labelling is for NMR spectroscopic assignments. Scheme 2. Synthesis of 5,6'-Me2bpy. Conditions: (i) nBuLi followed by ZnBr2, THF, −78 °C, 2h; (ii) 2- bromo-5-methyl-2,2'-bipyridine, 5 mol% [Pd(PPh3)4], THF, 80 °C, 60 h. Ring labelling is for NMR spectroscopic assignments. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy ORTEP representation of the structure of 5,6′-Me2bpy with ellipsoids plotted at a 40% probability level. Selected bond lengths: N1–C1 = 1.343(2), N1–C6 = 1.347(2), N2–C7 = 1.341(2), N2–C12 = 1.338(2), C1–C2 = 1.503(2), C10–C11 = 1.504(2) Å. 4 of 15 4 Molecules 2020, 25, 2760 O E Figure 2. (a) Centrosymmetric pairs of 5,6'-Me2bpy molecules, colored by symmetry operation; see ext for reference to the labels A, B, A′ and B′. (b) View down the a-axis showing zigzag chain arrangements of 5,6'-Me2bpy. Figure 2. (a) Centrosymmetric pairs of 5,6′-Me2bpy molecules, colored by symmetry operation; see text for reference to the labels A, B, A′ and B′. (b) View down the a-axis showing zigzag chain arrangements of 5,6′-Me2bpy. gure 2. (a) Centrosymmetric pairs of 5,6'-Me2bpy molecules, colored by symmetry operation; xt for reference to the labels A, B, A′ and B′. (b) View down the a-axis showing zigzag ch rangements of 5,6'-Me2bpy. Figure 2. (a) Centrosymmetric pairs of 5,6′-Me2bpy molecules, colored by symmetry operation; see text for reference to the labels A, B, A′ and B′. (b) View down the a-axis showing zigzag chain arrangements of 5,6′-Me2bpy. S th i d Ch t i ti f [C (POP)(5 6' M b )][PF ] d [C ( t h )(5 6' 2.2. Synthesis and Characterization of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] Synthesis and Characterization of [Cu(POP)(5,6 Me2bpy)][PF6] and [Cu(xantphos)(5,6 bpy)][PF6] The syntheses of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'-Me2bpy)][PF6] w ried out by either sequential (for POP) or simultaneous (for xantphos) addition of phosphane and 5,6'-Me2bpy ligands to [Cu(MeCN)4][PF6]. This is a standard procedure for erent P^P ligands, and the reasons for the different strategies have previously been deta 15]. The POP and xantphos-containing complexes were isolated in yields of 74.4% and 89. pectively The electrospray mass spectrum of each compound exhibited a base peak correspond The syntheses of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] were carried out by either sequential (for POP) or simultaneous (for xantphos) addition of the bisphosphane and 5,6′-Me2bpy ligands to [Cu(MeCN)4][PF6]. This is a standard procedure for the diﬀerent PˆP ligands, and the reasons for the diﬀerent strategies have previously been detailed [14,15]. The POP and xantphos-containing complexes were isolated in yields of 74.4% and 89.6%, respectively. The electrospray mass spectrum of each compound exhibited a base peak corresponding to the [M–PF6]+ ion (m/z 785.2 for [Cu(POP)(5,6′-Me2bpy)]+ and m/z 825.3 for [Cu(xantphos)(5,6′-Me2bpy)] (Figures S1 and S2; see the Supplementary Materials). pectively. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy The electrospray mass spectrum of each compound exhibited a base peak correspond he [M–PF6]+ ion (m/z 785.2 for [Cu(POP)(5,6'-Me2bpy)]+ and m/z 825.3 for [Cu(xantphos)(5 2bpy)] (Figures S1 and S2; see the Supplementary Materials). Single crystals of [Cu(POP)(5,6'-Me2bpy)][PF6].Me2CO and Cu(xantphos)(5 2bpy)][PF6].0.5CH2Cl2.0.75Et2O were grown by diffusion of Et2O into an acetone hloromethane solution of the POP- and xantphos-containing compounds, respectively. T mpounds crystallize in the monoclinic space groups P21/c and P21/n, respectively, and uctures of the cations are presented in Figure 3. Bond parameters for the copper(I) coordinat eres are listed in Table 1 along with Houser’s τ4 parameter [23]. The latter may be used to ass distortion of the copper(I) centre away from Td symmetry (τ4 = 1.00) towards C3v (τ4 = 0.85), saw geometries (τ4  0.64) and finally square-planar (τ4 = 0.00). The large P–Cu–N angles .84(6)° in [Cu(POP)(5,6'-Me2bpy)]+ and 120.61(7)° and 116.05(7)° in [Cu(xantphos)(5,6'-Me2bp tribute to values of τ4 (Table 1) that are close to τ4 = 0.85, calculated for a C3v (trigonal pyramid metry. In both complex cations, the 5,6'-Me2bpy ligand is positioned with the 5-methylpyrid g facing the backbone of the bisphosphane ligand, and, in [Cu(xantphos)(5,6'-Me2bpy)]+, t ults in the C51–H51 unit being accommodated within the bowl-shaped cavity of the xanthene u gure 4a). Considering the centroids of the two outer rings of the xanthene unit, the C 1…centroid distances are 3.01 and 3.22 Å, consistent with C–H…π interactions [24]. We note th many of the crystal structures of [Cu(xantphos)(6-Xbpy)][PF6], in which 6-Xbpy is a 6-substitut -bipyridine, the asymmetric 6-Xbpy ligand is oriented with the X group (X = Me, Et, OMe, O h, SMe, SEt, SPh, Br [14,17,25,26]) lying over the xanthene bowl, rather than being remote from mples of this alternative configuration are less commonly encountered in the solid state [14,27, in several structures disordering indicates little energy difference between the two possi ( g pp y ) Single crystals of [Cu(POP)(5,6′-Me2bpy)][PF6]·Me2CO and Cu(xantphos)(5,6′- Me2bpy)][PF6]·0.5CH2Cl2 0.75Et2O were grown by diﬀusion of Et2O into an acetone or dichloromethane solution of the POP- and xantphos-containing compounds, respectively. The compounds crystallize in the monoclinic space groups P21/c and P21/n, respectively, and the structures of the cations are presented in Figure 3. Bond parameters for the copper(I) coordination spheres are listed in Table 1 along with Houser’s τ4 parameter [23]. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy The latter may be used to assess the distortion of the copper(I) centre away from Td symmetry (τ4 = 1.00) towards C3v (τ4 = 0.85), C2v see-saw geometries (τ4 ≤0.64) and ﬁnally square-planar (τ4 = 0.00). The large P–Cu–N angles of 131.84(6)◦in [Cu(POP)(5,6′-Me2bpy)]+ and 120.61(7)◦and 116.05(7)◦in [Cu(xantphos)(5,6′-Me2bpy)]+ contribute to values of τ4 (Table 1) that are close to τ4 = 0.85, calculated for a C3v (trigonal pyramidal) geometry. In both complex cations, the 5,6′-Me2bpy ligand is positioned with the 5-methylpyridine ring facing the backbone of the bisphosphane ligand, and, in [Cu(xantphos)(5,6′-Me2bpy)]+, this results in the C51–H51 unit being accommodated within the bowl-shaped cavity of the xanthene unit (Figure 4a). Considering the centroids of the two outer rings of the xanthene unit, the C51–H51 . . . centroid distances are 3.01 and 3.22 Å, consistent with C–H . . . π interactions [24]. We note that, in many of the crystal structures of [Cu(xantphos)(6-Xbpy)][PF6], in which 6-Xbpy is a 6-substituted-2,2′-bipyridine, the asymmetric 6-Xbpy ligand is oriented with the X group (X = Me, Et, OMe, OEt, OPh, SMe, SEt, SPh, Br [14,17,25,26]) lying over the xanthene bowl, rather than being remote from it. Examples of this alternative conﬁguration are less commonly encountered in the solid state [14,27,28], and, in several structures, disordering indicates little energy diﬀerence between the two possible orientations [25,29]. Thus, it is noteworthy that the 5,6′-Me2bpy ligand exhibits a preference for an orientation with the 6-methyl group remote from the xanthene unit. A possible explanation is the combined eﬀects of the C–H . . . π interactions described above and weak interactions between the 5-methyl substituent and the arene rings of the xanthene unit. The closest CMe . . . Carene distance is 4.12 Å which, although greater than 5 of 15 Molecules 2020, 25, 2760 the sum of the CMe and Csp2 van der Waals radii [22], may indicate a stabilizing inﬂuence. As in the earlier discussion of the structure of the free ligand, we choose to use the CMe . . . Carene distance rather than the HMe . . . Carene separation because the H atoms of the methyl group are in calculated positions. Intramolecular π-stacking interactions are considered to be important in [Cu(PˆP)(NˆN)]+ complexes in terms of contributing towards enhanced PLQY values [30], and, in [Cu(xantphos)(NˆN)]+ cations, face-to-face π-stacking interactions are commonly observed between two phenyl rings of diﬀerent PPh2 units [15]. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy However, in [Cu(xantphos)(5,6′-Me2bpy)]+, the angle between the planes of the phenyl rings containing atoms C7 and C34 (Figure 3b) is too large (39.5◦) for a meaningful interaction. In contrast, in [Cu(POP)(NˆN)]+ cations, π-stacking between a phenyl ring and one arene ring of the O(C6H4)2-unit in the POP ligand is often a feature in the solid state, and [Cu(POP)(5,6′-Me2bpy)]+ is no exception. Figure 4b shows the stacking of the rings containing atoms C3 and C9; the angle between the ring-planes is 14.7◦, and the distance between the ring-centroids is 3.80 Å. Molecules 2020, 25, x FOR PEER REVIEW 5 of 15 the CMe…Carene distance rather than the HMe…Carene separation because the H atoms of the methyl group are in calculated positions. Intramolecular π-stacking interactions are considered to be important in [Cu(P^P)(N^N)]+ complexes in terms of contributing towards enhanced PLQY values [30], and, in [Cu(xantphos)(N^N)]+ cations, face-to-face π-stacking interactions are commonly observed between two phenyl rings of different PPh2 units [15]. However, in [Cu(xantphos)(5,6'- Me2bpy)]+, the angle between the planes of the phenyl rings containing atoms C7 and C34 (Figure 3b) is too large (39.5°) for a meaningful interaction. In contrast, in [Cu(POP)(N^N)]+ cations, π-stacking between a phenyl ring and one arene ring of the O(C6H4)2-unit in the POP ligand is often a feature in the solid state, and [Cu(POP)(5,6'-Me2bpy)]+ is no exception. Figure 4b shows the stacking of the rings containing atoms C3 and C9; the angle between the ring-planes is 14.7°, and the distance between the ring-centroids is 3.80 Å. Figure 3. The structures of (a) the [Cu(POP)(5,6'-Me2bpy)]+ cation in [Cu(POP)(5,6'- Me2bpy)][PF6].Me2CO and (b) the [Cu(xantphos)(5,6'-Me2bpy)]+ cation in [Cu(xantphos)(5,6'- Me2bpy)][PF6].0.5CH2Cl2.0.75Et2O. Ellipsoids are plotted at a 40% probability level, and H atoms are omitted for clarity. Figure 3. The structures of (a) the [Cu(POP)(5,6′-Me2bpy)]+ cation in [Cu(POP)(5,6′- Me2bpy)][PF6]·Me2CO and (b) the [Cu(xantphos)(5,6′-Me2bpy)]+ cation in [Cu(xantphos)(5,6′- Me2bpy)][PF6]·0.5CH2Cl2·0.75Et2O. Ellipsoids are plotted at a 40% probability level, and H atoms are omitted for clarity. Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 Figure 3. The structures of (a) the [Cu(POP)(5,6'-Me2bpy)]+ cation in [Cu(POP)(5,6'- Me2bpy)][PF6].Me2CO and (b) the [Cu(xantphos)(5,6'-Me2bpy)]+ cation in [Cu(xantphos)(5,6'- Me2bpy)][PF6].0.5CH2Cl2.0.75Et2O. Ellipsoids are plotted at a 40% probability level, and H atoms are omitted for clarity. Figure 3. The structures of (a) the [Cu(POP)(5,6′-Me2bpy)]+ cation in [Cu(POP)(5,6′- Me2bpy)][PF6]·Me2CO and (b) the [Cu(xantphos)(5,6′-Me2bpy)]+ cation in [Cu(xantphos)(5,6′- Me2bpy)][PF6]·0.5CH2Cl2·0.75Et2O. Ellipsoids are plotted at a 40% probability level, and H atoms are omitted for clarity. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy Parameter [Cu(POP)(5,6′-Me2bpy)]+ [Cu(xantphos)(5,6′-Me2bpy)]+ Cu–N/Å 2.075(2), 2.089(2) 2.053(2), 2.105(2) Cu–P/Å 2.2967(7), 2.2517(7) 2.2488(8), 2.2555(8) P–Cu–P/o 113.48(3) 117.36(3) N–Cu–N/o 79.90(8) 80.02(9) N–Cu–P/o 131.84(6), 108.61(6), 110.64(6), 103.82(6) 120.61(7), 116.05(7), 103.55(7), 110.47(7) τ4 1 0.83 0.87 1 τ4 parameter, see reference [23]. The solid-state structures discussed above assist in an interpretation of the solution NMR spectroscopic properties of the copper(I) compounds. 1H, 13C{1H}, COSY, NOESY, HMBC and HMQC-NMR spectra were recorded, allowing full assignment of the 1H and 13C{1H} NMR resonances in the complexes. 1D and 2D spectra of the compounds are shown in Figures 5 and 6, and in Figures S3–S10 in the Supplementary Materials. Figure 5 displays the aromatic regions of the 1H NMR spectra of the two compounds. Figure 5 reveals a substantial shift to lower frequency for the signal for proton A6 on going from [Cu(POP)(5,6′-Me2bpy)][PF6] to [Cu(xantphos)(5,6′-Me2bpy)][PF6]. This is consistent with the 5,6′-Me2bpy ligand adopting the same conﬁguration in solution as in the solid state, such that proton A6 is aﬀected by the ring currents of the two arene rings of the xanthene unit. The appearance of two sets of signals for the PPh2 phenyl D-rings (labelled D and D’ in Figure 5 and Figure S5) is consistent with 1H and 13C-NMR spectroscopic data for related POP and xantphos-containing copper(I) complexes; see, for example, [14,15,28,31,32]. In [Cu(POP)(5,6′-Me2bpy)][PF6], exchange (EXSY) peaks are observed between pairs of protons D2/D2’ and D3/D3’ (Figure 6). Corresponding peaks are not observed in the xantphos-containing complex (Figure S10). NOESY crosspeaks are observed in [Cu(POP)(5,6′-Me2bpy)][PF6] between protons D2/A6 and D2’/A6, and between D2/Me-B6 and D2’/Me-B6, indicating that the ﬂexible POP backbone undergoes dynamic behavior on the NMR timescale at 298 K, which exchanges the axial and equatorial phenyl rings of each PPh2 unit [14,28]. Molecules 2020, 25, x FOR PEER REVIEW 7 of 15 Figure 5. Comparison of the aromatic regions of the 1H NMR spectra (500 MHz, acetone-d6, 298 K) of (a) [Cu(POP)(5,6'-Me2bpy)][PF6] and (b) [Cu(xantphos)(5,6'-Me2bpy)][PF6]. See Schemes 1 and 2 for the atom labelling. Scale: δ/ppm. Figure 5. Comparison of the aromatic regions of the 1H NMR spectra (500 MHz, acetone-d6, 298 K) of (a) [Cu(POP)(5,6′-Me2bpy)][PF6] and (b) [Cu(xantphos)(5,6′-Me2bpy)][PF6]. See Schemes 1 and 2 for the atom labelling. Scale: δ/ppm. Figure 5. Comparison of the aromatic regions of the 1H NMR spectra (500 MHz, acetone-d6, 298 K) of (a) [Cu(POP)(5,6'-Me2bpy)][PF6] and (b) [Cu(xantphos)(5,6'-Me2bpy)][PF6]. See Schemes 1 and 2 for the atom labelling. Scale: δ/ppm. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy cules 2020, 25, x FOR PEER REVIEW 6 Figure 3. The structures of (a) the [Cu(POP)(5,6'-Me2bpy)]+ cation in [Cu(POP)(5,6'- Me2bpy)][PF6].Me2CO and (b) the [Cu(xantphos)(5,6'-Me2bpy)]+ cation in [Cu(xantphos)(5,6'- Me2bpy)][PF6].0.5CH2Cl2.0.75Et2O. Ellipsoids are plotted at a 40% probability level, and H atoms are omitted for clarity. Figure 3. The structures of (a) the [Cu(POP)(5,6′-Me2bpy)]+ cation in [Cu(POP)(5,6′- Me2bpy)][PF6]·Me2CO and (b) the [Cu(xantphos)(5,6′-Me2bpy)]+ cation in [Cu(xantphos)(5,6′- Me2bpy)][PF6]·0.5CH2Cl2·0.75Et2O. Ellipsoids are plotted at a 40% probability level, and H atoms are omitted for clarity. ecules 2020, 25, x FOR PEER REVIEW 6 tion sphere of each [Cu(P^P)(5,6'-M [Cu(xantphos)(5,6'-Me 2.053(2), 2.105(2) 2.2488(8), 2.2555(8 117.36(3) 80.02(9) 2(6) 120.61(7), 116.05(7), 103.55(7 0.87 ee reference [23]. Table 1. Bond lengths and angles in the coordination sphere of each [Cu(P^P)(5,6'-Me2bpy)]+ cation, and values of the τ4 parameter. Parameter [Cu(POP)(5,6'-Me2bpy)]+ [Cu(xantphos)(5,6'-Me2bpy)]+ Cu–N/Å 2.075(2), 2.089(2) 2.053(2), 2.105(2) Cu–P/Å 2.2967(7), 2.2517(7) 2.2488(8), 2.2555(8) P–Cu–P/o 113.48(3) 117.36(3) N–Cu–N/o 79.90(8) 80.02(9) N–Cu–P/o 131.84(6), 108.61(6), 110.64(6), 103.82(6) 120.61(7), 116.05(7), 103.55(7), 110.47(7) τ4 1 0.83 0.87 1 τ4 parameter, see reference [23]. Figure 4. Space-filling representations are used to highlight (a) the accommodation of the C51–H51 unit (next to the 5-methyl substituent) within the bowl-shaped cavity of the xanthene unit in [Cu(xantphos)(5,6′-Me2bpy)]+, and (b) face-to-face π-stacking of the arene rings containing C3 and C9 in [Cu(POP)(5,6′-Me2bpy)]+. Figure 4. Space-ﬁlling representations are used to highlight (a) the accommodation of the C51–H51 unit (next to the 5-methyl substituent) within the bowl-shaped cavity of the xanthene unit in [Cu(xantphos)(5,6′-Me2bpy)]+, and (b) face-to-face π-stacking of the arene rings containing C3 and C9 in [Cu(POP)(5,6′-Me2bpy)]+. engths and angles in the coordin e τ4 parameter. [Cu(POP)(5,6'-Me2bpy)]+ 2.075(2), 2.089(2) 2.2967(7), 2.2517(7) 113.48(3) 79.90(8) 131.84(6), 108.61(6), 110.64(6), 103 0.83 1 τ4 parameter, Figure 4. Space-filling representations are used to highlight (a) the accommodation of the C51–H51 unit (next to the 5-methyl substituent) within the bowl-shaped cavity of the xanthene unit in [Cu(xantphos)(5,6′-Me2bpy)]+, and (b) face-to-face π-stacking of the arene rings containing C3 and C9 in [Cu(POP)(5,6′-Me2bpy)]+. Figure 4. Space-ﬁlling representations are used to highlight (a) the accommodation of the C51–H51 unit (next to the 5-methyl substituent) within the bowl-shaped cavity of the xanthene unit in [Cu(xantphos)(5,6′-Me2bpy)]+, and (b) face-to-face π-stacking of the arene rings containing C3 and C9 in [Cu(POP)(5,6′-Me2bpy)]+. 6 of 15 Molecules 2020, 25, 2760 Table 1. Bond lengths and angles in the coordination sphere of each [Cu(PˆP)(5,6′-Me2bpy)]+ cation, and values of the τ4 parameter. g g ( ) p p 2.3. Electrochemical and Photophysical Properties 2.3. Electrochemical and Photophysical Properties Both [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'-Me2bpy)][PF6] are redox active and their electrochemical behavior was investigated in propylene carbonate solution using cyclic voltammetry (Figure 7). Each of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'-Me2bpy)][PF6] exhibits an irreversible copper-centered oxidation with Epc values of +0.81 and 0.89 V, respectively, and a reversible bpy-centered reductive process with E1/2 values of −2.12 V and −2.10 V, respectively (Epa − Epc = 100 V in each case). Data for the oxidative process are presented in Table 2 and are compared with oxidation potentials for closely related compounds. Note that a common solvent was not used for all the compounds in Table 2. Nonetheless, the data for the xantphos-containing Both [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] are redox active and their electrochemical behavior was investigated in propylene carbonate solution using cyclic voltammetry (Figure 7). Each of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] exhibits an irreversible copper-centered oxidation with Epc values of +0.81 and 0.89 V, respectively, and a reversible bpy-centered reductive process with E1/2 values of −2.12 V and −2.10 V, respectively (Epa −Epc = 100 V in each case). Data for the oxidative process are presented in Table 2 and are compared with oxidation potentials for closely related compounds. Note that a common solvent was not used for all the compounds in Table 2. Nonetheless, the data for the xantphos-containing complexes are consistent with a trend of copper(I) oxidation occurring at the highest potential for [Cu(xantphos)(6,6′-Me2bpy)]+ and following a sequence according to the NˆN ligand of 6,6′-Me2bpy > 5,6′-Me2bpy > 6-Mebpy > bpy. Copper(I)-to-copper(II) oxidation is accompanied by a geometrical change from tetrahedral to square-planar, and the trend in the oxidation potentials is consistent with decreasing steric hindrance in the coordination sphere of the copper center along the series from 6,6′-Me2bpy to 5,6′-Me2bpy to 6-Mebpy to bpy. The 5,6′-Me2bpy also ﬁts into this pattern for the POP-containing compounds, although (from the literature data) the trend from 6-Mebpy to bpy is less well deﬁned than for the xantphos family of compounds (Table 2). Molecules 2020, 25, x FOR PEER REVIEW 8 of 15 complexes are consistent with a trend of copper(I) oxidation occurring at the highest potential for [Cu(xantphos)(6,6'-Me2bpy)]+ and following a sequence according to the N^N ligand of 6,6'-Me2bpy > 5,6'-Me2bpy > 6-Mebpy > bpy. 2.1. Synthesis and Structural Characterization of 5,6'-Me2bpy 2.1. Synthesis and Structural Characterization of 5,6′-Me2bpy Figure 5. Comparison of the aromatic regions of the 1H NMR spectra (500 MHz, acetone-d6, 298 K) of (a) [Cu(POP)(5,6′-Me2bpy)][PF6] and (b) [Cu(xantphos)(5,6′-Me2bpy)][PF6]. See Schemes 1 and 2 for the atom labelling. Scale: δ/ppm. 7 of 15 for 7 of 15 for Molecules 2020, 25, 2760 (a) [Cu(POP)(5,6 the ato labelli Figure 6. Part of the NOESY spectrum (500 MHz, acetone-d6, 298 K) of [Cu(POP)(5,6'-Me2bpy)][PF6] showing exchange (EXSY) peaks between pairs of phenyl rings D2/D2' and D3/D3'. Figure 6. Part of the NOESY spectrum (500 MHz, acetone-d6, 298 K) of [Cu(POP)(5,6′-Me2bpy)][PF6] showing exchange (EXSY) peaks between pairs of phenyl rings D2/D2’ and D3/D3’. Figure 6. Part of the NOESY spectrum (500 MHz, acetone-d6, 298 K) of [Cu(POP)(5,6'-Me2bpy)][PF6] showing exchange (EXSY) peaks between pairs of phenyl rings D2/D2' and D3/D3' Figure 6. Part of the NOESY spectrum (500 MHz, acetone-d6, 298 K) of [Cu(POP)(5,6′-Me2bpy)][PF6] showing exchange (EXSY) peaks between pairs of phenyl rings D2/D2’ and D3/D3’. g g ( ) p p 2 3 El h i l d Ph h i l P i 2.3. Electrochemical and Photophysical Properties g g ( ) p p El h l d h h l 2.3. Electrochemical and Photophysical Properties g g ( ) p p 2.3. Electrochemical and Photophysical Properties Copper(I)-to-copper(II) oxidation is accompanied by a geometrical change from tetrahedral to square-planar, and the trend in the oxidation potentials is consistent with decreasing steric hindrance in the coordination sphere of the copper center along the series from 6,6'- Me2bpy to 5,6'-Me2bpy to 6-Mebpy to bpy. The 5,6'-Me2bpy also fits into this pattern for the POP- containing compounds, although (from the literature data) the trend from 6-Mebpy to bpy is less well defined than for the xantphos family of compounds (Table 2). Figure 7. Cyclic voltammograms of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'- Me2bpy)][PF6] in propylene carbonate at a scan rate of 0.1 V s−1 referenced to internal Fc/Fc+ = 0 V. The second of three reproducible cycles is shown. Figure 7. Cyclic voltammograms of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] in propylene carbonate at a scan rate of 0.1 V s−1 referenced to internal Fc/Fc+ = 0 V. The second of three reproducible cycles is shown. Figure 7. Cyclic voltammograms of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'- Me2bpy)][PF6] in propylene carbonate at a scan rate of 0.1 V s−1 referenced to internal Fc/Fc+ = 0 V. The second of three reproducible cycles is shown. Figure 7. Cyclic voltammograms of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] in propylene carbonate at a scan rate of 0.1 V s−1 referenced to internal Fc/Fc+ = 0 V. The second of three reproducible cycles is shown. 8 of 15 Molecules 2020, 25, 2760 Table 2. Copper(I)/(II) oxidation potentials in [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] in propylene carbonate solutions (ca. 10−4 mol dm–3); values are referenced to internal Fc/Fc+ = 0.0 V; [nBu4N][PF6] as supporting electrolyte and a scan rate of 0.1 V s–1. Values are compared to literature data (versus Fc/Fc+) for related compounds; see footnotes for solvents. Compound E1/2 /V Epc–Epa/mV Epca/V Reference [Cu(POP)(5,6′-Me2bpy)][PF6] +0.81 This work [Cu(xantphos)(5,6′-Me2bpy)][PF6] +0.89 This work [Cu(POP)(6,6′-Me2bpy)][BF4] +0.82 b – d [33] [Cu(xantphos)(6,6′-Me2bpy)][PF6] +0.90 150 [29] [Cu(POP)(6-Mebpy)][PF6] +0.69 – d [13] [Cu(xantphos)(6-Mebpy)][PF6] +0.85 c 100 [34] [Cu(POP)(bpy)][PF6] +0.72 110 [29] [Cu(POP)(bpy)][BF4] +0.72 b – d [33] [Cu(xantphos)(bpy)][PF6] +0.76 110 [29] [Cu(xantphos)(bpy)][BF4] +0.67 b – d [33] a The value of Epc is stated when the process is irreversible. b Recorded in MeCN. c Recorded in CH2Cl2. d No Epc −Epa value reported. The solution absorption spectra of the heteroleptic copper(I) compounds (Figure 8) show intense absorptions below 330 nm arising from ligand-based π*←π transitions. g g ( ) p p 2.3. Electrochemical and Photophysical Properties Values of λemmax were 626 and 622 nm (yellow emitters), respectively, PLQY values were around 9 of 15 Molecules 2020, 25, 2760 1%, and excited state lifetimes (τ) ≤1 µs. Low PLQYs in solution, even in non-coordinating solvents, have been attributed to the copper(I) complex undergoing exciplex formation, which favors non-radiative decay [35]. Excitation of powdered samples of the compounds (λexc = 365 nm) leads to emission maxima at higher energies than the solution samples while maintaining a yellow emission color. Such a blue-shift is typical of [Cu(PˆP)(NˆN)]+ complexes [36,37]. Table 3 summarizes the solid-state emission data, and the emission spectra are shown in Figure 8. Values of PLQY and τ for [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] are lower than for their 6,6′-Me2bpy-containing analogues (Table 3). The blue-shift in the emission maximum on going from the heteroleptic complexes containing bpy to those with 6-Mebpy, 5,6′-Me2bpy or 6,6′-Me2bpy is consistent with the electron-donating character of the methyl substituents. However, comparing values of λemmax for [Cu(PˆP)(5,6′-Me2bpy)][PF6] and [Cu(PˆP)(6,6′-Me2bpy)][PF6] indicates that a red-shift in the emission accompanies a change from 6,6′-Me2bpy to 5,6′-Me2bpy, and this in turn is consistent with the introduction of an electron-donating methyl group in a position meta to the N-donor stabilizing the LUMO of the complex. The PLQY values in Table 3 underline the importance of a second methyl group adjacent to the coordination site of the bpy ligand. Table 3. Solid-state emission data (298 K) for the copper(I) compounds (λexc = 365 nm) compared to data for related compounds. Compound λemmax/nm PLQY/% a τ/µs a Reference [Cu(POP)(5,6′-Me2bpy)][PF6] 553 12 6 b This work [Cu(xantphos)(5,6′-Me2bpy)][PF6] 555 11 5 b This work [Cu(POP)(6,6′-Me2bpy)][PF6] 535 43.2 10.5 c [13] [Cu(xantphos)(6,6′-Me2bpy)][PF6] 539 37 11 c [14] [Cu(POP)(6-Mebpy)][PF6] 567 9.5 2.6 b [13] [Cu(xantphos)(6-Mebpy)][PF6] 547 34 9.6 b [14] [Cu(POP)(bpy)][PF6] 580 3.0 1.5 c [17] [Cu(xantphos)(bpy)][PF6] 587 1.7 1.5 c [17] a Values from the literature are quoted as reported hence the inconsistency in the number of signiﬁcant ﬁgures Table 3. Solid-state emission data (298 K) for the copper(I) compounds (λexc = 365 nm) compared to data for related compounds. g g ( ) p p 2.3. Electrochemical and Photophysical Properties The broad absorption band with λmax at 376 nm in [Cu(POP)(5,6′-Me2bpy)][PF6] and at 374 nm in [Cu(xantphos)(5,6′-Me2bpy)][PF6] arises from MLCT transitions. The MLCT λmax values for the 5,6′-Me2bpy complexes are similar to those for [PF6]−or [BF4]– salts of [Cu(POP)(6,6′-Me2bpy)]+ (372 nm [33]) and [Cu(xantphos)(6,6′-Me2bpy)]+ (375 nm [33], 378 nm [14]), but are blue-shifted with respect to those of salts of [Cu(POP)(bpy)]+ (389 nm [12], 385 nm [33]), [Cu(xantphos)(bpy)]+ (383 nm [29], 382 nm [33]), [Cu(POP)(6-Mebpy)]+ (380 nm [13]) and [Cu(xantphos)(6-Mebpy)]+ (379 nm [14]). Since the LUMO of a [Cu(PˆP)(NˆN)]+ complex is localized on the NˆN ligand [14], the general trends in λmax are consistent with a destabilization of the LUMO upon introducing electron-donating methyl substituents into the bpy domain. Molecules 2020, 25, x FOR PEER REVIEW 9 of 15 consistent with a destabilization of the LUMO upon introducing electron-donating methyl substituents into the bpy domain. Figure 8. Solution absorption spectra of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'- Me2bpy)][PF6] in CH2Cl2 (2.5 × 10–5 mol dm–3) (solid lines) and solid-state emission spectra (dotted lines) Figure 8. Solution absorption spectra of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] in CH2Cl2 (2.5 × 10–5 mol dm–3) (solid lines) and solid-state emission spectra (dotted lines) Figure 8. Solution absorption spectra of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'- Me2bpy)][PF6] in CH2Cl2 (2.5 × 10–5 mol dm–3) (solid lines) and solid-state emission spectra (dotted lines). Figure 8. Solution absorption spectra of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] in CH2Cl2 (2.5 × 10–5 mol dm–3) (solid lines) and solid-state emission spectra (dotted lines). Figure 8. Solution absorption spectra of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'- Me2bpy)][PF6] in CH2Cl2 (2.5 × 10–5 mol dm–3) (solid lines) and solid-state emission spectra (dotted lines). Figure 8. Solution absorption spectra of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] in CH2Cl2 (2.5 × 10–5 mol dm–3) (solid lines) and solid-state emission spectra (dotted lines). Both aerated and deaerated solutions of [Cu(POP)(5,6'-Me2bpy)][PF6] and [Cu(xantphos)(5,6'- Me2bpy)][PF6] are weakly emissive when excited into the MLCT band at 375 and 374 nm, respectively. Values of λemmax were 626 and 622 nm (yellow emitters), respectively, PLQY values were around 1%, d it d t t lif ti ( ) 1 L PLQY i l ti i di ti l t h Both aerated and deaerated solutions of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′- Me2bpy)][PF6] are weakly emissive when excited into the MLCT band at 375 and 374 nm, respectively. a Values from the literature are quoted as reported, hence the inconsistency in the number of signiﬁcant ﬁgures. b A biexponential ﬁt to the decay was used, using the equation: τ = ΣAiτi/ΣAi where Ai is the pre-exponential factor for the lifetime. Values of A1, A2, τ1 and τ2 are given in Table S1. c A monoexponential ﬁt to the decay was used. 3.1. General 1H, 13C{1H} and 31P{1H} NMR spectra were recorded at 298 K on a Bruker Avance III-500 NMR spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). 1H and 13C-NMR chemical shifts were referenced to residual solvent peaks with respect to δ(TMS) = 0 ppm and 31P NMR chemical shifts with respect to δ(85% aqueous H3PO4) = 0 ppm. A Shimadzu LCMS-2020 (Shimadzu Schweiz GmbH, 4153 Reinach, Switzerland) was used to record electrospray ionization (ESI) mass spectra. Solution absorption and emission spectra were recorded using Shimadzu UV-2600 and Shimadzu RF-6000 instruments (Shimadzu Schweiz GmbH, 4153 Reinach, Switzerland), respectively. A Hamamatsu absolute photoluminescence quantum yield spectrometer C11347 Quantaurus-QY was used to measure PLQYs, and emission lifetimes and powder emission spectra were measured using a Hamamatsu Compact Fluorescence Lifetime Spectrometer C11367 Quantaurus-Tau with an LED light source (λexc = 365 nm). Electrochemical measurements were carried out using a CH Instruments 900B potentiostat (CH Instruments, Texas 78738, USA) with [nBu4N][PF6] (0.1 M) as supporting electrolyte and at a scan rate of 0.1 V s–1. The working electrode was glassy carbon, the reference electrode was a leakless 10 of 15 10 of 15 Molecules 2020, 25, 2760 Ag+/AgCl (eDAQ ET069-1) (eDAQ Europe, 01-471 Warszawa, Poland), and the counter-electrode was a platinum wire. Final potentials were internally referenced with respect to the Fc/Fc+ couple. p p y p p POP and xantphos were purchased from Acros (Fisher Scientiﬁc AG, 4153 Reinach Switzerland) and Fluorochem (Chemie Brunschwig AG, 4052 Basel, Switzerland), respectively. [Cu(MeCN)4][PF6] was prepared by the literature route [38]. 3.2. Synthesis of 5,6′-Me2bpy nBuLi (4.66 mL, 11.7 mmol, 2.5 M in n-hexane, 1.1 eq.) was added to a degassed solution of 2-bromo-6-methylpyridine (2.01 g, 11.7 mmol, 1.1 eq.) in dry THF (10 mL) at −78 ◦C under a nitrogen atmosphere. The mixture was stirred for 30 min during which time it turned deep red. Then, a solution of ZnCl2 in Et2O (10.6 mL, 10.6 mmol, 1.0 M in Et2O, 1.0 eq.) was added dropwise and the reaction mixture was stirred for 1.5 h. It was then allowed to warm to ambient temperature (ca. 22 ◦C) and was added dropwise to a solution of 2-bromo-5-methylpyridine (20.1 g, 11.7 mmol, 1.1 eq.) in dry THF (30 mL) containing [Pd(PPh3)4] (612 mg, 0.53 mmol, 0.05 eq.). This reaction mixture was heated to 80 ◦C and this temperature was maintained with stirring for 60 h. After cooling to ambient temperature, an aqueous solution of NaOH (ca. 100 mL, 3 M) was added until most of the precipitate had dissolved. The mixture was extracted with CH2Cl2 (3 × 50 mL) and the combined organic fractions were washed with aqueous NaOH, and then dried over MgSO4. The solvent was then removed under reduced pressure. The crude material was puriﬁed by column chromatography (neutral alumina, cyclohexane:ethyl acetate 40:1, followed by a second column with silica, cyclohexane:ethyl acetate 20:1) to give 5,6′-Me2bpy (937 mg, 5.08 mmol, 48%) as a white solid. 1H NMR (500 MHz, CDCl3) δ/ppm: 8.52–8.48 (m, 1H, HA6), 8.29 (d, J = 8.1 Hz, 1H, HA3), 8.13 (d, J = 7.9 Hz, 1H, HB3), 7.68 (t, J = 7.7 Hz, 1H, HB4), 7.61 (ddd, J = 8.1, 2.2, 0.7 Hz, 1H, HA4), 7.14 (d, J = 7.6 Hz, 1H, HB5), 2.63 (s, 3H, HMe-B6), 2.39 (s, 3H, HMe-A5). 13C{1H} NMR (126 MHz, CDCl3) δ/ppm: 158.0 (CB6), 155.8 (CB2), 154.0 (CA2), 149.7 (CA6), 137.6 (CA4), 137.2 (CB4), 133.3 (CA5), 123.1 (CB5), 120.9 (CA3), 118.0 (CB3), 24.8 (CMeB6), 18.5 (CMeA5). ESI MS: m/z 184.1 [M + H]+ (base peak, calc. 184.1). Found: C 78.01, H 6.75, N 15.40; C12H12N2 requires C 78.23, H 6.57, N 15.21%. 3.3. [Cu(POP)(5,6′-Me2bpy)][PF6] 3.4. [Cu(xantphos)(5,6′-Me2bpy)][PF6] [Cu(MeCN)4][PF6] (93.2 mg, 0.250 mmol, 1.0 eq.) was dissolved in CH2Cl2 (15 mL). A solution of xantphos (145 mg, 0.250 mmol, 1.0 eq.) and 5,6′-Me2bpy (46.1 mg, 0.250 mmol, 1.0 eq.) was added and the mixture turned orange then yellow while it was stirred for 1.5 h at room temperature (ca. 22 ◦C). The yellow solution was ﬁltered, and the solvent volume was reduced under vacuum. Et2O was then added to precipitate the product, and the solid was collected by ﬁltration, washed with Et2O (4 × 10 mL) using sonication, and dried under vacuum. [Cu(5,6′-Me2bpy)(xantphos)][PF6] was isolated as a yellow solid (218 mg, 0.224 mmol, 89.6%). 1H NMR (500 MHz, acetone-d6) δ/ppm: 8.45 (d, J = 8.0 Hz, 1H, HA3), 8.43 (d, J = 7.5 Hz, 1H, HB3), 8.13 (t, J = 7.8 Hz, 1H, HB4), 7.90−7.86 (overlapping m, 3H, HA4+C5), 7.62 (s, 1H, HA6), 7.52 (d, J = 7.7 Hz, 1H, HB5), 7.38 (t, J = 7.6 Hz, 2H, HD4/D4’), 7.35 (t, J = 7.2 Hz, 2H, HD4/D4’), 7.29 (t, J = 7.7 Hz, 2H, HC4), 7.23 (m, 4H, HD3’), 7.20 (m, 4H, HD3), 7.08 (m, 4H, HD2), 7.01 (m, 4H, HD2’), 6.60 (dtd, J = 7.6, 3.8, 1.4 Hz, 2H, HC3), 2.40 (s, 3H, HMe-B6), 2.05 (s, 3H, HMe-A5), 1.87 (s, 3H, HMe-xantphos), 1.77 (s, 3H, HMe-xantphos). 13C{1H} NMR (126 MHz, acetone-d6) δ/ppm: 159.2 (CB6), 155.9 (t, JPC = 6 Hz, CC1), 152.5 (t, JPC = 2 Hz, CB2), 151.0 (t, JPC = 2 Hz, CA2), 149.6 (CA6), 140.3 (CB4), 140.2 (CA4), 137.1 (CA5), 135.1 (t, JPC = 2 Hz, CC6), 133.8 (t, JPC = 8 Hz, CD2’), 133.6 (t, JPC = 8 Hz, CD2), 132.6 (t, JPC = 17 Hz, CD1’), 132.3 (t, JPC = 17 Hz, CD1), 131.6 (CC3), 131.2 (CD4’), 131.0 (CD4), 129.8 (t, JPC = 5 Hz, CD3’), 129.8 (t, JPC = 5 Hz, CD3), 128.4 (CC5), 126.6 (CB5), 126.4 (t, JPC = 2.4 Hz, CC4), 123.3 (CA3), 121.4 (overlapping d, JPC = 14 Hz, CC2), 120.7 (CB3), 37.0 (CCq-xantphos), 28.7 (CMe-xantphos), 27.8 (CMe-xantphos), 26.9 (CMe-B6), 18.1 (CMe-A5). 31P{1H} NMR (202 MHz, acetone-d6) δ/ppm: −12.1 (broad, FWHM ≈330 Hz), −144.2 (septet, JPF = 707 Hz, [PF6]−). ESI-MS: m/z 825.25 [M −PF6]+ (base peak, calc. 825.22), 641.10 [Cu(xantphos)]+ (calc. 641.12). UV-Vis (CH2Cl2, 2.5 × 10–5 mol dm–3): λ/nm (ε/dm3 mol–1 cm–1) 251 (35,000), 287 (29,700), 315 (14,000), 374 (3900). 3.6. 5,6′-Me2bpy C12H12N2, Mr = 184.24, colorless plate, monoclinic, space group P21/n, a = 6.4300(2), b = 19.2147(4), c = 8.0931(2) Å, β = 91.147(2)o, V = 999.71(4) Å3, Dc = 1.224 g cm–3, T = 130 K, Z = 4, µ(GaKα) = 0.369 mm−1. Total 36,616 reﬂections, 2030 unique (Rint = 0.0291). Reﬁnement of 1952 reﬂections (130 parameters) with I > 2σ(I) converged at ﬁnal R1 = 0.0564 (R1 all data = 0.0574), wR2 = 0.1686 (wR2 all data = 0.1698), gof = 1.190. CCDC 2005673. 3.4. [Cu(xantphos)(5,6′-Me2bpy)][PF6] Found: C 62.82, H 4.54, N 2.97; C51H44CuF6N2OP3 requires C 63.06, H 4.57, N 2.88% 3.3. [Cu(POP)(5,6′-Me2bpy)][PF6] [Cu(MeCN)4][PF6] (93.2 mg, 0.250 mmol, 1.0 eq.) and POP (148 mg, 0.275 mmol, 1.1 eq.) were dissolved in CH2Cl2 (30 mL) and the reaction mixture was stirred for 1.5 h at room temperature (ca. 22 ◦C). 5,6′-Me2bpy (46.1 mg, 0.250 mmol, 1.0 eq.) was then added, and stirring was continued for another 1.5 h. The yellow solution was ﬁltered, and the solvent volume of the ﬁltrate was reduced (under reduced pressure) and added to n-hexane (ca. 40 mL) to precipitate the product. The precipitate was separated and was washed with n-hexane (4 × 10 mL) using sonication and dried under vacuum. [Cu(POP)(5,6′-Me2bpy)][PF6] was isolated as a yellow solid (173 mg, 0.186 mmol, 74.4%). 1H NMR (500 MHz, acetone-d6) δ/ppm: 8.39 (d, J = 8.4 Hz, 1H, HA3), 8.36 (d, J = 8.3 Hz, 1H, HB3), 8.21 (s, 1H, HA6), 8.06 (t, J = 7.8 Hz, 1H, HB4), 7.86 (dd, J = 8.4, 2.1 Hz, 1H, HA4), 7.48–7.42 (m, 3H, HB5+C5), 7.42–7.33 (m, 4H, HD4+D4’), 7.31 (t, J = 7.5 Hz, 4H, HD3), 7.28–7.18 (m, 10H, HC6+D2+D3’), 7.15 (td, J = 7.5, 1.1 Hz, 2H, HC4), 7.05–6.97 (m, 4H, HD2’), 6.90 (m, 2H, HC3), 2.48 (s, 3H, HMe-B6), 2.11 (s, 3H, HMe-A5). 13C{1H} NMR (126 MHz, acetone-d6) δ/ppm: 159.6 (CB6), 158.9 (t, JPC = 6 Hz, CC1), 152.8 (t, JPC = 2 Hz, CB2), 150.9 (t, JPC = 2 Hz, CA2), 150.4 (CA6), 140.0 (CB4), 139.9 (CA4), 137.0 (CA5) 135.0 (CC3), 134.2 (t, JPC = 8 Hz, CD2), 133.7 (t, JPC = 8 Hz, CD2’), 133.2 (CC5), 132.1 (overlapping CD1’+A3D1), 131.2 (CD4), 130.8 (CD4’), 129.8 (t, JPC = 4 Hz, CD3), 129.6 (t, JPC = 4 Hz, CD3’), 126.6 (CB5), 126.1 (t, JPC = 2 Hz, CC4), 125.0 (overlapping d, JPC = 14 Hz, CC2), 123.0 (CA3), 121.2 (CC6), 120.5 (CB3), 26.8 (CMe-B6), 18.2 (CMe-A5). 31P{1H} NMR (202 MHz, acetone-d6) δ/ppm: –12.5 (broad, FWHM ≈ 450 Hz), –144.2 (septet, JPF = 707 Hz, [PF6]–). ESI-MS: m/z 785.20 [M–PF6]+ (base peak, calc. 785.19), 601.05 [Cu(POP)]+ (calc. 601.09). UV-Vis (CH2Cl2, 2.5 × 10–5 mol dm–3): λ/nm (ε/dm3 mol–1 cm–1) 253 (29,600), 293 (23,000), 316 (16,000), 376 (3,400). Found: C 62.03, H 4.87, N 2.68; C48H40CuF6N2OP3 requires C 61.90, H 4.33, N 3.01%. 11 of 15 11 of 15 Molecules 2020, 25, 2760 3.5. Crystallography Single-crystal data for the ligand were collected on a STOE StadiVari diﬀractometer equipped with a Pilatus300K detector and a Metaljet D2 source (GaKα radiation). Data reduction used STOE software [39] and the structure was solved using Olex2 [40], ShelXT [41], and ShelXL v. 2014/7 [42]. For the copper(I) compounds, single-crystal data were collected on a Bruker APEX-II diﬀractometer (CuKα radiation) with data reduction, solution and reﬁnement using the programs APEX2 [43], Superﬂip [44,45] and CRYSTALS [46]. Structure analysis including the ORTEP diagrams employed the program Mercury CSD v. 4.1.1 [47,48]. For [Cu(xantphos)(5,6′-Me2bpy)][PF6], SQUEEZE [49] was used to treat part of the solvent region, and an additional 0.75 Et2O was found that sums with the 0.5 CH2Cl2 that could be reﬁned. 4. Conclusions We have reported the synthesis and structural characterization of the 5,6′-Me2bpy ligand, and the preparations and solution and solid-state characterizations of the heteroleptic [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] compounds. Crystallographic structure determinations of the complexes conﬁrmed distorted tetrahedral copper(I) coordination environments with the 5,6′-Me2bpy ligand oriented with the 5-methylpyridine ring directed towards the (C6H4)2O unit of POP or the xanthene unit of xantphos. In the latter, this preference appears to arise from C–H . . . π interactions involving both the 6-CH unit and the 5-methyl substituent in the 5-methylpyridine ring and the arene rings of the xanthene unit. 1H NMR spectroscopic data are consistent with this same orientation being preferred in solution. The electrochemical behavior of [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] was investigated and the copper(I) oxidation occurs at a lower potential than in corresponding complexes with 6,6′-Me2bpy. [Cu(POP)(5,6′-Me2bpy)][PF6] and [Cu(xantphos)(5,6′-Me2bpy)][PF6] are yellow emitters in both solution and the solid-state, and, for powdered samples, PLQY values of 12 and 11%, respectively, and excited-state lifetimes of 5 and 6 µs, respectively, were observed. These values are lower than the PLQY and τ values for [Cu(POP)(6,6′-Me2bpy)][PF6] and [Cu(xantphos)(6,6′-Me2bpy)][PF6] [13,14]. Our results underline the importance of the 6,6′-dimethyl substitution pattern in the bpy ligand for enhancement of the PLQY in particular. Supplementary Materials: The following are available online. Figures S1 and S2: mass spectra. Figures S3–S10: NMR spectra. Table S1: Parameters for biexponential ﬁt to the lifetime decays. Author Contributions: Project conceptualization, administration, supervision and funding acquisition, E.C.C. and C.E.H.; investigation, data analysis, F.B.; crystallography, A.P.; writing, C.E.H., F.B.; manuscript editing, F.B., E.C.C., A.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by the Swiss National Science Foundation, grant number 200020_182000. Funding: This research was funded in part by the Swiss National Science Foundation, grant number 200020_182000. Acknowledgments: We thank the University of Basel for ﬁnancial support. ding: This research was funded in part by the Swiss National Science Foundation, grant number 200020_182000 Acknowledgments: We thank the University of Basel for ﬁnancial support. Acknowledgments: We thank the University of Basel for ﬁnancial support. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 3.8. [Cu(xantphos)(5,6′-Me2bpy)][PF6] .0.5CH2Cl2.0.75Et2O 3.8. [Cu(xantphos)(5,6′-Me2bpy)][PF6] .0.5CH2Cl2.0.75Et2O 3.8. [Cu(xantphos)(5,6′-Me2bpy)][PF6] .0.5CH2Cl2.0.75Et2O C54.50H52.50ClCuF6N2O1.75P3, Mr = 1069.38, yellow block, monoclinic, space group P21/n, a = 17.6502(15), b = 16.8504(15), c = 18.4582(16) Å, β = 104.685(3)o, V = 5310.4(8) Å3, Dc = 1.338 g cm–3, T = 130 K, Z = 4, µ(CuKα) = 2.422 mm–1. Total 55,278 reﬂections, 9603 unique (Rint = 0.0319). Reﬁnement of 8839 reﬂections (608 parameters) with I > 2σ(I) converged at ﬁnal R1 = 0.0544 (R1 all data = 0.0575), wR2 = 0.1728 (wR2 all data = 0.1768), gof = 1.025. CCDC 2005675. C54.50H52.50ClCuF6N2O1.75P3, Mr = 1069.38, yellow block, monoclinic, space group P21/n, a = 17.6502(15), b = 16.8504(15), c = 18.4582(16) Å, β = 104.685(3)o, V = 5310.4(8) Å3, Dc = 1.338 g cm–3, T = 130 K, Z = 4, µ(CuKα) = 2.422 mm–1. Total 55,278 reﬂections, 9603 unique (Rint = 0.0319). Reﬁnement of 8839 reﬂections (608 parameters) with I > 2σ(I) converged at ﬁnal R1 = 0.0544 (R1 all data = 0.0575), wR2 = 0.1728 (wR2 all data = 0.1768), gof = 1.025. CCDC 2005675. 3.7. [Cu(POP)(5,6′-Me2bpy)][PF6].Me2CO C51H46CuF6N2O2P3, Mr = 989.39, orange needle, monoclinic, space group P21/c, a = 9.5613(9), b = 14.8515(13), c = 32.472(3) Å, β = 90.338(4)o, V = 4611.0(7) Å3, Dc = 1.425 g cm–3, T = 130 K, Z = 4, µ(CuKα) = 2.227 mm–1. Total 31,246 reﬂections, 8211 unique (Rint = 0.029). Reﬁnement of 7611 reﬂections (586 parameters) with I > 2σ(I) converged at ﬁnal R1 = 0.0392 (R1 all data = 0.0420), wR2 = 0.0529 (wR2 all data = 0.0536), gof = 0.9967. CCDC 2005674. 12 of 15 12 of 15 Molecules 2020, 25, 2760 3.8. [Cu(xantphos)(5,6′-Me2bpy)][PF6] .0.5CH2Cl2.0.75Et2O References 1. Shaping Europe’s digital future. Lighting the Future. Policy. 2018. Shaping Europe’s digital future. Available online: https://ec.europa.eu/digital-single-market/en/lighting-future (accessed on 25 May 2020). 1. Shaping Europe’s digital future. Lighting the Future. Policy. 2018. Shaping Europe’s digital future. Available online: https://ec.europa.eu/digital-single-market/en/lighting-future (accessed on 25 May 2020). 1. Shaping Europe’s digital future. Lighting the Future. Policy. 2018. Shaping Europe’s digital future. Available online: https://ec.europa.eu/digital-single-market/en/lighting-future (accessed on 25 May 2020). 2. Costa, R.D. 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https://openalex.org/W3177185479	https://hal.sorbonne-universite.fr/hal-03285709/file/fpsyt-12-537383.pdf	English	null	Gender Differences Related to Spirituality, Coping Skills and Risk Factors of Suicide Attempt: A Cross-Sectional Study of French Adolescent Inpatients	Frontiers in psychiatry	2,021	cc-by	7,887	To cite this version: Bojan Mirkovic, Vincent Belloncle, Hugues Pellerin, Jean-Marc Guilé, Priscille Gérardin. Gender Dif- ferences Related to Spirituality, Coping Skills and Risk Factors of Suicide Attempt: A Cross-Sectional Study of French Adolescent Inpatients. Frontiers in Psychiatry, 2021, 12, pp.537383. 10.3389/fp- syt.2021.537383. hal-03285709 Gender Differences Related to Spirituality, Coping Skills and Risk Factors of Suicide Attempt: A Cross-Sectional Study of French Adolescent Inpatients Bojan Mirkovic, Vincent Belloncle, Hugues Pellerin, Jean-Marc Guilé, Priscille Gérardin ORIGINAL RESEARCH published: 25 June 2021 doi: 10.3389/fpsyt.2021.537383 ORIGINAL RESEARCH published: 25 June 2021 doi: 10.3389/fpsyt.2021.537383 Edited by: Alan Apter, Schneider Children’s Medical Center, Israel Reviewed by: Gianluca Seraﬁni, San Martino Hospital (IRCCS), Italy Enrique Baca-Garcia, University Hospital Fundación Jiménez Díaz, Spain Reviewed by: Gianluca Seraﬁni, San Martino Hospital (IRCCS), Italy Enrique Baca-Garcia, University Hospital Fundación Jiménez Díaz, Spain Background: Suicide attempts in adolescence represent a major public health concern, since these behaviors are associated with psychosocial burden and an increased risk of suicide. This cross-sectional study aimed to explore possible gender differences related to protective and risk factors in adolescents who have attempted suicide. Methods: Participants were French adolescents hospitalized for attempt suicide in ﬁve French pediatric departments. The participants were evaluated on 12 instruments measuring individual risk and protective factors. Correspondence: Bojan Mirkovic bojan.mirkovic@nh-navarre.fr Specialty section: This article was submitted to Child and Adolescent Psychiatry, a section of the journal Frontiers in Psychiatry Results: Our sample included 320 adolescents aged 13–17 years (M = 14.43, SD = 1.29), of whom 82% were female and 35% were repeat attempters. Boys had greater difﬁculties at school and used more lethal means such as strangulation. We failed to ﬁnd any differences between the two groups as regards the main Axis I psychiatric diagnoses. Boys tend to use more non-productive coping skills such as tension reduction or wishful thinking and girls use more reference to other strategies such as seeking social support. Although spirituality scores were low overall sample, they were signiﬁcantly higher among girls. Received: 23 February 2020 Accepted: 31 May 2021 Published: 25 June 2021 Bojan Mirkovic 1,2, Vincent Belloncle 3, Hugues Pellerin 4, Jean-Marc Guilé 5 and Priscille Gérardin 3,6 Bojan Mirkovic 1,2, Vincent Belloncle 3, Hugues Pellerin 4, Jean-Marc Guilé 5 and Priscille Gérardin 3,6 1 Department of Child and Adolescent Psychiatry, Nouvel Hopital de Navarre, Normandie Université, Evreux, France, 2 Équipe INSERM ≪Psychiatrie du Développement ≫, Centre de Recherche en Épidémiologie et Santé des Populations, UMR 1018, Université Paris-Saclay - UVSQ, Versailles, France, 3 Department of Child and Adolescent Psychiatry, CHU Rouen/CH-Le Rouvray, Normandie Université, Rouen, France, 4 Department of Child and Adolescent Psychiatry, Pitié-Salpêtrière AP-HP, UPMC, Sorbonne Université, Paris, France, 5 Department of Child and Adolescent Psychiatry, University Hospital of Amiens, Université Jules Verne, Amiens, France, 6 Laboratoire CRFDP, Normandie Université, Rouen, France Keywords: adolescent, attempt suicide, coping, gender, spirituality HAL Id: hal-03285709 https://hal.sorbonne-universite.fr/hal-03285709v1 Submitted on 13 Jul 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. INTRODUCTION rates of externalized behavior among men could lead to a higher death rate by suicide (9, 24–26). A recent meta-analysis by Miranda-Mendizabal et al. (26) identiﬁes eating disorders, post-traumatic stress disorder, bipolar disorder and depressive disorders as being risk factors for SA which are speciﬁc to women. However, the authors also report that previous mental or substance-abuse disorders and exposure to interpersonal violence were common risk factors for both sexes. Protective factors involved in moderating suicidal risk during adolescence include cognitive characteristics such as productive coping strategies, spirituality or religiosity, reasons for living, social and family support, doing well at school or regular physical activity (27–30). We selected productive coping skills and spirituality because these individual factors have been identiﬁed as being proximal to suicidal behavior, modiﬁable, and measurable (31). Regarding coping skills, a distinction is usually made between two coping styles: functional (productive) and dysfunctional (non-productive) (32). Non-functional coping strategies appear to be associated with depressive symptoms and suicidal ideation in adolescent clinical (29, 33) and community populations (28). Little data is available regarding gender diﬀerences because most studies have failed to take gender into consideration. It has been suggested that women were more likely than men to use emotion- based rather than problem-based coping strategies (33, 34). Spiritual and religious behaviors are diﬃcult to deﬁne and are subject to controversy. Spirituality refers to an individualistic, open, liberating and ultimately subjective quest, while religiosity requires a somewhat narrower characterization (35). Research on the impact of spirituality on adolescent developmental outcomes suggests that high levels of spirituality are associated with a better overall development (36). Spirituality seems to be a moderator for depression, hopelessness and adolescent SB (37). Rasic et al. (38) showed that religion had a protective eﬀect for depression and suicide ideation in adolescent populations. However, few studies have examined gender diﬀerences in clinical populations. Breton et al. (39) showed that self-discovery from the Spirituality Scale is a signiﬁcant protective factor for both girls and boys in the community but reached the signiﬁcance level only for boys in the adolescent clinical population. Suicide is the second leading cause of death among adolescents and young adults and represents a major public health concern (1). Epidemiological studies of adolescent suicide report higher rates of non-fatal suicidal behavior in women, suggesting that there is a gender diﬀerence in suicidal behavior during adolescence (2–6). INTRODUCTION In general, suicidal thoughts and suicide attempts (SA) are more common in women than in men, but the suicide death rate is higher in men. This characteristic is known as the “gender paradox” in suicide (7, 8). It is recognized that social factors play an important role in the diﬀerences in mental health between the sexes (9, 10). Several hypotheses have been proposed to explain this diﬀerence, such as the diversity of gender-based cultural norms (7) or the greater exposure of women to negative psychosocial experiences. In line with this, the recent study by Chang et al. (11) based on World Health Organization data showed that gender inequality (measured by the Gender Inequality Index) contributed to the overall variations in suicide in the world. Similarly, Sigurdson et al. (12) showed in a longitudinal study that women who were victims of bullying showed a decrease in suicidal thoughts between adolescence and adulthood, while men who were victims of bullying showed an increase in SA during the same period. Indeed, it is important to remember that exposure to childhood abuse and neglect has been associated with increased depression and suicidal behavior (13, 14). Another explanation for the gender paradox is that adolescent boys are more likely than adolescent girls to choose more lethal suicide methods such as hanging or ﬁrearms (15). The choice of method may be related to the intention to commit suicide. The Europe-wide study by Freeman et al. (16) showed a signiﬁcant association between suicidal intention and gender, with “serious suicide attempts” being assessed as signiﬁcantly more common in men than in women. On a neurobiological level, some recent studies have shown gender diﬀerences for glutaminergic activity (17) physical activity (18), sensory processing (19, 20) or sleep time (21). It has been reported that the direct eﬀect of lack of sleep on increased suicidal ideation is greater in adolescent girls than in adolescent boys (21). Lastly, diﬀerences were observed in the use of health care services. The study by Gontijo Guerra et al. (22) found that girls were more likely than boys to have used health services in the year before they died by suicide. Given the reality of such gender diﬀerences, the accepted approaches to suicide prevention should also be expected to take gender diﬀerences into account. Unfortunately, gender diﬀerences in terms of risk and protective factors for SA in adolescent have received insuﬃcient attention. Frontiers in Psychiatry \| www.frontiersin.org Citation: Mirkovic B, Belloncle V, Pellerin H, Guilé J-M and Gérardin P (2021) Gender Differences Related to Spirituality, Coping Skills and Risk Factors of Suicide Attempt: A Cross-Sectional Study of French Adolescent Inpatients. Front. Psychiatry 12:537383. doi: 10.3389/fpsyt.2021.537383 Conclusions: In the end, we ﬁnd little difference between the two groups in terms of risk factors. However, we have shown gender differences in spirituality and some coping strategies. These results should be taken into consideration when designing suicide prevention programs. June 2021 \| Volume 12 \| Article 537383 Frontiers in Psychiatry \| www.frontiersin.org 1 Gender Differences Related Suicide Attempt Mirkovic et al. METHODS Several studies have evaluated gender diﬀerences in mental disorders and suicidality. The Europe-wide study by Boyd et al. (23) shows in a consistently way that women have more internalized disorders (for example, mood disorders) while men have more externalized disorders (for example, behavioral disorders). The higher rates of internalized disorders among women could result in higher rates of SA, while the higher INTRODUCTION In this cross-sectional study, we propose to partially ﬁll this gap. The main objective of this descriptive study was to examine gender diﬀerences as regards the main risk and protective factors associated with SA in adolescence. We formulated the following hypotheses: (i) that boys would show more externalized disorders while girls would show more internalized disorders; (ii) that boys would use more non-productive coping strategies while girls would make more use of Reference to Others strategies. We did not formulate a hypothesis for spirituality. Risk Factors for SB Axis I psychiatric disorders were evaluated with the Scale of Mood Disorders and Schizophrenia for Children and Adolescents of School Age, Current and Past Episodes version (Kiddie-SADS- PL) (42). Depressive and hopelessness severity were measured, respectively, with the Beck Depression Inventory (BDI) second edition (43) and the Beck Hopelessness Scale (BHS) (44). Borderline personality disorders were assessed by Abbreviated-DIB (Ab-DIB) (45). It is a 10-min Diagnosis Interview for Borderline-Revised (DIB-R) derived 26- item self-report inventory used to assess impulsiveness, aﬀective and cognitive components of borderline personality symptomatology. The ﬁve pediatric departments involved belonged to two university hospitals and three peripheral hospitals. All ﬁve hospitals cover both urban and rural geographical areas in three French regions. Suicide characteristics were based on: the clinician- administered Columbia-Suicidal Severity Rating Scale (C-SSRS) (46) to assess the severity of suicidal ideations in the past month, actual attempt behavior severity, the number and characteristics of past attempts and Non-Suicidal Self Injury. Protective Factors The Adolescent Coping Scale (ACS) was used to assess speciﬁc behaviors adopted to deal with stressful life events. The ACS was designed and validated for adolescents aged 12–18 (32). The ACS assesses the speciﬁc behaviors used to cope with a situation or to solve a problem. According to the instrument manual, the three coping styles (productive coping, non-productive coping and reference to others) show suﬃcient internal coherence to justify their separate subscales (with alphas ranging from 0.62 to 0.75). The ACS has also demonstrated its reliability among 146 suicidal adolescents (31). Data-Analytic Strategy Descriptive statistics of sample characteristics, SA methods and risk and protective factors for SA were analyzed. We compared sociodemographic and clinical diﬀerences between boys and girls, using an independent t-test, Wilcoxon Rank-Sum test or ANOVA for continuous variables and the chi-square or Fisher’s exact tests for categorical variables. In statistical analyses, a signiﬁcance level of 0.05 was applied. We modeled the number of suicidal attempts using a Poisson regression (Model formula was: SA-number ∼Total_BDI + Gender + Total_spirituality + Gender∗Total_spirituality). The statistical package SPSS Release 16.0.2 (SPSS Inc., Chicago, IL, USA; 2008) and R 3.4.0 were used for the entire analyses. The Spirituality Scale (41) was used to score spiritual beliefs, self-discovery, self-awareness and collective consciousness, and respect for others and environment. The construct of spirituality proposed by Delaney (41) is broad. It goes beyond religious practices and encompasses three key relational aspects: connection with self (personal), with others (interpersonal), and with the divine (transpersonal). Possible scoring on the 23-item Spirituality scale ranged from 23 to 138. The scores indicated how important the phenomenon of spirituality is to, or is manifested by, the person. Scores between 23 and 60 indicated very low levels of spirituality, 61–91 indicated low Frontiers in Psychiatry \| www.frontiersin.org Participants Our study sample was made up of adolescents aged 13–17 admitted to a pediatric department following a suicide attempt and included in a longitudinal multisite study designed to assess the risk and protective factors associated with suicidal recidivism from January 2011 to December 2014 [detailed protocol in June 2021 \| Volume 12 \| Article 537383 2 Gender Differences Related Suicide Attempt Mirkovic et al. Mirkovic et al. (29)]. According to the French recommendations of good practice [issued by the HAS–Haute Autorité de Santé 1998 (40)], all adolescents who visit an emergency department for suicidal behavior are hospitalized. We considered the event as a SA only if intent to die was manifest. Subjects committing non-suicidal self-injury without intent to die were excluded. The adolescents also had to be able to understand French, live in the geographic area of the recruiting center, have no mental retardation or neurological disorder and have given their written consent. To participate in the study, under-age subjects needed to have the agreement of both parents or guardians. The exclusion criteria included an inability to provide written informed consent (for example, moderate to severe cognitive impairment) or a current medical condition or residence outside of the geographical area of each center. spirituality, 92–117 indicated moderate spirituality and 118– 138 suggested high levels of spirituality or spiritual wellness. Labelle et al. (31) have shown that this instrument is appropriate for assessing protective factors against depression and suicidal behavior in French-speaking adolescents in community (n = 283) and clinical settings (n = 146). Procedures During hospitalization, participation in the study was proposed systematically, by their referent psychiatrist, to all adolescents who met the inclusion criteria. After obtaining the consent of the adolescent and both parents, the research team contacted the patient and his/her family. The evaluation was conducted during hospitalization by psychiatrists trained in conducting semi- structured interviews and questionnaires as deﬁned in the study protocol. To avoid tiring the patients, the evaluation was carried out over more than one interview session. The investigators were senior psychiatrists and psychiatry residents. All the evaluation reports were analyzed individually by our study group. Impulsivity was assessed using the impulsivity section of the Eysenck Questionnaire (47). This 24-item self-administered questionnaire, completed by 8–17-year-olds, makes use of the norms established in two studies in the general Canadian and British population. The Life Events Questionnaire (48) is a 39-item instrument used to assess recent stressful situations experienced by adolescents (14–18 years-olds). Self-esteem was assessed with the Rosenberg Self-Esteem Rating Questionnaire (49) and attachment style with the Relationship Scales Questionnaire (RSQ) (50). RESULTS Characteristics Total Girls Boys p (N = 320) (N = 265) (N = 55) Mean age (SD) 14.43 (1.29) 14.65 (1.89) 14.14 (1.52) ns Number of children in family (SD) 2.46 (1.27) 2.44 (1.3) 2.44 (1.3) ns School-year repeaters N (%) 101 (31.5) 77 (29.06) 24 (43.64) 0.025 Residence with both parents N (%) 132 (41.2) 107 (40.38) 25 (45.45) ns Normal school career N (%) 271 (84.6) 228 (86.04) 43 (78.18) ns Mother’s level of education N (%) 1 32 (10) 29 (10.94) 3 (5.45) ns 2 88 (27.5) 74 (27.92) 14 (25.45) 3 94 (29.3) 78 (29.43) 16 (29.09) 4 65 (20.3) 52 (19.62) 13 (23.64) 5 41 (12.8) 32 (12.08) 9 (16.36) Father’s level of education N (%)* 1 39 (12.1) 33 (12.45) 6 (10.91) ns 2 96 (30) 81 (30.57) 15 (27.27) 3 74 (23.1) 59 (22.26) 15 (27.27) 4 63 (16.7) 53 (20) 10 (18.18) 5 48 (15) 39 (14.72) 9 (16.36) Had previous psychiatric care N (%) 100 (31.2) 82 (30.94) 18 (32.73) ns 1, primary and lower secondary school; 2, higher secondary school; 3, secondary professional school; 4, tertiary education (around 2–3 years more after high school diploma); 5, tertiary education (around 5–6 years more after high school diploma). TABLE 2 \| Characteristics of suicidal behaviors evaluated on the Columbia-suicide severity rating scale and comparisons by gender. 1, primary and lower secondary school; 2, higher secondary school; 3, secondary professional school; 4, tertiary education (around 2–3 years more after high school diploma); 5, tertiary education (around 5–6 years more after high school diploma). The 78 eligible adolescents who did not participate in the study made this choice for the following reasons: adolescent refused (n = 41), parent refused (n = 20), consent withdrawn during hospitalization (n = 8), others (n = 9). The sociodemographics of those who declined participation were roughly comparable to those of the participants regarding mean age (participants = 14.6 years vs. non-participants = 15.1 years) and gender (girl participants = 82% vs. girl non-participants = 79%). implies a desire to die, but without a speciﬁc plan to carry out death. implies an existing desire to die accompanied by a plan for how to carry out death. SA, Suicide Attempt. RESULTS Participation in the study was proposed to 398 eligible adolescents between February 2011 and December 2015. Three hundred twenty subjects were included, with 265 girls (82%) and 55 boys (18%), and the mean age was 14.43 (SD 1.29). June 2021 \| Volume 12 \| Article 537383 3 Gender Differences Related Suicide Attempt Mirkovic et al. TABLE 2 \| Characteristics of suicidal behaviors evaluated on the Columbia-suicide severity rating scale and comparisons by gender. Characteristics Total Girls Boys p (N = 320) (N = 265) (N = 55) Ideations N (%) None 89 (27.8) 73 (27.55) 16 (29.0) ns Passive 89 (27.8) 75 (28.3) 14 (25.6) Active** 142 (14.4) 117 (44.15) 25 (45.4) Intentionality N (%) Hidden 53 (16.56) 63 (23.77) 12 (21.82) ns Ambivalent 149 (46.5) 139 (52.45) 22 (40) Deﬁnitive 107 (33.4) 63 (23.77) 21 (38.18) Thoughts N (%) None 181 (56.5) 151 (56.98) 30 (54.55) ns Phobia 57 (17.8) 52 (19.62) 5 (9.09) Suicide-related 82 (25.6) 62 (23.4) 20 (36.36) Method N (%) Electrocution 5 (1.5) 4 (1.51) 1 (1.82) 0.007 Poisoning 242 (75.6) 205 (77.36) 37 (67.27) Laceration 20 (6.2) 18 (6.79) 2 (3.64) Jumping 13 (4) 13 (4.91) 0 (0) Strangulation 28 (8.7) 17 (6.42) 11 (20) Other 12 (3.7) 8 (3.02) 4 (7.27) Number of SAs N (%) 1 208 (65) 170 (64.15) 38 (69.09) 0.024 2 59 (18.6) 52 (19.62) 7 (12.73) 3 26 (8.1) 25 (9.43) 1 (1.82) 4 13 (4) 8 (3.02) 5 (9.09) 5 8 (2.5) 7 (2.64) 1 (1.82) 6 3 (0.9) 1 (0.38) 2 (3.64) 7 3 (0.9) 2 (0.75) 1 (1.82) Mean number of SAs N (%) 1.67 (1.18) 1.65 (1.11) 1.8 (1.51) ns Mean age at ﬁrst SA (SD) 14.48 (1.42) 14.65 (1.45) 15.07 (1.39) 0.048 implies a desire to die, but without a speciﬁc plan to carry out death. *implies an existing desire to die accompanied by a plan for how to carry out death. SA, Suicide Attempt. TABLE 1 \| Socio-demographic characteristics and comparisons by gender. Differences in Spirituality p y Both groups had low spirituality scores (<91). Girls had a signiﬁcantly higher overall spirituality score than boys W(317) = 7,208, p = 0.027. The results indicate that girls reported more self-awareness and collective consciousness W(317) = 7,263, p = 0.02 and respect of others and environment W(317) = 7434.5, p = 0.008 than boys did. Both groups had low spirituality scores (<91). Girls had a signiﬁcantly higher overall spirituality score than boys W(317) = 7,208, p = 0.027. The results indicate that girls reported more self-awareness and collective consciousness W(317) = 7,263, p = 0.02 and respect of others and environment W(317) = 7434.5, p = 0.008 than boys did. Differences in Coping Styles and Strategies We performed the Wilcoxon Rank-Sum test to explore diﬀerences in coping styles and strategies between girls and boys. Girls reported more frequent use of the Reference to others coping style than boys did W(302) = 7541.5, (p = 0.045). Of the strategies of the Reference to others style, only seeking social RESULTS p p g p p The number of adolescents by center was as follows: CHU Rouen: 177 (55%); CHU Amiens: 80 (25%); CH Compiègne: 32 (10%); CH Creil: 22 (7%); CH Maux: 9 (3%). Of the 320 subjects included, 112 (35%) had already made a ﬁrst SA. Table 1 summarizes the main sociodemographic characteristics of the entire sample and comparisons made by gender. We found only one sociodemographic variable which diﬀered according to gender. Boys have greater diﬃculties at school or more precisely, they have more often repeated a school year χ2 (319) = 5.03, (p = 0.025). Table 2 give the principal SA characteristics on the basis of the Columbia Suicide Severity Rating Scale. Boys more often resort to strangulation and girls to laceration. Girls are signiﬁcantly younger at their ﬁrst suicide attempt (p = 0.048) and they make more repeat attempts (p = 0.024). support was signiﬁcant W(302) = 7692.5, (p = 0.023). As regards unproductive coping, we identiﬁed two signiﬁcant diﬀerences between the two groups: wishful thinking W(302) = 5,013 (p = 0.049) and tension reduction W(317) = 7,553, (p = 0.042). Dimensional Characteristics Regarding protective factors, we showed that girls make more use of the Reference to Others coping style, mainly the seeking social support strategy, while men make more use of the non-productive coping style, mainly wishful thinking and tension reduction strategies. Lastly, regarding spirituality, although the scores were generally low in both groups, the girls had signiﬁcantly higher scores, especially for questions related to self-awareness and collective consciousness. We found no signiﬁcant diﬀerences between boys and girls for: the Beck Depression Inventory-II, the Beck Hopelessness Scale, the Life Events Questionnaire for Adolescents, and the dependence, impulsivity, self-esteem and attachment style (Table 3). DISCUSSION the two groups (Table 3). As regards borderline personality disorder assessed with the Ab-DIB, girls ﬁtted the diagnosis signiﬁcantly better than boys, respectively, n = 191 (73.7%) and n = 32 (60.4%), χ2 (312) = 3.86, p = 0.05. In this study we examined gender diﬀerence for the risk and protective factors associated with adolescent SAs. We included 320 adolescents, 82% of whom were girls, during hospitalization for AS. In this study we examined gender diﬀerence for the risk and protective factors associated with adolescent SAs. We included 320 adolescents, 82% of whom were girls, during hospitalization for AS. Axis I Diagnoses and Borderline Personality Disorders For the two groups, we compared all the diagnoses assessed with the Kiddie-SADS-PL. We found no signiﬁcant diﬀerence between June 2021 \| Volume 12 \| Article 537383 Frontiers in Psychiatry \| www.frontiersin.org 4 Gender Differences Related Suicide Attempt Mirkovic et al. TABLE 3 \| Individual risk factors and comparisons by gender. Characteristics Total Girls Boys p (N = 320) (N = 265) (N = 55) Axis I diagnoses (DSM-IV-R) Yes, N (%) Major depressive disorders 131 (41%) 105(41.5%) 25(48.1%) ns Anxiety disorders 88 (28%) 70(27.7%) 12(22.6%) ns Psychotic disorders 3 (1%) 3(1.2%) 2(3.8%) ns Substance and alcohol-related disorders 41 (13%) 72(28.5%) 20(38.5%) ns Disruptive and oppositioal behavior disorder 66 (21%) 55(21.7%) 8(15.1%) ns Attention deﬁcit hyperactivity disorder 17 (5%) 12(4.7%) 4(7.5%) ns Borderline disorder (Ad-DIB) N (%) No 89 (28%) 68 (26.3%) 21 (39.6%) 0.05 Yes 224 (70%) 191 (73.7%) 32 (60.4%) Dimensional assessments Beck Depression Inventory-II, Mean (SD) 25.3 (13.9) 25.7 (13.6) 23.15 (14.1) ns Beck Hopelessness Scale, Mean (SD) 9.35 (5.5) 9.28 (5.55) 9.62 (4.77) ns Life Events Questionnaire for Adolescents, Mean (SD) 3.5 (2.3) 3.62 (2.22) 3.27 (1.89) ns Dependance, Mean (SD) 7.3 (7.2) 7.38 (6.93) 6.49 (6.1) ns Impulsivity, Mean (SD) 12.2 (4.75) 12.34 (4.75) 11.58 (4.83) ns Secure attachment, Mean (SD) 13.4 (3.11) 13.7 (3.49) 13.4 (3.26) ns Fearful attachment, Mean (SD) 11.6 (3.42) 11.78 (3.72) 11.55 (3.44) ns Dimissing attachment, Mean (SD) 12.05 (3.08) 12.11 (3.28) 11.81 (3.21) ns Preoccupied attachment, Mean (SD) 16.4 (3.5) 16.84 (3.94) 16.32 (3.5) ns Self estime, Mean (SD) 5.95 (2.3) 6.21(2.41) 4.93(2.2) ns TABLE 3 \| Individual risk factors and comparisons by gender. Characteristics TABLE 3 \| Individual risk factors and comparisons by gender. Regression Models Total Girls Boys (N = 320) (N = 265) (N = 55) p Mean SD Mean SD Mean SD Spirituality scale Spiritual beliefs 17.84 8.09 18.08 8.01 16.73 8.44 ns Self-discovery 18.97 4.78 19.06 4.72 18.51 5.12 ns Self-awareness and 14.52 5.03 14.87 4.89 12.9 5.42 0.008 collective consciousness Respect of others and 18.61 3.87 18.83 3.79 16.59 4.1 0.02 environment Total 69.97 16.63 70.85 16.27 65.73 17.78 0.027 Adolescent coping scale Productive coping (total) 59.35 13.93 59.44 13.75 58.71 14.97 ns Focus on solving problem 51.12 16.06 51.41 15.85 49.1 16.82 ns Work hard and achieve 62.35 17.41 62.85 17.07 59.92 19.14 ns Focus on the positive 50.80 17.63 51.01 17.55 49.4 18.06 ns Seek relaxing diversions 73.45 19.66 73.23 19.56 74.34 20.44 ns Physical recreation 60.62 24.17 60.27 23.19 62.59 28.84 ns Non-productive coping 54.42 12.10 54.65 12.27 53.22 11.39 ns (total) Worry 52.66 17.42 52.7 17.9 52.63 17.13 ns Seek to belong 57.03 15.35 57.42 15.31 54.64 15.17 ns Wishful thinking 49.95 17.45 48.25 17.44 53.76 17.05 0.049 Not coping 50.07 16.69 50.8 17.22 46.27 14.84 ns Tension reduction 52.50 17.28 53.42 17.56 48.08 15.46 0.042 Ignore the problem 49.10 17.13 48.66 17.03 51.1 17.7 ns Self-blame 60.46 19.72 60.98 19.9 57.65 18.8 ns Keep to self 67.38 20.60 67.91 20.61 64.22 20.31 ns Reference to others (total) 41.35 10.82 42.19 10.49 40.23 12.36 0.045 Invest in close friends 58.51 17.61 59.25 17.5 56.94 19.95 ns Seek social support 49.9 18.42 51 18.5 44.63 17.37 0.023 Seek spiritual support 30.33 17.71 30.82 18.25 28.14 14.86 ns Social action 30.92 11.23 30.64 10.64 32.2 13.89 ns Seek professional help 39.66 18.99 39.59 19.04 39.7 18.96 ns TABLE 4 \| Individual protective factors and comparisons by gender (Wilcoxon Rank-Sum test). in what they perceive as barriers to seeking help. For example, stigma and embarrassment are more dissuasive for men than for women (54). This is particularly important because several studies have shown the protective role of the perception of social or family support in the face of suicidal behavior (55, 56). Yet support can only be provided if there is a prior request for help. Finally, concerning spirituality, our results contribute to furthering the relatively limited knowledge of gender diﬀerences. Girls had higher scores, especially for self-awareness, collective awareness and respect of others and environment. Regression Models Our epidemiological data agree with the data from the literature. Our sample is composed mainly of girls (82%) and 35% of repeat attempters (3, 9, 51). Although the repeat attempter rate is higher than that reported by other teams, it is consistent with other studies including high-risk adolescents, such as King et al. (51). Regarding risk factors, we found very few diﬀerences by gender. We failed to ﬁnd any diﬀerence for the main psychiatric diagnoses between the two groups. This result does not support recent studies which have shown a predominance of internalized disorders in women (23). Our divergent results may be explained in part by the nature of our sample. Indeed, our cohort consists only of adolescents hospitalized for attempted suicide. It is therefore a severe clinical sample with a high prevalence of depressive disorders and severe overall functioning. We conducted linear regressions to see how spirituality inﬂuences the number of suicides attempts according to gender and adjusting for depression (Beck Depression inventory). On average, Spirituality (total score) eﬀect on the mean number of suicidal attempts is signiﬁcantly increased for males (vs. females) by a factor 1.023, p = 0.002 (Estimate = 0.023, Std. Error = 0.007, z-value = 3.151). Spirituality eﬀect on the mean number of suicidal attempts in females is not signiﬁcant, p = 0.810 (Estimate = −0.001, Std. Error = 0.003, z-value = −0.241). An increase of one point of total spirituality in males increases the mean number of suicidal attempts by a factor 1.022, p = 0.001, (Estimate = 0.022, Std. Error = 0.007, z-value = 3.393) see Table 4. June 2021 \| Volume 12 \| Article 537383 Frontiers in Psychiatry \| www.frontiersin.org 5 Gender Differences Related Suicide Attempt Mirkovic et al. TABLE 4 \| Individual protective factors and comparisons by gender (Wilcoxon Rank-Sum test). Frontiers in Psychiatry \| www.frontiersin.org Regression Models p f In our regression model, spirituality can be considered a risk factor for suicidality only for boys. For girls there was no signiﬁcant eﬀect. Our results are not in agreement with other similar population-based studies that have shown spirituality to be a protective factor (27, 39). However, in the study by Breton et al. (39) only the subscale Self-discovery was a signiﬁcant protective factor for suicide attempt. In adolescents, spirituality seems to have a versatile eﬀect on suicidality and this eﬀect is diﬀerent for girls and boys. The construct of spirituality proposed by Delaney encompasses three key relational aspects: connection with self (personal), with others (interpersonal), and with he divine (transpersonal) and goes far beyond religious practices. In contrast to spirituality, the protective eﬀect of religion against suicidal risk has been reported in many studies (57–59). Among the underlying reasons for this eﬀect, various factors have been suggested: religious beliefs, church attendance and participation in public religious practices, moral objections to suicide, lower levels of aggressiveness, and fewer addictions. The study by Kralovec et al. (57), the only one to have studied gender-speciﬁc associations between religion/spirituality and suicide risk in a clinical sample, showed that religion/spirituality was correlated protectively with suicide risk, with stronger associations among women. More precisely, the authors found that most dimensions of religiousness were inversely correlated with many of the factors described in suicide models. This indicates that women’s risk of suicide may not be primarily aﬀected by church attendance, but by a wide range of religious dimensions, including belief/ideology, religious experience and prayer. Previous studies have reported protective eﬀects of other religious dimensions in addition to church attendance. In adolescents, private religiousness (prayer and the importance of religion) was signiﬁcantly associated with fewer suicidal thoughts and fewer suicide attempts (58). Moreover, Caribe et al. 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Examining gender diﬀerences in risk factors for suicide attempts made 1 and 7 years later in a nationally representative sample. J Adolesc Health. (2011) 48:391–7. doi: 10.1016/j.jadohealth.2010.07.018 4. Soor GS, Vukin I, Bridgman-Acker K, Marble R, Barnﬁeld P, Edwards J, et al. The eﬀects of gender on adolescent suicide in Ontario, Canada. J Can Acad Child Adolesc Psychiatry. (2012) 21:179–85. 14. Pompili M, Innamorati M, Lamis DA, Erbuto D, Venturini P, Ricci F, et al. The associations among childhood maltreatment, “male depression” and suicide risk in psychiatric patients. Psychiatry Res. (2014) 220:571–8. Regression Models Furthermore, this result is in agreement with the recent study by Miranda- Mendizabal et al. (52) which showed that mood disorders are the only risk factors for suicidal thoughts that are common to both sexes. We found diﬀerences in SA patterns that have been widely reported by other teams (4, 5, 25). Overall, boys use more lethal methods such as strangulation while girls use mostly drug poisoning. The large number of participants constitutes a strength of the present study. However, it has some limitations. The major limitation of the study is the fact that it was a cross-sectional study. This is important, as no causal claims can be inferred from the statistical associations identiﬁed in the study. Moreover, adolescents hospitalized for attempted suicide may diﬀer from those who are not (Berkson’s selection bias). Added to that, the self-assessment methodology used may be subject to a registration bias, such as social desirability. As regards coping skills, we showed that girls are more likely to seek help from their entourage (seeking social support) than boys. Gould et al. (53), in a self-report survey on the self-management of suicidal behavior in the general population (among secondary school students), showed that girls had better results for help- seeking strategies. In addition, men and women show diﬀerences June 2021 \| Volume 12 \| Article 537383 6 Gender Differences Related Suicide Attempt Mirkovic et al. CONCLUSIONS and Medical Research Committee (2010 A00 330 - 39). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Our results highlight diﬀerences between boys and girls as regards spirituality and certain coping strategies. Speciﬁcally, girls are more likely to use seeking social support strategies than boys. Boys are more likely to use unproductive coping strategies such as tension reduction (substance abuse) and wishful thinking (avoidance strategies. Individuation of interventions represents a promising strategy to counter the misuse of gender roles to tailor suicide prevention programs. School mental health classes can be adapted to consider diﬀerent approaches for males and females adolescent, for example, by valuing help-seeking strategies for young males. By ensuring the personalization of suicide prevention programs, mental health professionals can expect greater responsiveness and, in theory, a decrease in suicide attempts and suicides among youth. ETHICS STATEMENT Ethics approval was obtained for the study from the North West I (Charles Nicolle CHU -University Hospital) Group Ethics AUTHOR CONTRIBUTIONS BM and VB participated in the conception of the manuscript, coordinated the data collection, conducted the data analyses, provided drafts of the manuscript, and ﬁnalized the manuscript. J-MG participated in the design and coordination of the study and performed the measurements. HP performed the statistical analysis. PG conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the ﬁnal manuscript. FUNDING The datasets generated for this study are available on request to the corresponding author. This work was supported by Center Hospitalier Le Rouvray (sponsor), CHU Rouen, Fondation Pﬁzer and Fondation de France. None of the sponsors or the promoters interfered in any way with the study design, the interpretation of the data, or the writing of the draft. REFERENCES Seraﬁni G, Gonda X, Canepa G, Pompili M, Rihmer Z, Amore M, et al. Extreme sensory processing patterns show a complex association with depression, and impulsivity, alexithymia, and hopelessness. J Aﬀect Disord. (2017) 210:249–57. doi: 10.1016/j.jad.2016.12.019 40. HAS. Haute Autorité de Santé. Prise en charge hospitalière des adolescents après une tentative de suicide. Dépression et suicide. Paris (FR): Masson (1998). 41. Delaney C. The Spirituality Scale: development and psychometric testing of a holistic instrument to assess the human spiritual dimension. J Holist Nurs. (2005) 23:145–67. doi: 10.1177/0898010105276180 21. Park W-S, Kim S, Kim H. 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Frydenberg E. 59. Caribe AC, Nunez R, Montal D, Ribeiro L, Sarmento S, Quarantini LC, et al. Religiosity as a protective factor in suicidal behavior: a case-control study. J Nerv Ment Dis. (2012) 200:863–7. doi: 10.1097/NMD.0b013e31826b6d05 Copyright © 2021 Mirkovic, Belloncle, Pellerin, Guilé and Gérardin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. REFERENCES The Adolescent Coping Scale. 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(2017) 56:784– 95. doi: 10.1007/s10943-015-0133-3 38. Rasic D, Kisely S, Langille DB. Protective associations of importance of religion and frequency of service attendance with depression risk, suicidal behaviours and substance use in adolescents in Nova Scotia, Canada. J Aﬀect Disord. (2011) 132:389–95. doi: 10.1016/j.jad.2011. 03.007 58. Nonnemaker JM, McNeely CA, Blum RW. Public and private domains of religiosity and adolescent health risk behaviors: evidence from the National Longitudinal Study of Adolescent Health. Soc Sci Med. (2003) 57:2049–54. doi: 10.1016/S0277-9536(03)00096-0 June 2021 \| Volume 12 \| Article 537383 Frontiers in Psychiatry \| www.frontiersin.org 8 Gender Differences Related Suicide Attempt Mirkovic et al. Copyright © 2021 Mirkovic, Belloncle, Pellerin, Guilé and Gérardin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Psychiatry \| www.frontiersin.org Gender Differences Related Suicide Attempt June 2021 \| Volume 12 \| Article 537383 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. June 2021 \| Volume 12 \| Article 537383 Frontiers in Psychiatry \| www.frontiersin.org 9
https://openalex.org/W2790387000	https://europepmc.org/articles/pmc5867307?pdf=render	English	null	Editorial: Vestibular Contributions to Health and Disease	Frontiers in neurology	2,018	cc-by	2,674	Editorial published: 19 March 2018 doi: 10.3389/fneur.2018.00117 Editorial on the Research Topic Vestibular Contributions to Health and Disease INTRODUCTION The inter-related functions of several systems that evolved hundreds of millions of years ago, the vestibular, sympathetic, and cardiovascular systems, have important clinical implications in mod- ern neurology and neuro-otology. Operating largely out of consciousness, the vestibular system responds to head and body movement in any direction, stabilizes the head and body with regard to gravity in three-dimensional space, and maintains vital bodily functions through an interaction of vestibular-related components of the brainstem and cerebellum with the cardiovascular and sympathetic systems. We conceived this volume as an opportunity to highlight some of the basic science and clinical findings that have arisen over the first years of the twenty-first century with regard to these systems. Our concept was to have several broad topics: 1. fundamental vestibular, brainstem, and cerebellar science; 2. new approaches to vestibular diagnosis, pathology, and treatment; and 3. investigations of the vestibular impact on the cardiovascular and sympathetic systems. Topics include the func- tion of the otolith organs, evaluation of the vestibulo-ocular reflex (VOR) in pathologic conditions, and the vestibular input to sympathetic functions such as the vestibulo-sympathetic reflex (VSR). Motion sickness, a vestibulo-sympathetic interaction, is considered both historically and in terms of its typical clinical presentation. Also included are summaries of a little known receptor system, the skin sympathetic nerve activity (SSNA), which forms an important part of the sympathetic “fight or flight” response, and a new hypothesis about the neural basis for the Mal de Debarquement syndrome (MdDS). Edited and Reviewed by: Michael Strupp, Ludwig-Maximilians-Universität München, Germany Correspondence: Richard Lewis richard_lewis@meei.harvard.edu Edited and Reviewed by: Michael Strupp, Ludwig-Maximilians-Universität München, Germany Specialty section: This article was submitted to Neuro-Otology, a section of the journal Frontiers in Neurology Specialty section: This article was submitted to Neuro-Otology, a section of the journal Frontiers in Neurology Received: 06 February 2018 Accepted: 19 February 2018 Published: 19 March 2018 Citation: Cohen B and Lewis R (2018) Editorial: Vestibular Contributions to Health and Disease. Front. Neurol. 9:117. doi: 10.3389/fneur.2018.00117 Bernard Cohen1 and Richard Lewis 2,3 1 Department of Neurology, Mount Sinai School of Medicine, New York, NY, United States, 2 Department of Otolaryngology, Harvard Medical School, Boston, MA, United States, 3 Department of Neurology, Harvard Medical School, Boston, MA, United States Keywords: vestibular, physiology, pathophysiology, labyrinth, disease Keywords: vestibular, physiology, pathophysiology, labyrinth, disease VESTIBULAR PHYSIOLOGY—BASIC SCIENCE Ward et al. demonstrate that large Tesla magnets used in MRI scanners activate the peripheral end organ in a manner that replicates a constant angular acceleration. Thus, the MRI magnet allows one to study adaptation to this type of stimulus and simultaneously provides a way to identify, modify, or remove the vestibular activation that contaminates “resting” functional MRI scans. Received: 06 February 2018 Accepted: 19 February 2018 Published: 19 March 2018 Citation: Curthoys et al. demonstrate that otolith activity can be functionally segregated into static or sustained responses and transient responses, with the latter transduced primarily by type I hair cells. They show how this static-dynamic dichotomy could be clinically relevant, with particular reference to vestibular responses measured in the extra-ocular muscles and evoked with skull vibration (ocular vestibular-evoked myogenic potential). Cohen B and Lewis R (2018) Editorial: Vestibular Contributions to Health and Disease. Front. Neurol. 9:117. doi: 10.3389/fneur.2018.00117 March 2018 \| Volume 9 \| Article 117 1 Frontiers in Neurology \| www.frontiersin.org Vestibular System in Health and Disease Cohen and Lewis The history of the superior canal dehiscence syndrome (SCD), which is now approaching two decades after its initial description by Lloyd Minor, is reviewed by Ward et al. This paper summarizes the clinical presentation, diagnosis, and therapeutic options for SCD. Because it can be treated surgically by inactivating the abnormal canal or closing the defect in its roof, it is essential that this diagnosis be considered when the clinical signs suggest its presence. McCall et al. provide an extensive review of the inputs and outputs of the vestibular system, particularly directed toward ves- tibulospinal and reticulospinal contributions. A large literature demonstrates significant differences in the functional character- istics of vestibulospinal and reticulospinal projections in alert and anesthetized cats and non-human primates and emphasizes the complexity of the terminations in neural groups adjacent to the motor neurons. Karmali et al. examine how self-motion perceptual thresholds relate to age and fall risk, and find that while all of the tested angular, linear, and tilt thresholds that were measured increase with age, only roll tilt thresholds have a meaningful correlation with fall risk. Yakushin et al. (1) found that vestibular only (VO) neurons in the vestibular nuclei are responsible for the shift of the axis of eye rotation toward gravity. The VO neurons did not modulate with head position and most likely underlie the direct pathway link- ing the vestibular end organ with the ocular motor nuclei. When tested in three dimensions, the VO neurons on either side of the brainstem responded differently to ipsilateral and contralateral rotations and were insensitive to drowsiness, in contrast to oculomotor-related vestibular neurons. Dai et al. review the results of treatment of the MdDS in 141 patients. Citation: Their discovery of the first successful treatment for the MdDS, reported in 2014, has led to extensive experience treating this disorder which they report here. They found that the treat- ment was initially successful in 75% of patients, although efforts still must be made to stabilize the patients better in the time after treatment (Dai et al.).h VESTIBULAR PATHOPHYSIOLOGY— UNDERSTANDING, DIAGNOSING, AND TREATING VESTIBULAR DISORDERS The neural mechanism underlying the MdDS is considered by Cohen et al. They posit that MdDS had arises after prolonged exposure to roll tilts causes a lateral shift of the pitch orientation vector in the cerebellar nodulus. This activates VO neurons in the vestibular nuclei and causes them to oscillate at 0.2 Hz, resulting in the continuous sensation of rocking, swaying, and/or bobbing, which are characteristic of MdDS. The pathophysiology of vestibular compensation is considered in a paper by Batuecas-Caletrio et al. They studied outcomes in patients undergoing surgical resection of vestibular nerve schwannomas and observed that the characteristics of re-fixation saccades correlated with compensation. Frejo et al. address the pathophysiology of Meniere’s disease, classifying patients with bilateral disease based on co-morbidities such as migraine or autoimmune disease. They propose that this form of phenotyping may help segregate the different mecha- nisms that result in the Meniere’s syndrome and could eventually be linked to specific genetic syndromic diagnoses. VESTIBULO-SYMPATHETIC INTERACTIONS A major theme of vestibular research in the twenty-first cen- tury has been expanding the realm of studies to include the frequently overlooked vestibular effects on the sympathetic nervous system, which helps control blood pressure, heart rate, sweating, and other basic behaviors. There are two distinctly different components to the VSR. One component, using gluta- matergic transmission, involves the otolith system and the verti- cal semicircular canals [Holstein et al.; (3); Yakushin et al.; (4)]. Vestibular stimulation, i.e., upward, linear acceleration of the head and body produced by standing causes constriction of peripheral blood vessels in the legs by activating muscle sympa- thetic nerve activity (MSNA) (5–8).f van den Burg et al. demonstrated an exciting new develop- ment in the diagnosis of Meniere’s disease, namely imaging that identified anatomic abnormalities in this illness. Specifically, enlargement of the semicircular canals was observed, a finding that mirrors the earlier results of Hallpike 80 years ago (2). Dumas et al. show that vibration can elicit evidence of vestibular asymmetry in patients with peripheral deficits, even when they are fully compensated. The underlying physiology is not certain, but the authors argue that both canal and otolith afferents are activated by skull vibration. This technique could offer a quick new way to estimate the presence of semicir- cular canal lesions without rotational apparatus or caloric stimulation. Studying the effect of sinusoidal galvanic vestibular stimula- tion (sGVS) on anesthetized rats, Cohen and colleagues found that such stimulation initially caused a drop in BP and HR, a vasovagal response that is the precursor of neurogenic syncope (9). Interestingly, the vasovagal response disappeared with con- tinued bouts of stimulation with sGVS, which was associated with a change in the response of BP and HR. The authors raise the possibility that such vestibular stimulation potentially could be used to thwart the occurrence of neurogenic syncope. Halmagyi et al. review their technique for characterizing canal function and analyzing lesions of individual semicircular canals using the video head impulse test. Rapid head move- ment and observation of the evoked eye movements are much closer to the natural function of the VOR. 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Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow. Am J  Physiol Regul Integr Comp Physiol (2007) 292:R1532–41. doi:10.1152/ ajpregu.00633.2006 j 15. Cohen B, Dai M, Ogorodnikov D, Laurens J, Raphan T, Muller P, et al. Motion sickness on tilting trains. FASEB J (2011) 25:3765–74. doi:10.1096/fj.11-184887 fi 5. Wallin BG, Sundlöf G. Sympathetic outflow to muscles during vaso- vagal syncope. J Auton Nerv Syst (1982) 6:287–91. doi:10.1016/0165- 1838(82)90001-7 j 16. Joseph JA, Griffin MJ. CONCLUSION This collection of papers provides insight into the current state of vestibular research at levels ranging from basic physiology to clinical treatment. In particular, we feel that vestibular-autonomic interactions, which have been relatively neglected by much of the vestibular community, will ultimately prove to be an area of much significance for both the normal and pathologic influences of the vestibular system on behavior. Recent experiments have come closer to understanding the nature of the provocative motions responsible for producing nausea and motion sickness. The causes of motion sickness on tilting trains (15) were revisited, with refined apparatus by Bertolini et al. who argue that specific motion patterns, namely AUTHOR CONTRIBUTIONS BC and RL contributed equally to this manuscript. VESTIBULO-SYMPATHETIC INTERACTIONS A second VSR system originates largely in the lateral and vertical semicircular canals and the otolith organs and projects into other aspects of the sympathetic system, using serotonin as the transmitter to initiate anxiety, sweating, and fear (10–14). March 2018 \| Volume 9 \| Article 117 2 Vestibular System in Health and Disease Cohen and Lewis Hammam and Macefield studied SSNA, a little known set of receptors activated by vestibular input that are critical for gen- erating phenomena associated with motion sickness. Activated by weak linear acceleration along the horizontal plane, the SSNA is responsible for pallor and sweating. In comparison with the MSNA, which activates BP upon standing, the SSNA is also activated by linear acceleration, but the strength of the activa- tion is orders of magnitude weaker than that elicited by MSNA. A striking finding was that SSNA is associated with the symptoms of motion sickness, namely nausea and vomiting, as well as with skin pallor and sweating. those that elicit Coriolis forces (which are generated when one moves within a moving reference frame), are particularly likely to elicit symptoms of motion sickness. An earlier study on tilting trains (15) concluded that motion sickness in the passengers came when the roll tilt of the cars was initiated after the train had entered into the curve. This implicated roll as the exciting source of the motion sickness, confirming the findings of Griffin and colleagues (16, 17). Bertolini et al. were able to show that motion sickness was dependent on starting the roll after the rotation had begun, as in the earlier study (15), showing that it was a combination of linear acceleration and roll that had caused the motion sickness. Motion sickness, produced by activation of this system, has been an accompanying feature of travel for centuries. Huppert et al. provide a comprehensive review of motion sickness in their historical context. It is clear that the ancient Chinese and Greeks were well aware that body motion was responsible for “cart” sick- ness and seasickness, but the actual provocative motions were still a mystery. The authors review the incidents of both Chinese and European history when such seasickness proved to be a vital force that helped decide the course of battles and the fates of various nations. REFERENCES Motion sickness from combined lateral and roll oscil- lation: effect of varying phase relationships. Aviat Space Environ Med (2007) 78:944–50. doi:10.3357/ASEM.2043.2007 fif 6. Macefield VG, Wallin BG. Effects of static lung inflation on sympathetic activity in human muscle nerves at rest and during asphyxia. J Auton Nerv Syst (1995) 53:148–56. doi:10.1016/0165-1838(94)00174-I i 17. Donohew BE, Griffin MJ. Motion sickness: effect of the frequency of lateral oscillation. Aviat Space Environ Med (2004) 75:649–56. 7. Macefield VG, Gandevia SC, Henderson LA. Neural sites involved in the sus- tained increase in muscle sympathetic nerve activity induced by inspiratory capacity apnea: a fMRI study. J Appl Physiol (2006) 100:266–73. doi:10.1152/ japplphysiol.00588.2005 i Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 8. Yates BJ, Bolton PS, Macefield VG. Vestibulo-sympathetic responses. Compr Physiol (2014) 4:851–87. doi:10.1002/cphy.c130041 Copyright © 2018 Cohen and Lewis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 9. Cohen B, Martinelli GP, Ogorodnikov D, Xiang Y, Raphan T, Holstein GR, et al. Vestibular activation habituates the vasovagal response in the rat. Exp Brain Res (2011) 1:45–55. doi:10.3389/fneur.2017.00083 10. Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci (2006) 7:335–46. doi:10.1038/nrn1902 10. Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci (2006) 7:335–46. doi:10.1038/nrn1902 March 2018 \| Volume 9 \| Article 117 Frontiers in Neurology \| www.frontiersin.org
https://openalex.org/W2328604686	https://europepmc.org/articles/pmc4861542?pdf=render	English	null	Projecting Month of Birth for At-Risk Infants after Zika Virus Disease Outbreaks	Emerging infectious diseases	2,016	cc-by	3,708	Projecting Month of Birth for At-Risk Infants after Zika Virus Disease Outbreaks Jennita Reefhuis, Suzanne M. Gilboa, Michael A. Johansson, Diana Valencia, Regina M. Simeone, Susan L. Hills, Kara Polen, Denise J. Jamieson, Lyle R. Petersen, Margaret A. Honein potential link between Zika virus infection and microceph- aly, on February 1, 2016, WHO declared a public health emergency of international concern (9,10). potential link between Zika virus infection and microceph- aly, on February 1, 2016, WHO declared a public health emergency of international concern (9,10). The marked increase in infants born with microcephaly in Brazil after a 2015 outbreak of Zika virus disease suggests an association between maternal Zika virus infection and congenital microcephaly. To project the timing of delivery of infants born to mothers infected during early pregnancy in 1 city in Bahia State, Brazil, we incorporated data on re- ported Zika cases and microcephaly cases into a graphical schematic of weekly birth cohorts. We projected that these births would occur through February 2016. Applying simi- lar projections to a hypothetical location at which Zika virus transmission started in November, we projected that full- term infants at risk for Zika virus infection would be born dur- ing April–September 2016. We also developed a modifiable spreadsheet tool that public health officials and researchers can use for their countries to plan for deliveries of infants to women who were infected with Zika virus during different pregnancy trimesters. g y ( ) As of February 26, 2016, WHO reported 31 countries and territories (11) in the Americas in which local vector- borne transmission of Zika virus was ongoing (12). With expanding local transmission and the possible link between infection during pregnancy and congenital microcephaly, projecting the effects of Zika virus infections for other countries and understanding the gestational time when risk is greatest are critical. As Zika virus has spread through the Americas, questions have arisen about the remarkably high numbers of infants with microcephaly reported in Brazil and the absence of reported microcephaly cases in some other countries where transmission is high. To help answer these questions, assessment of the timing of transmission and its relation to gestational week of pregnancy for the cohort of women who were pregnant during the outbreak is necessary. Our report illustrates the expected periods of exposure and weeks of delivery for the cohorts of pregnant women potentially infected with Zika virus during out- breaks in Bahia State, Brazil. Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (J. Reefhuis, S.M. Gilboa, M.A. Johansson, D. Valencia, R.M. Simeone, K. Polen, D.J. Jamieson, M.A. Honein); Centers for Disease Control and Prevention, Fort Collins, Colorado, USA (S.L. Hills, L.R. Petersen) DOI: http://dx.doi.org/10.3201/eid2205.160290 RESEARCH RESEARCH Projecting Month of Birth for At-Risk Infants after Zika Virus Disease Outbreaks Public health officials and re- searchers in areas with local transmission could apply these methods to country-specific data to produce more precise models and predictions. I n May 2015, the World Health Organization (WHO) re- ported an outbreak of Zika virus disease in Brazil (1). Zika virus is a single-stranded RNA virus spread primarily by Aedes aegypti mosquitoes; maternal–fetal transmission of Zika virus has been reported (2). Infection is asymptomatic in many patients; when clinical illness does occur, it is gener- ally mild, with exanthematous rash, fever, conjunctivitis, or arthralgia (3). An association with Guillain-Barré syndrome is under investigation; on rare occasion, death of patients with chronic disease has been reported (4). I Projecting Month of Birth for At-Risk Infants Projecting Month of Birth for At-Risk Infants To project the probable timing of births with adverse effects associated with Zika virus infection in early preg- nancy, we then applied this approach to a hypothetical country. We assumed that transmission in Country A began on October 4, 2015, and followed the patterns that were seen in Salvador (14) and Yap Island (3). That is, we as- sumed that the level of transmission during October was low, during early November 2015 through mid-February 2016 was high, and from mid-February through mid-March 2016 was lower (Figure 2). across years). We also graphed the reported cases of mi- crocephaly by month of report, assuming that the month of report reflected the month of birth (15). In Bahia, ≈4,000 infants are born each week (16); therefore, each bar represents ≈4,000 pregnancies. We de- rived epidemiologic data from a published report on ex- anthematous illness in the city of Salvador, Bahia State, Brazil (14). We assumed that the epidemic curve of exan- thematous illness was representative of the epidemic curve of Zika virus infection and that the epidemic curve for the city of Salvador could be extrapolated to Bahia State. Be- cause exact numbers of cases were not available, we de- rived estimates from the published epidemic curve, which was sufficient to identify the period of high Zika activity as being from March through June 2015. From the Live Birth Information System in Brazil (16), we obtained the monthly reports of infants born with microcephaly during August 2015–February 2016; information on births from January 2016 on were probably incomplete or were not yet available. The expected baseline prevalence of micro- cephaly is 6 cases per 10,000 births; for a state with 16,000 births per month, 10 cases of microcephaly would be ex- pected each month. Methods Using published data for Bahia State and assuming that all pregnancies lasted 40 weeks (full term), we created figures demonstrating cohorts of pregnant women by week of de- livery and then extrapolated to the beginning of pregnancy. Live-birth data from Brazil showed small differences in the proportions of infants born at full term (37–41 weeks) with microcephaly (76.7%) compared with those born at full term without birth defects (83.6%) (13). We considered the first 2 weeks of pregnancy to be the time from last men- strual period to conception (Figure 1). We also assumed the number of births to be constant across months of the year. To indicate the probable high-risk period for Zika vi- rus transmission, we graphed the number of reported cases of Zika disease or Zika-like illness by epidemiologic week (the standardized method to enable comparison of weeks In October 2015, Brazil started to report higher than expected rates of microcephaly among infants born in the same states where Zika outbreaks had occurred several months before (5). Laboratory tests later confirmed Zika virus infection in several infants born with microcephaly, and several case series have reported that mothers who de- livered an infant with microcephaly had experienced Zika symptoms during early pregnancy (5–8). Because of the 828 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 Results In the city of Salvador, Zika virus transmission was highest during March–June 2015 (Figure 1) (14). During this pe- riod, a cohort of pregnant women could have been infected, and these infections would have occurred at different times during their pregnancies. The period of highest Zika activ- ity was March 22–May 31, 2015 (Figure 1) across all co- horts. Pregnancies that began during November 2014–June 2015 correspond to births anticipated during August 2015– March 2016. For pregnancies that began in December 2014 or January 2015, the highest likelihood of Zika virus infec- tion would have been late in the first trimester or during Figure 1. Projection of birth months after Zika virus transmission and occurrence of microcephaly, Salvador, Bahia State, Brazil. Weekly pregnancy cohorts are based on 40-week pregnancies and monthly reports of infants with microcephaly in Bahia State, Brazil, in relation to periods of high risk for Zika virus transmission. The epidemic curve shows cases treated for illness with rash in Salvador, Brazil, estimated from (14). Complete monthly report data for January–March 2016 are not yet available. Figure 1. Projection of birth months after Zika virus transmission and occurrence of microcephaly, Salvador, Bahia State, Brazil. Weekly pregnancy cohorts are based on 40-week pregnancies and monthly reports of infants with microcephaly in Bahia State, Brazil, in relation to periods of high risk for Zika virus transmission. The epidemic curve shows cases treated for illness with rash in Salvador, Brazil, estimated from (14). Complete monthly report data for January–March 2016 are not yet available. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 829 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 RESEARCH the second trimester of pregnancy, and these pregnancies would have resulted in term births during September and October 2015. For pregnancies that began during late Feb- ruary 2015–May 2015, the highest likelihood of Zika virus infection would have been during the first trimester, and term births would have occurred during November 2015– February 2016. pregnancy. Women whose pregnancies began in July 2015 would be expected to deliver in late March and early April 2016, and risk for infection would have been highest during the second trimester. The highest likelihood of first trimester and early second trimester infection would be among wom- en who became pregnant during September 2015–January 2016, which corresponds to births from mid-May through early October 2016. Results The increased number of reported cases of microceph- aly in Bahia State began with October births; reported cases rose sharply during November 2015–January 2016. For the city of Salvador, these November 2015–January 2016 births corresponded to the highest likelihood of infection occur- ring in the first trimester or early in the second trimester of pregnancy, assuming that the date of report approximates the date of birth. There are no reports to indicate whether the city of Salvador experienced the Zika virus disease out- break earlier or later than the rest of Bahia State. To enable readers to project months when births with exposure in different trimesters can be expected, we de- veloped a modifiable spreadsheet tool (online Technical Appendix, http://wwwnc.cdc.gov/EID/article/22/5/16-0290- Techapp1.xlsx). Users may enter start and end dates of hy- pothetical outbreaks. Discussion Understanding the timing of Zika virus infection of pregnant women is key because the effects of infection on pregnancy and fetal and infant outcomes is likely to vary by gestational timing, as has been demonstrated for other congenital infections such as rubella and cytomegalovirus; transmission risk may also vary according to gestational timing (17,18). For rubella, risk for adverse fetal effects is highest during the first trimester; for cytomegalovirus, risk is highest during the first trimester but is also present after exposure during the second or third trimesters (17,19). For countries currently experiencing Zika outbreaks, it will be several months before the first pregnancies during which exposure could have occurred will reach term, particularly if the critical period of pregnancy is in the first or second trimester, as our data suggest. Some of the reported cases of microcephaly included in the graph are still being assessed, and some might not meet the final case definition for microcephaly in Brazil (i.e., head circumference <32 cm) (20); increased attention to the pos- sible association between Zika virus infection and micro- cephaly may have led to overascertainment. However, the rate of false-positive reports was lower in Bahia than in other states in Brazil (21). Data on births of infants with micro- cephaly were available for September 2015–February 2016, and although the data from January and February 2016 are probably not complete, they do show a decline in the number of infants born with microcephaly. Maternal–fetal transmis- sion might result in other adverse pregnancy outcomes, and the full range of these outcomes is of interest; however, our study accounts for microcephaly only. Also, our assumption of 40-week pregnancies does not account for possible differ- ences in gestational age or for fetal losses and miscarriages, although early case reports do not indicate high rates of pre- maturity (5). If infants with microcephaly were consistently born premature, the relevant exposure period would be de- layed to include more of the second trimester. Our hypothetical data (Figure 2) demonstrate the time between high levels of Zika virus transmission during preg- nancy and pregnancy outcomes for each weekly cohort of pregnant women. With some shifting of dates, these projec- tions could apply to many countries in South and Central America that are currently experiencing outbreaks of Zika virus disease. Discussion We found ecologic evidence of a temporal relationship between maternal Zika virus infection during pregnancy and congenital microcephaly in Bahia State and the possible ges- tational time when risk is highest (Figure 1). This relation- ship does not necessarily imply causality, but it does give additional credence to the pathological findings and case re- ports that suggest a link between Zika virus infection and mi- crocephaly (1,5). Assessing this relationship in other states in Brazil or other locations would have been informative, but very limited data on the spread of Zika virus are avail- able. One limitation of the projections was that the estimated epidemic curve for Bahia State was based on Salvador, the capital city, which contains only ≈18% of the population of Bahia State. It is unknown whether the timing of the out- break in Salvador was similar to that in the remainder of the state, which served as the basis for the microcephaly case numbers. Also, the epidemic curve for Zika virus disease is not based solely on laboratory-confirmed cases, but rather it includes both suspected and confirmed cases determined primarily on the basis of clinical presentation. The micro- cephaly data probably include some reporting delays, es- pecially for January and February. Moreover, these projec- tions assume a true association between maternal Zika virus We assumed that the birth rates in these models remain constant throughout the year, which is not true for all loca- tions. The data for Zika virus infection and infants with mi- crocephaly are based on dates of report, which are probably later than actual occurrence. Despite these limitations, our assessments provide some indication that the period of highest risk might be during the first trimester or early in the second trimester of pregnancy. This assessment can help inform public health officials about risks for microcephaly and help them plan for deliveries in areas where Zika virus disease outbreaks occur. Conducting surveillance for microcephaly but also other pregnancy outcomes such as pregnancy loss and oth- er birth defects will enable continued evaluation of any ef- fects of Zika virus disease might have on pregnancy. These data also emphasize the role of arboviral disease–tracking activities for informing public health planning. Discussion Our projections, based on ecologic data, indicate that in Ba- hia State, Brazil, Zika virus infection during the first trimester or early in the second trimester of pregnancy is temporally associated with the observed increase in infants born with microcephaly; this projection is consistent with the observed In Country A (Figure 2), for the cohort of women whose pregnancies began in May 2015, corresponding to births dur- ing February–early March 2016, the likelihood of Zika virus infection would have been limited to the third trimester of Figure 2. Projection of anticipated birth months after Zika virus transmission in a hypothetical country. Projected birth months for weekly pregnancy cohorts are based on 40-week pregnancies in a hypothetical country in which the highest level of Zika activity was from November 2015 through mid-February 2016. Figure 2. Projection of anticipated birth months after Zika virus transmission in a hypothetical country. Projected birth months for weekly pregnancy cohorts are based on 40-week pregnancies in a hypothetical country in which the highest level of Zika activity was from November 2015 through mid-February 2016. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 830 Projecting Month of Birth for At-Risk Infants infection and infant microcephaly; other maternal cofactors, such as other infections or environmental exposures, might account for some or all of the observed temporal relation- ship. The effects of the imprecision of some of the factors just described are unknown. Countries that can repeat this exercise with more precise prospective data will be better able to describe the expected critical exposure window, and if risk estimates for outcomes such as microcephaly and Guillain-Barré syndrome after Zika virus infection become available, the expected number of individuals who will be affected during a certain period can be predicted. reported decline for January and February 2016. This finding adds to pathologic findings documenting Zika virus infection in several infants with microcephaly (7,8). To create a more precise projection of when to expect the first full-term births to mothers who were infected during their second trimester of pregnancy, readers can refine our model by using our modi- fied spreadsheet tool (online Technical Appendix) and local data from countries in which Zika virus is transmitted. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 References 1. 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Update: interim guidelines for health care providers caring for pregnant women and women of reproductive age with possible Zika virus exposure—United States, 2016. MMWR Morb Mortal Wkly Rep. 2016;65:122–7. http://dx.doi.org/10.15585/mmwr.mm6505e2 6. European Centre for Disease Prevention and Control. Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barré syndrome [cited 2016 Jan 31]. http://ecdc.europa.eu/en/publications/Publications/zika-virus- americas-association-with-microcephaly-rapid-risk-assessment.pdf 23. Discussion The US Centers for Disease Control and Prevention has prepared interim guidelines for US healthcare providers who care for women who are pregnant during a Zika outbreak (22) as well as interim guidelines for the evaluation and testing of 831 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 5, May 2016 RESEARCH infants whose mothers might have been infected with Zika virus during pregnancy (23). 10. Cha AE, Dennis B, Murphy B. Zika virus: WHO declares global public health emergency, says causal link to brain defects ‘strongly suspected.’ Washington Post. 2016 [cited 2016 Feb 2]. https://www.washingtonpost.com/news/to-your-health/ wp/2016/02/01/zika-virus-who-declares-global-public-health- emergency-given-rapid-spread-in-americas/ The consequences of Zika virus infection during preg- nancy are not fully understood. Given the growing evi- dence of an association with microcephaly (5,7,8), and ac- counting for the time lapse between disease outbreaks and the birth of any affected infants as highlighted here, it can be expected that the number of infants born with micro- cephaly and other adverse pregnancy outcomes will con- tinue to rise. 11. Pan American Health Organization, World Health Organization. Countries and territories with autochthonous transmission in the Americas reported in 2015–2016 [cited 2016 Feb 26]. http://www.paho.org/hq/index.php?option=com_content&view= article&id=11603&Itemid=41696&lang=en 12. Hennessey M, Fischer M, Staples JE. ZIKAV spreads to new areas—region of the Americas, May 2015–January 2016. MMWR Morb Mortal Wkly Rep. 2016;65:55–8. http://dx.doi.org/10.15585/ mmwr.mm6503e1 Acknowledgments We thank all members of the Pregnancy and Birth Defects Task Force from the 2016 Centers for Disease Control and Prevention Zika Response. 13. Live Birth Information System Brazil (SINASC). Characteristics of microcephaly and other defects. Panel 3 [cited 2016 Feb 6]. https://public.tableau.com/profile/bruno.zoca#!/ https://public.tableau.com/profile/bruno.zoca#!/ 14. Cardoso CW, Paploski IA, Kikuti M, Rodrigues MS, Silva MM, Campos GS, et al. Outbreak of exanthematous illness associated with Zika, chikungunya, and dengue viruses, Salvador, Brazil. Emerg Infect Dis. 2015;21:2274–6. http://dx.doi.org/10.3201/ eid2112.151167 Dr. Reefhuis is a senior health scientist and team lead in the Birth Defects Branch, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta. Her research has focused on identifying modifiable risk factors for birth defects. 15. Live Birth Information System Brazil (SINASC). Microcephaly in Brazil, 2000–2016. Panel 2 [cited 2016 Feb 26]. https://public.tableau.com/profile/bruno.zoca#!/ https://public.tableau.com/profile/bruno.zoca#!/ 16. Live Birth Information System Brazil (SINASC). Live birth data for states in Brazil [cited 2016 Feb 4]. http://tabnet.datasus.gov.br/ cgi/tabcgi.exe?sinasc/cnv/nvBA.def Address for correspondence: Jennita Reefhuis, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop E86, Atlanta, GA 30329-4027, USA; email: nzr5@cdc.gov References Fleming-Dutra KE, Nelson JM, Fischer M, Staples JE, Karwowski MP, Mead P, et al. Update: interim guidelines for health care providers caring for infants and children with possible Zika virus infection—United States, February 2016. MMWR Morb Mortal Wkly Rep. 2016;65:182–7. http://dx.doi.org/10.15585/ mmwr.mm6507e1 7. Oliveira Melo AS, Malinger G, Ximenes R, Szejnfeld P, Alves Sampaio S, Bispo de Filippis A. ZIKAV intrauterine infec- tion causes fetal brain abnormality and microcephaly: tip of the iceberg? Ultrasound Obstet Gynecol. 2016;47:6–7. http://dx.doi. org/10.1002/uog.15831 8. 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https://openalex.org/W4367181657	https://link.springer.com/content/pdf/10.1007/s12210-023-01139-3.pdf	English	null	Assessment of anticancer, antibacterial, antifungal, and antioxidant activities of Micractinium reisseri (Chlorophyta, Trebouxiophyceae) methanolic extract	Rendiconti lincei. Scienze fisiche e naturali	2,023	cc-by	5,051	Abstract This study looked at the anticancer, antimicrobial, and antioxidant properties of a methanolic extract of Micractinium reis- seri. In vitro antitumor activity of M. reisseri methanolic extract revealed an inhibitory effect against MCF-7 and HCT-116 (breast carcinoma and colon carcinoma) cell lines. Antimicrobial activity of M. reisseri methanolic extract was estimated against Gram-negative bacteria (Pseudomonas aeruginosa RCMB 010,049 and Escherichia coli RCMB 010,052), Gram- positive bacteria (Streptococcus pneumonia RCMB 010,010 and Bacillus subtilis RCMB 010,067) and fungi (Aspergillus fumigatus RCMB 02,568 and Candida albicans RCMB 05,036). The extract showed promising results against all the tested microbes except C. albicans which gave negative results. The algal extract showed 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing and hydroxyl radical scavenging activity in a concentration-dependent manner with maximum scavenging activity at concentrations (0.001, 0.001, and 5 mg/ml) for each assay, respectively. High-performance liquid chromatography (HPLC) revealed that the active compounds are phenols, alkaloids, and flavonoids. In conclusion, M. reisseri methanolic extract exhibited effective anticancer, antimicrobial, and antioxidant activities. Keywords Micractinium reisseri · Methanolic extract · MCF-7 · HCT-116 · Colon cancer · Breast cancer · Antioxidant activity · Antibacterial activity · Antifungal activity Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 https://doi.org/10.1007/s12210-023-01139-3 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 https://doi.org/10.1007/s12210-023-01139-3 RESEARCH PAPER Assessment of anticancer, antibacterial, antifungal, and antioxidant activities of Micractinium reisseri (Chlorophyta, Trebouxiophyceae) methanolic extract Mofida E. M. Makhlof1 · Doaa A. Ghareeb2 · Eman T. El‑Kenany3,4 Received: 30 September 2022 / Accepted: 2 February 2023 / Published online: 27 April 2023 © The Author(s) 2023 2.4 M. reisseri methanolic extract One hundred grams of M. reisseri biomass were extracted in one liter of 100% methanol. The mixtures were kept at room temperature in a shaking incubator for 3 days. After 3 days, the samples were centrifuged for 20 min at 7200 g. The clear extract was concentrated with a rotary evaporator at 40 °C under reduced pressure (72 mbar) and dried with a lyophilizer. The dried extract was collected and then dis- solved in 5 mg/ml Di-methyl sulfoxide (DEMSO) (Chernane et al. 2014). 1 Introduction Unicellular microalgae are microorganisms found in fresh or saltwater and have varied shapes with a diameter or length of about 3–10 µm (Ferreira et al. 2013). Many studies have been conducted to investigate the products of microalgal metabolism, not only to identify their nature but also to look for ingredients with potential applications to humans in a variety of important fields (De Morais et al. 2015). They contain amino acids, steroids, phenolic com- pounds, terpenoids, phlorotannins, halogenated ketones, alkenes, and cyclic polysulfides, all of which have different physiological (antioxidant, antibacterial, antiviral, antifun- gal, enzyme inhibiting, immunosuppressive, cytotoxic, and algicide activity) effects (Taskin et al. 2007 and Rao et al. 2007). Trends in natural drug research suggest that algae are a promising source of new biochemically active substances (Mayer and Hamann 2005). Meanwhile, screening extracts or separation of metabolites from various microalgae is a collaborative method for expressing the biological activity of these components (Herrero et al. 2013). Micractinium * Eman T. El‑Kenany emantahaali@gmail.com Mofida E. M. Makhlof mofida_makhlof@yahoo.com Doaa A. Ghareeb doaa_ghareeb76@yahoo.com 1 Botany and Microbiology Department, Faculty of Science, Damanhour University, Damanhour, Egypt 2 Bio‑Screening and Preclinical Trial Lab, Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt 3 Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt 4 Oral Biology Department, Faculty of Dentistry, Pharos University, Alexandria, Egypt * Eman T. El‑Kenany emantahaali@gmail.com Mofida E. M. Makhlof mofida_makhlof@yahoo.com Doaa A. Ghareeb doaa_ghareeb76@yahoo.com * Eman T. El‑Kenany emantahaali@gmail.com Mofida E. M. Makhlof mofida_makhlof@yahoo.com Doaa A. Ghareeb doaa_ghareeb76@yahoo.com 1 Botany and Microbiology Department, Faculty of Science, Damanhour University, Damanhour, Egypt 2 Bio‑Screening and Preclinical Trial Lab, Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt 3 Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt 4 Oral Biology Department, Faculty of Dentistry, Pharos University, Alexandria, Egypt 2 Bio‑Screening and Preclinical Trial Lab, Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt 2 Bio‑Screening and Preclinical Trial Lab, Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt 3 Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt 4 Oral Biology Department, Faculty of Dentistry, Pharos University, Alexandria, Egypt 4 Oral Biology Department, Faculty of Dentistry, Pharos University, Alexandria, Egypt (01234 1 3 (0121 3456789) 3 Rendiconti Lincei. 1 Introduction Scienze Fisiche e Naturali (2023) 34:483–489 484 reisseri is a member of the Chlorellaceae family and is dis- tinguished by spherical or oval cells with a single chloroplast that is parietal with a pyrenoid (Krienitz et al. 2004; Chae et al. 2019). This alga is well known for being a promising source of biofuels (El-Sheekh et al. 2020), but for unknown reasons, the vital activities of M. reisseri methanolic extract have not been tested yet. Therefore, for the first time, this work aims to evaluate the effectiveness of M. reisseri metha- nolic extract cytotoxicity against (MCF-7 & HCT-116) cell lines, antimicrobial activity with MIC against P. aeruginosa, E. coli, S. pneumonia, B. subtilis, C. albicans, A. fumigatus with the evaluation of its antioxidant power using 2,2-diphe- nyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and Hydroxyl radical scavenging assays. spectrophotometric method recommended by Jensen and Liaaen (1959). 2.5 Phytochemical analysis of M. reisseri methanolic extract The total flavonoid content was determined using the alu- minum chloride colorimetric method with aluminum chlo- ride hexahydrate (AlCl3), and the results were expressed in milligram quercetin equivalents (QE)/g lyophilized powder (Chang et al. 2002). The total phenolic content was calcu- lated using the Folin-Ciocalteu method and sodium carbon- ate (Na2CO3). The data were converted to milligrams gallic acid equivalents (GAE) per 100 mL of algal extract. Accord- ing to Ayoola et al. (2008), total alkaloid content was deter- mined by reacting alkaloid with bromocresol green (BCG), resulting in a yellow-colored product. The method uses bro- mocresol green solution (110–4), NaOH/2N, distilled water, phosphate buffer solution (pH 4.7), and atropine standard solution to provide sensitivity and stability. 2.3 Large‑scale cultivation and culture harvesting M. reisseri culture was previously grown in a 2000 ml Erlenmeyer flask containing 1400 ml of cultivation medium and a 50 ml known inoculum (with initial turbidity: 0.07 at 450 nm). This inoculum was divided into five groups of 280 ml each, and each inoculum was transferred to a steri- lized 2000 ml Erlenmeyer flask containing 1400 ml steri- lized cultivation medium, yielding five flasks. The indoor batch cultures were grown in the same manner as the indoor airlift cultures. The cultures were aerated using an air injec- tion device connected to a glass pipe equipped with a steri- lized bacterial filter (0.2 m), which released air bubbles from the flask bottoms. The air-flow rate was adjusted to ensure proper culture mixing through the upward movement of air bubbles. Centrifugation at 3606 g for 20 min was used to harvest the algal cells. The supernatants were removed, and the remaining pellets were used to make the methanolic extract. 2.1 Microalga and growth condition Micractinium reisseri R. Hoshina, M. Iwataki, and N. Ima- mura, a unicellular green alga, was chosen for this study. A routine bacterial test, however, was performed on a regu- lar basis by inoculating one drop of the culture at the start and end of the experiment into previously autoclaved 10 ml vials containing a bacterial growth medium, F. medium (Feeley et al. 1978). M. reisseri axenic cultures were grown in 500 ml Erlenmeyer flasks with 200 ml of the selected medium in Bold’s Basal Medium (BBM). The experiment was carried out in a culturing chamber under controlled lab- oratory conditions (28 °C temperature, 80 µmol m−2S−1 light intensity). This temperature was chosen based on the results of Ginzburg and Ginzburg (1981). Cultures were illuminated using a 16-h light/eight-hour dark cycle. All culture flasks were manually swirled daily to detach adhered algal cells from the flask walls. The research period lasted 18 days. To achieve suitable biomass for bioactivity measurements, large-scale cultivation was prepared under the same culture conditions. 2.2 Growth measurements The optical density and pigment content of M. reisseri were used to estimate growth (chlorophyll a and carotenoids). These analyses were carried out every 2 days for the next 18 days. The optical density was determined using a Perkin Elmer (Lambda 1) Visible–UV (ultraviolet) spectrophotom- eter at 450 nm, as described by Robert (1979). Chlorophyll pigment absorption was measured at wavelengths of 664 nm, 647 nm, and 630 nm. The trichromatic equation of Jeffrey and Humphrey (1975) was used to calculate chlorophyll a and b. carotenoids at 450 nm were determined using the 1 3 3 485 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 2.8 Evaluation of antimicrobial activity Pseudomonas aeruginosa RCMB 010049, Escherichia coli RCMB 010052, Streptococcus pneumoniae RCMB 010010, Bacillus subtilis RCMB 010067, Aspergillus fumigatus RCMB 02568, and Candida albicans RCMB 05036 were the strains tested in this study. The standard disc diffusion assay (Balouiri et al. 2016) was used to test antimicrobial activity in vitro against the pathogenic bac- teria and fungi mentioned. The experiment was repeated three times, and the average zone of inhibition was cal- culated. According to Doughari (2006), the minimum inhibitory concentration (MIC) of M. reisseri extract was determined in triplicate for each of the tested organisms. The MIC values were calculated as the lowest dose of antibacterial substances that prevented the visible growth of microbes (EL-Saadony et al. 2021). The algal methanolic extracts were separated using an HPLC device Agilent Technologies1200 with an HP 1200 UV vari- able wavelength detector on a C18 Zorbax Hypersil column (150 mm 4.6 mm, i.d. 5 m). The extraction and HPLC analy- sis conditions were set according to (Rodríguez-Bernaldo et al. 2010); however, partial modifications were required to optimize the results. 2.9 Evaluation of antioxidant activity The antioxidant compound that prevents the oxidation of DPPH was used to test the free radical scavenging activities (Blois 1958). The following equation was used to calculate the ability of diet extract to scavenge DPPH radicals: DPPH radical scavenging activity = Acontrol −Asample Acontrol × 100 Hydroxyl radical scavenging activity was assayed as described by Elizabeth and Rao (1990). The inhibition percentage was estimated as follows: 2.7.2 Cytotoxicity evaluation using viability assay The inhibition percentage (%) = [ (test −control)∕control ] × 100 Tumor cell lines were suspended in the medium at a concen- tration of 5 × 104 cell/well in Corning® 96-well tissue cul- ture plates, and then incubated for 24 h. After that, M. reis- seri extract was added to 96-well plates in three replicates to achieve ten concentrations. As a control, six vehicle controls with media or 0.5% DMSO were run for each 96-well plate. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra- zolium bromide test was used to determine the number of viable cells after 24 h of incubation (MTT test). The viability percentage was calculated as [(ODt/ODc)] × 100%, where ODt represents the mean optical density of wells treated with M. reisseri extract and ODc represents the mean optical density of untreated cells. The survival curve of each tumor cell line after M. reisseri extract treatment is obtained by plotting the relationship between surviving cells and drug concentration. The 50% inhibitory concentration (IC50), or the concentration required to cause toxic effects in 50% of intact cells, was calculated using Graphpad Prism software (San Diego, CA, USA) and graphic plots of the quantity- response curve for each concentration (Mosmann 1983). The antioxidant capacity of M. reisseri extract is meas- ured using ferric reducing antioxidant power (FRAP) according to Banerjee and Maulik (2002) and Sutharsingh et al. (2011). The reducing power is due to the conversion of ferric cyanide into ferrous form via the donation of an e- from the antioxidant molecule found in M. reisseri extract. After blank subtraction, the reducing power was expressed as an increase in absorbance at 700 nm. 2.7.1 Mammalian cell lines ATCC Culture collection provided MCF-7 and HCT-116 cells (breast and colon carcinoma). Sigma sold dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye (St. Louis, Mo., USA). Lonza provided foetal bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% Trypsin–EDTA. Crystal violet stain (1%) is made up of 0.5% (w/v) crystal violet and 50% methanol and filtered through a Whatmann No.1 filter paper (Mosmann 1983). Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 g/ ml gentamycin was used to grow the cells. All cells were cultured twice a week and kept at 37 oC in a humidified atmosphere with 5% CO2. 3.1 Growth measurements Figures 1 and 2 depict M. reisseri growth measurements by optical density, growth rate, chlorophyll a, chloro- phyll b, and carotenoid content. The maxima of these 1 3 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 486 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 12 14 16 18 Time (days) Optical density Growth rate Fig. 1 Growth measured as optical density and growth rate of M. reisseri 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 14 16 18 Concentration (mg/l) Time (days) Ch a Ch b Carotenoids Fig. 2 Chlorophyll a, Chlorophyll b, and Carotenoids content of M. reisseri 3.2 Cytotoxic activity of M. reisseri methanolic extract -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 12 14 16 18 Time (days) Optical density Growth rate Fig. 1 Growth measured as optical density and growth rate of M. reisseri -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 12 14 16 18 Time (days) Optical density Growth rate Fig. 1 Growth measured as optical density and growth rate of M. reisseri Table 1 demonstrates the cytotoxic activity of M. reis- seri extract against MCF-7 and HCT-116 cell lines, the results showed that M. reisseri extract exhib- its cytotoxicity in all concentrations (ranging from 1.56 to 50 g/ml), the cytotoxic activity increased by increasing the extract concentration in a concentra- tion-dependent manner, showing inhibition percent- ages ranging from (1.78 ± 1.34%–83.68 ± 1.57%) and (10.55 ± 2.05%–86.33 ± 0.35%). Thus, M. reisseri metha- nolic extract was more effective against colon cancer than breast cancer. According to Gabriel et al. (2022), geran- iol has been widely investigated as a potential anticancer agent, and the same is true for M. reisseri extract (Table 2, Fig. 3). Gallic acid and quercetin, which are found in the extract, have also been linked to the prevention and pro- gression of various types of cancer (Zhang et al. 2019 and Hong et al. 2021). Fig. 1 Growth measured as optical density and growth rate of M. reisseri Fig. 1 Growth measured as optical density and growth rate of M. reisseri 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 14 16 18 Concentration (mg/l) Time (days) Ch a Ch b Carotenoids Fig. 2 Chlorophyll a, Chlorophyll b, and Carotenoids content of M. reisseri 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 14 16 18 Concentration (mg/l) Time (days) Ch a Ch b Carotenoids Fig. 2 Chlorophyll a, Chlorophyll b, and Carotenoids content of M. reisseri Concentration (mg/l) 3.3 Antimicrobial activity With the evaluation of their MIC, a strong and remarka- ble activity of M. reisseri methanolic extract was detected against all tested Gram-positive, Gram-negative bacterial pathogens and fungi (Table 3). Antibacterial activity was classified as no activity (−: inhibition diameter 10 mm), low (+ : inhibition diameter between 10 and 15 mm), moder- ate (+ + : inhibition diameter between 15 and 20 mm), and high activity (+ + + : inhibition diameter 20 mm). However, as shown in Table 3 and Fig. 4, the majority of inhibition zones produced by M. reisseri extract against all tested pathogens were highly promising, ranging from moderate to high activity levels. The highest antibacterial activity levels were reported against B. subtilis RCMB 010,067 and E. coli RCMB 010,052, with mean inhibition zones and standard deviations of (20.60.58, 22.41.2) and MICs of 1.95 g/ml and 0.98 g/ml, respectively. According to Table 3 and Fig. 4, the extract has moderate antibacterial activity against P. aer- uginosa RCMP 010,049 and S. pneumonia RCMP 010,010, with a mean inhibition zone and SD (18.3 0.72–18.3 0.63) and MIC 7.81 for the two bacterial strains. These findings were supported by the findings of Mc Gee et al. (2020), who examined extracts from 80 newly isolated marine and freshwater microalgae strains such as Micractinium and tested them for antimicrobial activity against 6 pathogens (Escherichia coli, Pseudomonas aeruginosa, Bacillus sub- tilis, Enterococcus faecalis, Staphylococcus aureus, and Candida albicans). Our findings also show that M. reisseri extract has moderate antifungal activity against Aspergillus fumigatus but no activity against Candida albicans. Fig. 2 Chlorophyll a, Chlorophyll b, and Carotenoids content of M. reisseri Table 1 Cytotoxic activity of Micractinium reisseri methanolic extract against (MCF-7 and HCT-116) cell lines Sample conc. (µg/ ml) HCT-116 cell line IC50 = 9.8 ± 1.29 µg MCF-7 cell line IC50 = 16.1 ± 1.09 µg Viability% Inhibitory% Viability% Inhibitory% 0 100 0 100 0 1.56 89.45 10.55 ± 2.05 98.22 1.78 ± 1.3 3.125 78.72 21.28 ± 2.03 91.47 8.53 ± 0.98 6.25 59.48 40.52 ± 1.23 78.39 21.61 ± 1.22 12.5 42.81 57.19 ± 0.98 56.21 43.79 ± 0.23 25 26.59 73.41 ± 1.24 34.85 65.15 ± 0.59 50 13.67 86.33 ± 0.35 16.32 83.68 ± 1.57 Table 1 Cytotoxic activity of Micractinium reisseri methanolic extract against (MCF-7 and HCT-116) cell lines measurements were observed on the 14th, 6th, 12th, 14th, and 12th days of cultivation, respectively. 3.3 Antimicrobial activity As a result, the harvesting date was set for the 14th day of cultivation to obtain the highest yield of algal cells. measurements were observed on the 14th, 6th, 12th, 14th, and 12th days of cultivation, respectively. As a result, the harvesting date was set for the 14th day of cultivation to obtain the highest yield of algal cells. 1 3 3 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 487 487 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483 489 Table 2 Phytochemical ingredients of Micractinium reisseri methanolic extract Compounds Total Alkaloids (mg/ml) 20.19 ± 0.67 Total Flavonoids (µg/ml) 2.428 ± 1.7 Total Phenolics (mg/ml) 0.0118 ± 0.2 Compounds (mg/ml) Retention time (Min) Amount (mg/ml) Phenolic compounds Tanic acid 5.400 Nil Phloroidzin 5.202 Nil Cinnamic acid 12.982 Nil Gallic acid 2.274 3.9 2,5dihydroxy benzoic acid 3.249 Nil Chlorogenic acid 2.147 Nil Flavonoid compounds Catecin 3.980 Nil Rutin 4.600 Nil Quercetin 9.052 6.6e−3 Alkaloid Geranoil 7.743 4.71e−2 Fig. 3 HPLC analysis of M. reisseri methanolic extract Table 3 antimicrobial activity of Micractinium reisseri methanolic extract Tested organisms Mean Inhibition Zone mm ± SD Minimum Inhibition Concentration (µg/ ml) Candida albicans RCMB 05036 NA NA Aspergillus fumigatus RCMB 02568 16.8 ± 1.2 15.63 Streptococcus pneumonia RCMB 010010 18.3 ± 0.63 7.81 Bacillus subtilis RCMB 010067 20.6 ± 0.58 1.95 Pseudomonas aeruginosa RCMB 010049 18.3 ± 0.72 7.81 Escherichia coli RCMB 010052 22.4 ± 1.2 0.98 Table 2 Phytochemical ingredients of Micractinium reisseri methanolic extract Compounds Total Alkaloids (mg/ml) 20.19 ± 0.67 Total Flavonoids (µg/ml) 2.428 ± 1.7 Total Phenolics (mg/ml) 0.0118 ± 0.2 Compounds (mg/ml) Retention time (Min) Amount (mg/ml) Phenolic compounds Tanic acid 5.400 Nil Phloroidzin 5.202 Nil Cinnamic acid 12.982 Nil Gallic acid 2.274 3.9 2,5dihydroxy benzoic acid 3.249 Nil Chlorogenic acid 2.147 Nil Flavonoid compounds Catecin 3.980 Nil Rutin 4.600 Nil Quercetin 9.052 6.6e−3 Alkaloid Geranoil 7.743 4.71e−2 Fig. 3 HPLC analysis of M. 3.4 Antioxidant activity The antioxidant activity of M. reisseri methanolic extract in DPPH scavenging and FRAP is shown in Fig. 5. The activity was highest at the lowest concentrations (0.001 mg/ml) and decreased as the algal extract concentration increased. The algal extract demonstrated the greatest antioxidant activity in the hydroxyl radical scavenging test at the highest tested concentration (5 mg/ml). The extract’s reducing power is primarily due to the presence of flavonoids, alkaloids, and phenolic compounds (Table 2), which is supported by previ- ous findings of Prasad and Muralidhara (2017), Gao et al. (2019), and Xu et al. (2019) who tested the antioxidant activ- ity of geranoil (alkaloid), gallic acid (phenolic), and querce- tin (flavonoid), yielding promising results. 3.3 Antimicrobial activity reisseri methanolic extract a against Escherichia coli and b against Bacillus subtilis used for algal extraction has unique properties, optimizing growth factors such as pH, culture media component, and solvent type may increase the amount and effectiveness of the extracted compounds. used for algal extraction has unique properties, optimizing growth factors such as pH, culture media component, and solvent type may increase the amount and effectiveness of the extracted compounds. 0 10 20 30 40 50 60 70 0.001 0.005 0.1 0.5 1 2.5 5 Activity (%) Concentration (mg/ml) DPPH FRAP Fig. 5 The antioxidant activity of M. reisseri methanolic extract in DPPH scavenging and FRAP Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyp- tian Knowledge Bank (EKB). Declarations Conflict of interest No funding was received from any organization for the completion of this research article. Fig. 5 The antioxidant activity of M. reisseri methanolic extract in DPPH scavenging and FRAP Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Ayoola G, Coker H, Adesegun S, Adepoju-Bello A, Obaweya K, Ezennia E, Atangbayila T (2008) Phytochemical screening and antioxidant activities of some selected medicinal plants used for malaria therapy in Southwestern Nigeria. Trop J Pharm Res 7(3):1019–1102 Balouiri M, Sadiki M, Ibnsouda KS (2016) Methods for in vitro evalu- ating antimicrobial activity: a review. J Pharm Anal 6(2):71–79f 3.3 Antimicrobial activity reisseri methanolic extract Table 3 antimicrobial activity of Micractinium reisseri methanolic extract Tested organisms Mean Inhibition Zone mm ± SD Minimum Inhibition Concentration (µg/ ml) Candida albicans RCMB 05036 NA NA Aspergillus fumigatus RCMB 02568 16.8 ± 1.2 15.63 Streptococcus pneumonia RCMB 010010 18.3 ± 0.63 7.81 Bacillus subtilis RCMB 010067 20.6 ± 0.58 1.95 Pseudomonas aeruginosa RCMB 010049 18.3 ± 0.72 7.81 Escherichia coli RCMB 010052 22.4 ± 1.2 0.98 Table 2 Phytochemical ingredients of Micractinium reisseri methanolic extract Compounds Total Alkaloids (mg/ml) 20.19 ± 0.67 Total Flavonoids (µg/ml) 2.428 ± 1.7 Total Phenolics (mg/ml) 0.0118 ± 0.2 Compounds (mg/ml) Retention time (Min) Amount (mg/ml) Phenolic compounds Tanic acid 5.400 Nil Phloroidzin 5.202 Nil Cinnamic acid 12.982 Nil Gallic acid 2.274 3.9 2,5dihydroxy benzoic acid 3.249 Nil Chlorogenic acid 2.147 Nil Flavonoid compounds Catecin 3.980 Nil Rutin 4.600 Nil Quercetin 9.052 6.6e−3 Alkaloid Geranoil 7.743 4.71e−2 Fig. 3 HPLC analysis of M. reisseri methanolic extract Table 3 antimicrobial activity of Micractinium reisseri methanolic extract Tested organisms Mean Inhibition Zone mm ± SD Minimum Inhibition Concentration (µg/ ml) Candida albicans RCMB 05036 NA NA Aspergillus fumigatus RCMB 02568 16.8 ± 1.2 15.63 Streptococcus pneumonia RCMB 010010 18.3 ± 0.63 7.81 Bacillus subtilis RCMB 010067 20.6 ± 0.58 1.95 Pseudomonas aeruginosa RCMB 010049 18.3 ± 0.72 7.81 Escherichia coli RCMB 010052 22.4 ± 1.2 0.98 HPLC analysis of M. i methanolic extract Fig. 3 HPLC analysis of M. reisseri methanolic extract Table 3 antimicrobial activity of Micractinium reisseri methanolic extract Tested organisms Mean Inhibition Zone mm ± SD Minimum Inhibition Concentration (µg/ ml) Candida albicans RCMB 05036 NA NA Aspergillus fumigatus RCMB 02568 16.8 ± 1.2 15.63 Streptococcus pneumonia RCMB 010010 18.3 ± 0.63 7.81 Bacillus subtilis RCMB 010067 20.6 ± 0.58 1.95 Pseudomonas aeruginosa RCMB 010049 18.3 ± 0.72 7.81 Escherichia coli RCMB 010052 22.4 ± 1.2 0.98 Fig. 3 HPLC analysis of M. reisseri methanolic extract Table 3 antimicrobial activity of Micractinium reisseri methanolic extract Tested organisms 488 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 used for algal extraction has unique properties, optimizing growth factors such as pH, culture media component, and solvent type may increase the amount and effectiveness of the extracted compounds. Fig. 4 The antibacterial activity of M. reisseri methanolic extract a against Escherichia coli and b against Bacillus subtilis Fig. 4 The antibacterial activity of M. 4 Conclusion Banerjee SK, Maulik SK (2002) Effect of garlic on cardiovascular disorders. Nutr J 19:1–4 Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181:1199–1200 M. reisseri could be a promising treatment for cancer, microbes, and oxidative damage diseases after the neces- sary clinical studies. According to the findings, M. reis- seri methanolic extract has promising cytotoxicity against MCF-7 and HCT-116 cell lines, as well as antioxidant, antibacterial, and antifungal properties. Because each tool Chae H, Lim S, Kim HS, Choi H, Kim JH (2019) Morphology and phylogenetic relationships of Micractinium (Chlorellaceae, Tre- bouxiophyceae) taxa, including three new species from Antarc- tica. Algae 34(4):267–275 3 3 Rendiconti Lincei. Scienze Fisiche e Naturali (2023) 34:483–489 489 Chang CC, Yang MH, Wen HM, Chern JC (2002) Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 10:178–182 algae, and natural phycoplankton. Biochem Physiol Pflanzen 167:191–194 Jensen A, Liaaen S (1959) Quantitative paper chromatography of carot- enoids. Acta Chem Scand 13:1863–1868 Chernane H, Mansori M, Latique S, ElMaoua M (2014) Evaluation of antioxidant capacity of methanol extract, and its solvent fractions obtained from four moroccan macro algae species. Europ Sci J 10(15):35–48 Krienitz L, Hegewald EH, Hepperle D, Huss VAR, Rohr T, Wolf M (2004) Phylogenetic relationship of Chlorella and Parachlo- rella gen. nov. (Chlorophyta, Trebouxiophyceae). Phycologia 43:529–542 De Morais MG, Vaz Bda S, de Morais EG, Costa JAV (2015) Biologi- cally active metabolites synthesized by microalgae. Biomed Res Int. https://doi.org/10.1155/2015/835761. (Article ID 835761) Mayer AM, Hamann MT (2005) Marine pharmacology in 2001–2002: marine compounds with antihelmintic, antibacterial, anticoagu- lant, antidiabetic, antifungal, anti-inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activi- ties; affecting the cardiovascular, immune, and nervous systems, and other miscellaneous mechanisms of action. Compar Biochem Physiol 140:265–286 De Rodriguez-Bernaldo AQ, Lage-Yusty MA, López-Hernández J (2010) Determination of phenolic compounds in macroalgae for human consumption. Food Chem 121:634–638 Doughari JH (2006) Antimicrobial activity of Tamarindus indica L. 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Afr J Biotech 6(24):2746–2751 Ginzburg M, Ginzburg BZ (1981) Interrelationships of light, tem- perature, sodium chloride, and carbon source on growth of halotolerent, and halophilic strains of Dunaliella. Brit Phycol J 16:313–324 Xu D, Hu MJ, Wang YQ, Cui YL (2019) Antioxidant activities of quercetin and its complexes for medicinal application. 4 Conclusion Molecules 24(6):1123–1237 Zhang T, Ma L, Wu P, Li W, Li T, Gu R, Dan X, Li Z, Fan X, Xiao Z (2019) Gallic acid has anticancer activity and enhances the anticancer effects of cisplatin in non-small cell lung cancer A549 cells via the JAK/STAT3 signaling pathway. Oncol Rep 41(3):1779–1788 Herrero M, Mendiola JA, Plaza M, Ibañez E (2013) Screening for bio- active compounds from algae. Advanced biofuels, and bioprod- ucts. 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https://openalex.org/W4310989008	https://www.frontiersin.org/articles/10.3389/fevo.2022.1090859/pdf	English	null	Editorial: Biologically-informed approaches to design processes and applications	Frontiers in ecology and evolution	2,022	cc-by	1,741	TYPE Editorial PUBLISHED 29 November 2022 DOI 10.3389/fevo.2022.1090859 TYPE Editorial PUBLISHED 29 November 2022 DOI 10.3389/fevo.2022.1090859 KEYWORDS biomimetic, bioinspiration, robotics, architectural design, materials, biophotonics Pugnale A, Stuart-Fox D, Elgar MA, Laschi C and Dumanli AG (2022) Editorial: Biologically-informed approaches to design processes and applications. Editorial on the Research Topic Biologically-informed approaches to design processes and applications OPEN ACCESS Alberto Pugnale1, Devi Stuart-Fox2, Mark A. Elgar2, Cecilia Laschi3 and Ahu Gumrah Dumanli4 1Faculty of Architecture, Building and Planning, The University of Melbourne, Melbourne, VIC, Australia, 2School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia, 3Department of Mechanical Engineering, National University of Singapore, Singapore, Singapore, 4Department of Materials, The University of Manchester, Manchester, United Kingdom KEYWORDS Editorial: Biologically-informed approaches to design processes and applications OPEN ACCESS EDITED AND REVIEWED BY Elise Huchard, UMR5554 Institut des Sciences de l’Evolution de Montpellier (ISEM), France CORRESPONDENCE Devi Stuart-Fox d.stuart-fox@unimelb.edu.au SPECIALTY SECTION This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution RECEIVED 06 November 2022 ACCEPTED 11 November 2022 PUBLISHED 29 November 2022 CITATION Pugnale A, Stuart-Fox D, Elgar MA, Laschi C and Dumanli AG (2022) Editorial: Biologically-informed approaches to design processes and applications. Front. Ecol. Evol. 10:1090859. doi: 10.3389/fevo.2022.1090859 frontiersin.org Editorial on the Research Topic the aerodynamic forces and demonstrate how tandem wings can generate higher lift and improve stability compared with a single pair of wings. The mechanism could be used for the design of a future biomimetic micro air vehicle. diversity and understanding the ecological and evolutionary context of the biological models. They also highlight that interdisciplinary engagement is a two-way street—for example, application of engineering approaches can improve biological understanding just as biology can oﬀer new engineering solutions. A bio-informed approach to the design process is exempliﬁed by the article by Mirra et al. The authors implement a novel artiﬁcial intelligence (AI) agent to design 3D tree forms with complexity and features that are attractive to arboreal wildlife. Although the generated artiﬁcial designs deviate from natural structural forms, they are easy to build, and their form preserves those visual features that are meaningful to birds and other wildlife. Several of the articles in this Research Topic focused on expanding biological knowledge for bio-informed applications. Ensikat and Weigend explored the diversity and distribution of biominerals (organic/inorganic composite materials) that comprise the “hairs” (trichomes) on leaf surfaces of many plants. Biomineralization is an area of considerable interest for biomedical applications and plant trichomes provide a more tractable model to enable in-vivo study of complex composite materials comprising diﬀerent minerals and organic compounds. Finally, a bio-informed approach encompasses not only artiﬁcial materials and technologies, but also natural materials. Mycelium—the vegetative body of a mushroom or fungus— is one of the most promising natural materials for a wide range of applications. Like polystyrene, it is light weight with excellent thermal insulation and ﬁre retardance, but unlike polystyrene, it is sustainable and biodegradable. McGaw et al. argue that the slow uptake of mycelium-based materials is due to psychological, aesthetic and economic barriers, rather than technical challenges. McGaw et al. show the importance of considering social, cultural and economic factors in the design of bioinspired sustainable materials, and identify opportunities for future zero-waste mycelium- based products. Turning from plants to animals, Freyer et al. compared the structures responsible for diﬀerent types of iridescent plumage in two bird species. Their study shows how adjusting the thickness of one layer in the complex feather microstructure can tune the iridescence from blue-purple in the European starling to blue-green in the Cape starling. This discovery has potential applications in bioinspired materials that tune color through a single sensing layer. Frontiers in Ecology and Evolution Concluding remarks The articles in this Research Topic span an extraordinary breadth of biological systems and disciplines. They showcase the utility of a biologically informed approach to enhance not only design processes and applications, but also biological understanding and sustainability. The focus of the articles ranges from expanding biological knowledge and simulating complex biological processes for bioinspired applications, designing new technologies and human-made habitat structures, to exploring the social and economic context for the uptake of products based on novel biomaterials. We hope that this collection of articles inspires new ideas and helps to foster deeper collaboration between biologists, physical and social scientists, engineers and designers to solve the signiﬁcant challenges of our time. Sellers et al. also simulated a biological process, namely locomotion, borrowing from robotics to improve our understanding of animal biomechanics. Rather than typical steady state locomotion such as walking or running at constant speed, the study focused on non-steady-state movements such as starting, stopping and turning. The study shows how gait controllers used in robotics can be used to realistically simulate non-steady-state gaits of a chimpanzee. Editorial on the Research Topic Two studies in this Research Topic provided elegant demonstrations of how complex biological processes can be simulated, simultaneously enhancing biological understanding and paving the way for future applications. Howard et al. showed that honeybees are able to process and categorize odd and even numbers of elements and that this learning task can be simulated with a simple neural network comprising 5 neurons. On one hand, this study suggests that complex cognitive-like behaviors can be seen in honeybees and other assumed simple biological systems. On the other hand, it demonstrates that it is possible to design computing solutions with very simpliﬁed mechanisms that resemble simple animal brains. Editorial on the Research Topic Editorial on the Research Topic Biologically-informed approaches to design processes and applications Front. Ecol. Evol. 10:1090859. doi: 10.3389/fevo.2022.1090859 COPYRIGHT © 2022 Pugnale, Stuart-Fox, Elgar, Laschi and Dumanli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Practitioners across domains—from architectural design (Zari, 2010; Ha and Lu, 2020), medical interventions (Chen et al., 2021), and robotics (Coyle et al., 2018; Ahmed et al., 2022) to materials science (Wegst et al., 2015)—are increasingly drawing inspiration from biology to address a wide range of challenges. This is perhaps unsurprising because life on earth represents over 3.8 billion years of evolution, whereby natural selection ruthlessly purges design failures. Despite the growth and promise of biomimetic and bioinspired approaches, the analogy to biological form or function is often superﬁcial or largely ﬁgurative. This Research Topic was born from the topic editors’ shared belief that to capitalize on insights from biology, we need to move beyond ﬁgurative referencing toward functional analogy informed by accurate biological knowledge. For this reason, we advocate a biologically-informed (or bio-informed) approach that captures key properties of living organisms and systems, such as sustainability, multifunctionality and self-assembly. The articles collected in this interdisciplinary Research Topic illustrate the strengths of a bio-informed approach to design processes and applications. Ultimately, the goal is to demonstrate how simple concepts can be abstracted from highly complex and inter-connected biological materials, structures and processes to be applied or manufactured at scale. A bio-informed approach requires deep collaboration between biologists and practitioners in other ﬁelds. To gauge the current extent of engagement with biologists in biomimetic and bioinspired research, Ng et al. surveyed the literature over 30 years (1990–2020) to reveal that only 41% of research papers published in the ﬁeld included an author aﬃliated to a biology-related department, and most of them focus on a limited range of popular model species. The authors show the value of capitalizing on biological Frontiers in Ecology and Evolution frontiersin.org 01 Frontiers in Ecology and Evolution 10.3389/fevo.2022.1090859 Pugnale et al. Acknowledgments that could be construed as a potential conﬂict of interest. that could be construed as a potential conﬂict of interest. The Research Topic was conceived through the Bioinspiration Hallmark Research Initiative at the University of Melbourne. Author contributions Turning to applications, Salami et al. used a bio-informed approach to design a novel tandem ﬂapping wing mechanism based on dragonﬂies, which achieve remarkable ﬂying agility with four wings arranged in a tandem conﬁguration. The tandem ﬂapping mechanism enabled the team to study AP and DS-F wrote the ﬁrst draft. All authors edited and contributed intellectually to the work and approved it for publication. 02 frontiersin.org Pugnale et al. Pugnale et al. 10.3389/fevo.2022.1090859 Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Zari, M. P. (2010). Biomimetic design for climate change adaptation and mitigation. Architect. Sci. Rev. 53, 172–183. doi: 10.3763/asre.200 8.0065 Coyle, S., Majidi, C., LeDuc, P., and Hsia, K. J. (2018). Bio-inspired soft robotics: material selection, actuation, and design. Extreme Mech. Lett. 22, 51–59. doi: 10.1016/j.eml.2018.05.003 Wegst, U. G. K., Bai, H., Saiz, E., Tomsia, A. P., and Ritchie, R. O. (2015). Bioinspired structural materials. Nat. Mater. 14, 23–36. doi: 10.1038/nm at4089 Ha, N. S., and Lu, G. (2020). A review of recent research on bio-inspired structures and materials for energy absorption applications. Compos. Part B: Eng. 181, 107496. doi: 10.1016/j.compositesb.2019.107496 Ha, N. S., and Lu, G. (2020). A review of recent research on bio-inspired structures and materials for energy absorption applications. Compos. Part B: Eng. 181, 107496. doi: 10.1016/j.compositesb.2019.107496 Wegst, U. G. K., Bai, H., Saiz, E., Tomsia, A. P., and Ritchie, R. O. (2015). Bioinspired structural materials. Nat. Mater. 14, 23–36. doi: 10.1038/nm at4089 Chen, H., Zhang, Y., Zhang, L., Ding, X., and Zhang, D. (2021). Applications of bioinspired approaches and challenges in medical devices. Bio-Design Manuf. 4, 146–148. doi: 10.1007/s42242-020-00103-6 References Ahmed, F., Waqas, M., Jawed, B., Soomro, A. M., Kumar, S., Hina, A., et al. (2022). Decade of bio-inspired soft robots: a review. Smart Mater. Struct. 31, 073002. doi: 10.1088/1361-665X/ac6e15 Chen, H., Zhang, Y., Zhang, L., Ding, X., and Zhang, D. (2021). Applications of bioinspired approaches and challenges in medical devices. Bio-Design Manuf. 4, 146–148. doi: 10.1007/s42242-020-00103-6 03 frontiersin.org Frontiers in Ecology and Evolution Frontiers in Ecology and Evolution
https://openalex.org/W2992745800	https://www.frontiersin.org/articles/10.3389/fonc.2019.01421/pdf	English	null	Predisposition to Apoptosis in Hepatocellular Carcinoma: From Mechanistic Insights to Therapeutic Strategies	Frontiers in oncology	2,019	cc-by	8,506	REVIEW published: 13 December 2019 doi: 10.3389/fonc.2019.01421 REVIEW Edited by: Luis Enrique Gomez-Quiroz, Universidad Autónoma Metropolitana, Mexico Reviewed by: Isabel Fabregat, Biomedical Research Institute of Bellvitge, Spain Barbara Marengo, University of Genoa, Italy Correspondence: Jens U. Marquardt marquarj@uni-mainz.de Frank Edlich frank.edlich@ biochemie.uni-freiburg.de Specialty section: This article was submitted to Cancer Metabolism, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Cancer Metabolism, a section of the journal Frontiers in Oncology Received: 16 September 2019 Accepted: 29 November 2019 Published: 13 December 2019 Predisposition to Apoptosis in Hepatocellular Carcinoma: From Mechanistic Insights to Therapeutic Strategies Jens U. Marquardt 1,2 and Frank Edlich 3,4* 1 Department of Medicine I, University Medical Center Schleswig-Holstein, Lübeck, Germany, 2 Department of Medicine, Lichtenberg Research Group, University Mainz, Mainz, Germany, 3 Heisenberg Research Group “Regulation von Bcl-2-Proteinen Durch Konformationelle Flexibilität,” Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany, 4 CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany Hepatocellular carcinoma (HCC) ranks among the most rapidly evolving cancers in the Western world. The majority of HCCs develop on the basis of a chronic inﬂammatory liver damage that predisposes liver cancer development and leads to deregulation of multiple cellular signaling pathways. The resulting dysbalance between uncontrolled proliferation and impaired predisposition to cell death with consecutive failure to clear inﬂammatory damage is a key driver of malignant transformation. Therefore, resistance to death signaling accompanied by metabolic changes as well as failed immunological clearance of damaged pre-neoplastic hepatocytes are considered hallmarks of hepatocarcinogenesis. Hereby, the underlying liver disease, the type of liver damage and individual predisposition to apoptosis determines the natural course of the disease as well as the therapeutic response. Here, we will review common and individual aspects of cell death pathways in hepatocarcinogenesis with a particular emphasis on regulatory networks and key molecular alterations. We will further delineate the potential of targeting cell death-related signaling as a viable therapeutic strategy to improve the outcome of HCC patients. Keywords: hepatocellular carcinoma, cell death, inﬂammation, BCL-2 family, BH3, primed to death, BH3 proﬁling, mitochondrial apoptosis INTRODUCTION The common hallmark of the vast majority of Hepatocellular carcinomas (HCC) is a chronic inﬂammatory liver damage induced by a diverse spectrum of etiological risk factors (1). Depending on the type of liver injury and persistence of the underlying inﬂammatory stimulus, HCCs are particularly characterized by a signiﬁcant phenotypic and molecular heterogeneity. Therefore, HCCs are oncogenic paradigms for inﬂammation-induced cancers (2). Herein, the underlying causes of the chronic liver disease range from chronic hepatitis B (HBV) and C viruses (HCV) infections over excessive alcohol abuse to metabolic liver diseases. Importantly, the obesity-associated alterations of the hepatic microenvironment resembling non-alcoholic fatty liver disease and, more importantly, steatohepatitis (NAFLD/NASH) are now among the most prominent etiological risk factors for HCC in several Western countries (3). The particular type of inﬂammatory liver damage induced by NASH is also responsible for a high number of Citation: Marquardt JU and Edlich F (2019) Predisposition to Apoptosis in Hepatocellular Carcinoma: From Mechanistic Insights to Therapeutic Strategies. Front. Oncol. 9:1421. doi: 10.3389/fonc.2019.01421 Marquardt JU and Edlich F (2019) Predisposition to Apoptosis in Hepatocellular Carcinoma: From Mechanistic Insights to Therapeutic Strategies. Front. Oncol. 9:1421. doi: 10.3389/fonc.2019.01421 December 2019 \| Volume 9 \| Article 1421 Frontiers in Oncology \| www.frontiersin.org 1 Apoptosis Predisposition in HCC Marquardt and Edlich a key factor for malignant transformation and specify factors that aﬀect diﬀerential predisposition to apoptotic stimuli during liver cancer development and therapy. Finally, the impact for personalized medicine and precision oncology will be discussed. HCCs without underlying cirrhosis (4, 5). Given the rising incidence of the metabolic syndrome worldwide, it is not surprising that HCC currently ranks among the most rapidly evolving and deadliest cancers in the Western world. Further, the impaired liver function and observed molecular heterogeneity renders eﬀective treatments of HCCs particularly challenging (6, 7). MECHANISMS OF CELL DEATH IN HEPATOCARCINOGENESIS In the context of HCC development and progression, special importance can be assigned to the type of liver damage and associated changes to the hepatic micromilieu that create a pro- oncogenic ﬁeld eﬀect and precede malignant transformation of hepatocytes (8, 9). Cell death is intrinsically associated with chronic inﬂammation in various organs including the liver (10). Herein, infectious and metabolic changes induced by the underlying etiological agent prone hepatocytes for further damage. Liver ﬁbrogenesis and carcinogenesis are signiﬁcantly accelerated by oxidative stress, cell death and inﬂammation. Thus, HCC is the ﬁnal and most deadly consequence of all major chronic liver diseases (2). Consistently, continuous inﬂammatory cell death is one of the hallmarks of hepatocarcinogenesis. Almost all HCC patients show signs of cell death in sera and tissue and their emergence is indicative of adverse biological traits (18). Various types of liver injury and associated chronic cell death responses have been identiﬁed to trigger inﬂammatory liver diseases, ﬁbrosis development and, ultimately, hepatocarcinogenesis (10, 11). Accordingly, major cell death processes as well as signaling pathways are associated with liver cancer development and mainly involve apoptosis and necrosis. However, other forms of cell death, such as autophagy, necroptosis, pyroptosis, ferroptosis, or combinations of these death programs, have also been linked to HCC development and progression (11). Damaged hepatocytes induce activation and cross-talk of other non-parenchymal, immune and stromal cells with subsequent release of cytokine that fuel inﬂammation-induced damage and prone cancer development (12). Abnormalities in glucose and lipid metabolisms as well as microbiota composition further aggravate the oncogenic process. While the mentioned cell death mechanisms are relevant for hepatocarcinogenesis, regardless of the underlying etiological risk factors, oxidative stress and consecutive impairment of mitochondrial function seem to particularly induce hepatocyte death during metabolic liver damage and lead to signaling through B-cell lymphoma-2 (BCL-2) family proteins and activation of caspases and c-Jun N-terminal kinase during NASH-induced HCC (13). Besides prominent roles of cell death pathways in HCC development, cell death regulation and associated changes are also important for diagnosis and therapy. Several surrogate methods to assess and quantify liver injury, predominant mode of cell death and activation of inﬂammatory processes have been successfully evaluated in the context of acute and chronic liver diseases (14, 15). However, reliable and non- invasive cell death markers are not available in clinical routine. MECHANISMS OF CELL DEATH IN HEPATOCARCINOGENESIS Cell death and inﬂammatory markers have also been assessed as prognostic markers or to facilitate monitoring of therapy response in the context of liver cancer (16, 17). In addition, inhibitors of apoptosis, particularly inhibitors of BCL-2 family members or caspases, have recently been introduced to target several chronic inﬂammatory diseases including NASH. These inhibitors might not only prevent malignant transformation and, thus, be eﬀective as preventive compounds, but also be viable therapeutic strategies for HCC. Together, inﬂammatory cell death is particular relevant for mechanistic and clinical applications in liver cancer. g ( ) The apoptosis program governs the cell-autonomous removal of superﬂuous, infected, or damaged cells (19, 20) and thus constitutes the most prominent defense mechanism against hepatocarcinogenesis. During chronic damage, apoptosis is regulated on the outer mitochondrial membrane (OMM) by BCL-2 proteins. The pro-apoptotic BCL-2 proteins, BCL-2- associated X protein (BAX) and BCL-2 antagonist killer 1 (BAK) permeabilize the OMM and release intermembrane space proteins, such as cytochrome c, into the cytoplasm in order to activate the caspase cascade (21). Therefore, BAX and BAK can commit the cell to apoptosis. The cell is protected from BAX and BAK activity by functionally redundant pro-survival BCL- 2 proteins. Although, BAX/BAK activation is usually followed by irreversible cellular commitment to apoptosis, cell survival is possible after limited OMM permeabilization (22). Even cells with the capacity to undergo death receptor-dependent apoptosis without mitochondrial apoptosis signaling enhance their apoptotic response by BAX/BAK activation (23). Therefore, therapeutic success of anti-tumor strategies, including targeted strategies, immune therapies as well as chemotoxic stress, rely on eﬃcient BAX/BAK engagement in targeted cells. Several molecular alterations could be associated with induction or imbalance of pro- and anti-apoptotic BCL-2 proteins in liver cancer. They play an essential role in maintaining genomic integrity of hepatocytes. Disruption of the apoptotic program is frequently observed already during chronic liver diseases (12). Activation of BCL-xL is further observed at high frequencies in human HCC, whereas concomitant downregulation of BAX is a common feature of HCC with p53 alterations and observed at progressed stages of the disease (24). Moreover, inhibition of caspases e.g., by XIAP is also common in human HCC and associated with TGFβ signaling and subsequent acquisition of pro-metastatic properties. In addition to the inhibition of pro- apoptotic proteins or caspases, activation of pro-survival genes as well as pathways contributes to liver cancer development and progression (25). Frontiers in Oncology \| www.frontiersin.org MECHANISMS OF CELL DEATH IN HEPATOCARCINOGENESIS The here presented review aims to summarize key cellular and molecular mechanisms involved in liver cell death during hepatocarcinogenesis with a main focus on apoptosis. We will also delineate the importance of predisposition to apoptosis as December 2019 \| Volume 9 \| Article 1421 2 Apoptosis Predisposition in HCC Marquardt and Edlich A prominent molecular alteration detected in a sizable number of HCC patients is NF-kB pathway that is also particularly important in metabolic liver diseases and NASH- induced HCC (26–28). The pathway controls diverse functions in a cell type and context-dependent manner and activity is observed during chronic inﬂammation, ﬁbrogenesis as well as development and progression of HCC (29, 30). In hepatocytes, NF-κB mainly mediates survival during chronic damage in response to e.g., oxidative stress while suppression contributes to malignant transformation. However, NF-κB activation in non- parenchymal and immune cells can aggravate inﬂammation and ﬁbrogenesis (31). Tumor necrosis factor-α and interleukin-6 are among the major inﬂammatory cytokines that induce this pathway. NF-κB downstream signaling resembling c-Jun N- terminal kinase (JNK), and signal transducer and activator of transcription 3 play a major role in inﬂammation-associated HCC (32). NF-κB activation can also be critically linked to several anti-apoptotic molecules including (cIAP1, cIAP2), XIAP, the BCL-2 family members A1 and BCL- xL, cFLIP, TRAF1, TRAF2, and GADD45β (33). Besides JNK, NF-κB also activates other pro-survival and pro-proliferative pathways, resembling p38 MAPK (mitogen-activated protein kinase) kinase (34, 35). In this context, upstream regulators, such as the NF-κB essential modulator (NEMO), the IKK kinase complex as well as death- domain kinase receptor-interacting protein kinase 1 (RIPK1) are of particular importance. The central regulators of cell death resembling TAK1 and RIPK1 are, consequently, other common ﬁndings mechanistically linked to malignant transformation in the liver. TAK1 (MAP3-kinase TGF-β-activated kinase 1) is critically involved in the modulation of innate and adaptive immune responses. Activation of TAK1 in parenchymal cells signiﬁcantly inhibits apoptosis and demonstrated anti- tumorigenic eﬀects mediated by NF-κB activation via TNF (36). Conversely, deﬁciency of TAK1 impaired NF-κB activity and induced hepatocyte apoptosis, inﬂammation as well as HCC development in a NEMO-dependent manner (36). Consistently, alteration of the immune cell composition and impairment of immune-mediated clearance of damaged hepatocytes is an important driver of liver cancer. MECHANISMS OF CELL DEATH IN HEPATOCARCINOGENESIS It has recently been shown that dysregulation of lipid metabolism in NAFLD induces selective ablation of intrahepatic CD4+ cells, which impairs mitochondrial function and generates high levels of oxidative damage, thus, corroborating lipid dysregulation with impaired anti-tumor immune-surveillance (37). Accordingly, impaired senescence surveillance by myeloid cells also induced failure in immune-mediated clearance of damaged hepatocytes and accelerated hepatocarcinogenesis. ER stress induced by metabolic liver damage following a high fat diet further enhanced resulting liver damage, increased immune inﬁltration, and lipogenesis and, ultimately, led to HCC development (28). Another form of cell death recently linked to HCC development is necroptosis Again the mentioned TAK1 model These results indicate the diverse molecular functions of key cell death pathways in mediating apoptotic, necroptotic or other forms of cell death. Detailed dissection of the relative contribution and mechanistic hallmarks are urgently needed (12). An improved biological understanding of the exact mechanisms driving hepatocyte cell death and, ultimately, cancer growth are not only of particular scientiﬁc interest, but also directly imply translational applications. Besides identiﬁcation of patients at risk for cancer development, biomarkers of cell death might also be instrumental to delineate the biological trait, i.e., prognosis, of a tumor but also for prediction and monitoring of treatment response. Nevertheless, excessive cell death was successfully identiﬁed predict the development as well as progression of liver cancer. Furthermore, expression of key markers in cell death and surrogate characteristics were associated with clinical outcome. As such, the new checkpoint molecules RIPK1 and TRAF2 were recently conﬁrmed as independent prognostic markers in liver cancer (38). Furthermore, the serum cell death parameter M65, which detects cleaved and uncleaved CK-18 fragments, was also demonstrated to possess clinical utility as a non-invasive marker for tumor initiation as well as prognosis, corroborating the potential as a new diagnostic tool for HCC (16). Finally, it is well-established that transcriptome proﬁles conferring to cell death resistance are signiﬁcantly enriched in HCCs with low diﬀerentiation, high invasion and a particularly poor outcome (39). In summary, imbalance of a broad range of molecules with critical function of cell death, including dysregulation of cytokines and inﬂammatory as well as survival pathways during chronic liver disease, possess high relevance for clinical application and harbor potential as translational biomarkers of malignant transformation as well as progression. Frontiers in Oncology \| www.frontiersin.org ANALYSIS OF BCL-2 PROTEINS TO PREDICT TUMOR CELL APOPTOSIS While liver tumors possess molecular characteristics that set them apart from other types of tumors, general mechanisms of apoptosis regulation apply as they have been shown in many diﬀerent cell types. The discovery of opposing BCL- 2 protein activities led to the rheostat model to describe regulatory interactions in mitochondrial apoptosis signaling (40, 41). The model postulates that pro-survival BCL-2 proteins act anti-apoptotic by binding to BAX and BAK. Therefore, mitochondrial apoptosis would largely dependent on diﬀerent protein expression and degradation rates. In fact, platelets contain a molecular timer that commits them to apoptosis when BAK levels exceed the levels of the predominant pro-survival BCL-2 (42). The rheostat model sparked a body of work suggesting prediction of therapeutic success based on measuring BAX levels (Figure 1). The reﬁned version of this approach investigated the BAX/BCL-2 ratio. However, subsequent research expanded our knowledge on protein localizations and interactions, revealing the absence of the prerequisite of the rheostat model: stable protein Another form of cell death recently linked to HCC development is necroptosis. Again, the mentioned TAK1 model with liver-speciﬁc ablation was employed to clarify the relative contribution of necroptosis during hepatocarcinogenesis. While response to apoptosis in the model promoted inﬂammation and tumorigenesis, necroptotic response had opposing eﬀects and conferred anti-inﬂammatory and tumor-suppressive functions. December 2019 \| Volume 9 \| Article 1421 3 Apoptosis Predisposition in HCC Marquardt and Edlich FIGURE 1 \| Strategies to analyze apoptotic predisposition based on BCL-2 proteins. (A) BAX level. The right cell contains more BAX (blue) than the left and is therefore considered to have a higher apoptosis predisposition. Other relevant factors are not measured. (B) BAX vs. BCL-xL level. The ratio between BAX and a single pro-survival BCL-2 protein (BCL-xL, red) is similar in both cells. Therefore, both cells would be judged to have the same tendency to initiate apoptosis. The redundancy of the BCL-2 family would require this analysis to be expanded to all BCL-2 proteins in order to be insightful. (C) BH3 proﬁling. Permeabilized and cultured cells are incubated with BH3 peptides (green star) in order to titrate the amount of free BH3 binding sites on the outer mitochondrial membrane (OMM). BH3-only proteins (green) associated with the OMM following prior cell stress reduce the amount of free BH3 binding sites and thus increase the sensitivity toward BH3 mimetics. ANALYSIS OF BCL-2 PROTEINS TO PREDICT TUMOR CELL APOPTOSIS The analysis can be supplemented with measuring the functionally redundant and similarly regulated BAK, which is usually shifted toward the mitochondria but shows a similar range of localizations in human samples. FIGURE 1 \| Strategies to analyze apoptotic predisposition based on BCL-2 proteins. (A) BAX level. The right cell contains more BAX (blue) than the left and is therefore considered to have a higher apoptosis predisposition. Other relevant factors are not measured. (B) BAX vs. BCL-xL level. The ratio between BAX and a single pro-survival BCL-2 protein (BCL-xL, red) is similar in both cells. Therefore, both cells would be judged to have the same tendency to initiate apoptosis. The redundancy of the BCL-2 family would require this analysis to be expanded to all BCL-2 proteins in order to be insightful. (C) BH3 proﬁling. Permeabilized and cultured cells are incubated with BH3 peptides (green star) in order to titrate the amount of free BH3 binding sites on the outer mitochondrial membrane (OMM). BH3-only proteins (green) associated with the OMM following prior cell stress reduce the amount of free BH3 binding sites and thus increase the sensitivity toward BH3 mimetics. The increased capacity of the left cell would translate into a reduced sensitivity toward BH3 mimetics. (D) Relative BAX localization. Determination of the cytosolic and mitochondrial BAX pools in intact cells describes the position of the BAX localization equilibrium and thus the cellular predisposition to apoptosis. While single contributing factors cannot be dissected, all contributing factors, e.g., BCL-2 protein interactions with BH3 motifs and other segments, interacting proteins outside the BCL-2 family, are included. BH3-only proteins reduce the shuttling rate and thus the cytosolic BAX pool. The larger cytosolic pool of the left cell shows reduced predisposition to apoptosis. The analysis can be supplemented with measuring the functionally redundant and similarly regulated BAK, which is usually shifted toward the mitochondria but shows a similar range of localizations in human samples. to the OMM, including DNA damage, ER stress, death receptor signaling and other types of stress (43). BH3-only proteins are thought to either inhibit pro-survival BCL-2 proteins and/or directly activate BAX and BAK (44). Inhibition of pro-survival BCL-2 proteins by BH3-only proteins is structurally well- characterized and has led to the development of low molecular weight inhibitors. These targeted anti-cancer small molecule inhibitors called BH3 mimetics bind to and inhibit pro-survival BCL-2 proteins in a manner similar to BH3-only proteins. complexes. Frontiers in Oncology \| www.frontiersin.org ANALYSIS OF BCL-2 PROTEINS TO PREDICT TUMOR CELL APOPTOSIS The increased capacity of the left cell would translate into a reduced sensitivity toward BH3 mimetics. (D) Relative BAX localization. Determination of the cytosolic and mitochondrial BAX pools in intact cells describes the position of the BAX localization equilibrium and thus the cellular predisposition to apoptosis. While single contributing factors cannot be dissected, all contributing factors, e.g., BCL-2 protein interactions with BH3 motifs and other segments, interacting proteins outside the BCL-2 family, are included. BH3-only proteins reduce the shuttling rate and thus the cytosolic BAX pool. The larger cytosolic pool of the left cell shows reduced predisposition to apoptosis. The analysis can be supplemented with measuring the functionally redundant and similarly regulated BAK, which is usually shifted toward the mitochondria but shows a similar range of localizations in human samples. FIGURE 1 \| Strategies to analyze apoptotic predisposition based on BCL-2 proteins. (A) BAX level. The right cell contains more BAX (blue) than the left and is therefore considered to have a higher apoptosis predisposition. Other relevant factors are not measured. (B) BAX vs. BCL-xL level. The ratio between BAX and a single pro-survival BCL-2 protein (BCL-xL, red) is similar in both cells. Therefore, both cells would be judged to have the same tendency to initiate apoptosis. The redundancy of the BCL-2 family would require this analysis to be expanded to all BCL-2 proteins in order to be insightful. (C) BH3 proﬁling. Permeabilized and cultured cells are incubated with BH3 peptides (green star) in order to titrate the amount of free BH3 binding sites on the outer mitochondrial membrane (OMM). BH3-only proteins (green) associated with the OMM following prior cell stress reduce the amount of free BH3 binding sites and thus increase the sensitivity toward BH3 mimetics. The increased capacity of the left cell would translate into a reduced sensitivity toward BH3 mimetics. (D) Relative BAX localization. Determination of the cytosolic and mitochondrial BAX pools in intact cells describes the position of the BAX localization equilibrium and thus the cellular predisposition to apoptosis. While single contributing factors cannot be dissected, all contributing factors, e.g., BCL-2 protein interactions with BH3 motifs and other segments, interacting proteins outside the BCL-2 family, are included. BH3-only proteins reduce the shuttling rate and thus the cytosolic BAX pool. The larger cytosolic pool of the left cell shows reduced predisposition to apoptosis. ANALYSIS OF BCL-2 PROTEINS TO PREDICT TUMOR CELL APOPTOSIS The porin voltage-dependent anion channel 2 (VDAC 2, purple) acts as mitochondrial BAX/BAK receptor and as platform for the retrotranslocation of BAX and BAK back into the cytosol dependent on the activities of pro-survival BCL-2 proteins (red). The equilibrium between BAX/BAK translocation and retrotranslocation determines the cellular predisposition to apoptosis. Intrinsic stress as well as death receptor signaling is mediated by BH3-only proteins (yellow) that inhibit BAX/BAK retrotranslocation shifting BAX and BAK toward the mitochondria. The BH3-only proteins tBID, BIM and PUMA are also thought to directly activate BAX and BAK initiating OMM permeabilization and the release of cytochrome c (cyt c) and SMAC into the cytosol. This function can be inhibited by pro-survival BCL-2 proteins. Cytosolic cyt c initiates in turn the formation of the apoptosome (green), an APAF-1 complex activating Caspase 9 (Cas 9). Subsequently, Caspases 3 and 7 are activated that can be inhibited by IAPs in the absence of SMAC in the cytosol. Caspase 3/7 activation leads to the efﬁcient dismantling of the cell into apoptotic bodies that are later phagocytosed. as the corresponding BAK pool is variable because both proteins are inhibited by a dynamic shuttling equilibrium between cytosol and mitochondria (57). Pro-survival BCL-2 proteins constantly retrotranslocate BAX and BAK from the mitochondria and cell stress mediated by BH3-only proteins shifts both pro- apoptotic BCL-2 proteins back onto the mitochondria. The importance of mitochondrial BAX for apoptosis induction implies that (i) the total cell protein population is not critical for apoptosis induction and (ii) accurately measuring mitochondrial BAX (or BAK) fractions or shuttling rates could predict apoptotic outcome in response to stress (Figure 1). Experimental observations have shown that the ratio between cytosolic and mitochondrial BAX/BAK is the best available representation of the average localization dynamics of BAX/BAK molecules (58). The paradigm that relevant protein pool and total protein level are not necessarily connected is true for BAX, BAK, pro-survival proteins, like BCL-2 and BCL-xL, and BH3-only proteins, like BID (53, 59, 60). Relative BAX/BAK localization reﬂects the combined contributions of all players, known and unknown, to the cellular predisposition to apoptosis. Similar diﬀerences in the cellular BAX localization could also be present in HCC and could be associated with distinct molecular and clinical characteristics of the tumors. priming” in this context is the resulting stress capacity of cells dependent on the presence of pro-survival BCL-2 proteins, OMM-accumulated BH3-only proteins and BAX/BAK (Figure 1). ANALYSIS OF BCL-2 PROTEINS TO PREDICT TUMOR CELL APOPTOSIS Actually, BH3 proﬁling is again based on the rheostat model. It expands the model by emphasizing the potential role of BH3-only proteins, but does not take into account the transient nature of BCL-2 protein interactions and interactions among BCL-2 proteins other than through the BH3 motif. Extensive work shows the feasibility of “BH3 proﬁling” in diﬀerent cellular settings (45–49). The analysis involves the short culturing of cells, limited cell lysis, incubation with peptides corresponding to BH3 domains and the analysis of OMM permeabilization through a membrane potential-sensitive dye. Cell culturing is prone to changes the apoptotic predisposition of a given tumor clone despite relative genetic stability. In addition, recent research has provided evidence of several secondary binding sites in BCL- 2 protein interactions that BH3 proﬁling cannot account for (50–53). Therefore, BH3 proﬁling can particularly identify the contribution of pro-survival BCL-2 activities to the survival and therefore support selection of the potentially most eﬀective BH3 mimetic. On the other hand, the appropriate BH3 mimetics could be tested directly, as procedure and readout would be similar. ANALYSIS OF BCL-2 PROTEINS TO PREDICT TUMOR CELL APOPTOSIS Prediction of apoptotic outcome based on protein levels encountered another major problem with the discovery of new members of the BCL-2 family. Their functional redundancies forsake all educated guesses, whether pro-survival BCL-2 proteins outnumber pro-apoptotic BCL-2 proteins. Therefore, apoptosis predictions based on protein levels, although occasionally attempted, are unrewarding. A group of proteins that has inﬂuenced revised paradigms for apoptosis signaling and predictions of apoptotic outcome contains BH3-only proteins (Figure 2). BH3-only proteins are deﬁned by harboring a single BH3 motif, while the remaining protein structures diverge as much as the type of stress signaled The concept that BH3-only proteins loaded on the OMM could determine the cellular response to apoptosis has led to the strategy to proﬁle BH3-only proteins. “Mitochondrial December 2019 \| Volume 9 \| Article 1421 4 Apoptosis Predisposition in HCC Marquardt and Edlich FIGURE 2 \| Mitochondrial apoptosis signaling. Mitochondrial apoptosis is regulated by members of the BCL-2 protein family on the outer mitochondrial membrane (OMM). The pro-apoptotic BCL-2 proteins BAX and BAK (blue) constantly translocate to the OMM undergoing a conformational change. The porin voltage-dependent anion channel 2 (VDAC 2, purple) acts as mitochondrial BAX/BAK receptor and as platform for the retrotranslocation of BAX and BAK back into the cytosol dependent on the activities of pro-survival BCL-2 proteins (red). The equilibrium between BAX/BAK translocation and retrotranslocation determines the cellular predisposition to apoptosis. Intrinsic stress as well as death receptor signaling is mediated by BH3-only proteins (yellow) that inhibit BAX/BAK retrotranslocation shifting BAX and BAK toward the mitochondria. The BH3-only proteins tBID, BIM and PUMA are also thought to directly activate BAX and BAK initiating OMM permeabilization and the release of cytochrome c (cyt c) and SMAC into the cytosol. This function can be inhibited by pro-survival BCL-2 proteins. Cytosolic cyt c initiates in turn the formation of the apoptosome (green), an APAF-1 complex activating Caspase 9 (Cas 9). Subsequently, Caspases 3 and 7 are activated that can be inhibited by IAPs in the absence of SMAC in the cytosol. Caspase 3/7 activation leads to the efﬁcient dismantling of the cell into apoptotic bodies that are later phagocytosed. FIGURE 2 \| Mitochondrial apoptosis signaling. Mitochondrial apoptosis is regulated by members of the BCL-2 protein family on the outer mitochondrial membrane (OMM). The pro-apoptotic BCL-2 proteins BAX and BAK (blue) constantly translocate to the OMM undergoing a conformational change. TARGETING OF CELL DEATH AS A THERAPEUTIC STRATEGY FOR HCC Prior stress and stress response inﬂuence the apoptotic predisposition but are also reﬂected in the cellular localization of the pro-apoptotic BCL-2 proteins. Despite their functional redundancy, BAK is found largely on the OMM in many cell types, while BAX resides primarily in the cytoplasm (54, 55). This apparent diﬀerence is important, since the sizes of the mitochondrial protein pools prior apoptotic stress determines apoptotic response (56). The mitochondrial BAX pool as much Hepatocyte damage and consecutive activation of cell death signaling plays a pivotal role for liver cancer initiation, but is also of particular importance for modulating treatment eﬀects during established therapies. Herein, cell death can be induced by chemotherapeutic as well as targeted approaches (61). Sorafenib and lenvatinib are the only approved ﬁrst line therapies for advanced stages in liver cancer (62). Both December 2019 \| Volume 9 \| Article 1421 Frontiers in Oncology \| www.frontiersin.org 5 Apoptosis Predisposition in HCC Marquardt and Edlich TABLE 1 \| Selected targets of cell death in liver diseases and cancer. Drug Target, function, pathway Target population Phase clinical development PRIMA-1 Restoration of p53 function Pre-clinical N/A Emricasan/IDN-6556 GS9450 Pan-Caspase Inhibitor NASH, liver cirrhosis Phase II (e.g., NCT02960204, NCT02686762, NCT03205345) Venetoclax/ABT-199 BH3 mimetic Pre-clinical N/A GSK2982772 RIPK1/RIPK3 Inhibitor Pre-clinical N/A Etanercept TNF Inhibitor Alcoholic hepatitis, chronic viral hepatitis, NAFLD/NASH, AIH, PBC Phase I-II AEG35156 XIAP Antisense HCC Phase I-II (e.g., NCT00882869) Curcumin NF-kB, RIPK Inhibitor HCC, NAFLD/NASH Phase I-II (e.g., NCT03864783) lymphocytic leukemia (CLL) has shown the potential of this strategy, as response rates of about 80% can be achieved with single-agent venetoclax even in a relapsed/refractory setting (69). Current eﬀorts explore the combination of venetoclax with rituximab, obinutuzumab or ibrutinib in order to suppress acquired resistance observed during monotherapy (70, 71). Venetoclax in combination with hypomethylating agents (HMAs) has also received special attention for the treatment of acute myeloid leukemia (AML) (72, 73). The combination has been shown to target leukemia stem cells (74). In the liver, recent evidence suggest that BH3-only protein BID signiﬁcantly contributes to the development of liver cancer (75). Loss of BID was shown to delay hepatocarcinogenesis by reducing cell death, liver inﬂammation, and compensatory proliferation (76). Thus, modulation of the BCL-2 protein interplay might be a promising therapeutic strategie for liver cancer. compounds are multi-tyrosine kinase inhibitors with anti- angiogenic properties. TARGETING OF CELL DEATH AS A THERAPEUTIC STRATEGY FOR HCC It is well-known, that sorafenib is a strong inducer of apoptosis and exposure to hepatoma cells leads to BAX/BAK activation, at least in part through the BH3-only protein PUMA (63). Furthermore, high numbers of objective response rates observed in HCC patients further indicate that considerable cell death follows lenvatinib treatment (64). However, several recent reports suggest that in addition to induction of cell death the treatment eﬀect is also signiﬁcantly induced by immunomodulation through targeted therapies (65). In consequence, several combination therapies with PD1/PD-L1 therapies are currently under clinical evaluation (62). p y Direct targeting of cell death pathways and modulation of the apoptotic response might be a viable preventive strategy in chronic liver diseases but also exert direct anti-tumorigenic properties in HCC (Table 1). Given central role of p53 as a regulator of cell death, restoration of its function was attempted to induce anti-tumor activity in several studies. While adenoviral delivery of recombinant p53 did not reveal promising results, modulation of p53 activity by e.g., ubiquitination through inhibition of COP1, was recently explored (66). Blockade of COP1 by systemic delivery of RNAi decreased in vivo cancer growth and signiﬁcantly induced apoptosis in several HCC cell lines. Furthermore, several compounds were identiﬁed to restore p53 functions. Prominently, p53 reactivation and induction of massive apoptosis (PRIMA-1) and PRIMA-1Met are currently evaluated in several clinical trials (67). In the liver, application of the compounds is currently restricted to preclinical data and shows promising anti-tumor eﬀects when mutant p53 is silenced by siRNA. Other therapeutic strategies aimed to directly target proteins involved in apoptosis to enhance the apoptotic response of cancer therapies. Interestingly, XIAP antisense therapy in combination with sorafenib showed synergistic anti- tumor eﬀects in a recent phase II clinical trial (68). Results showed a moderate increase in progression-free survival (4.0 months vs. 2.6 months), overall survival (6.5 months vs. 5.4 months), and objective response rates (16.1% vs. 9.7%) compared with Sorafenib monotherapy. Notably, drug-related adverse events were moderate. In addition to the therapeutic targeting of critical apoptosis regulators, pan-caspase inhibitors, e.g., Emricasan/IDN- 6556, or selective caspase-1,8,9 inhibitors, e.g., GS9450, have been explored in preclinical models as well as clinical trials, mainly in the context of chronic liver diseases (77). Frontiers in Oncology \| www.frontiersin.org REFERENCES 10. Kondylis V, Pasparakis M. RIP Kinases in Liver Cell Death, Inﬂammation and Cancer. Trends Mol Med. (2019) 25:47– 63. doi: 10.1016/j.molmed.2018.10.007 1. Yang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A, Roberts LR. A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol. (2019) 16:589–60. doi: 10.1038/s41575-019-0186-y 1. Yang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A, Roberts LR. A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol. (2019) 16:589–60. doi: 10.1038/s41575-019-0186-y 11. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Diﬀer. (2018) 25:486–541. doi: 10.1038/s41418-017-0012-4 2. Marquardt JU, Andersen JB, Thorgeirsson SS. Functional and genetic deconstruction of the cellular origin in liver cancer. Nature reviews. Cancer. (2015) 15:653–67. doi: 10.1038/nrc4017 12. Schwabe RF, Luedde T. Apoptosis and necroptosis in the liver: a matter of life and death. Nat Rev. (2018) 15:738–52. doi: 10.1038/s41575-018- 0065-y 3. Xiao J, Wang F, Wong NK, He J, Zhang R, Sun R, et al. Global liver disease burdens and research trends: Analysis from a Chinese perspective. J Hepatol. (2019) 71:212–21. doi: 10.1016/j.jhep.2019.03.004 13. Garcia-Ruiz C, Fernandez-Checa JC. Mitochondrial oxidative stress and antioxidants balance in fatty liver disease. Hepatol Commun. (2018) 2:1425– 39. doi: 10.1002/hep4.1271 4. Marquardt JU, Galle PR, Teufel A. Molecular diagnosis and therapy of hepatocellular carcinoma (HCC): an emerging ﬁeld for advanced technologies. J Hepatol. (2012) 56:267–75. doi: 10.1016/j.jhep.2011.07.007 14. Ku NO, Strnad P, Bantel H, Omary MB. Keratins: biomarkers and modulators of apoptotic and necrotic cell death in the liver. Hepatology. (2016) 64:966– 76. doi: 10.1002/hep.28493 5. Liu Z, Jiang Y, Yuan H, Fang Q, Cai N, Suo C, et al. The trends in incidence of primary liver cancer caused by speciﬁc etiologies: results from the Global Burden of Disease Study 2016 and implications for liver cancer prevention. J Hepatol. (2019) 70:674–83. doi: 10.1016/j.jhep.2018.12.001 15. Mazzolini G, Sowa JP, Canbay A. Cell death mechanisms in human chronic liver diseases: a far cry from clinical applicability. Clin Sci. (2016) 130:2121– 38. doi: 10.1042/CS20160035 16. Waidmann O, Koberle V, Bettinger D, Trojan J, Zeuzem S, Schultheiss M, et al. Diagnostic and prognostic signiﬁcance of cell death and macrophage activation markers in patients with hepatocellular carcinoma. J Hepatol. (2013) 59:769–79. doi: 10.1016/j.jhep.2013. 06.008 6. TARGETING OF CELL DEATH AS A THERAPEUTIC STRATEGY FOR HCC While the majority of these trials showed improved liver enzymes as a surrogate for hepatocyte protection, eﬀect on degree of hepatitis and ﬁbrogenesis is still unclear and is currently under evaluation in large phase III trials for the treatment NASH with and without liver cirrhosis (NCT02960204, NCT02686762, NCT03205345). Importantly, caspase inhibition might induce necroptosis or other complications and, thus, require further investigations addressing the safety of long-term administration (12). Although no clinical trials have yet been initiated to test the clinical eﬃcacy of necroptosis inhibition in liver disease, preclinical studies and early phase clinical trials in inﬂammatory (auto-immune) disease indicate that inhibition of RIPK1 kinase activity might also be a promising therapeutic strategy and prevent apoptosis in chronic liver diseases (78). However, while the importance of several key proteins including RIPK1, TAK1, and NEMO has been shown, the therapeutic potential for HCC remains to be demonstrated. Based on the regulatory functions of RIPK1, inhibition might even cause paradox reactions depending on the context of inhibition and aﬀected cell type (30). Finally, given the redundancy in the diﬀerent pathways, combination of Pro-survival BCL-2 proteins are also under intensive preclinical and clinical evaluation as cancer therapy targets. The use of the BH3 mimetic venetoclax or ABT-199 in chronic December 2019 \| Volume 9 \| Article 1421 Frontiers in Oncology \| www.frontiersin.org 6 Apoptosis Predisposition in HCC Marquardt and Edlich diﬀerent anti-tumor therapies with one or several modulators of cell death pathways might be of particular therapeutic potential. diﬀerent anti-tumor therapies with one or several modulators of cell death pathways might be of particular therapeutic potential. conclusively dissected to advance the ﬁeld and before application of speciﬁc modulators of cell death in human is warranted. Furthermore, intensive translational research is needed to characterize the molecular hallmarks that operate on the intersection between cell death and inﬂammation. In this context, individual predisposition to apoptosis of cancer cells or cells within the hepatic microenvironment might be of particular relevance and might require distinct therapeutic strategy, thus, precision oncological approaches. Nevertheless, targeting of apoptosis is a promising avenue of HCC treatment that might yield to novel treatment strategies for this deadly inﬂammatory-driven cancer. FUNDING JM was supported by grants from the German Research Foundation (MA 4443/2-2; SFB1292), the Volkswagen Foundation (Lichtenberg program) and by a grant from the Wilhelm-Sander Foundation (2017.007.1). FE was supported by the DFG Heisenberg program, the Collaborative Research Cluster (CRC) 746, the Else Kröner-Fresenius-Stiftung, the Wilhelm-Sander Foundation, and Germany’s Excellence Strategy (CIBSS – EXC-2189 – Project ID 390939984). JM was supported by grants from the German Research Foundation (MA 4443/2-2; SFB1292), the Volkswagen Foundation (Lichtenberg program) and by a grant from the Wilhelm-Sander Foundation (2017.007.1). FE was supported by the DFG Heisenberg program, the Collaborative Research Cluster (CRC) 746, the Else Kröner-Fresenius-Stiftung, the Wilhelm-Sander Foundation, and Germany’s Excellence Strategy (CIBSS – EXC-2189 – Project ID 390939984). SUMMARY AND CONCLUSIONS Hepatocyte death is a key driver of chronic inﬂammatory liver diseases and hepatocarcinogenesis. Several lines of evidence suggest that apoptosis and other types of cell death are critically linked to initiation and progression of liver cancer. They participate in shaping the biological trait of the tumor, thus, ultimately determining patient prognosis. Herein, existence and degree of cell death infers several mechanistic and translational implications. While detection of the apoptotic predisposition might be a powerful diagnostic tool, direct targeting of mitochondrial apoptosis might complement the limited therapeutic strategies for HCC. In light of recent advances in immune-oncological approaches, targeting of cell death might also exert synergistic immuno- modulatory properties that could be explored in combination treatment strategies. However, our understanding of the detailed mechanisms and triggers of activation underlying the diverse mechanisms of cell death remains limited. Thus, deﬁnition of the actual state of cell death signaling eﬀect in the distinct parenchymal and non-parenchymal cell types within the liver is urgently needed. Furthermore, the relative importance for distinct disease stages, i.e., chronic liver disease, tumor initiation as well as progression should be AUTHOR CONTRIBUTIONS JM and FE conceptualized, wrote, and edited the review. 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https://openalex.org/W2046960085	https://europepmc.org/articles/pmc4361758?pdf=render	English	null	Childhood behaviour problems predict crime and violence in late adolescence: Brazilian and British birth cohort studies	Social psychiatry and psychiatric epidemiology	2,014	cc-by	8,663	Abstract Maughan MRC Social, Developmental and Genetic Psychiatry Centre, Institute of Psychiatry, King’s College London, London, UK Joseph Murray • Ana M. B. Menezes • Matthew Hickman • Barbara Maughan • Erika Alejandra Giraldo Gallo • Alicia Matijasevich • Helen Gonc¸alves • Luciana Anselmi • Maria Cecı´lia F. Assunc¸a˜o • Fernando C. Barros • Cesar G. Victora Joseph Murray • Ana M. B. Menezes • Matthew Hickman • Barbara Maughan • Erika Alejandra Giraldo Gallo • Alicia Matijasevich • Helen Gonc¸alves • Luciana Anselm Maria Cecı´lia F. Assunc¸a˜o • Fernando C. Barros • Cesar G. Victora Received: 9 June 2014 / Accepted: 7 October 2014 / Published online: 16 October 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Methods A comparison was made of birth cohorts in Brazil and Britain, including measures of behaviour prob- lems based on parental report at age 11, and self-reports of crime at age 18 (N = 3,618 Brazil; N = 4,103 Britain). Confounders were measured in the perinatal period and at age 11 in questionnaires completed by the mother and, in Brazil, searches of police records regarding parental crime. Results Conduct problems, hyperactivity and violent crime were more prevalent in Brazil than in Britain, but nonviolent crime was more prevalent in Britain. Sex dif- ferences in prevalence rates were larger where behaviours were less common: larger for conduct problems, hyperac- tivity, and violent crime in Britain, and larger for nonvio- lent crime in Brazil. Conduct problems and hyperactivity predicted nonviolent and violent crime similarly in both countries; the effects were partly explained by perinatal health factors and childhood family environments. Childhood behaviour problems predict crime and violence in late adolescence: Brazilian and British birth cohort studies Joseph Murray • Ana M. B. Menezes • Matthew Hickman • Barbara Maughan • Erika Alejandra Giraldo Gallo • Alicia Matijasevich • Helen Gonc¸alves • Luciana Anselmi • Maria Cecı´lia F. Assunc¸a˜o • Fernando C. Barros • Cesar G. Victora Abstract Purpose Most children live in low- and middle-income countries (LMICs), many of which have high levels of violence. Research in high-income countries (HICs) shows that childhood behaviour problems are important precur- sors of crime and violence. Evidence is lacking on whether this is also true in LMICs. This study examines prevalence rates and associations between conduct problems and hyperactivity and crime and violence in Brazil and Britain. Results Conduct problems, hyperactivity and violent crime were more prevalent in Brazil than in Britain, but nonviolent crime was more prevalent in Britain. Sex dif- ferences in prevalence rates were larger where behaviours were less common: larger for conduct problems, hyperac- tivity, and violent crime in Britain, and larger for nonvio- lent crime in Brazil. Conduct problems and hyperactivity predicted nonviolent and violent crime similarly in both countries; the effects were partly explained by perinatal health factors and childhood family environments. Electronic supplementary material The online version of this article (doi:10.1007/s00127-014-0976-z) contains supplementary material, which is available to authorized users. J. Murray (&) Department of Psychiatry, University of Cambridge, Douglas House, 18b Trumpington Road, Cambridge CB2 8AH, UK e-mail: jm335@cam.ac.uk Conclusions Conduct problems and hyperactivity are similar precursors of crime and violence across different social settings. Early crime and violence prevention pro- grammes could target these behavioural difﬁculties and associated risks in LMICs as well as in HICs. A. M. B. Menezes E. A. G. Gallo A. Matijasevich H. Gonc¸alves L. Anselmi M. C. F. Assunc¸a˜o F. C. Barros C. G. Victora Post-graduate Program in Epidemiology, Federal University of Pelotas, Pelotas, Brazil A. M. B. Menezes E. A. G. Gallo A. Matijasevich H. Gonc¸alves L. Anselmi M. C. F. Assunc¸a˜o F. C. Barros C. G. Victora Post-graduate Program in Epidemiology, Federal University of Pelotas, Pelotas, Brazil Keywords Conduct problems Hyperactivity Crime Cohort study Middle-income country ALSPAC M. Hickman School of Social and Community Medicine, University of Bristol, Bristol, UK M. Hickman School of Social and Community Medicine, University of Bristol, Bristol, UK B. Maughan MRC Social, Developmental and Genetic Psychiatry Centre, Institute of Psychiatry, King’s College London, London, UK A. Matijasevich Department of Preventive Medicine, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil F. C. Barros Postgraduate Program in Health and Behavior, Universidade Cato´lica de Pelotas, Pelotas, Brazil B. Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 DOI 10.1007/s00127-014-0976-z Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 DOI 10.1007/s00127-014-0976-z ORIGINAL PAPER 1993 Pelotas Birth Cohort Study, Brazil The 1993 Pelotas Birth Cohort Study is an ongoing pop- ulation-based study designed to investigate the effects of a wide range of inﬂuences on health and development. Pe- lotas is a city located in the extreme south of Brazil, with an estimated population of 345,179 inhabitants, 93 % of whom live in the urban area. All births occurring in the ﬁve maternity clinics in the town were monitored in 1993 (99 % of births in Pelotas occurred in hospital). For the 5,265 children born alive, only 16 mothers could not be interviewed or refused to participate in the study. The 5,249 newborns, whose mothers lived in the urban area, were included in the cohort. The detailed methodology of this study can be found elsewhere [17]. During the peri- natal study, mothers were interviewed to collect demo- graphic, health and socioeconomic information about the family. Follow-up home visits were conducted in 2004–2005 (age 11) and in clinic sessions in 2011–2012 (age 18) [18]. The perinatal study and each follow-up were approved by the Research Ethics Committee of the Federal University of Pelotas School of Medicine. After being informed of the details of the study, participants signed a term of informed consent. Previous studies compared the effects of childhood behaviour problems on crime between the US, Canada, Britain, New Zealand, and Australia [7, 13, 14]. All found similarity in the effects of conduct problems [7], impul- sivity [13, 14], concentration problems [13], and hyper- activity [7] on crime between sites. However, we are not aware of any previous comparison between contexts as different as Brazil and Britain. Although 90 % per cent of the world’s 2.2 billion children and adolescents live in LMICs [15], and many LMICs have high rates of vio- lence [16], it is not known whether childhood behaviour problems have similar effects on crime and violence in LMICs. We examined associations between conduct problems and hyperactivity at age 11, and nonviolent and violent crime at age 18 in a large, prospective study of a popu- lation sample in Pelotas, Brazil, and compared results with a well-matched study in Britain (ALSPAC). Pelotas is a relatively poor city in a relatively rich state of southern Brazil. 1993 Pelotas Birth Cohort Study, Brazil When crime data were collected for this study in 2011, there were 18.9 homicides in Pelotas per 100,000 population, lower than the national rate of 27.1, but considerably higher than in England and Wales (1.1) and Avon and Somerset (1.1), where the British study is set. This is the ﬁrst major longitudinal survey of self- reported offending in Brazil [16] and, to our knowledge, the ﬁrst comparison of prospective risk factors for crime between any LMIC and a HIC. The study had three main questions: Introduction Childhood conduct disorder and attention deﬁcit hyperac- tivity disorder are important contributors to the global burden of disease [1]. Prominent theories suggest that these behaviour problems play an important role in the devel- opment of interpersonal violence [2, 3], which is itself a major global cause of healthy life years lost, particularly in A. Matijasevich Department of Preventive Medicine, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil F. C. Barros Postgraduate Program in Health and Behavior, Universidade Cato´lica de Pelotas, Pelotas, Brazil F. C. Barros Postgraduate Program in Health and Behavior, Universidade Cato´lica de Pelotas, Pelotas, Brazil 123 12 3 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 580 Method Method low- and middle-income countries (LMICs) in Africa and Latin America [4, 5]. Conduct problems might increase the risk for participation in crime because they contribute to poor psychosocial functioning, for example poor relation- ships with parents and peers, poor educational perfor- mance, or drug use [2]. Prospective studies in high-income countries (HICs) show robust associations between conduct problems, crime and violence [6–10], but less consistent associations with hyperactivity [6–12] when controlling for prior social and biological risk factors. The effects of behaviour problems on crime appear similar for males and females [6, 9], or slightly stronger for males [7, 11]. Avon Longitudinal Study of Parents and Children (ALSPAC), Britain ALSPAC is a separate, ongoing population-based study in Britain. ALSPAC recruited 14,541 pregnant women resi- dents in Avon, Britain with expected dates of delivery from 1st April 1991 to 31st December 1992; and, from age 7, continued to recruit children born in that area at that time until age 18. The total sample size for analyses using any data collected after the age of seven is 15,247 pregnancies, resulting in 15,458 children. We used data on 14,762 live- born singleton or twin children; triplets and quads were excluded for reasons of conﬁdentiality. The detailed methodology of ALSPAC can be found elsewhere [19, 20] and the study website contains details of all the data that is available through a fully searchable data dictionary (http://www.bris.ac.uk/alspac/researchers/data-access/data- dictionary/). When compared to 1991 National Census Data, the ALSPAC sample was found to be similar to the UK population as a whole, but had a slightly higher pro- portion of married or cohabiting mothers and families who were owner occupiers, and (consistent with the area where the study is based), a smaller proportion of mothers from ethnic minorities (2.2 versus 7.6 %) [20]. When cohort members were 11 years old, mothers completed 1. What is the prevalence of conduct problems and hyperactivity at age 11, and self-reported crime and violence at age 18 in Pelotas, Brazil, and how do prevalence rates compare with ALSPAC, Britain? 2. Do conduct problems and hyperactivity at age 11 predict increased risk of crime and violence in both Brazil and Britain, and are these associations similar for females and males? 3. Do conduct problems and hyperactivity predict crime and violence independently of confounders in Brazil and Britain? 123 3 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 581 questionnaires about the children. When cohort members were 18 years old, adolescents participated in focus clinic sessions. Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. extremely similar to the majority with valid crime data on all perinatal characteristics (see the Online Supplement, Table S1). Behaviour problems at age 11 When children were 11 years old, parents (usually mothers) completed the Strengths and Difﬁculties Ques- tionnaire (SDQ) for 4,423 children in Pelotas and 7,307 children in ALSPAC. The SDQ is a screening question- naire that assesses child mental health symptoms in the previous 6 months. It includes sub-scales measuring two types of child behaviour problems: conduct problems (symptoms of oppositional deﬁant and conduct disorders) and hyperactivity (symptoms of inattention and hyperac- tivity disorders). The SDQ was developed by Goodman [21] and validated in Brazil by Fleitlich-Bilyk and Goodman [22]. A previous study in Pelotas compared the SDQ with a diagnostic instrument [Development and Well-Being Assessment (DAWBA)]. In relation to diag- noses on DAWBA as the gold standard, the psychometric properties of the SDQ were 78.2 % sensitivity, 70.4 % speciﬁcity, and 74.0 % area under the curve [23]. The same cut-points were used in Pelotas and ALSPAC to identify ‘‘abnormal’’ levels of conduct problems ([3) and hyperactivity ([6). Avon Longitudinal Study of Parents and Children (ALSPAC), Britain We used two summary crime variables as outcomes in our analyses: (1) reported at least one of nine types of nonviolent crimes: stole from shops/stores, damaged property, stole from vehicle, stole vehicle, sold drug, bur- gled, sold stolen good, arson, stole from person without threat/force; (2) reported at least one of four types of vio- lent crime: stole from person with threat/force, assault, carried a weapon for ﬁghts or self-defence, used weapon. Police and justice system records were also searched in Pelotas. In the main analyses we use only self-reported crime data, to maximise comparability with the British study, but we note here that, in Pelotas, the association between self-reported crime and ofﬁcially recorded crime at age 18 was strong (risk ratio = 4.4 for nonviolent crime and 5.2 for violent crime). Confounding variables Numerous biological, psychological and social variables from pregnancy through late childhood predict the devel- opment of antisocial behaviour [6, 12, 25–27]. In both studies, we included confounders measured with mothers in the perinatal period, and indicators of parental crime and mental health up to age 11; variables were dichotomised to maximise comparability between studies. The following perinatal characteristics were measured in both studies: unplanned pregnancy (yes/no), mother ever smoked during pregnancy (yes/no), mother used alcohol during pregnancy (yes/no), maternal urinary infection during pregnancy (yes/ no), intrauterine growth restriction (yes/no; referring to \10th percentile/C10th percentile for gestational age and gender, according to the reference curve developed by Kramer et al. [28]), premature birth \37 weeks (yes/no). Measures Behaviour problems at age 11 Crime and violence at age 18 Crime and violence at age 18 A conﬁdential self-reported crime questionnaire, originally developed in the Edinburgh Study of Youth Transitions and Crime [24], was completed by 4,102 adolescents in AL- SPAC clinic sessions at age 18. Thirteen questions from this instrument were then included in the conﬁdential questionnaire in the Pelotas study clinic sessions at age 18, referring to crimes committed by the adolescents in the previous 12 months. For the Pelotas study, questions were ﬁrst translated into Brazilian Portuguese, then pilot tested among adolescent offenders in Pelotas (in a young offenders’ institution) and among adolescents in the com- munity (in a public health clinic), adjusted by bilingual researchers, further pilot tested, and then back translated into English. Due to a printing error, the ﬁrst 325 ques- tionnaires (8 % of 4,106 participants at age 18) in Pelotas were not usable. The current analyses of criminal behav- iour in Pelotas included the vast majority of participants (N = 3,618) with complete crime data from these ques- tionnaires. The Pelotas sub-sample without crime data is The following socio-demographic characteristics were measured in both studies in the perinatal period: maternal age (\20/C20 years), low maternal education (yes/no; referring in Pelotas to 0–8 versus C9 years of schooling; referring in ALSPAC to qualiﬁed up to certiﬁcate of sec- ondary qualiﬁcation level, versus qualiﬁed to at least vocational level, O-level, or A-level), marital status (single mother/with partner), three or more siblings (yes/no), family income (lowest quintile/second–ﬁfth quintiles). All health and socio-demographic variables in the perinatal period have been carefully compared between Pelotas and ALSPAC in previous work and related to childhood con- duct problems and adolescent violence [29, 30]. No variable was correlated with any other variable more than phi = 0.3. Parental crime (between the child’s birth and age 11), and maternal mental health were also included as con- founding variables in this study. In Pelotas, parental crime 123 12 3 3 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 582 was measured by searching state-wide police registries to identify whether the mother or father had committed a crime between the child’s birth and 11th birthday. In AL- SPAC, parental crime was measured by asking both mothers and their partners about whether either person had been in trouble with the law/convicted since the previous interview on eight occasions between when children were 8 months and 11 years old. Crime and violence at age 18 Any indication that either parent had been in trouble with the law from the child’s birth to 11 years was coded as positive for parental crime. mind that our cross-national comparisons are based on risk ratios, and we conceptualise a sex difference of 10 versus 5 % in one country as larger than a difference of 25 versus 20 % in another country. In Pelotas, participants with valid crime data at age 18 had very similar perinatal characteristics compared with participants without crime data; however, this was not true in ALSPAC (see Online Supplement Table S1). To reduce bias caused by missing data, we estimated associations between childhood behavioural problems and adolescent crime using multiple imputation for missing data in both studies. Fifty data sets (each with 2,645 females and 2,603 males in Pelotas, and 5,937 females and 6,242 males in ALSPAC) were created by imputing missing predictor and outcome data using the mi impute-chained command in STATA 12.1. All variables in the multivariate models (crime at 18, age at crime measurement, conduct problems and hyperactivity at 11, and confounding variables) were used in the imputation process. A minimum requirement for inclusion in the multiple imputation analyses was that at least half of the confounding variables were valid or childhood behaviour data were valid or crime data were valid. Logistic regression was used to impute binary vari- ables and OLS regression to impute continuous variables (age). In the main text, results are presented based on multiple imputation; the Online Supplement shows that results based on complete case analyses are very similar. Maternal mental health was measured in Pelotas using the self report questionnaire (SRQ) when children were 11 years. The SRQ measures depression and anxiety and was validated in a Brazilian sample of 485 subjects [31]. We used a cut-off of eight points to classify mothers as having probable minor psychiatric disorders. In ALSPAC, maternal mental health was measured using the 10-item Edinburgh Postnatal Depression Scale (EPDS [32]) at 11 years. We used the recommended cut-off of 13 points to identify mothers with probable depression. Statistical analyses The prevalence of conduct problems, hyperactivity, crime and violence were compared between Pelotas and ALSPAC, and between females and males within each study, using risk ratios and 95 % conﬁdence intervals. Associations between behaviour problems (conduct problems and hyperactivity) and nonviolent and violent crime were also examined using risk ratios and 95 % conﬁdence intervals. To investigate possible differences in risk ratios (interactions) between studies or between sexes, the ratio of risk ratios with 95 % conﬁdence intervals was calculated [33]. To calculate adjusted risk ratios, we used Poisson regression with robust standard errors, as proposed by Barros and Hirakata [34]. Results Children in Pelotas had about four times higher risk for both conduct problems and hyperactivity measured on the SDQ, compared with children in ALSPAC (Table 1). Interestingly, although the prevalence of self-reported violent crime was higher in Pelotas, the prevalence of nonviolent crime was higher in ALSPAC. There was no difference in the probability of reporting ‘‘any crime’’ between the two sites. Note that the violent behaviour most commonly reported in both studies (assault) was not trivial. The question speciﬁed that the assault should have been done with the intention of hurting the victim and excluded ﬁghting with siblings. The majority of adolescents who reported committing assault also stated that they did indeed cause an injury in their most serious ﬁght in the last year (83 % Pelotas; 77 % ALSPAC). Comparisons between countries can be conceptualised in different ways. Our use of risk ratios to compare asso- ciations between countries has a particular meaning, which can be illustrated in an example of comparing sex differ- ences in violence between countries. Imagine in Country One, the prevalence of violence is 10 % among males and 5 % among females; this equals a male–female risk ratio of 2.0 (10 7 5 %), and a risk difference between males and females of 5 % (10 - 5 %). In Country Two, the preva- lence of violence might be 25 % among males and 20 % among females, which equals a lower risk ratio of 1.25 (25 7 20 %), but the same risk difference of 5 % (25 - 20 %). In this hypothetical example, the risk ratio in Country One is larger than in Country Two, although the risk difference is the same in both countries. Both ﬁndings, based on risk ratios and risk differences, are correct, but conceptualise the comparison between countries (and sexes) in different ways. Thus, it is important to bear in Conduct problems and hyperactivity were strongly related to each other in both Pelotas and ALSPAC. In Pelotas, 56.4 % of males with conduct problems had high hyperactivity scores versus 18.5 % of males without con- duct problems (RR = 3.0; CI = 2.7–3.4) ; for females equivalent rates were 45.0 versus 11.8 % (RR = 3.8; CI = 3.3–4.5). Results In ALSPAC, 40.5 % of males with conduct 123 123 583 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 Table 1 Prevalence of childhood behaviour problems (age 11) and adolescent crime (age 18) in Pelotas, Brazil and ALSPAC, Britain Pelotas females (%) ALSPAC females (%) RR (95 % CI) p value Pelotas males (%) ALSPAC males (%) RR (95 % CI) p value Behaviour problems N = 2,245 N = 3,656 N = 2,178 N = 3,651 Conduct 28.7 6.0 4.8 (4.1–5.5) \0.001 33.9 8.5 4.0 (3.5–4.5) \0.001 Hyperactive 21.3 4.2 5.1 (4.3–6.1) \0.001 31.4 9.9 3.2 (2.8–3.6) \0.001 Individual nonviolent Crimes N = 1,847 N = 2,252 N = 1,771 N = 1,760 Stole from shops/stores 0.7 7.0 0.1 (0.1–0.2) \0.001 2.4 8.9 0.3 (0.2–0.4) \0.001 Damaged property 0.6 1.2 0.5 (0.2–1.0) 0.034 4.2 6.4 0.7 (0.5–0.9) 0.005 Stole from vehicle 0.1 0.1 0.6 (0.1–6.7) 0.683 0.7 0.7 0.9 (0.4–2.0) 0.829 Stole vehicle 0.1 0.8 0.1 (0.0–0.5) \0.001 0.7 2.5 0.3 (0.2–0.5) \0.001 Sold drug 0.9 2.3 0.4 (0.2–0.7) \0.001 3.5 6.1 0.6 (0.4–0.8) \0.001 Burgled 0.1 0.1 0.6 (0.1–6.7) 0.683 0.5 0.4 1.3 (0.5–3.4) 0.625 Sold stolen good 0.7 0.3 2.4(0.9–6.5) 0.065 3.1 1.8 1.7 (1.1–2.6) 0.018 Arson 0.1 0.4 0.3 (0.1–1.4) 0.111 1.0 1.4 0.7 (0.4–1.3) 0.263 Stole from person—no threat/force 0.5 0.5 1.1 (0.5–2.6) 0.813 0.9 0.7 1.3 (0.6–2.8) 0.458 Individual violent crimes N = 1,847 N = 2,252 N = 1,771 N = 1,760 Assault with intention to injure 7.8 2.4 3.3 (2.4–4.5) \0.001 18.0 9.2 2.0 (1.6–2.3) \0.001 Carried weapon 2.2 0.8 2.6 (1.5–4.4) \0.001 10.2 3.0 3.4 (2.5–4.6) \0.001 Used weapon 0.2 0.0 N/A 0.027 2.7 0.6 4.8 (2.4–9.4) \0.001 Stole from person—with threat/force 0.1 0.0 2.4 (0.2–26.9) 0.452 1.0 0.1 8.9 (2.1–38.5) \0.001 Summary crime variables N = 1,847 N = 2,252 N = 1,771 N = 1,760 Any crime 10.2 11.2 0.9 (0.8–1.1) 0.325 25.7 23.5 1.1 (1.0–1.2) 0.135 Nonviolent crime 2.7 9.4 0.3 (0.2–0.4) \0.001 9.7 18.0 0.5 (0.5–0.6) \0.001 Violent crime 8.9 3.0 3.0 (2.3–4.0) \0.001 22.6 11.0 2.1 (1.8–2.4) \0.001 RR risk ratio comparing Pelotas and ALSPAC, with ALSPAC as the reference category y p ( ) 12 3 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 584 problems had high hyperactivity scores, versus 7.0 % of (Pelotas = 3.6, CI = 2.6–4.9; ALSPAC = 1.9, CI = Table 2 Childhood behaviour problems and adolescent nonviolent crime Row percents Pelotas Nonviolent crime ALSPAC Nonviolent crime N Yes No N Yes No Females Conduct p \ 0.001 p = 0.009 Yes 517 5.2 % 94.8 % 97 16.5 % 83.5 % No 1,284 1.6 % 98.4 % 1,743 8.7 % 91.3 % Hyperactive p = 0.009 p = 0.733 Yes 397 4.5 % 95.5 % 58 10.3 % 89.7 % No 1,404 2.1 % 97.9 % 1,782 9.0 % 91.0 % Males Conduct p \ 0.001 p = 0.001 Yes 556 13.3 % 86.7 % 85 31.8 % 68.2 % No 1,127 8.2 % 91.8 % 1,370 17.7 % 82.3 % Hyperactive p = 0.006 p = 0.079 Yes 522 12.8 % 87.2 % 95 25.3 % 74.7 % No 1,161 8.5 % 91.5 % 1,360 18.0 % 82.0 % Table 3 Childhood behaviour problems and adolescent violent crime Row percents Pelotas Violent crime ALSPAC Violent crime N Yes No N Yes No Females Conduct p \ 0.001 p = 0.381 Yes 517 14.3 % 85.7 % 97 4.1 % 95.9 % No 1,284 6.6 % 93.4 % 1,743 2.6 % 97.4 % Hyperactive p \ 0.001 p = 0.728 Yes 397 16.1 % 83.9 % 58 3.5 % 96.5 % No 1,404 6.8 % 93.2 % 1,782 2.7 % 97.3 % Males Conduct p \ 0.001 p = 0.002 Yes 556 28.4 % 71.6 % 85 21.2 % 78.8 % No 1,127 20.7 % 79.3 % 1,370 10.2 % 89.8 % Hyperactive p \ 0.001 p \ 0.001 Yes 522 29.3 % 70.7 % 95 22.1 % 77.9 % No 1,161 20.5 % 79.5 % 1,360 10.1 % 89.9 % Table 2 Childhood behaviour problems and adolescent nonviolent crime problems had high hyperactivity scores, versus 7.0 % of males without conduct problems (RR = 5.8; CI = 4.8–6.9); for females equivalent rates were 24.6 versus 2.9 % (RR = 8.6; CI = 6.4–11.7). Results (Pelotas = 3.6, CI = 2.6–4.9; ALSPAC = 1.9, CI = 1.6–2.3; p for the comparison of ratios = 0.054). However, the male–female ratio was larger in ALSPAC than in Pelotas for violent crime (ALSPAC = 3.7, CI = 2.8–4.9; Pelotas = 2.5, CI = 2.1–3.0; p = 0.054), conduct problems (AL- SPAC = 1.4, CI = 1.2–1.7; Pelotas = 1.2, CI = 1.1–1.3; p = 0.154), and hyperactivity (ALSPAC = 2.4, CI = 2.0–2.9; Pelotas = 1.5, CI = 1.3–1.6; p = 0.010). In other words, male–female differences were larger in settings where behaviours were less common––that is, for nonviolent crime in Pelotas, and for violent crime, conduct problems, hyperactivity and in ALSPAC. As one would expect from previous studies, males were more likely than females to show conduct problems, hyperactivity, non-violent, and violent crime, and this was true in both Pelotas and ALSPAC (p \ 0.001 for all sex comparisons in both studies). However, these sex differ- ences, expressed as male–female ratios, were not equal across sites and types of behaviour. The male–female ratio was larger in Pelotas than in ALSPAC for nonviolent crime 123 123 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 585 % Non-violent Crime % Violent Crime Notes. Total samples (males and females combined). All Chi-square tests of linear trends p<.001 0 10 20 30 40 0-1 2-3 4-5 6-10 Conduct Problem Score Pelotas ALSPAC 0 5 10 15 20 25 0-1 2-3 4-5 6-10 Hyperacvity Score Pelotas ALSPAC 0 5 10 15 20 25 0-1 2-3 4-5 6-10 Conduct Problem Score Pelotas ALSPAC 0 5 10 15 20 25 0-1 2-3 4-5 6-10 Hyperacvity Score Pelotas ALSPAC Fig. 1 Prevalence of adolescent crime by behaviour problem scores in Pelotas, Brazil and ALSPAC, Britain 0 5 10 15 20 25 0-1 2-3 4-5 6-10 Hyperacvity Score Pelotas ALSPAC % Non-violent Crime 0 10 20 30 40 0-1 2-3 4-5 6-10 Conduct Problem Score Pelotas ALSPAC Conduct Problem Score Hyperacvity Score 0 5 10 15 20 25 0-1 2-3 4-5 6-10 Hyperacvity Score Pelotas ALSPAC % Violent Crime 0 5 10 15 20 25 0-1 2-3 4-5 6-10 Conduct Problem Score Pelotas ALSPAC Hyperacvity Score Notes. Total samples (males and females combined). Results All Chi-square tests of linear trends p<.001 Table 4 Unadjusted associations between childhood behaviour problems and adolescent crime Table 4 Unadjusted associations between childhood behaviour problems and adolescent crime Pelotas ALSPAC Pelotas–ALSPAC interaction RR (95 % CI) p value RR (95 % CI) p value RRR (95 % CI) p value Females Behavioural predictor Crime outcome Conduct Non-violent 2.7 (1.6–4.5) \0.001 1.8 (1.2–2.6) 0.002 1.5 (0.8–2.8) 0.223 Hyperactive Non-violent 1.9 (1.1–3.3) 0.030 1.3 (0.8–2.1) 0.278 1.4 (0.7–3.0) 0.337 Conduct Violent 1.9 (1.5–2.5) \0.001 1.8 (1.0–3.2) 0.049 1.1 (0.6–2.1) 0.789 Hyperactive Violent 2.2 (1.6–3.0) \0.001 1.8 (0.9–3.5) 0.075 1.2 (0.6–2.5) 0.609 Males Behavioural predictor Crime outcome Conduct Non-violent 1.7 (1.3–2.3) \0.001 1.7 (1.4–2.1) \0.001 1.0 (0.7–1.5) 0.961 Hyperactive Non-violent 1.5 (1.1–2.0) 0.004 1.4 (1.1–1.8) 0.005 1.1 (0.7–1.6) 0.698 Conduct Violent 1.4 (1.2–1.7) \0.001 1.9 (1.5–2.4) \0.001 0.8 (0.6–1.0) 0.097 Hyperactive Violent 1.5 (1.3–1.8) \0.001 1.8 (1.4–2.4) \0.001 0.8 (0.6–1.1) 0.183 Based on 50 data sets using multiple imputation of missing data as described in methods section; N = 2,645 females and 2,603 males in Pelotas, and 5,937 females and 6,242 males in ALSPAC RR risk ratio, comparing risk of crime outcome between children with behaviour problem and children without behaviour problem, controlling only for child age in months at time of crime assessment CI conﬁdence interval RRR = ratio of risk ratios = RR for Pelotas divided by RR for ALSPAC Table 4 Unadjusted associations between childhood behaviour problems and adolescent crime RR risk ratio, comparing risk of crime outcome between children with behaviour problem and children without behaviour problem, controlling only for child age in months at time of crime assessment CI conﬁdence interval RRR = ratio of risk ratios = RR for Pelotas divided by RR for ALSPAC CI conﬁdence interval RRR = ratio of risk ratios = RR for Pelotas divided by RR for ALSPAC adolescence (Tables 2 and 3). To consider how prediction varied with severity of behaviour problems, we also examined the prevalence of crime according to four dif- ferent levels of behaviour problems. Discussion Behaviour problems and violence are major global health problems. In 2010, 5.8-million healthy life years were lost worldwide due to conduct disorder, 0.5 million due to attention deﬁcit hyperactivity disorder, and 25.5 million due to interpersonal violence [35]. In LMICs in the Americas, one-third of all deaths among 15–29-year-olds were caused by interpersonal violence in 2011 [36]. It is important to establish whether key risk factors identiﬁed in HICs, such as childhood behaviour problems, inﬂuence the development of crime and violence in similar ways in LMIC settings. We found that childhood conduct problems and hyperactivity were similarly associated with crime and violence in two large, population-based, longitudinal studies in Brazil and Britain. Results Note that, at all levels of child behaviour problems, adolescents in Pelotas were more likely to report violent crime than in ALSPAC, and ado- lescents in ALSPAC were more likely to report nonviolent crime than in Pelotas (Fig. 1). This pattern was true for both females and males when analysed separately, as well as for both sexes pooled (see Online Supplement Table S2). All associations between behaviour problems and crime were positive, for girls and boys, in both Pelotas and AL- SPAC (Table 4). There was no signiﬁcant difference in the strength of these associations between Pelotas and AL- SPAC (all tests of interaction p [ 0.05). Comparing asso- ciations between females and males within each study, only one interaction out of eight was signiﬁcant: the association between hyperactivity and violent crime was larger for girls (RR = 2.2, CI = 1.6–3.0) than boys (RR = 1.5, CI = 1.3–1.8) in Pelotas (p = 0.027 for interaction). conduct problems and hyperactivity, and nonviolent and violent crime (Fig. 1). Note that, at all levels of child behaviour problems, adolescents in Pelotas were more likely to report violent crime than in ALSPAC, and ado- lescents in ALSPAC were more likely to report nonviolent crime than in Pelotas (Fig. 1). This pattern was true for both females and males when analysed separately, as well as for both sexes pooled (see Online Supplement Table S2). All associations between behaviour problems and crime were positive, for girls and boys, in both Pelotas and AL- SPAC (Table 4). There was no signiﬁcant difference in the strength of these associations between Pelotas and AL- SPAC (all tests of interaction p [ 0.05). Comparing asso- ciations between females and males within each study, only one interaction out of eight was signiﬁcant: the association between hyperactivity and violent crime was larger for girls (RR = 2.2, CI = 1.6–3.0) than boys (RR = 1.5, CI = 1.3–1.8) in Pelotas (p = 0.027 for interaction). Results The relationship was almost linear in both studies, between the four levels of Bivariate associations In both Pelotas and ALSPAC, children with high conduct problem and high hyperactivity scores were more likely to self-report both nonviolent and violent crime in late 12 3 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 586 Table 5 Associations between childhood behaviour problems and crime, adjusted for confounders Pelotas ALSPAC Pelotas–ALSPAC interaction RR (95 % CI) p value RR (95 % CI) p value RRR (95 % CI) p value Females Behavioural predictor Crime outcome Conduct Non-violent 2.4 (1.3–4.3) 0.005 1.6 (1.1–2.3) 0.022 1.5 (0.7–3.1) 0.272 Hyperactive Non-violent 1.3 (0.7–2.4) 0.434 1.0 (0.6–1.7) 0.872 1.2 (0.5–2.8) 0.612 Conduct Violent 1.4 (1.0–1.9) 0.050 1.4 (0.8–2.6) 0.272 1.0 (0.5–1.9) 0.930 Hyperactive Violent 1.8 (1.3–2.5) 0.001 1.5 (0.8–3.0) 0.243 1.2 (0.6–2.5) 0.651 Males Behavioural predictor Crime outcome Conduct Non-violent 1.4 (1.0–2.0) 0.044 1.4 (1.1–1.8) 0.003 1.0 (0.6–1.5) 0.909 Hyperactive Non-violent 1.2 (0.9–1.7) 0.192 1.2 (0.9–1.5) 0.227 1.1 (0.7–1.6) 0.796 Conduct Violent 1.2 (1.0–1.5) 0.051 1.4 (1.1–1.8) 0.019 0.9 (0.6–1.2) 0.421 Hyperactive Violent 1.3 (1.1–1.6) 0.001 1.5 (1.2–2.0) 0.003 0.9 (0.6–1.2) 0.445 Based on 50 data sets using multiple imputation of missing data as described in methods section; N = 2,645 females and 2,603 males in Pelotas, and 5,937 females and 6,242 males in ALSPAC RR risk ratio adjusted for conduct and hyperactive problems age 11; unplanned pregnancy, ever smoked in pregnancy, alcohol use in pregnancy, urinary infection in pregnancy, intrauterine growth restriction, premature birth, maternal age, maternal education, marital status, 3? siblings, low family income, parental crime birth-age 11, maternal mental health age 11, child age in months at time of crime assessment CI conﬁdence interval RRR = ratio of adjusted risk ratios = RR for Pelotas divided by RR for ALSPAC RRR = ratio of adjusted risk ratios = RR for Pelotas divided by RR for ALSPAC difference in the strength of associations between Pelotas and ALSPAC (all tests of interaction p [ 0.05). There was also no signiﬁcant difference in the strength of association comparing females and males within each study (all tests of interaction p [ 0.05). In sensitivity analyses, we conducted the same analyses using only cases with complete data, rather than using multiple imputation for missing data, and results were very similar (Online Supplement, Table S3). conduct problems and hyperactivity, and nonviolent and violent crime (Fig. 1). Strengths and limitations This study has several important strengths. A major strength is the collection of prospective cohort data on behaviour problems and violence in Brazil. To our knowledge, this is the ﬁrst Latin American study that has assessed the effects of childhood behaviour problems on later crime and violence. The current study is also novel in its direct comparisons of prospectively measured risk fac- tors for crime between LMIC and HIC settings. Additional strengths are that the study is based on two large, pro- spective, population-based surveys that are well matched in terms of year of birth, ages at follow-up, and instruments used for key predictor and outcome variables. Also, although most major studies of antisocial behaviour have included only boys, both our Brazilian and British studies included females as well as males. In both Pelotas and ALSPAC, childhood behaviour problems were measured using parental reports and crime was measured using self- reports, reducing the problem of common informant bias, which might otherwise inﬂate the size of associations. Furthermore, there was a wide range of comparable con- founding variables included in both studies. The speciﬁcity of results for violent and nonviolent crime suggest that differences in prevalence rates between Brazil and Britain were not an artefact of generic over or under-reporting. The size of sex differences in behaviour problems and crime varied between our Brazilian and British samples. The male–female ratio was larger in Britain than in Brazil for conduct problems, hyperactivity, and violent crime, but the male–female ratio was larger in Brazil for nonviolent crime. In other words, sex differences were more pro- nounced in settings where the particular problem behaviour was less common. This is consistent with the most com- prehensive analysis to date of sex differences in antisocial behaviour, based on the Dunedin cohort, New Zealand: this showed that the male–female ratio in antisocial behaviour was especially large for less frequent and more serious behaviours in that setting [11]. Although prevalence rates varied substantially between Brazil and Britain, conduct problems and hyperactivity were similarly associated with crime and violence in both sites, speaking to the cross-cultural signiﬁcance of early behaviour problems in the development of more serious crime and violence. However, in both Brazil and Britain, associations were attenuated when taking into account the co-occurrence of conduct problems and hyperactivity, and other risk factors measured up to age 11. Multivariate models After controlling for confounding variables in multivariate models, associations between childhood behaviour prob- lems and crime were reduced in both Pelotas and ALSPAC (Table 5). Nonetheless, four associations remained signif- icant in Pelotas (with risk ratios ranging from 1.3 to 2.4), and four remained signiﬁcant in ALSPAC (with risk ratios ranging from 1.4 to 1.6). Again, there was no signiﬁcant Homicide rates are very high in Brazil compared with HICs such as Britain [16], but there is a lack of reliable 123 123 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 587 data on non-lethal crime and violence in Brazil to compare with other countries. In the ﬁrst study of its type, we found interesting patterns of self-reported crime: higher rates of violent crime in Brazil than in Britain, but higher rates of nonviolent crime in Britain compared with Brazil. This is consistent with evidence that England and Wales lie in a cluster of Anglo-Saxon countries with high levels of property crime and drug use, but average levels of violence [37]. Brazil may be more similar to a cluster of Eastern European countries, which have high levels of violence but not high levels of property crime or drug use [16, 37]. It has been speculated that, in those countries, increased rates of violence are caused by social inequality, low levels of social control, and widespread material poverty [37], and this may also be true in Brazil [16]. other settings [38]. Given the strong association between poverty and adolescent behaviour problems in Pelotas [39], poverty reduction strategies are also needed. Strengths and limitations Strengths and limitations Therefore, conduct problems and hyperactivity predict crime partly because they mark other inﬂuences in childhood, including health and socioeconomic factors in the perinatal period, and maternal depression and parental criminality during childhood. Although previous studies have found mixed results for the effects of hyper- activity on crime and violence [6–12], it is notable that, in our samples, hyperactivity was predictive of violence for both females and males in Brazil, and for males in Britain, even after adjustment for confounders. g p g The following limitations of the study should also be considered. There was signiﬁcant attrition in ALSPAC and missing data were associated with childhood risk factors. Given selective attrition, the prevalence of behaviour prob- lems and crime are probably underestimated in ALSPAC. It was reassuring that we found similar associations between behaviour problems and crime using both complete cases and using multiple imputation for missing data; also some evi- dence suggests that predictive models are quite robust to missing data [40]. However, multiple imputation cannot guarantee lack of bias caused by selective attrition. Multiple imputation eliminate bias only if enough variables that pre- dict missing values are included in the imputation model. If participants with missing data differ from other participants in ways not reﬂected by the variables included in the study, the missing at random assumption does not hold and bias cannot be eliminated. Although we included quite an extensive range of predictor variables in this study, we can- not rule out the possibility that results would have been different if there were no missing data. Our ﬁndings suggest that childhood conduct problems and hyperactivity could be signiﬁcant factors contributing to crime and violence in Brazil as well as in Britain. Early interventions to prevent childhood behaviour problems that can lead to crime should be evaluated in Brazil where rates of violence are particularly high, for example parent- training programmes which have been found effective in While corroborative evidence from ofﬁcial records makes it very plausible that rates of violence in Brazil are higher than in Britain, we urge particular caution regarding 12 3 588 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 the extremely high rates of conduct problems and hyper- activity reported for Brazilian children (four times the rates reported in Britain). References 1. Vos T, Flaxman AD, Naghavi M, Lozano R, Michaud C, Ezzati M et al (2012) Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990–2010: a systematic analysis for the global burden of disease study 2010. Lancet 380(9859):2163–2196. doi:10.1016/S0140-6736(12)61729-2 It would have been ideal to include multiple measures of both child behaviour problems (e.g., also diagnostic assessments) and crime (e.g., also ofﬁcial records), which we did not have in both studies. As not all crime is reported to the police, it would have been ideal to also include self-reported criminal behaviours of parents. Finally, while both studies used large community popu- lations, neither used national samples and results reﬂect each local population. 2. Mofﬁtt TE (1993) Adolescence-limited and life-course-persistent antisocial behavior: a developmental taxonomy. Psychol Rev 100(4):674–701. doi:10.1037/0033-295X.100.4.674 3. Loeber R, Wung P, Keenan K, Giroux B, Southamer-Loeber M, van Kammen WB (1993) Developmental pathways in disruptive child behavior. Dev Psychopathol 5(1–2):101–132. doi:10.1017/ S0954579400004296 4. Viner RM, Coffey C, Mathers C, Bloem P, Costello A, Santelli J, Patton GC (2011) 50-year mortality trends in children and young people: a study of 50 low-income, middle-income, and high- income countries. Lancet 377(9772):1162–1174 5. Gore FM, Bloem PJN, Patton GC, Ferguson J, Joseph V, Coffey C, Sawyer SM, Mathers CD (2011) Global burden of disease in young people aged 10–24 years: a systematic analysis. Lancet 377(9783):2093–2102 Strengths and limitations Although other questionnaire-based studies in Brazil also document very high levels of child behaviour problems [41, 42], there is debate as to whether this reﬂects overreporting of symptoms by Brazilian par- ents [42]. It is possible that speciﬁc procedures (for example, reading questionnaires to Brazilian parents with less education) or cultural differences in interpreting questionnaire items causes overestimation of children’s behaviour problems in Brazil. If indeed problem behav- iours are overreported in Brazilian studies, cut-off points to identify ‘‘abnormal’’ levels of problem behaviour could be raised. However, we believe this methodological issue does not affect our key ﬁndings on the associations between childhood behaviour problems and risk for adolescent crime. We found that these relationships were linear across different levels of behaviour problem scores in both Brazil and Britain, suggesting that the speciﬁc cut-off point used did not affect the observed strength of associations. receptionists and nurses. The UK Medical Research Council and the Wellcome Trust (Grant: 092731) and the University of Bristol provide core support for ALSPAC. Additional support for the data collected at age 18 in this paper was provided by UK Medical Research Council (Grants G0800612 and G0802736). This publication is the work of the authors and Joseph Murray will serve as guarantor for the contents of this paper. The research for this speciﬁc article was funded by the Wellcome Trust (Grant: 089963/Z/09/Z). receptionists and nurses. The UK Medical Research Council and the Wellcome Trust (Grant: 092731) and the University of Bristol provide core support for ALSPAC. Additional support for the data collected at age 18 in this paper was provided by UK Medical Research Council (Grants G0800612 and G0802736). This publication is the work of the authors and Joseph Murray will serve as guarantor for the contents of this paper. The research for this speciﬁc article was funded by the Wellcome Trust (Grant: 089963/Z/09/Z). Conﬂict of interest On behalf of all authors, the corresponding author states that there is no conﬂict of interest. Conﬂict of interest On behalf of all authors, the corresponding author states that there is no conﬂict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. Conclusion Mofﬁtt TE, Caspi A, Rutter M, Silva PA (2001) Sex differences in antisocial behaviour: conduct disorder, delinquency, and vio- lence in the Dunedin longitudinal study. Cambridge University Press, Cambridge 28. Kramer MS, Platt RW, Wen SW, Joseph KS, Allen A, Abra- hamowicz M, Blondel B, Bre´art G (2001) A New and improved population-based Canadian reference for birth weight for gesta- tional age. Pediatrics 108(2):e35. doi:10.1542/peds.108.2.e35 12. Murray J, Farrington DP (2010) Risk factors for conduct disorder and delinquency: key ﬁndings from longitudinal studies. Can J Psychiatry 55(10):633–642 29. 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Victora CG, Hallal PC, Arau´jo CL, Menezes AM, Wells JC, Barros FC (2008) Cohort proﬁle: the 1993 Pelotas (Brazil) birth cohort study. Int J Epidemiol 37(4):704–709. doi:10.1093/ije/ dym177 34. Conclusion In summary, childhood conduct problems and hyperactiv- ity are similar precursors of both nonviolent and violent crime across two very different social contexts. Conduct problems and hyperactivity potentially represent both markers of other childhood risk factors, and possible risk mechanisms increasing the chances of engagement in adolescent crime and violence. These ﬁndings speak to the need to evaluate early intervention programmes to reduce childhood behaviour problems in LMICs, as well as in high-income settings. 6. Murray J, Irving B, Farrington DP, Colman I, Bloxsom CAJ (2010) Very early predictors of conduct problems and crime: results from a national cohort study. J Child Psychol Psychiatry 51(11):1198–1207. doi:10.1111/j.1469-7610.2010.02287.x 7. Broidy LM, Nagin DS, Tremblay RE, Bates JE, Brame B, Dodge KA et al (2003) Developmental trajectories of childhood dis- ruptive behaviors and adolescent delinquency: a six-site, cross- national study. Dev Psychol 39(2):222–245. doi:10.1037/0012- 1649.39.2.222 8. Copeland WE, Miller-Johnson S, Keeler G, Angold A, Costello EJ (2007) Childhood psychiatric disorders and young adult crime: a prospective, population-based study. Am J Psychiatry 164(11): 1668–1675. doi:10.1176/appi.ajp.2007.06122026 9. Fergusson DM, Boden JM, Horwood LJ (2010) Classiﬁcation of behavior disorders in adolescence: scaling methods, predictive validity, and gender differences. J Abnorm Psychol 119(4): 699–712. doi:10.1037/a0018610 Acknowledgments The 1993 Pelotas Birth Cohort Study is cur- rently supported by the Wellcome Trust through the program entitled Major Awards for Latin America on Health Consequences of Popu- lation Change (Grant: 086974/Z/08/Z). The European Union, National Support Program for Centers of Excellence (PRONEX), the Brazilian National Research Council (CNPq), and the Brazilian Ministry of Health supported previous phases of the study. We are extremely grateful to all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory techni- cians, clerical workers, research scientists, volunteers, managers, 10. Loeber R, Farrington DP, Stouthamer-Loeber M, van Kammen WB (1998) Multiple risk factors for multiproblem boys: co- occurrence of delinquency, substance use, attention deﬁcit, con- duct problems, physical aggression, covert behavior, depressed mood, and shy/withdrawn behavior. In: Jessor R (ed) New per- spectives on adolescent risk behavior. Cambridge University Press, Cambridge, pp 90–149 12 3 123 Soc Psychiatry Psychiatr Epidemiol (2015) 50:579–589 589 27. 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Boyd A, Golding J, Macleod J, Lawlor DA, Fraser A, Henderson J et al (2013) Cohort proﬁle: the ‘Children of the 90s’—the index offspring of the Avon longitudinal study of parents and children. Int J Epidemiol 42(1):111–127. doi:10.1093/ije/dys064 36. World Health Organization (2013) Global burden of disease: disease and injury regional estimates for 2011. http://www.who. int/healthinfo/global_burden_disease/en/. Accessed 19 November 2013 20. Fraser A, Macdonald-Wallis C, Tilling K, Boyd A, Golding J, Davey Smith G et al (2013) Cohort Proﬁle: the Avon longitudinal study of parents and children: ALSPAC mothers cohort. Int J Epidemiol 42(1):97–110. doi:10.1093/ije/dys066 37. Eisner M (2002) Crime, problem drinking, and drug use: patterns of problem behavior in cross-national perspective. Ann Am Acad Politi Soc Sci 580(1):201–225. doi:10.1177/000271620258000109 21. Goodman R (2001) Psychometric properties of the strengths and difﬁculties questionnaire. J Am Acad Child Adolesc Psychiatry 40(11):1337–1345. doi:10.1097/00004583-200111000-00015 38. Farrington DP, Welsh BC (2007) Saving children from a life of crime: early risk factors and effective interventions. Oxford University Press, Oxford 22. Fleitlich-Bilyk B, Goodman R (2004) Prevalence of child and adolescent psychiatric disorders in southeast Brazil. J Am Acad Child Adolesc Psychiatry 43(6):727–734. doi:10.1097/01.chi. 0000120021.14101.ca 39. Anselmi L, Menezes AMB, Hallal PC, Wehrmeister F, Gonc¸alves H, Barros FC, Murray J, Rohde LA (2012) Socioeconomic changes and adolescent psychopathology in a Brazilian birth cohort study. J Adolesc Health 51(6):S5–S10. doi:10.1016/j. jadohealth.2012.06.026 23. Anselmi L, Fleitlich-Bilyk B, Menezes A, Arau´jo C, Rohde L (2010) Prevalence of psychiatric disorders in a Brazilian birth cohort of 11-year-olds. Soc Psychiatry Psychiatr Epidemiol 45(1):135–142. doi:10.1007/s00127-009-0052-2 40. Wolke D, Waylen A, Samara M, Steer C, Goodman R, Ford T, Lamberts K (2009) Selective drop-out in longitudinal studies and non-biased prediction of behaviour disorders. Br J Psychiatry 195(3):249–256. doi:10.1192/bjp.bp.108.053751 24. Conclusion McAra L, McVie S (2010) Youth crime and justice: key mes- sages from the Edinburgh study of youth transitions and crime. Criminol Crim Justice 10(2):179–209. doi:10.1177/1748895 809360971 41. Rescorla L, Achenbach T, Ivanova MY, Dumenci L, Almqvist F, Bilenberg N et al (2007) Behavioral and emotional problems reported by parents of children ages 6 to 16 in 31 societies. J Emot Behav Disord 15(3):130–142. doi:10.1177/10634 266070150030101 25. Linnet KM, Dalsgaard S, Obel C, Wisborg K, Henriksen TB, Rodriguez A et al (2003) Maternal lifestyle factors in pregnancy risk of attention deﬁcit hyperactivity disorder and associated behaviors: review of the current evidence. Am J Psychiatry 160(6):1028–1040. doi:10.1176/appi.ajp.160.6.1028 42. Murray J, Anselmi L, Gallo E, Fleitlich-Bilyk B, Bordin I (2013) Epidemiology of childhood conduct problems in Brazil: sys- tematic review and meta-analysis. Soc Psychiatry Psychiatr Ep- idemiol 48(10):1527–1538. doi:10.1007/s00127-013-0695-x 26. Liu J (2011) Early health risk factors for violence: conceptuali- zation, evidence, and implications. Aggress Violent Behav 16(1):63–73. doi:10.1016/j.avb.2010.12.003 12 123
https://openalex.org/W4301043175	https://zenodo.org/records/6854850/files/803-806.pdf	English	null	References to '' Recent Researches in the Biochemistry of the Nitrogen Cycle.''	Zenodo (CERN European Organization for Nuclear Research)	1,929	cc-by	812	• Vide This Journal, p. 1, (1929). BY GILBBRT J. FowLBR. D.Se., F.I.C. 1. Lipman, "Soils and Men," Proceedings o£ First Inter- national Congress of Soil Science. Washington, 1928, Vol. L p. 19. 2. Economic Symposium on Nitrogen, Journ. Ind. Eng. Chern. Vol. 20. 1928, pp. 1128-1147. 3. King, "Farmers of For~y Centuries ". 4. Russel, " Modern Applications of Chemistry to Crop Pro- duction." Inst. o£ Chem. (Lecture). 5. Viswa Nath and Suryanarayana, with Mr;Carrison, "The Effect of Manuring a Crop on the Vegetative and Repro- ductive Capacity of the Seed." Pusa Memoirs, Chem. Series, Vol. IX. No. 4. 6. Fowler, " Methods and Aims of the Biochemist in India," Business World, Madras, March, 1922. 7. Fowler, " C.onservation of Nitrogen," Journ. 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https://openalex.org/W2102691551	https://digital.library.adelaide.edu.au/dspace/bitstream/2440/106095/3/hdl_106095.pdf	English	null	Apolipoprotein E gene polymorphism: effects on plasma lipids and risk of type 2 diabetes and coronary artery disease	Cardiovascular diabetology	2,012	cc-by	11,345	PUBLISHED VERSION Rajesh Chaudhary, Atip Likidlilid, Thavatchai Peerapatdit, Damras Tresukosol, Sorachai Srisuma, Suphachai Ratanamaneechat and Charn Sriratanasathavorn Apolipoprotein E gene polymorphism: effects on plasma lipids and risk of type 2 diabetes and coronary artery disease Cardiovascular Diabetology, 2012; 11(1):36-1-36-11 © 2012 Chaudhary et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Originally published at http://dx.doi.org/10.1186/1475-2840-11-36 g y p p g PERMISSIONS https://creativecommons.org/licenses/by/2.0/ 20 June 2017 PERMISSIONS 20 June 2017 20 June 2017 * Correspondence: atip.lik@mahidol.ac.th 1Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Full list of author information is available at the end of the article ORIGINAL INVESTIGATION Open Access Abstract Background: The most common apolipoprotein E (apoE) gene polymorphism has been found to influence plasma lipid concentration and its correlation with coronary artery disease (CAD) has been extensively investigated in the last decade. It is, however, unclear whether apoE gene polymorphism is also associated with increased risk of type 2 diabetes mellitus (T2DM). The knowledge of this study may provide the primary prevention for T2DM and CAD development before its initiation and progression. Therefore, this study was carried out to determine the association between apoE gene polymorphism and T2DM with and without CAD and its role in lipid metabolism. Methods: The case-control study was carried out on a total of 451 samples including 149 normal control subjects, 155 subjects with T2DM, and 147 subjects with T2DM complicated with CAD. The apoE gene polymorphism was tested by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Univariable and multivariable logistic regression analyses were used to identify the possible risks of T2DM and CAD. Results: A significantly increased frequency of E3/E4 genotype was observed only in T2DM with CAD group (p = 0.0004), whereas the ε4 allele was significantly higher in both T2DM (p = 0.047) and T2DM with CAD (p = 0.009) as compared with controls. E3/E4 genotype was also the independent risk in developing CAD after adjusting with established risk factors with adjusted odds ratio (OR) 2.52 (95%CI 1.28-4.97, p = 0.008). The independent predictor of individuals carrying ε4 allele still remained significantly associated with both CAD (adjusted OR 2.32, 95%CI 1.17- 4.61, p = 0.016) and T2DM (adjusted OR 2.04, 95%CI 1.07-3.86, p = 0.029). After simultaneously examining the joint association of E3/E4 genotype combined with either obesity or smoking the risk increased to approximately 5-fold in T2DM (adjusted OR 4.93, 95%CI 1.74-13.98, p = 0.003) and 10-fold in CAD (adjusted OR 10.48, 95%CI 3.56-30.79, p < 0.0001). The association between apoE genotypes on plasma lipid levels was compared between E3/E3 as a reference and E4-bearing genotypes. E4-bearing genotypes showed lower HDL-C and higher VLDL-C and TG, whereas other values of plasma lipid concentrations showed no significant difference. Conclusions: These results indicate that ε4 allele has influence on lipid profiles and is associated with the development of both T2DM with and without CAD, and furthermore, it increased the risk among the subjects with obesity and/or smoking, the conditions associated with high oxidative stress. http://hdl.handle.net/2440/106095 http://hdl.handle.net/2440/106095 CARDIO VASCULAR DIABETOLOGY Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 CARDIO VASCULAR DIABETOLOGY © 2012 Chaudhary et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Apolipoprotein E gene polymorphism: effects on plasma lipids and risk of type 2 diabetes and coronary artery disease Rajesh Chaudhary1, Atip Likidlilid1, Thavatchai Peerapatdit2, Damras Tresukosol2, Sorachai Srisuma3, Suphachai Ratanamaneechat4 and Charn Sriratanasathavorn5 Correspondence: atip.lik@mahidol.ac.th 1Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Full list of author information is available at the end of the article © 2012 Chaudhary et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction The prevalence of T2DM and CAD is increasing in Thai population according to the cross-sectional ECG survey of 1991 in Thai population, which found that the age-standardized prevalence rate of CAD was 9.9/1000 subjects (men 9.2/1000, women 10.7/1000) [19]. World Health Organization (WHO) global prevalence of dia- betes report estimated that by the year 2030, 366 million people, particularly in developing countries, will be affected by diabetes [20]. Genetic factors like apoE are Type 2 diabetes mellitus (T2DM) is one of the most com- mon diseases with a high incidence and prevalence throughout the world. It affects nearly 4% of the world’s population and this percentage will supposedly be increas- ing up to 5.4% by year 2025 [1]. Prevalence of diabetes in Thai adults as shown by the previous study on Thai popu- lation was 9.6% (2.4 million population) and the impaired fasting glucose was 5.4% (1.4 million people). Mean fasting plasma glucose level by age, sex, and area of residence was found to be substantially higher in urban population group than rural [2]. T2DM is also known as a major independent risk factor for coronary artery disease (CAD) and is the major cause of morbidity and mortality affecting people with diabetes. To date, several mechanisms such as dyslipoproteinemia, obesity, oxidative stress, smoking, exercise, alcohol intake, and genetic factors have been identified as risk factors of both T2DM and CAD. Lack of apolipoprotein E (apoE) gene has been clearly demon- strated as a leading cause of severe hyperlipidemia and spontaneous development of atherosclerosis in mammals [3,4]. However, few studies have been able to demonstrate an association between T2DM and various single nucleo- tide polymorphisms (SNPs) [5]. Recently, Zeljko et al. 2011 [6] indicated that apoE gene polymorphism is also associated with obesity in normal Croatian Roma popula- tion. Adipocytes in an obese person which are the central and causal components in T2DM can generate high amount of biologically active molecules called adipokines or adipocytokines such as plasminogen activator inhibitor- 1 (PAI-1), resistin, leptin, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-a) [7]. These inflammatory cytokines inhibit insulin-stimulated glucose metabolism in skeletal muscles and stimulate gluconeogenesis in hepato- cytes causing hyperglycemia [8]. Hyperglycemia-induced oxidative stress results in reducing glucose uptake from blood by muscle cells and develops into insulin resistance by decreasing insulin secretion from pancreatic b-cells [9]. Abstract In addition, apo ε2 homozygote in com- bination with certain additional disorders may develop type III familial hyperlipidemia and premature athero- sclerosis [12]. Consequently, with their ability to affect lipid levels, the apoE gene polymorphism could be one of the factors influencing development of both T2DM and CAD. The prevalence of T2DM and CAD is increasing in Thai population according to the cross-sectional ECG survey of 1991 in Thai population, which found that the age-standardized prevalence rate of CAD was 9.9/1000 subjects (men 9.2/1000, women 10.7/1000) [19]. World Health Organization (WHO) global prevalence of dia- b d h b h ll Abstract Keywords: Apolipoprotein E, Polymorphism, Type 2 diabetes mellitus, Hyperglycemia, Coronary artery disease, Restriction fragment length polymorphism Page 2 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 lipoproteins in our body and also responsible for the development of CAD [10,11]. ApoE acts as a high affinity ligand for several hepatic lipoprotein receptors such as low-density lipoprotein receptor (LDLR) and LDL-related protein (LRP) and is involved in the process of cellular incorporation of several lipoproteins for transport and digestion [12]. ApoE is a plasma glycoprotein of 34 kDa with 299-amino acids associated with several other plasma glycoproteins, such as high density lipoprotein (HDL), very low density lipoprotein (VLDL), and chylo- microns [13]. In humans, apoE gene located on the chro- mosome at position 19q13.2 has been known to be polymorphic. SNPs at positions 112 (rs 429358) and 158 (rs 7412) determine three major alleles: ε2 (T to C substi- tution at position 158), the most common ε3, and ε4 (C to T substitution at position 112); 3 isoforms: ApoE2 (Cys112, 158Cys), ApoE3 (Cys112, 158Arg), and ApoE4 (Arg112, 158Arg); and 6 genotypes having 3 homozygous: E2/E2, E3/E3, E4/E4, and 3 heterozygous: E2/E3, E2/E4, E3/E4 [13]. Previous studies have shown that apoE alleles have influence on the lipid clearance and metabolism in humans. ApoE ε2 allele has been reported to be asso- ciated with higher plasma levels of apoE, decreased plasma levels of LDL cholesterol (LDL-C) and lower risk of CAD [14] while apoE ε4 is associated with lower plasma level of apoE, increased plasma levels of total cho- lesterol (TC), LDL-C, VLDL cholesterol (VLDL-C), and greater risk of CAD when compared to apoE3 homozy- gotes [15]. One reason for this impaired clearance by apoE ε4 leading to pathogenesis of CAD might be that apoE ε4 binds strongly to LDLR compared to other geno- types. The resulting high amount of lipid can suppress the synthesis of LDLR leading to lower clearance of lipo- protein from our body through LDLR [15]. Other studies have also supported that apoE ε4 allele is associated with the risk of CAD [16]. However, according to a recent meta-analysis, the cardiovascular role of apoE2 is uncer- tain [17] because of its tendency to increase triglyceride (TG) level [18]. Introduction lipoproteins in our body and also responsible for the development of CAD [10,11]. ApoE acts as a high affinity ligand for several hepatic lipoprotein receptors such as low-density lipoprotein receptor (LDLR) and LDL-related protein (LRP) and is involved in the process of cellular incorporation of several lipoproteins for transport and digestion [12]. ApoE is a plasma glycoprotein of 34 kDa with 299-amino acids associated with several other plasma glycoproteins, such as high density lipoprotein (HDL), very low density lipoprotein (VLDL), and chylo- microns [13]. In humans, apoE gene located on the chro- mosome at position 19q13.2 has been known to be polymorphic. SNPs at positions 112 (rs 429358) and 158 (rs 7412) determine three major alleles: ε2 (T to C substi- tution at position 158), the most common ε3, and ε4 (C to T substitution at position 112); 3 isoforms: ApoE2 (Cys112, 158Cys), ApoE3 (Cys112, 158Arg), and ApoE4 (Arg112, 158Arg); and 6 genotypes having 3 homozygous: E2/E2, E3/E3, E4/E4, and 3 heterozygous: E2/E3, E2/E4, E3/E4 [13]. Previous studies have shown that apoE alleles have influence on the lipid clearance and metabolism in humans. ApoE ε2 allele has been reported to be asso- ciated with higher plasma levels of apoE, decreased plasma levels of LDL cholesterol (LDL-C) and lower risk of CAD [14] while apoE ε4 is associated with lower plasma level of apoE, increased plasma levels of total cho- lesterol (TC), LDL-C, VLDL cholesterol (VLDL-C), and greater risk of CAD when compared to apoE3 homozy- gotes [15]. One reason for this impaired clearance by apoE ε4 leading to pathogenesis of CAD might be that apoE ε4 binds strongly to LDLR compared to other geno- types. The resulting high amount of lipid can suppress the synthesis of LDLR leading to lower clearance of lipo- protein from our body through LDLR [15]. Other studies have also supported that apoE ε4 allele is associated with the risk of CAD [16]. However, according to a recent meta-analysis, the cardiovascular role of apoE2 is uncer- tain [17] because of its tendency to increase triglyceride (TG) level [18]. In addition, apo ε2 homozygote in com- bination with certain additional disorders may develop type III familial hyperlipidemia and premature athero- sclerosis [12]. Consequently, with their ability to affect lipid levels, the apoE gene polymorphism could be one of the factors influencing development of both T2DM and CAD. Materials and methods Subjects T2DM complicated with CAD (T2DM + CAD) subjects included 147 subjects. Subjects were confirmed CAD by coronary angiography, with at least 50% stenosis in a major coronary artery or one of their branches. Subjects were diagnosed to have diabetes with FPG≥126 mg/dL or those with drug-treated T2DM. Exclusion criteria included those having renal disease, hepatic disease, type 1 diabetes mellitus, any form of endocrine disease or metabolic disease. The studied subjects were recruited from Siriraj Hospital in Bangkok province. Full consent forms were signed by the subjects after the nature and motif of the study was clearly explained to them. The study protocol was approved by the Ethics Committee of Clinical Study in Humans, Faculty of Medicine Siriraj Hospital, Mahidol University. Questionnaires were used to collect the infor- mation of family and medical history, alcohol consump- tion, smoking habits and physical activity. Other clinical and biochemical data such as dyslipidemia, systolic blood pressure (SBP) and diastolic blood pressure (DBP) from all subjects were obtained from clinical and laboratory exami- nations. Anthropometric data (weight, height) were col- lected and used for BMI calculation. Obesity was defined as BMI ≥25 kg/m2 according to WHO suggested criteria for Asian populations [21]. Dyslipidemic or hyperlipidemic were defined as when one has level of TC >200 mg/dL, TG >150 mg/dL, LDL-C >130 mg/dL, HDL-C <40 mg/dL, TC/HDL-C ratio >4.0 or under medication of lipid lower- ing drugs. Cigarette smokers were allowed into the study if they had once smoked even if they were no longer smo- kers. Alcohol drinkers were defined as those who drank at least two times a week for more than a year. Physical activ- ities were defined as exercise for at least 2 to 3 days/week for at least 30 minutes. Hypertension was defined as blood pressure above 140/90 mmHg or taking antihypertensive drugs. These subjects were categorized into three groups: normal healthy controls, T2DM with and T2DM without CAD, according to the criteria of American Diabetes Association Classification 2010 [22], with an age range from 40-65 years. APOE Genotyping Blood samples were collected in EDTA containing tubes. Guanidine-HCl precipitation method was performed for genomic DNA extraction. Genomic DNA was subjected to polymerase chain reaction (PCR) with primers specific to apoE gene, sense: 5’ AACAACTGACCCCGGTGGCG 3’, antisense: 5’ ATGGCGCTGAGGCCGCGCTC 3’, sense: 5’ CCCACCTGCGCAAGCTGCGC 3’, using thermal cycler with thermal profile according to Richard et al [23]. In brief, PCR reaction mixture included 20 pmol of each pri- mer, 0.3 μg genomic DNA, 10 mM of each dNTP, 10X PCR buffer, 10% DMSO in a final volume of 50 μL. 10 μL of PCR products was digested with 0.3 unit of Hha1 enzyme according to the supplier’s recommended proce- dure (Biolabs New England). The resulted fragments were then separated on 8% polyacrylamide gel and stained with ethidium bromide. Bands were compared with 25 bp DNA marker and the different individual genotypes were sepa- rated and categorized based on the following band length criteria: E2/E2: 91, 83, 61; E3/E3: 91, 61, 48, 35; E4/E4: 72, 61, 48, 35; E2/E3: 91, 83, 61, 48, 35; E2/E4: 91, 83, 72, 61, 48, 35 and E3/E4: 91, 72, 61, 48, 35. Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Page 3 of 11 T2DM subjects without CAD included 155 subjects. All subjects fulfilled the diabetes mellitus diagnostic cri- teria of FPG≥126 mg/dL or were under treatment with oral antidiabetic drugs. The subjects had no electrocar- diogram (ECG) and/or angiography abnormalities, no documented history of CAD and no sign of myocardial ischemia during exercise. Other exclusion criteria for this group were possession of type 1 diabetes mellitus, renal disease, hepatic disease, endocrine disease, and other metabolic diseases. also considered to be genetic determinants of plasma lipoprotein levels and play a central role in the develop- ment of CAD. However, it is unclear whether apoE gene is associated with T2DM. The primary aim of this study was to demonstrate the influence of the apoE gene poly- morphism on plasma lipids is notable and is an impor- tant determinant of T2DM and CAD. Secondarily, this genetic study might also add more information about a Thai population beyond traditional risk factors. Introduction Taken these together, increased oxidative stress in hyper- glycemia and reduced lipid clearance because of apoE gene polymorphism are effective in developing insulin resistance and T2DM. In addition, high oxidative stress can also cause vascular inflammation leading to athero- sclerosis through several cytokines such as NF-B, TNF-a, IL-1b, and other proinflammatory cytokines [10]. All of these factors are one of the underlying causes of metabolic syndrome [7]. Grundy reported that subjects suffering from the metabolic syndrome are at 2-fold higher risk of developing CAD and at 5-fold higher risk of developing T2DM; however, persons suffering from T2DM are at 3-fold higher risk of developing CAD [11]. ApoE gene is one of the most studied genes which is responsible for stabilizing and solubilizing circulating ApoE gene is one of the most studied genes which is responsible for stabilizing and solubilizing circulating Lipid analysis and biochemical determination The normal healthy control group consisted of 149 subjects with fasting plasma glucose (FPG) < 100 mg/dL from among those who were randomly selected after rou- tine health check-up to screen out those having hyperli- pidemia, hypertension, history of chest pain, family history of CAD or any forms of cardiovascular disease, diabetes mellitus, hepatic and renal diseases, inflamma- tion, general illness, traumatic injury, endocrine disease, and other metabolic disorders. Subjects under medication or drug abusers were also excluded. p y Venous blood samples were collected from the patients after 12 hours of overnight fast. Plasma (TC), TG, HDL- C, glucose and glycosylated hemoglobin (HbA1c) were quantified using an automated clinical chemistry analy- zer and enzyme-based colorimetric lists supplied by Roche Diagnostics, Germany. LDL-C levels were calcu- lated using Friedewald formula [24]. VLDL-C level was obtained by using the following equation, VLDL-C = (TC - LDL-C - HDL-C) and non-HDL-C level was Page 4 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 levels of physical activity (p = 0.030) as compared to controls and T2DM patients. Lipid profile data demon- strated significantly higher levels of TC, LDL-C, VLDL- C, TG, non-HDL-C and lower level of HDL-C in T2DM and CAD patients when compared to controls. Patients with CAD had diabetes for a longer period (p < 0.0001) and showed no significant difference in HbA1c (p = 0.151) as compared to the patients without CAD. Blood glucose levels were also significantly higher in both groups as compared to the controls (p < 0.0001). calculated by subtracting HDL-C value from total cho- lesterol value as a candidate biometrical equivalent to apoB 100 in diabetes [25]. levels of physical activity (p = 0.030) as compared to controls and T2DM patients. Lipid profile data demon- strated significantly higher levels of TC, LDL-C, VLDL- C, TG, non-HDL-C and lower level of HDL-C in T2DM and CAD patients when compared to controls. Patients with CAD had diabetes for a longer period (p < 0.0001) and showed no significant difference in HbA1c (p = 0.151) as compared to the patients without CAD. Blood glucose levels were also significantly higher in both groups as compared to the controls (p < 0.0001). Association of apoE gene polymorphism and diseases p g p y p Low lipid clearance property of ε4 allele may possibly make it an independent risk factor for the development of CAD and T2DM. Thus, E3/E4 genotype may be the risk factor for development of CAD and/or diabetes. The univariate analysis was used to determine these associa- tions according to the genotype and allele frequencies of apoE gene polymorphism. Statistical analysis All statistical analyses were performed with IBM SPSS v1 (IBM corporation, Armonk, New York, US) and Micro- soft 2010 based Excel (Microsoft, US). All data were expressed as mean (SEM). One-way analysis of variance (ANOVA) followed by posthoc Bonferroni multiple com- parison test was applied to evaluate the mean difference of the data between three groups (control, T2DM with- out CAD, and T2DM with CAD). Independent samples t-tests were applied to evaluate the mean difference in HbA1c, diabetes duration between T2DM and T2DM with CAD and lipid profiles between groups. Categorical data such as sex, hypertension, history of smoking, his- tory of alcohol drinking, physical activity and dyslipide- mia were evaluated by Chi-square tests or Fisher’s exact tests. Allele and genotype difference between groups and deviations from Hardy-Weinberg equilibrium were tested by Chi-square tests. The association between diseases and polymorphism was provided by crude or univariable logistic regression analysis with unadjusted odds ratio (OR) and 95% confidence interval (95% CI). The adjusted OR with 95% CI was used to determine the independent risk factor for development of diabetes and CAD by mul- tivariable logistic regression analysis after adjusting for age, sex, BMI, smoking habits, and physical activity. The multivariable logistic regression analysis was also repeated for adjusted OR with 95% CI to determine the risk of apoE polymorphism combined with obesity and smoking (joint association) after adjusting for age, sex, and physical activity. Statistical significance was consid- ered as p < 0.05. ApoE genotype and allele frequencies The genotype distribution of both controls and CAD patients were in Hardy-Weinberg equilibrium except for T2DM that deviated from the basic norm (p = 0.0001). This was due to the lower presence of E2/E3 genotype in diabetic group (Table 2). However, since it was unlikely that any of the assumptions for Hardy-Weinberg equili- brium were violated, such a departure was attributed to chance. E3/E3 is also the most common genotype in Thai general population. E3/E3 genotype and ε3 allele were significantly lower (p = 0.003 and 0.010 respectively) while E3/E4 genotype was significantly higher only in T2DM with CAD subjects (p = 0.0004). In T2DM sub- jects, E3/E4 genotype had a tendency to be higher (19.35% vs. 14.09%) but showed no significant difference (p = 0.219). In addition, E2/E3 genotype and ε2 allele were also significantly lower in T2DM patients (p = 0.005 and 0.010, respectively). However, ε4 allele frequency manifested itself as significantly higher in both T2DM and CAD patients as compared to the controls (p = 0.047 and 0.009, respectively) (Table 3). The baseline characteristics of the study population The baseline characteristics of the study population All enrolled subjects were Thais recruited from Bangkok area. The anthropometric and demographic data were summarized along with the clinical and biochemical data as shown in Table 1. The data from normal con- trols were used to compare with the data from T2DM with and without CAD. A significant age difference was found between control and T2DM with CAD groups (p < 0.0001) which particularly signifies that increase in age could possibly lead to higher chances of developing CAD. There was also significant sex difference between controls and CAD (p < 0.0001) due to the high preva- lence of males among CAD patients. Other clinical data such as BMI, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly higher in both groups when compared to controls. CAD patients had higher frequencies of smoking (p = 0.033) and lower p g p y p Interestingly, E3/E4 genotype increased the risk of CAD with unadjusted OR 2.78 (95%CI 1.50-5.16, p = 0.0004) and showed no association with T2DM with unadjusted OR 1.46 (95%CI 0.76-2.81, p = 0.219). The ε4 allele appeared to increase risk of both T2DM and CAD with unadjusted OR 1.72 (95%CI 0.97-3.06, p = 0.047) and 2.37 (95%CI 1.36-4.15, p = 0.0009, respectively) (Table 4). After being adjusted for age, sex, BMI, smoking habits, and physical activity using multivariable binary logistic regression analysis as shown in Table 5 the E3/E4 genotype appeared to be the independent risk factor for development of CAD with adjusted OR 2.52 (95%CI 1.28-4.97, p = 0.008). However, the E3/E4 genotype was Chaudhary et al. The baseline characteristics of the study population not found to be an independent risk factor for diabetes, but the ε4 allele was the independent risk factor of both T2DM and CAD with adjusted OR 2.04 (95%CI 1.07- 3.86, p = 0.029) and OR 2.32 (95%CI 1.17-4.61, p = 0.016), respectively. The multivariable analysis of apoE4 polymorphism and the risk of T2DM and CAD were also determined according to anthropometric and demo- graphic characteristics. Sex (particularly male gender), age and BMI were also independent risks for CAD but only BMI was the independent risk for T2DM (Table 5). not found to be an independent risk factor for diabetes, but the ε4 allele was the independent risk factor of both T2DM and CAD with adjusted OR 2.04 (95%CI 1.07- 3.86, p = 0.029) and OR 2.32 (95%CI 1.17-4.61, p = 0.016), respectively. The multivariable analysis of apoE4 polymorphism and the risk of T2DM and CAD were also determined according to anthropometric and demo- graphic characteristics. Sex (particularly male gender), age and BMI were also independent risks for CAD but only BMI was the independent risk for T2DM (Table 5). From joint association analysis, E3/E4 genotype was found to further increase the risk for development of diabetes and CAD when combined with either obesity or smoking after being adjusted for age, sex, and physical activity. The risk factor for T2DM increased from adjusted OR 1.42 (95%CI 0.72-2.78, p = 0.310) (Table 5) to adjusted OR 4.93 (95%CI 1.74-13.98, p = 0.003) (Table 6), whereas CAD risk increased from adjusted OR 2.52 (95%CI 1.28-4.97, p = 0.008) (Table 5) to adjusted OR 10.48 (95%CI 3.56-30.79, P < 0.0001) (Table 6). However, the number of E3/E4 From joint association analysis, E3/E4 genotype was found to further increase the risk for development of diabetes and CAD when combined with either obesity or smoking after being adjusted for age, sex, and physical activity. The risk factor for T2DM increased from adjusted OR 1.42 (95%CI 0.72-2.78, p = 0.310) (Table 5) to adjusted OR 4.93 (95%CI 1.74-13.98, p = 0.003) (Table 6), whereas CAD risk increased from adjusted OR 2.52 (95%CI 1.28-4.97, p = 0.008) (Table 5) to adjusted OR 10.48 (95%CI 3.56-30.79, P < 0.0001) (Table 6). The baseline characteristics of the study population Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Page 5 of 11 Table 1 Anthropometric, demographic and clinical data of T2DM with and without CAD compared to normal healthy controls Variables Controls (n = 149) T2DM (n = 155) T2DM + CAD (n = 147) Age (Years) 52.01 (0.62) 51.95 (0.53) 57.56 (0.47) Sex (male/female) 49/100 57/98 95/52 BMI (kg/m2) 23.84 (0.27) 26.89 (0.31)* 27.49 (0.36)** SBP (mmHg) 111.54 (1.12) 147.31 (1.76)* 155.46 (1.96)** DBP (mmHg) 73.11 (0.86) 84.50 (1.01)* 92.26 (1.20)** Hypertension (%) - 73.54 93.19 Smokers (%) 3.35 4.51 9.52† Alcohol consumer (%) 23.80 25.50 32.70 Physical activity (%) 68.70 67.09 56.46† Diabetes duration (Years) - 6.09 (0.37) 9.07 (0.62) Glucose (mg/dL) 89.7 (0.64) 225.9 (6.41)* 192.9 (6.55)** HbA1c (%) - 8.94 (0.16) 8.60 (0.17) Triglyceride (mg/dL) 98.23 (3.68) 221.1 (10.35)* 204.3 (8.70)** TC (mg/dL) 191.52 (1.80) 237.76 (5.47)* 202.48(4.19)† LDL-C (mg/dL) 111.31(1.76) 146.65(4.84)* 122.36(3.93)† HDL-C (mg/dL) 60.17 (1.23) 50.42 (1.27)* 44.59 (0.95)** VLDL-C (mg/dL) 19.65 (0.73) 45.86 (2.67)* 40.86 (1.74)** Non-HDL-C (mg/dL) 131.35 (1.95) 182.26 (4.46)* 157.88 (4.09)** TC/HDL-C 3.4 (0.1) 4.9 (0.2)* 4.8 (0.1)** Dyslipidemia (%) - 85.2 100 Data are presented as mean values (SEM), or numbers (n) and percentage of subjects. p-value < 0.0001 in comparison between controls and T2DM, p-value < 0.0001 in comparison between controls and T2DM + CAD, †p-value < 0.05 in comparison between controls and T2DM + CAD. BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; TG: triglyceride; HDL-C: high density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol. Table 1 Anthropometric, demographic and clinical data of T2DM with and without CAD compare t l demographic and clinical data of T2DM with and without CAD compared to normal healthy Data are presented as mean values (SEM), or numbers (n) and percentage of subjects. p-value < 0.0001 in comparison between controls and T2DM, p-value < 0.0001 in comparison between controls and T2DM + CAD, †p-value < 0.05 in comparison between controls and T2DM + CAD. BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; TG: triglyceride; HDL-C: high density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol. Relationship between Lipid profiles and apoE4-bearing genotypes It has been reported that dyslipidemia or dyslipoproteine- mia might strongly contribute towards aggravating the problems of micro- and macroangiopathic complications in diabetic patients [26,27]. This implication was mainly characterized by higher prevalence of male gender, older age, smoking habits, and less physical activity. Other dif- ferences were duration of diabetes, lower levels of HDL-C and hypertension when compared to those without CAD which were similar to this study. However, some studies have also shown that elevation of TG and non-esterified fatty acid (NEFA) levels accelerated the pathogenesis of T2DM [28-30]. This suggests that dyslipidemia is asso- ciated with both T2DM and CAD. Genetic factors which have strong impact on the metabolism of plasma lipid have been studied regarding the potential effect of T2DM and cardiovascular outcomes in various diabetic and non- diabetic subjects. These studies have underscored that The significant differences in apo E distribution among these three groups are mainly due to the differences in frequencies of the ε4 allele. These distributions in apo ε4 allele frequencies may lead to the differences in plasma lipid levels. To test this hypothesis, we analyzed the correlation between apoE4 and plasma lipid levels (Table 7). There were no significant differences between E4 carriers (E2/E4, E3/E4, E4/E4) and E3/E3 genotype as a reference for all values of plasma lipid levels in both control and T2DM with CAD. In T2DM group, there were significant elevation in the values of VLDL-C and TG but there were no significant differences in TC, LDL-C, HDL-C and non-HDL-C levels in E4 carriers. The baseline characteristics of the study population However, the number of E3/E4 Table 2 Frequency distribution of apoE genotypes and alleles in Hardy-Weinberg Equilibrium Genotype Controls (n = 149) T2DM (n = 155) T2DM + CAD (n = 147) E2/E2 2 (1.34%) 1 (0.64%) 1 (0.68%) E3/E3 113 (75.83%) 117 (75.48%) 88 (59.86%) E4/E4 1 (0.67%) 4 (2.58%) 1 (0.68%) E2/E3 12 (8.05%) 2 (1.29%) 11 (7.48%) E2/E4 0 (0%) 1 (0.64%) 0 (0%) E3/E4 21 (14.09%) 30 (19.35%) 46 (31.29%) p-value 0.198 0.0001 0.157 Allele ε2(95% CI) 0.05(0.03 - 0.08) 0.02(0.01 - 0.04) 0.04(0.02 - 0.07) Allele ε3(95% CI) 0.87(0.82 - 0.90) 0.86(0.81 - 0.89) 0.79 (0.74 - 0.83) Allele ε4(95% CI) 0.07(0.05 - 0.11) 0.13 (0.09 - 0.16) 0.16 (0.12 - 0.21) Table 2 Frequency distribution of apoE genotypes and alleles in Hardy-Weinberg Equilibrium tribution of apoE genotypes and alleles in Hardy-Weinberg Equilibrium Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Page 6 of 11 Table 3 Genotype and Allele frequencies distribution of apoE gene polymorphism in controls, T2DM with and without CAD compared to healthy controls Genotype Groups Controls (n = 149) T2DM (n = 155) T2DM + CAD (n = 147) E2/E2 0.013 0.006 0.006 E3/E3 0.758 0.754 0.598 E4/E4 0.007 0.025 0.007 E2/E3 0.080 0.012* 0.074 E2/E4 0 0.006 0 E3/E4 0.140 0.193 0.321*** Allele ε2 0.05 0.02* 0.04 Allele ε3 0.87 0.86 0.79** Allele ε4 0.07 0.13* 0.16†† p-value < 0.01 compared between controls and T2DM, p-value < 0.01, *p-value < 0.001 compared between controls and T2DM + CAD. Table 3 Genotype and Allele frequencies distribution of apoE gene polymorphism in controls, T2 CAD compared to healthy controls Table 3 Genotype and Allele frequencies distribution of apoE gene polymorphism in controls, T2DM with and without CAD compared to healthy controls Genotype Groups Allele frequencies distribution of apoE gene polymorphism in controls, T2DM with and without lthy controls TG levels while HDL-C concentration was significantly decreased as compared to E3/E3. genotypes combined with both obesity and smoking in T2DM with and without CAD was small; thus the statisti- cal power of this joint association comparison is limited. Relationship between Lipid profiles and apoE4-bearing genotypes After pooling the overall subjects, the results showed that E4 carrier has significantly elevated VLDL-C and Table 4 Associations of apoE gene polymorphisms with the risk of T2DM and CAD compared to healthy controls represented as unadjusted OR Genotype Controls (n = 149) T2DM (n = 155) Unadjusted OR (95% CI) p-value T2DM + CAD (n = 147) Unadjusted OR (95% CI) p-value E2/E2 2 1 0.48 (0.02 - 6.78) 0.616 1 0.50 (0.02 - 7.16) 1.000 E3/E3 113 117 0.98 (0.56 - 1.71) 0.942 88 0.48 (0.28 - 0.81) 0.003 E4/E4 1 4 3.92 (0.41 - 93.19) 0.371 1 1.01(0.06 - 16.36) 1.000 E2/E3 12 2 0.15 (0.02 - 0.72) 0.005 11 0.92(0.36 - 2.33) 0.854 E2/E4 0 1 - 1.000 0 - - E3/E4 21 30 1.46 (0.76 - 2.81) 0.219 46 2.78(1.50 - 5.16) 0.0004 Allele ε2 16 5 0.29 (0.09 - 0.85) 0.011 13 0.81(0.36 - 1.81) 0.580 Allele ε3 259 266 0.91 (0.56 - 1.48) 0.691 234 0.57(0.36 - 0.90) 0.107 Allele ε4 23 39 1.72 (0.97 - 3.06) 0.047 49 2.37(1.36 - 4.15) 0.0009 Table 4 Associations of apoE gene polymorphisms with the risk of T2DM and CAD compared to healthy controls represented as unadjusted OR G C l T2DM U dj d OR l T2DM CAD U dj d OR l ns of apoE gene polymorphisms with the risk of T2DM and CAD compared to healthy controls adjusted OR ble 4 Associations of apoE gene polymorphisms with the risk of T2DM and CAD compared to hea presented as unadjusted OR Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Relationship between Lipid profiles and apoE4-bearing genotypes E3/E3 genotype was the most frequent isoform found in all groups compared to the other genotypes which corresponds to previous reports [13,41]. Additionally, the higher frequency of E3/E4 geno- type was observed only in CAD (p = 0.0004), whereas the higher frequency of ε4 allele was observed in both T2DM and CAD (p = 0.047, p = 0.0009, respectively). From this study we can conclude that among these selected apoE alleles (ε2, ε3, and ε4), ε4 allele is one of the predictors of both diseases in Thai subjects. In addition, the PDAY (Pathobiological Determinants of Atherosclerosis in Youth) study reported that individuals with E2/E3 geno- type had fewer atherosclerotic lesions, whereas those with the E3/E4 had more lesions in the abdominal aorta [42]. These observations strongly suggest that the ε2 allele has a protective role against atherosclerosis. However, in this study, we found that E2/E3 genotype and ε2 allele were also significantly lower in T2DM group indicating that the protective effect of ε2 allele on the development of hyperli- pidemia might be less in T2DM patients. It is probable that other environmental and genetic factors are involved in the pathogenesis of CAD. apoE gene encoding apolipoprotein E is associated with significant variation in lipid profile in our body [31]. ApoE gene is one of the most widely studied candidate genes for CAD or any other form of cardiovascular disease and/or diabetes. A significant relationship of apo E polymorphism and CAD has been observed in several ethnic groups, including Caucasian in the USA [32], Austrian [33], Fin- nish [34], Italian [35], Turkish [36], Indian [37] and Chi- nese [38] populations. Some studies have shown apoE ε4 allele as an independent risk factor after further adjust- ment of other established risk factors for development of CAD in T2DM [16] and myocardial infraction [37] patients. However, no independent association was observed after adjusting for age, sex, smoking, BMI, HDL- C and TG in African- Americans and Caucasians [39]. Therefore, it is of great interest to study the independent risk factors of this gene polymorphism in a Thai popula- tion. The present study also coincides with a previous study regarding the development of CAD in T2DM sub- jects indicating that the frequency of ε4 allele is signifi- cantly higher in CAD compared to controls and can be one of the factors for the progression of CAD disease [40]. †† After adjusting for age, sex and physical activity. Normal reference ranges of clinical and biochemical data are described in materials and methods. Relationship between Lipid profiles and apoE4-bearing genotypes The present study also coincides with a previo study regarding the development of CAD in T2DM su jects indicating that the frequency of ε4 allele is signi cantly higher in CAD compared to controls and can one of the factors for the progression of CAD disease [4 The frequencies of apoE allele and genotype vary betwe different populations [13]. In this study, ε3 allele was t most frequent allele in control, diabetes, and CAD su jects, while ε2 was less frequent. E3/E3 genotype was t most frequent isoform found in all groups compared the other genotypes which corresponds to previous repor Table 6 Joint association study to evaluate the risk of T Genotype Obesity/Smoking Controls T2DM Adjusted OR E3/E3 Neither 70 34 1 E3/E3 Either 40 73 3.65 (2.07 E3/E3 Both 2 10 10.05 (2.06 E3/E4 Neither 14 14 1.97 (0.84 E3/E4 Either 6 15 4.93 (1.74 E3/E4 Both 6 1 - †† After adjusting for age, sex and physical activity. Normal reference range apoE gene encoding apolipoprotein E is associated with significant variation in lipid profile in our body [31]. ApoE gene is one of the most widely studied candidate genes for CAD or any other form of cardiovascular disease and/or diabetes. A significant relationship of apo E polymorphism and CAD has been observed in several ethnic groups, including Caucasian in the USA [32], Austrian [33], Fin- nish [34], Italian [35], Turkish [36], Indian [37] and Chi- nese [38] populations. Some studies have shown apoE ε4 allele as an independent risk factor after further adjust- ment of other established risk factors for development of CAD in T2DM [16] and myocardial infraction [37] patients. However, no independent association was observed after adjusting for age, sex, smoking, BMI, HDL- C and TG in African- Americans and Caucasians [39]. Therefore, it is of great interest to study the independent risk factors of this gene polymorphism in a Thai popula- tion. The present study also coincides with a previous study regarding the development of CAD in T2DM sub- jects indicating that the frequency of ε4 allele is signifi- cantly higher in CAD compared to controls and can be one of the factors for the progression of CAD disease [40]. The frequencies of apoE allele and genotype vary between different populations [13]. In this study, ε3 allele was the most frequent allele in control, diabetes, and CAD sub- jects, while ε2 was less frequent. Relationship between Lipid profiles and apoE4-bearing genotypes Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Page 7 of 11 Page 7 of 11 Table 5 Association of apoE gene polymorphism with the risk of T2DM and CAD compared to healthy controls represented as adjusted OR T2DM T2DM + CAD Adjusted OR (95% CI)†† p-value Adjusted OR (95% CI)†† p-value E3/E4 genotype 1.42 (0.72 - 2.78) 0.310 2.52 (1.28 - 4.97) 0.008†† ε4 allele 2.04 (1.07 - 3.86) 0.029†† 2.32 (1.17 - 4.61) 0.016†† Age 1.00 (0.97 - 1.04) 0.887 1.13 (1.08 - 1.18) <0.0001 Sex 1.29 (0.77 - 2.14) 0.326 3.10 (1.75 - 5.49) <0.0001 BMI 3.68 (2.25 - 6.00) <0.0001 3.66 (2.09 - 6.39) <0.0001 Smoking 2.58 (0.87 - 7.66) 0.086 0.59 (0.13 - 2.50) 0.473 Physical activity 0.68 (0.40 - 1.19) 0.148 0.67 (0.36 - 1.24) 0.206 ††After adjusting for age, sex, BMI, smoking habit and physical activity. Normal reference ranges of clinical and biochemical data are described in materials and methods. Table 5 Association of apoE gene polymorphism with the risk of T2DM and CAD compared to he represented as adjusted OR tion of apoE gene polymorphism with the risk of T2DM and CAD compared to healthy controls adjusted OR apoE gene encoding apolipoprotein E is associated wi significant variation in lipid profile in our body [31]. Apo gene is one of the most widely studied candidate genes f CAD or any other form of cardiovascular disease and/ diabetes. A significant relationship of apo E polymorphis and CAD has been observed in several ethnic group including Caucasian in the USA [32], Austrian [33], Fi nish [34], Italian [35], Turkish [36], Indian [37] and Ch nese [38] populations. Some studies have shown apoE allele as an independent risk factor after further adjus ment of other established risk factors for development CAD in T2DM [16] and myocardial infraction [3 patients. However, no independent association w observed after adjusting for age, sex, smoking, BMI, HD C and TG in African- Americans and Caucasians [39 Therefore, it is of great interest to study the independe risk factors of this gene polymorphism in a Thai popul tion. Relationship between Lipid profiles and apoE4-bearing genotypes The frequencies of apoE allele and genotype vary between different populations [13]. In this study, ε3 allele was the most frequent allele in control, diabetes, and CAD sub- jects, while ε2 was less frequent. E3/E3 genotype was the most frequent isoform found in all groups compared to the other genotypes which corresponds to previous reports This present study is the first report to demonstrate that the ε4 allele containing genotypes is the major pre- dictor of development of both T2DM and CAD. Our study also shows strong association of E3/E4 genotype with development of CAD in T2DM patients (Table 4) as reported in previous studies [16,42]. E4-bearing genotypes = E3/E4, E4/E4, E2/E4. Independent sample t-test test was applied to compare between E3/E3 and E4-bearing genotypes. Data are presented as mean values (SEM). p-value < 0.05 in comparison between E3/E3 and E4-bearing genotypes in T2DM. p p g g yp p-value < 0.01 in comparison between E3/E3 and E4-bearing genotypes in total subjects. g g yp n between E3/E3 and E4-bearing genotypes in total subjects. s applied to compare between E3/E3 and E4-bearing genotypes. Data are presented as mean values (SEM). Relationship between Lipid profiles and apoE4-bearing genotypes Moreover, oxidative stress also increases vascular inflammation leading to CAD and/or any form of cardiovascular disease with or with- out combination of T2DM [46]. higher risk (p = 0.016) for developing CAD and ε4 allele containing genotypes led to a 2.04-fold higher risk (p = 0.029) in the development of T2DM as compared to the controls. This signifies that other risk factors have no influence in development or curbing the disease, suggest- ing that ε4 allele is the independent risk factor for both T2DM and CAD. However, E3/E4 genotype further increases the risk for the development of diabetes and CAD when combined with smoking and/or obesity (Table 6). This confirms the findings that progression and development of diabetes and CAD is the conse- quence of multifactorial parameters, for example, obesity, smoking, oxidative stress, defect in pancreatic b-cells, alcohol consumption, exercise, hypertension and genetic factors. In addition, incidence of T2DM is also subject to gene-environment interactions, for example, dietary fac- tors, intake of vegetable fat, polyunsaturated fatty acid, dietary fiber (particularly cereal fiber), magnesium, and caffeine were significantly inversely correlated and intakes of trans fat, saturated fatty acid and heme-iron, glycemic index, and glycemic load were significantly posi- tively correlated with the incidence of T2DM [43]. Simi- larly, regular exercise has been shown to reduce weight, BMI, HDL-C and insulin resistance. This indicates that exercise is also associated with decrease risk of T2DM and CAD. Furthermore, apoE gene polymorphism has been shown to modulate the effects of exercise on lipo- protein concentrations in plasma [44]. In this study, smoking and obesity are shown to be the two major con- tributing factors that can promote the development of T2DM and CAD or both when placed in joint association (Table 6). The reason for this is that smoking and obesity enhance the oxidative stress which results in decreased insulin secretion from pancreatic b-cell and decreased uptake of blood glucose into the muscle cells [9,10]. This evidence was supported by the experimental study on atherosclerosis-susceptible B6 (B6.apoE -/-) and athero- sclerosis-resistant BALB (BALB.apoE -/-) mice that showed defects in insulin secretion rather than defects in insulin resistance which explains the mark difference in susceptibility to T2DM [45]. Moreover, oxidative stress also increases vascular inflammation leading to CAD and/or any form of cardiovascular disease with or with- out combination of T2DM [46]. Relationship between Lipid profiles and apoE4-bearing genotypes After adjusting for age, sex, smoking, BMI, and physical activity, E3/E4 con- taining subjects showed 2.52-fold higher risk (p = 0.008) while ε4 allele containing genotypes led to a 2.32-fold Table 6 Joint association study to evaluate the risk of T2DM and CAD compared to controls Genotype Obesity/Smoking Controls T2DM Adjusted OR (95% CI)†† p-value T2DM + CAD Adjusted OR (95% CI)†† p-value E3/E3 Neither 70 34 1 - 30 1 - E3/E3 Either 40 73 3.65 (2.07 - 6.43) <0.0001 56 2.24 (1.15 - 4.35) 0.018 E3/E3 Both 2 10 10.05 (2.06 - 48.96) 0.004 1 2.02 (0.16 - 25.01) 0.585 E3/E4 Neither 14 14 1.97 (0.84 - 4.64) 0.118 7 1.02 (0.34 - 3.06) 0.970 E3/E4 Either 6 15 4.93 (1.74 - 13.98) 0.003 36 10.48 (3.56 - 30.79) <0.0001 E3/E4 Both 6 1 - - 3 - - †† Table 6 Joint association study to evaluate the risk of T2DM and CAD compared to controls Table 7 Fasting lipid concentration according to apoE4-bearing genotypes Control T2DM T2DM + CAD Total Variables (mg/dL) E3/E3 E4-bearing genotypes E3/E3 E4-bearing genotypes E3/E3 E4-bearing genotypes E3/E3 E4-bearing genotypes (n = 113) (n = 22) (n = 117) (n = 35) (n = 88) (n = 47) (n = 318) (n = 104) TC 191.58 (2.02) 194.09 (5.22) 232.80 (5.33) 234.77 (8.65) 206.52 (5.26) 192.04 (7.96) 210.88 (2.72) 206.86 (5.11) LDL-C 110.87 (1.97) 118.52 (4.45) 145.14 (4.54) 140.91 (8.43) 125.33 (4.84) 113.80 (7.33) 127.48 (2.39) 123.92 (4.59) HDL-C 60.07 (1.43) 55.77 (2.51) 50.84 (1.13) 48.63 (2.98) 45.49 (1.27) 42.70 (1.37) 52.64 (0.81) 47.46 (1.37) VLDL-C 20.04 (0.87) 20.18 (1.69) 41.38 (1.99) 55.77 (5.85)* 41.25 (2.37) 38.91 (3.04) 33.76 (1.17) 40.62 (2.72)** TG 100.20 (4.33) 100.91 (8.48) 206.92 (9.99) 278.83 (29.63)* 206.26 (11.78) 194.53 (15.21) 168.82 (5.88) 203.10 (13.62)** Non-HDL-C 131.50(2.16) 138.32(4.99) 181.97(5.36) 186.14(8.12) 161.03(5.24) 149.34(7.44) 158.24 (2.83) 159.39 (4.82) Table 7 Fasting lipid concentration according to apoE4-bearing genotypes Page 9 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 allele and lipid profile is still controversial [51]. The ε4 allele has been shown to be associated with high concen- tration of serum TC and LDL-C in Chinese population [38]. Relationship between Lipid profiles and apoE4-bearing genotypes Some studies also showed a significant relationship between E3/E4 genotype with lower HDL-C and higher LDL-C concentrations in CAD patients [52] and with higher TG levels in T2DM patients [48] as compared to the healthy controls. However, a study in the Tunisian population has suggested that HDL-C concentration and other lipid profiles are not associated with apoE gene poly- morphism in the total population studied, but has proven that ε4 allele increased LDL-C in type 2 diabetic men [53]. Association of ε4 allele with higher LDL-C and lower HDL-C levels was only found in type 2 diabetic women in Spanish population [54] indicating that gender affects the effect of apoE gene polymorphism. In this study, there is a mean comparison between E4 carriers and E3/E3 geno- type in the T2DM and total population studied which showed a significantly higher VLDL-C, TG and lower HDL-C levels in E4 carriers (Table 7) similar to the report of Knouff et al 1999 [15]. Nevertheless, the differences of lipid profiles were not observed in controls and T2DM with CAD. This may be due to dietary restrictions in the controls and lipid-lowering aggressive treatment in T2DM with CAD. Kolovou et al. 2006 [55] reported that the ε4 allele can increase LDL-C concentrations in the presence of an atherogenic diet, but a lower fat diet can suppress this effect. On the other hand, aerobically trained indivi- duals have high HDL and display enhanced glucose toler- ance [56]. A lipid-lowering-drug which raises HDL levels and decrease TG levels delays the onset of T2DM and reduces the development of atherosclerosis [57]. In addi- tion, loss of caspase-1 activity which involves the inflam- matory process in human atherosclerotic vessel has shown to reduce atherosclerosis lesion formation in Casp1-/- Apo E-/- mice [58]. Another reason for these variations of lipid profiles might be because of the differences in genetic background and the prevalence of high oxidative stress in the studied population. When considering the basis of gene-gene and gene-environment interactions, as described above, the polymorphism study at the genome level might not provide a real picture for development of diseases in respect to lipid profile. A more robust parallel study at the protein level, or of other candidate genes, may be necessary to evaluate the mechanism of T2DM and CAD development. Relationship between Lipid profiles and apoE4-bearing genotypes To demonstrate that apoE gene polymorphism is involved in the lipid clearance process and it has great influence on the lipid level in our body [47,48]. Although previous study has shown that non-HDL-C concentration is similar to or better than LDL-C alone in predicting car- diovascular disease (CVD) incidence [49], in this study, the results showed that the decrease in HDL-C and elevation of TG, VLDL-C and LDL-C levels (Table 1) are also the landmarks of diabetes and CAD development as found in an earlier study [50]. However, the association of apoE ε4 Relationship between Lipid profiles and apoE4-bearing genotypes However, this study will provide a good starting point for the screening of large populations before proceeding to the protein level study of apoE and other candidate genes in various races and, additionally, including pharmacogenomics study in the future. higher risk (p = 0.016) for developing CAD and ε4 allele containing genotypes led to a 2.04-fold higher risk (p = 0.029) in the development of T2DM as compared to the controls. This signifies that other risk factors have no influence in development or curbing the disease, suggest- ing that ε4 allele is the independent risk factor for both T2DM and CAD. However, E3/E4 genotype further increases the risk for the development of diabetes and CAD when combined with smoking and/or obesity (Table 6). This confirms the findings that progression and development of diabetes and CAD is the conse- quence of multifactorial parameters, for example, obesity, smoking, oxidative stress, defect in pancreatic b-cells, alcohol consumption, exercise, hypertension and genetic factors. In addition, incidence of T2DM is also subject to gene-environment interactions, for example, dietary fac- tors, intake of vegetable fat, polyunsaturated fatty acid, dietary fiber (particularly cereal fiber), magnesium, and caffeine were significantly inversely correlated and intakes of trans fat, saturated fatty acid and heme-iron, glycemic index, and glycemic load were significantly posi- tively correlated with the incidence of T2DM [43]. Simi- larly, regular exercise has been shown to reduce weight, BMI, HDL-C and insulin resistance. This indicates that exercise is also associated with decrease risk of T2DM and CAD. Furthermore, apoE gene polymorphism has been shown to modulate the effects of exercise on lipo- protein concentrations in plasma [44]. In this study, smoking and obesity are shown to be the two major con- tributing factors that can promote the development of T2DM and CAD or both when placed in joint association (Table 6). The reason for this is that smoking and obesity enhance the oxidative stress which results in decreased insulin secretion from pancreatic b-cell and decreased uptake of blood glucose into the muscle cells [9,10]. This evidence was supported by the experimental study on atherosclerosis-susceptible B6 (B6.apoE -/-) and athero- sclerosis-resistant BALB (BALB.apoE -/-) mice that showed defects in insulin secretion rather than defects in insulin resistance which explains the mark difference in susceptibility to T2DM [45]. Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 without CAD complication by influencing the plasma lipid levels that are important risk factors for both T2DM and CAD. No single factor can give a satisfactory explanation regarding development of diabetes and CAD as both diseases are complex diseases. Focusing, there- fore, on only one risk factor may be less than optimal in enabling researchers to predict who will develop unto- ward events in complex diseases like diabetes and CAD. Nonetheless, our study strongly supported that the apoE ε4 allele is an independent risk factor for development of both T2DM and CAD. Moreover, obesity and/or smoking, conditions associated with high oxidative stress, can aggravate the progression of both diseases. Genetic studies can thus provide information that may help to improve the ability to identify individuals, families and populations at increased risk, as well as to improve the clinical management of patients with T2DM and CAD. Such information may be useful in developing public health programs reinforcing primary and secondary prevention for T2DM and CAD. Further- more, T2DM and CAD patients identified to carry high risk genotype or allele should be treated aggressively to prevent the progression of disease. Abbreviations APOE A li 8. Boden G, Shulman GI: Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction. Eur J Clin Invest 2002, 32(Suppl 3):14-23. APOE: Apolipoprotein E; CAD: Coronary artery disease; T2DM: Type 2 diabetes mellitus; PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism; HDL-C: High-density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TC: Total cholesterol; TG: Triglyceride; non-HDL-C: Non-high- density lipoprotein cholesterol; SNPs: Single nucleotide polymorphisms; NF- κB: Nuclear factor kappa B; LDLR: Low density lipoprotein receptor; T: Thymine; C: Cytosine; ECG: Electrocardiogram; WHO: World health organization; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; BMI: Body mass index; FPG: Fasting plasma glucose; Guanidine-HCL: Guanidine- hydrochloride; APO B-100: Apolipoprotein B-100; HbA1c: Glycated hemoglobin A1c; NEFA: Non-esterified fatty acid; PDAY: Pathobiological determinants of atherosclerosis in youth. 9. Martyn JA, Kaneki M, Yasuhara S: Obesity-induced insulin resistance and hyperglycemia: etiologic factors and molecular mechanisms. Anesthesiology 2008, 109:137-148. 10. Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 2002, 23:599-622. 11. Grundy SM: Drug therapy of the metabolic syndrome: minimizing the emerging crisis in polypharmacy. Nat Rev Drug Discov 2006, 5:295-309. 12. Mahley RW, Rall SC Jr: Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet 2000, 1:507-537. 13. Singh PP, Singh M, Mastana SS: APOE distribution in world populations with new data from India and the UK. Ann Hum Biol 2006, 33:279-308. Author details 1 f 1Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 2Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 3Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 4Department of Preventive and Social Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 5Her Majesty’s Cardiac Center, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 16. Guang-da X, You-ying L, Zhi-song C, Yu-sheng H, Xiang-jiu Y: Apolipoprotein e4 allele is predictor of coronary artery disease death in elderly patients with type 2 diabetes mellitus. Atherosclerosis 2004, 175:77-81. 17. Wilson PW, Schaefer EJ, Larson MG, Ordovas JM: Apolipoprotein E alleles and risk of coronary disease. A meta-analysis. Arterioscler Thromb Vasc Biol 1996, 16:1250-1255. 18. Dallongeville J, Lussier-Cacan S, Davignon J: Modulation of plasma triglyceride levels by apoE phenotype: a meta-analysis. J Lipid Res 1992, 33:447-454. Acknowledgements h d 14. Siest G, Pillot T, Regis-Bailly A, Leininger-Muller B, Steinmetz J, Galteau MM, Visvikis S: Apolipoprotein E: an important gene and protein to follow in laboratory medicine. Clin Chem 1995, 41:1068-1086. This study was supported by the Graduate Thesis Scholarship grant from Faculty of Medicine Siriraj Hospital, Mahidol University. The authors wish to thank Dr. Saowalak Hunnangkul for excellent statistical advice and Dr. William M. Honsa for English language editorial support. 15. Knouff C, Hinsdale ME, Mezdour H, Altenburg MK, Watanabe M, Quarfordt SH, Sullivan PM, Maeda N: Apo E structure determines VLDL clearance and atherosclerosis risk in mice. J Clin Invest 1999, 103:1579-1586. References 1. 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Li Y, Li X, Shi L, Yang M, Yang Y, Tao W, Xiong Y, Zhang Y, Yao Y: Association of adiponectin SNP + 45 and SNP + 276 with type 2 diabetes in Han Chinese populations: a meta-analysis of 26 case-control studies. PLoS One 2011, 6:e19686. 6. Zeljko HM, Skaric-Juric T, Narancic NS, Tomas Z, Baresic A, Salihovic MP, Starcevic B, Janicijevic B: E2 allele of the apolipoprotein E gene polymorphism is predictive for obesity status in Roma minority population of Croatia. Lipids Health Dis 2011, 10:9. 6. Zeljko HM, Skaric-Juric T, Narancic NS, Tomas Z, Baresic A, Salihovic MP, Starcevic B, Janicijevic B: E2 allele of the apolipoprotein E gene polymorphism is predictive for obesity status in Roma minority population of Croatia. Lipids Health Dis 2011, 10:9. 7. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I: Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004, 114:1752-1761. Competing interests Received: 26 March 2012 Accepted: 23 April 2012 Published: 23 April 2012 Competing interests The authors declare that they have no competing interests. without CAD complication by influencing the plasma lipid levels that are important risk factors for both T2DM and CAD. No single factor can give a satisfactory explanation regarding development of diabetes and CAD as both diseases are complex diseases. Focusing, there- fore, on only one risk factor may be less than optimal in enabling researchers to predict who will develop unto- ward events in complex diseases like diabetes and CAD. Nonetheless, our study strongly supported that the apoE ε4 allele is an independent risk factor for development of both T2DM and CAD. Moreover, obesity and/or smoking, conditions associated with high oxidative stress, can aggravate the progression of both diseases. Genetic studies can thus provide information that may help to improve the ability to identify individuals, families and populations at increased risk, as well as to improve the clinical management of patients with T2DM and CAD. Such information may be useful in developing public health programs reinforcing primary and secondary prevention for T2DM and CAD. Further- more, T2DM and CAD patients identified to carry high risk genotype or allele should be treated aggressively to prevent the progression of disease. Conclusions This study tentatively supports the fact that variability in apoE gene locus is associated with diabetes with and Page 10 of 11 Page 10 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Kuusi T, Nieminen MS, Ehnholm C, Yki-Jarvinen H, Valle M, Nikkila EA, Taskinen MR: Apoprotein E polymorphism and coronary artery disease. Increased prevalence of apolipoprotein E-4 in angiographically verified coronary patients. 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Kraus WE, Houmard JA, Duscha BD, Knetzger KJ, Wharton MB, McCartney JS, Bales CW, Henes S, Samsa GP, Otvos JD, Kulkarni KR, Slentz CA: Effects of the amount and intensity of exercise on plasma lipoproteins. N Engl J Med 2002, 347:1483-1492. 37. Kumar P, Luthra K, Dwivedi M, Behl VK, Pandey RM, Misra A: Apolipoprotein E gene polymorphisms in patients with premature myocardial infarction: a case-controlled study in Asian Indians in North India. Ann Clin Biochem 2003, 40:382-387. 57. Tenenbaum A, Motro M, Fisman EZ, Schwammenthal E, Adler Y, Goldenberg I, Leor J, Boyko V, Mandelzweig L, Behar S: Peroxisome proliferator-activated receptor ligand bezafibrate for prevention of type 2 diabetes mellitus in patients with coronary artery disease. Circulation 2004, 109:2197-2202. 38. Yan S, Zhou X, Lin Q, Song Y: Association of polymorphism of apolipoprotein E gene with coronary heart disease in Han Chinese. Chin Med J (Engl) 1999, 112:224-227. 58. Gage J, Hasu M, Thabet M, Whitman SC: Caspase-1 Deficiency Decreases Atherosclerosis in Apolipoprotein E-Null Mice. Can J Cardiol 2012, 28:222-229. 39. Anuurad E, Yamasaki M, Shachter N, Pearson TA, Berglund L: ApoE and ApoC-I polymorphisms: association of genotype with cardiovascular disease phenotype in African Americans. J Lipid Res 2009, 50:1472-1478. doi:10.1186/1475-2840-11-36 Cite this article as: Chaudhary et al.: Apolipoprotein E gene polymorphism: effects on plasma lipids and risk of type 2 diabetes and coronary artery disease. Cardiovascular Diabetology 2012 11:36. 40. Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Inamdar PA, Kelkar SM, Devasagayam TP, Bapat MM: Apolipoprotein E polymorphism in non-insulin-dependent diabetics of Mumbai, India and its effect on plasma lipids and lipoproteins. Diabetes Res Clin Pract 2000, 47:217-223. 27. Jenkins AJ, Rowley KG, Lyons TJ, Best JD, Hill MA, Klein RL: Lipoproteins and diabetic microvascular complications. Curr Pharm Des 2004, 10:3395-3418. 28. Bitzur R, Cohen H, Kamari Y, Shaish A, Harats D: Triglycerides and HDL cholesterol: stars or second leads in diabetes? Diabetes Care 2009, 32(Suppl 2):S373-S377. 49. Fernandez ML, Webb D: The LDL to HDL cholesterol ratio as a valuable tool to evaluate coronary heart disease risk. J Am Coll Nutr 2008, 27:1-5. 29. DeFronzo RA: Insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis: the missing links. The Claude Bernard Lecture 2009. Diabetologia 2010, 53:1270-1287. 50. Washio M, Sasazuki S, Kodama H, Yoshimasu PK, Liu Y, Tanaka K, Tokunaga S, Kono PS, Arai H, Koyanagi S, Hiyamuta K, Doi Y, Kawano MT, Nakagaki MO, Takada K, Nii MT, Shirai K, Ideishi MM, Arakawa MK, Mohri MM, Takeshita A: Role of hypertension, dyslipidemia and diabetes mellitus in the development of coronary atherosclerosis in Japan. Jpn Circ J 2001, 65:731-737. g 30. Mooradian AD: Dyslipidemia in type 2 diabetes mellitus. Nat Clin Pract Endocrinol Metab 2009, 5:150-159. 31. Eichner JE, Dunn ST, Perveen G, Thompson DM, Stewart KE, Stroehla BC: Apolipoprotein E polymorphism and cardiovascular disease: a HuGE review. Am J Epidemiol 2002, 155:487-495. 51. Frikke-Schmidt R: Context-dependent and invariant associations between APOE genotype and levels of lipoproteins and risk of ischemic heart disease: a review. Scand J Clin Lab Invest Suppl 2000, 233:3-25. 32. Eichner JE, Kuller LH, Orchard TJ, Grandits GA, McCallum LM, Ferrell RE, Neaton JD: Relation of apolipoprotein E phenotype to myocardial infarction and mortality from coronary artery disease. Am J Cardiol 1993, 71:160-165. 52. Singh PP, Singh M, Bhatnagar DP, Kaur TP, Gaur SK: Apolipoprotein E polymorphism and its relation to plasma lipids in coronary heart disease. Indian J Med Sci 2008, 62:105-112. 33. van Bockxmeer FM, Mamotte CD: Apolipoprotein epsilon 4 homozygosity in young men with coronary heart disease. Lancet 1992, 340:879-880. S h h l C k ll kk l 53. Chaaba R, Attia N, Hammami S, Smaoui M, Ben Hamda K, Mahjoub S, Hammami M: Association between apolipoprotein E polymorphism, lipids, and coronary artery disease in Tunisian type 2 diabetes. J Clin Lipidol 2008, 2:360-364. 34. Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 20. Wild S, Roglic G, Green A, Sicree R, King H: Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004, 27:1047-1053. 42. McGill HC Jr, McMahan CA: Determinants of atherosclerosis in the young. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Am J Cardiol 1998, 82:30T-36T. 43. Murakami K, Okubo H, Sasaki S: Effect of dietary factors on incidence of type 2 diabetes: a systematic review of cohort studies. J Nutr Sci Vitaminol 2005, 51:292-310. . The Asia-Pacific Perspective: Redefining obesity and its treatme 21. The Asia-Pacific Perspective: Redefining obesity and its treatment. World Health Organization: WHO western pacific region; 2000, 1-56. World Health Organization: WHO western pacific region; 2000, 1-5 22. American Diabetes Association: Diagnosis and classification of diabetes mellitus. Diabetes Care 2010, 33(Suppl 1):S62-S69. 44. Bernstein MS, Costanza MC, James RW, Morris MA, Cambien F, Raoux S, Morabia A: Physical activity may modulate effects of ApoE genotype on lipid profile. Arterioscler Thromb Vasc Biol 2002, 22:133-140. 23. Richard P, Thomas G, de Zulueta MP, De Gennes JL, Thomas M, Cassaigne A, Bereziat G, Iron A: Common and rare genotypes of human apolipoprotein E determined by specific restriction profiles of polymerase chain reaction-amplified DNA. Clin Chem 1994, 40:24-29. 45. Li J, Wang Q, Chai W, Chen MH, Liu Z, Shi W: Hyperglycemia in apolipoprotein E-deficient mouse strains with different atherosclerosis susceptibility. Cardiovasc Diabetol 2011, 10:117. 24. Friedewald WT, Levy RI, Fredrickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972, 18:499-502. 46. Tosukhowong P, Sangwatanaroj S, Jatuporn S, Prapunwattana P, Saengsiri A, Rattanapruks S, Srimahachota S, Udayachalerm W, Tangkijvanich P: The correlation between markers of oxidative stress and risk factors of coronary artery disease in Thai patients. Clin Hemorheol Microcirc 2003, 29:321-329. 25. Hermans MP, Sacks FM, Ahn SA, Rousseau MF: Non-HDL-cholesterol as valid surrogate to apolipoprotein B100 measurement in diabetes: Discriminant Ratio and unbiased equivalence. Cardiovasc Diabetol 2011, 10:20. 47. Tan CE, Tai ES, Tan CS, Chia KS, Lee J, Chew SK, Ordovas JM: APOE polymorphism and lipid profile in three ethnic groups in the Singapore population. Atherosclerosis 2003, 170:253-260. 26. Krauss RM, Siri PW: Dyslipidemia in type 2 diabetes. Med Clin North Am 2004, 88:897-909, x. 48. Authors’ contributions All authors fulfill the criteria for authorship. RC carried out the SNPs analysis. AL conceived all of the study and coordination. RC and AL conducted statistical analysis and drafted manuscript. TP, DT, SR, and CS provided the samples for SNP analysis. AL, TP, DT, SS, and CS participated in study conception and design, interpretation of data and critical revision of manuscript for important intellectual content. All authors read and approved the final version of the manuscripts. All authors fulfill the criteria for authorship. RC carried out the SNPs analysis. AL conceived all of the study and coordination. RC and AL conducted statistical analysis and drafted manuscript. TP, DT, SR, and CS provided the samples for SNP analysis. AL, TP, DT, SS, and CS participated in study conception and design, interpretation of data and critical revision of manuscript for important intellectual content. All authors read and approved the final version of the manuscripts. 19. Tatsanavivat P, Klungboonkrong V, Chirawatkul A, Bhuripanyo K, Manmontri A, Chitanondh H, Yipintsoi T: Prevalence of coronary heart disease and major cardiovascular risk factors in Thailand. Int J Epidemiol 1998, 27:405-409. Page 11 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36 http://www.cardiab.com/content/11/1/36 Stengard JH, Pekkanen J, Ehnholm C, Nissinen A, Sing CF: Genotypes with the apolipoprotein epsilon4 allele are predictors of coronary heart disease mortality in a longitudinal study of elderly Finnish men. Hum Genet 1996, 97:677-684. 41. Kataoka S, Robbins DC, Cowan LD, Go O, Yeh JL, Devereux RB, Fabsitz RR, Lee ET, Welty TK, Howard BV: Apolipoprotein E polymorphism in American Indians and its relation to plasma lipoproteins and diabetes. The Strong Heart Study. Arterioscler Thromb Vasc Biol 1996, 16:918-925.
https://openalex.org/W4205122720	https://link.springer.com/content/pdf/10.1007/978-3-030-67818-0_5.pdf	English	null	Politicization of the Non-politicizable: The Collapse of the Ice Skating Rink in Bad Reichenhall on 2 January 2006	Springer eBooks	2,022	cc-by	4,699	This chapter is the revised and translated version of the related case study in Wolfgang Seibel, Kevin Klamann and Hannah Treis: Verwaltungsdesaster. Von der Loveparade zu den NSU-Ermittlungen. Frankfurt/New York: Campus Publ. 2017, 113–158. The German original was authored by Kevin Klamann and Wolfgang Seibel. 1 Landgericht Traunstein, 2. Strafkammer, Verdict of 18 November 2008, 2KLs 200 JS 865/06, paragraphs 95 and 233. Henceforth cited as LG 2008 RN plus number of Randnummer (RN = paragraph no.). 2 LG 2008 RN 96, 277. 3 LG 2008 RN 66–90. CHAPTER 5 129 © The Author(s) 2022 W. Seibel, Collapsing Structures and Public Mismanagement, https://doi.org/10.1007/978-3-030-67818-0_5 ( ) W. Seibel, Collapsing Structures and Public Mismanagement, https://doi.org/10.1007/978-3-030-67818-0_5 8 LG 2008 RN 190, 391–2, 462; Landgericht Traunstein, 6th Strafkammer, Verdict of 27.10.2011, 6KLs 200 JS 865/06 (3), paragraph 271. Cited below as LG 2011 + Randnummer (number of paragraph). 10 “Schlamperei, Ignoranz und Skrupellosigkeit”, Welt Online, 27.10.2011, http://www. welt.de/vermischtes/weltgeschehen/article13684199/Schlamperei-Ignoranz-und- Skrupellosigkeit.html, last accessed 23 March 2017. In this chapter, all quotes from German originals are author’s translations. 5 The judicial processing of the catastrophe of 2 January 2006 began with a criminal trial before the Traunstein Regional Court or Landgericht. This ended on 18 November 2008 with the conviction of an engineer involved in the construction. A project manager involved in the construction and an expert who had issued a positive certificate for the hall roof in 2003 were acquitted of negligent manslaughter charges. An appeal trial filed by the Public Prosecutor with the Traunstein Regional Court and the joint plaintiffs regarding the expert’s acquittal ended on 12 January 2010 with the revocation of the acquittal and referral to the Traunstein Regional Court (Federal Supreme Court, Verdict of 12 January 2010, 1 StR 272/09). A different chamber of the Regional Court confirmed the first instance acquittal of the expert by Verdict of 27 October 2011 (Landgericht Traunstein, 6. Strafkammer, Urteil vom 27.10.2011, 6KLs 200 JS 865/06 (3)). 5.1 Characteristics of the Case 1 2 3 129 130 W. SEIBEL 4 5 6 7 8 9 10 What the case has in common with other man-made disasters involving German governmental agencies is the absence of any investigation beyond criminal trial. No expert commission, no parliamentary investigation com mittee nor any otherwise independent investigation was undertaken. Among the four cases of collapsed bridges and buildings caused by public 4 LG 2008 RN 32–33. 5 The judicial processing of the catastrophe of 2 January 2006 began with a criminal trial before the Traunstein Regional Court or Landgericht. This ended on 18 November 2008 with the conviction of an engineer involved in the construction. A project manager involved in the construction and an expert who had issued a positive certificate for the hall roof in 2003 were acquitted of negligent manslaughter charges. An appeal trial filed by the Public Prosecutor with the Traunstein Regional Court and the joint plaintiffs regarding the expert’s acquittal ended on 12 January 2010 with the revocation of the acquittal and referral to the Traunstein Regional Court (Federal Supreme Court, Verdict of 12 January 2010, 1 StR 272/09). A different chamber of the Regional Court confirmed the first instance acquittal of the expert by Verdict of 27 October 2011 (Landgericht Traunstein, 6. Strafkammer, Urteil vom 27.10.2011, 6KLs 200 JS 865/06 (3)). 6 LG 2008 RN 102–135. 7 LG 2008 RN 139, 229, 452. 8 LG 2008 RN 190, 391–2, 462; Landgericht Traunstein, 6th Strafkammer, Verdict of 27.10.2011, 6KLs 200 JS 865/06 (3), paragraph 271. Cited below as LG 2011 + Randnummer (number of paragraph). 9 LG 2011 RN 272–277. 10 “Schlamperei, Ignoranz und Skrupellosigkeit”, Welt Online, 27.10.2011, http://www. welt.de/vermischtes/weltgeschehen/article13684199/Schlamperei-Ignoranz-und- Skrupellosigkeit.html, last accessed 23 March 2017. In this chapter, all quotes from German originals are author’s translations. 8 LG 2008 RN 190, 391–2, 462; Landgericht Traunstein, 6th Strafkammer, Verdict of 27.10.2011, 6KLs 200 JS 865/06 (3), paragraph 271. Cited below as LG 2011 + Randnummer (number of paragraph). 9 LG 2011 RN 272–277. 10“Schlamperei Ignoranz und Skrupellosigkeit” Welt Online 27 10 2011 http://www 4 LG 2008 RN 32–33. 5 The judicial processing of the catastrophe of 2 January 2006 began with a criminal trial before the Traunstein Regional Court or Landgericht. This ended on 18 November 2008 with the conviction of an engineer involved in the construction. A project manager involved in the construction and an expert who had issued a positive certificate for the hall roof in 2003 were acquitted of negligent manslaughter charges. An appeal trial filed by the Public Prosecutor with the Traunstein Regional Court and the joint plaintiffs regarding the expert’s acquittal ended on 12 January 2010 with the revocation of the acquittal and referral to the Traunstein Regional Court (Federal Supreme Court, Verdict of 12 January 2010, 1 StR 272/09). A different chamber of the Regional Court confirmed the first instance acquittal of the expert by Verdict of 27 October 2011 (Landgericht Traunstein, 6. Strafkammer, Urteil vom 27.10.2011, 6KLs 200 JS 865/06 (3)). 6 LG 2008 RN 102–135. 7 LG 2008 RN 139, 229, 452. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 131 administration mismanagement that are analyzed in the present volume, the German case with a death toll of twelve children and three mothers accompanying them to a pastime entertainment is the only one without official acknowledgment of responsibility. 11 LG 2008 RN 56, 292. 12 LG 2008 RN 277. 13 LG 2008 RN 44. 12 LG 2008 RN 277. 11 LG 2008 RN 56, 292. 14 LG 2008 RN 1–10. 15 LG 2011 RN 7, 30. 16 Bundesgerichtshof, Verdict of 12.01.2010, 1 StR 272/09, paragraph 1 and paragraphs 81 to 86. Cited below as BGH 2010. 5.2 Facts of the Matter 11 12 13 132 W. SEIBEL 14 15 The negligent attitude of the city administration was partly driven by an exemplary conflict of interests. The city was both the owner of the hall and the supervisory authority of building safety. The quest for saving taxpay er’s money and the necessity to ensure the safety of infrastructure began to collide when maintenance costs increased and urban planning ideas of the Lord Mayor envisaged abandoning the hall complex altogether. So the building authority was exposed to the Lord Mayor’s expectation not to invest too much efforts and money into the maintenance, let alone reno vation, of the hall complex which comprised both the ice rink and an indoor swimming pool. According to the Lord Mayor’s own admission before the Traunstein Regional Court, this culminated in the downright obstruction of a municipal parliament decision to substantially renovate the hall at estimated costs of 5.5 million Euros. 16 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 133 17 18 The death of twelve children and three mothers who lost their lives under the collapsing roof of the Bad Reichenhall ice rink on 2 January 2006 was not the result of a tragic chain of unfortunate circumstances but of the failure of the Bad Reichenhall city administration to meet elemen tary professional and ethical requirements. The Lord Mayor of the city and leading officials in the building authority pursued their own urban planning ambitions at the expense of the safety of the hall users and the operational staff. This happened despite warnings of water ingress that had been noticed for years but were ignored by the city administration. While these water infiltrations did not cause the collapse, a risk-conscious reac tion on the part of the city administration would have commissioned a thorough investigation into their origin which in turn, according to all likelihood, would have uncovered the basic construction and design errors of the roof that caused the collapse of the hall on 2 January 2006. 5.2.1 Design and Construction Errors 19 20 17 LG 2011 RN 251. 18 LG 2011 RN 3. 19 LG 2008 RN 98, 266–267. 20 LG 2008 RN 45, 176, 242. 134 134 W. SEIBEL W. SEIBEL 21 22 23 24 25 26 36 LG 2008 RN 462: In the first proceedings, the Traunstein Regional Court discussed breaches of duty by third parties and their possible influence on the responsibility of the three defendants. The Court did not make a final assessment of the actions or omissions of the Bad Reichenhall municipal administration as criminal charges against municipal officials were ulti mately dropped. 35 “E wie Eishalle”, in: DER SPIEGEL 14/2006, p. 58.i 29 LG 2008 RN 57–58, 436, 462. 30 LG 2008 RN 60–63. 31 LG 2008 RN 380–392. 32 LG 2008 RN 389. 33 LG 2008 RN 391. 34 LG 2008 RN 62, 385, 390. 35 “E wie Eishalle”, in: DER SPIEGEL 14/2006, p. 58. 36 LG 2008 RN 462: In the first proceedings, the Traunstein Regional Court discussed breaches of duty by third parties and their possible influence on the responsibility of the three defendants. The Court did not make a final assessment of the actions or omissions of the Bad Reichenhall municipal administration as criminal charges against municipal officials were ulti mately dropped. 5.2.2 Unbureaucratic Sloppiness 27 28 21 LG 2008 RN 37–41. 22 LG 2008 RN 428. 23 LG 2008 RN 37, 108–109, 253. 24 LG 2008 RN 109. 25 LG 2008 RN 242. 26 LG 2008 RN 125, 242, 433–439. 27 LG 2011 RN 277. 28 LG 2008 RN 273. 28 LG 2008 RN 273. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 135 29 30 31 32 33 34 35 36 34 LG 2008 RN 62, 385, 390. W. SEIBEL 136 37 38 5.2.3 Acknowledged Yet Unaddressed Safety Issues 5.2.3 Acknowledged Yet Unaddressed Safety Issues 39 40 41 42 43 44 37 Ibid. 38 LG 2008 RN 139. 39 LG 2008 RN 177, LG 2011 RN 163. 40 LG 2008 RN 177. 41 LG 2008 RN 179. 42 LG 2011 RN 166–176. 43 LG 2008, RN 378. 44 LG 2008, RN 184–185. 37 Ibid. 38 LG 2008 RN 139. 39 LG 2008 RN 177, LG 2011 RN 163. 40 LG 2008 RN 177. 41 LG 2008 RN 179. 42 LG 2011 RN 166–176. 43 LG 2008, RN 378. 44 LG 2008, RN 184–185. 43 LG 2008, RN 378. 44 LG 2008, RN 184–185. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 137 45 46 47 48 49 50 45 Ibid., LG 2011 RN 207–209. 46 LG 2008 RN 378. 47 LG 2008 RN 185, 378. 48 LG 2011 RN 275. 49 LG 2008 RN 187–8, LG 2011 RN 176. 50 LG 2011 RN 276. Also with regard to a “highlighted in bold” (LG 2008 RN 401) danger of a hanging wooden ceiling crashing in the indoor swimming pool in the expert report of February 2002, the City only reacted with remedial work after a piece of wood had actually fallen down. 45 Ibid., LG 2011 RN 207–209. 46 LG 2008 RN 378. 47 LG 2008 RN 185, 378. 48 LG 2011 RN 275. 49 LG 2008 RN 187–8, LG 2011 RN 176. 50LG 2011 RN 276 Al i h d 59 LG 2011, RN 231. 60 LG 2011 RN 244, see also LG 2008 RN 353. 61 LG 2008 RN 208, 358–359. 62 While the Traunstein Regional Court assumed in 2008 that the review of the original construction documents would only have become necessary in the course of an in-depth inspection that did not take place, a different Chamber of the same Court in 2011 assumed that the defendant had had the obligation to review the original documentation anyway. Cf. LG 2008 RN 361, LG 2011 RN 264. 63 LG 2011 RN 262. 64 LG 2011 RN 254–6. 65LG 2011 RN 160 63 LG 2011 RN 262. 5.2.4 Mobilizing Recognizably Insufficient Expert Opinions 50 LG 2011 RN 276. Also with regard to a “highlighted in bold” (LG 2008 RN 401) danger of a hanging wooden ceiling crashing in the indoor swimming pool in the expert report of February 2002, the City only reacted with remedial work after a piece of wood had actually fallen down. 138 W. SEIBEL 51 52 53 54 55 56 57 58 51 LG 2008 RN 190. 52 LG 2011 RN 271. 53 LG 2008 RN 190. 54 LG 2008 RN 375. 55 LG 2008 RN 411; LG 2011 RN 271. 56 LG 2008, RN 169, 206. 57 LG 2011 RN 129. 58 LG 2008 RN 336. 51 LG 2008 RN 190. 52 LG 2011 RN 271. 53 LG 2008 RN 190. 54 LG 2008 RN 375. 55 LG 2008 RN 411; LG 2011 RN 271. 56 LG 2008, RN 169, 206. 57 LG 2011 RN 129. 58 LG 2008 RN 336. 51 LG 2008 RN 190. 52 LG 2011 RN 271. 53 LG 2008 RN 190. 54 LG 2008 RN 375. 55 LG 2008 RN 411; LG 2011 RN 271. 56 LG 2008, RN 169, 206. 57 LG 2011 RN 129. 58 LG 2008 RN 336. 55 LG 2008 RN 411; LG 2011 RN 271. 56 LG 2008, RN 169, 206. 57 LG 2011 RN 129. 58 LG 2008 RN 336. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 139 59 59 60 61 62 63 64 65 64 LG 2011 RN 254–6. 65 LG 2011 RN 160. 140 W. SEIBEL 66 67 70 LG 2011 RN 214–215. 71 LG 2008 RN 94. 72 LG 2008 RN 236–238; LG 2011 RN 279. 73 LG 2011 RN 279. 74 LG 2008 RN 95, General Prosecutor’s Office Munich, refusal of 21.08.2007, p. 6. The document was temporarily available at: http://reichenhaller-pranger.de/resources/generals taatsanwalt+m$C3$BCnchen+29.08.07.pdf, last accessed on 27 February 2013. 75 With regard to the errors committed by the civil engineer convicted in 2008, the Regional Court 2008 declared: “All three established breaches of duty of the defendant G, construction contrary to the building authority approval, inadequate static calculation and careless monitoring of the manufacturing process were causal for the collapse. The existence of only one of the assumed breaches of duty in each case would possibly not have led to the collapse on 02.01.2006, but the simultaneous existence of these errors did have.” LG 2008 RN 450. 5.2.5 An Obstructed Municipal Parliament Decision and the Path to Disaster 5.2.5 An Obstructed Municipal Parliament Decision and the Path to Disaster 68 69 66 LG 2011 RN 173. 67 LG 2011 RN 271. 68 LG 2011 RN 270. 69 Ibid. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 141 70 70 71 72 73 71 74 142 W. SEIBEL 75 77 LG 2008 RN 190; LG 2011 RN 265–272. 76 LG 2011 RN 266, 271. 5.3.1 Turning Points and Critical Junctures 76 77 78 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 143 79 80 81 5.1 81 LG 2011 RN 163. 82 See the assessment of the Landgericht Traunstein: LG 2008 RN. 98, 452. 83 LG 2008 RN 98. 84 This was also the evaluation of the Landgericht (Regional Court) Traunstein, cf. LG 2008 RN 109, 450. 79 LG 2008 RN 139. 80 The recommendation to take measures to estimate the renovation costs for the roof of the ice rink, which was found in the summary report of March 2004 (LG 2011 RN 248, 271), suggests that a more detailed inspection of the roof could have been expected for the renovation planning if the administration had actually wanted to carry out the renovation itself (LG 2011 RN 248, 271). The city did not react to this recommendation either, as the Traunstein Regional Court stated: “Even when Witness L. in his 2004 study—as he credibly described it—pointed out urgently to the obviously unexamined and unevaluated roof con struction, there was still no reaction on the part of the city administration, neither the com missioning of an additional study nor a possible consultation with the defendant.” LG 2011 RN 248. 81 LG 2011 RN 163. 144 W. SEIBEL 144 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 145 85 LG 2008 RN 274, 290, 441. 86 LG 2008 RN 275. 87 LG 2008 RN 100. 5.3.2 Contributing Factors and Necessary Conditions The collapse of the skating rink in Bad Reichenhall was the cumulative effect of several independent causal factors, yet it did not result from the proverbial chain of unfortunate circumstances. It was not the circum stances that triggered the catastrophe of 2 January 2006 but decisions taken by officials of the Bad Reichenhall City administration who them selves could not fully anticipate the consequences of their decisions or omissions but nevertheless failed to act mindfully and with the necessary sense of responsibility. This pertains to the original design and construc tion errors of 1973, the faulty initial inspection and approval of the roof structure, the way in which the vitrification of the ice rink was carried out including the installation of exhaust air systems in 1977, the disregard of an expert’s recommendation of 2002 as well as the continued indications by users and operating personnel that water was entering the ice skating rink, the non-compliance with the municipal parliament’s decision of June 2005 to renovate the hall complex and the decision making concerning the closure of the ice skating rink on the day of the accident itself. 82 83 84 146 W. SEIBEL 85 86i An additional risk factor was the vitrification of the ice rink carried out in 1977 which inevitably increased the level of humidity in the hall, so that in the same year an exhaust air system was installed with the necessary equipment placed on the roof of the hall. This double measure—vitrifica tion and installation of the exhaust air system—was problematic on the one hand because an insufficiently moisture-resistant urea resin glue had been used to erect the wooden structure of the roof and, on the other hand, because the additionally installed exhaust system increased the roof load by a total of 2300 kilograms. With regard to the vitrification itself, the city of Bad Reichenhall acted flawlessly. This measure was already envisaged in the building contract of 1971, so the city could assume that the subsequent vitrification had been taken into account in the construc tion of the roof itself. 88 LG 2008 RN 138, 452, 460–462. 89 LG 2008 RN 452. 90 LG 2008 RN 99. 91 LG 2008 RN 462. 99 LG 2011, RN 163–165, 270–271. 100 LG 2011 RN 181, 271. 101 Cf. LG 2008 RN 373–406, 462, 482. 102 LG 2008 RN 396, 411. g y g NC2 Omission of the engineer in charge to apply for a special permit for the installation of wooden girders with an extended length; omission to check on the type of glue used in the erection of the roof, 1973. 5.3.2 Contributing Factors and Necessary Conditions 87 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 147 88 89 A stronger counterfactual scenario can be assumed under the premise that no documentation was available and that this very fact would have been detected in a certification process in accordance with due diligence standards when the vitrification of the hall in 1977 was in the making. Continued operation of the hall in the knowledge that there was no verifi able documentation of the building’s structural design would have been inconceivable. To that extent, the absence of a verification of the design can be defined a further necessary condition for the collapse of 2 January 2006. 90 91 148 W. SEIBEL 92 93 94 95 96 97 98 In this respect, the non-observance of the repeated complaints about water ingress by those responsible in the Bad Reichenhall City administra tion can also be defined as a necessary, albeit insufficient, condition for the later collapse of the hall roof. According to the Traunstein Regional Court, obvious water ingress would have been assessed differently by a risk- sensitive administration. Even if the administration, unaware of the 92 LG 2008 RN 229, 373. 93 LG 2008 RN 373. 94 LG 2011 RN 241. 95 LG 2008 RN 380. 96 LG 2008 RN 139. 97 LG 2008 RN 380. 98 LG 2008 RN 362; LG 2011 RN 266. 92 LG 2008 RN 229, 373. 97 LG 2008 RN 380. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 149 original construction errors which caused the collapse, could not have been fully aware of the consequences of its omission, a risk-sensitive atti tude could have prevented the catastrophe of 2 January 2006. 99 100 101 102i A mentality of municipal officials of accepting risks to the life and limb of the hall users was reflected in the lack of response to the warnings in the 2001 and 2002 reports and, to a lesser extent, in the indifference regard ing the water ingress and the recommendation of an architect consulted in 2002 who suggested an in-depth inspection of the roof structure. That mentality came to bear even on the day of the disaster itself. 150 W. 104 Cf. James D. Thompson: Organizations in Action: Social Science Bases of Administrative Theory. With a new preface by Meyer N. Zald and a new introduction by W. Richard Scott, London and New York: Routledge 2017 [1967], 132–143. 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 151 5.3.2 Contributing Factors and Necessary Conditions SEIBEL Table 5.1 Contributing Factors (CF) and Necessary Conditions (NC), collapse of Bad Reichenhall Ice Skating Rink Table 5.1 Contributing Factors (CF) and Necessary Conditions (NC), collapse of Bad Reichenhall Ice Skating Rink NC1 Design error and faulty construction of the ice skating rink roof, 1971–1973. NC1 Design error and faulty construction of the ice skating rink roof, 1971–1973. NC2 Omission of the engineer in charge to apply for a special permit for the installation of wooden girders with an extended length; omission to check on the type of glue used in the erection of the roof, 1973. CF1 Improper documentation of structural engineering details and calculations. NC3 Installation of exhaust air systems of the hall roof without building permit an without checking on relevant calculations of the roof statics, 1977. NC4 Failure to follow the recommendation of an expert opinion of July 2002 to conduct an in-depth inspection of the whole roof, January 2003. NC4 Failure to follow the recommendation of an expert opinion of July 2002 to conduct an in-depth inspection of the whole roof, January 2003. NC5 Purposeful omission of the Lord Mayor to implement the decision of the municip parliament of June 2005 to renovate the hall complex. NC5 Purposeful omission of the Lord Mayor to implement the decision of the municipal parliament of June 2005 to renovate the hall complex. CF2 Continuous carelessness and neglect of maintenance despite visible signs of water ingress. CF2 Continuous carelessness and neglect of maintenance despite visible signs of water ingress. 103 5.1 103 LG 2011 RN 279. 105 For the phenomenon of power in organizations and its use to exert pressure on the supposedly a-political “operative core” cf. Henry Mintzberg: The Power Game and the Players. In Steven J. Ott, Jay M. Shafritz and YongSuk Jang, eds., Classic Readings in Organization Theory. Belmont: Wadsworth 1983, 330–337. 106 Diane Vaughan: The Dark Side of Organizations: Mistake, Misconduct, and Disaster. Annual Review of Sociology 25 (1999): 271–305. 107 This was one of the suppositions of the Federal Supreme Court in its ruling of 2010 (BGH 2010 RN 85). On symbolic problem solving and the interest in ignorance about it cf. Wolfgang Seibel: Successful Failure. An Alternative View on Organizational Coping. 06 Diane Vaughan: The Dark Side of Organizations: Mistake, Misconduct, and Disaster nual Review of Sociology 25 (1999): 271–305. American Behavioral Scientist 39 (1996): 1011–1024. On delegation and diffusion of responsibility see Björn Bartling and Urs Fischbacher: Shifting the Blame: On Delegation and Responsibility. The Review of Economic Studies 79 (2012): 67–87. 108 Cf. George A. Akerlof: The Market for ‘Lemons’: Quality Uncertainty and the Market Mechanism. Quarterly Journal of Economics 84 (1970): 488–500; Michael C. Jensen and William H. Meckling: Theory of the Firm: Managerial Behavior, Agency Costs, and Ownership Structure. Journal of Financial Economics 3 (1979): 305–360. 109 LG 2011 RN 270. 110 LG 2011 RN 278. 5.3.3 Causal Mechanisms 104l From the basic conflict of interest within the City administration resulted further situational mechanisms. One pertained to information asymmetries and the control or principal agent problems that arose both in the relationship between the City administration and the expert con sulted in 2003 and between the municipal parliament and the administra tion, especially the Lord Mayor himself. For its part, the city administration had to rely on the expert’s assessment while the municipal parliament had to rely on the Lord Mayor as far as the comprehensive renovation of the hall complex was concerned which the parliament had agreed-upon on 14 June 2005. It was probably beyond the imagination of municipal parlia ment key-figures that the Lord Mayor not only had deliberately restrained the scope and precision of the expert inspection in 2003 and thus was acting on the basis of inadequate information but that he also was deter mined to obstruct the parliamentary decision anyway. 152 W. SEIBEL 105 The mechanisms promoting the one-sided determination to save costs at the expense of safety drove the city administration’s action. This was reflected in the indolence vis-à-vis regular reports from users and operat- ing personnel on water ingress. What is more, expert opinions submitted in 2001 and 2002 and the warning notices contained therein were not taken seriously. They pertained to the condition of the canopy of the hall, to fire protection deficiencies and presumed insufficient load-bearing capacity of the indoor swimming pool roof. The question is to what extent these omissions were intentional or the result of incompetence. 106 107 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 153 108 109 110 The mechanisms which created an incentive structure for the actions and omissions of the Bad Reichenhall city administration to downplay safety issues and, in contrast, upgrade aspects of urban planning and cost savings, can be defined as ‘situational’ in nature. One contribution to this constellation was the information deficits of the city administration with regard to the original construction defects. These situational mechanisms formed opportunity structures or permissive conditions but they obvi ously did not result in imminent risks for the safety of the hall complex. Rather, these mechanisms represented standard risks that could have been 154 W. SEIBEL brought under control by appropriate countermeasures. Which, however, did not happen. 111 Maurizio Catino: Organizational Myopia. Problems of Rationality and Foresight in Organizations. Cambridge: Cambridge University Press 2014. 112 Wolfgang Seibel: Successful Failure. 113 LG 2011 RN 279. 5.3.3 Causal Mechanisms Instead, the Bad Reichenhall city administration was dis tinctly negligent in handling risks to life and limb of hall users despite continued indications of possible construction defects. This concerned water ingress, the canopy roof of the hall complex, flawed fire protection and defects of the secondary (non-structural) parts of the swimming pool hall roof. In addition, an expert had recommended to the city in 2002 to conduct an in-depth inspection of the roof structure of the ice rink. However, the city did not commission such an inspection. 111 112 5.2 113 5 POLITICIZATION OF THE NON-POLITICIZABLE: THE COLLAPSE… 155 Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. 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https://openalex.org/W1987808907	https://europepmc.org/articles/pmc3898532?pdf=render	English	null	Comprehensive analysis of the transcriptional landscape of the human FMR1 gene reveals two new long noncoding RNAs differentially expressed in Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome	Human genetics	2,013	cc-by	7,281	Hum Genet (2014) 133:59–67 DOI 10.1007/s00439-013-1356-6 Hum Genet (2014) 133:59–67 DOI 10.1007/s00439-013-1356-6 ORIGINAL INVESTIGATION Comprehensive analysis of the transcriptional landscape of the human FMR1 gene reveals two new long noncoding RNAs differentially expressed in Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome Chiara Pastori • Veronica J. Peschansky • Deborah Barbouth • Arpit Mehta • Jose P. Silva • Claes Wahlestedt Received: 28 May 2013 / Accepted: 25 August 2013 / Published online: 5 September 2013 The Author(s) 2013. This article is published with open access at Springerlink.com FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 pro- moter, whereas FMR6 is an antisense transcript overlap- ping the 30 region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clin- ical outcomes. Abstract The majority of the human genome is tran- scribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, [200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains \55 CGG repeats in its 50-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called ‘‘Deep-RACE’’, which combines rapid ampliﬁcation of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the Electronic supplementary material The online version of this article (doi:10.1007/s00439-013-1356-6) contains supplementary material, which is available to authorized users. Introduction Trinucleotide repeat expansions give rise to more than 30 neurological and neuromuscular diseases, including Hun- tington’s disease (HD), Fragile X Syndrome (FXS) and Spinocerebellar Ataxia (Lopez Castel et al. 2010; Mirkin 2007). FXS, an X-linked genetic disorder, is the leading cause of inherited intellectual disability, and is otherwise characterized by behavioral problems and speciﬁc physical dysmorphisms. It is caused by an expansion of CGG repeats in the 50 untranslated region (50-UTR) of the Fragile X mental retardation 1 gene (FMR1). The 50-UTR of FMR1 contains 6–54 repeats in the general population; however, this region occasionally expands in subsequent generations. A size of 55–200 repeats is called a ‘‘premutation’’ (PM), while an expansion beyond 200 repeats is called a full mutation and results in Fragile X Syndrome in males. In C. Pastori V. J. Peschansky J. P. Silva C. Wahlestedt (&) Department of Psychiatry and Behavioral Sciences and Center for Therapeutic Innovation, Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA e-mail: cwahlestedt@med.miami.edu C. Pastori e-mail: cpastori@med.miami.edu D. Barbouth Division of Clinical and Translational Genetics, Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA D. Barbouth Division of Clinical and Translational Genetics, Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA A. Mehta Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA 12 123 3 Hum Genet (2014) 133:59–67 60 from introns, promoters and 30 end regions (Djebali et al. 2012; Mattick, 2005; Yan and Ma, 2012). Transcriptomic studies have revealed that antisense transcription is a common feature of mammalian genes that are actively transcribed from microsatellite disease loci (Cho et al. 2005; Ladd et al. 2007; Moseley et al. 2006). Furthermore, others and we have recently reported that lncRNAs emanate from the FMR1 gene locus and are differentially expressed in both FXS and premutation carriers (Khalil et al. 2008; Ladd et al. 2007). These FMR1-derived lncRNAs are pri- mate-speciﬁc, and animal models have not addressed their potential inﬂuence on the FXS phenotype (The Dutch- Belgian Fragile X Consortium 1994; Chen and Toth 2001; Fisch et al. 1999; Godfraind et al. 1996; Miller et al. 1999; Stafstrom et al. 2012). It is possible that ncRNAs produced from the FMR1 locus may modulate certain aspects of FXS/ FXTAS as has been demonstrated in other human diseases [reviewed in (Pastori and Wahlestedt 2012)]. We hypothe- size that ncRNA may contribute to and be reﬂective of clinical variability in humans and hence could be used as biomarkers for FXS/FXTAS. most patients with FXS, both an upstream CpG island and the expanded CGG/CCG repeats are hypermethylated (Hornstra et al. 1993; Sutcliffe et al. 1992). This hyper- methylation is associated with hypoacetylation of histones H3 and H4 in the promoter and the 50 UTR of FMR1 (Coffee et al. 2002; Coffee et al. 1999), leading to chro- matin condensation and transcriptional silencing. The protein encoded by FMR1, FMRP, is an RNA-binding protein involved in translational repression, synaptic mat- uration, dendritic mRNA localization and nucleoplasmic shuttling of mRNA (Antar et al. 2005; Brown et al. 1998; Weiler et al. 1997) lending support to the idea that its aberrant expression contributes to the intellectual disabili- ties (ID) associated with FXS (Hinton et al. 1991; Irwin et al. 2000; O’Donnell and Warren, 2002). FMRP may also have other important functions in the nucleus, according to a recent study showing that FMRP binds methylated H3K79 chromatin and mediates the DNA-damage response pathway (Shi et al. 2012). FMR1 premutation carriers are at risk for developing FXTAS, a neurodegenerative condition affecting approximately 46 % of males and 17 % of females (Garcia-Arocena and Hagerman 2010). A. Mehta FXTAS is characterized by ataxia, parkinsonism, intentional tremors, psychiatric symptoms and cognitive decline with onset usually after 50 years of age (Hagerman et al. 2001). Additionally, approximately 20 % of female premutation carriers are at an increased risk for FXPOI (Allingham- Hawkins et al. 1999). Although each Fragile X-associated condition does have a characteristic phenotype, great var- iability exists in severity and penetrance. Here, we employed a recently developed method called Deep-RACE (Olivarius et al. 2009) to comprehensively search the entire FMR1 locus for novel lncRNAs. The NCBI database reports several antisense-oriented ESTs (Expressed Sequence Tags) mapping to the FMR1 locus, suggesting the presence of as yet uncharacterized tran- scripts. By performing rapid ampliﬁcation of cDNA ends (RACE) on total human brain RNA followed by next generation sequencing, we have identiﬁed two new tran- scripts that we refer to as FMR5 and FMR6. The expression of these newly described RNA species was validated in several regions of unaffected human brain tissue as well as in brain samples from FXS and premutation carriers. Our work provides a systematic analysis of the complex tran- scriptional landscape of the FMR1 locus, uncovering two novel lncRNAs. Furthermore, the molecular mechanisms by which FXS and FXTAS/FXPOI arise are largely unrelated. In FXS, the symptoms are due to the silencing of FMR1 and ensuing lack of FMRP, while in FXTAS/FXPOI the expansion does not silence the FMR1 gene. In fact, FMRP levels in FXTAS are not or only slightly reduced compared to the normal population whereas FMR1 mRNA expression lev- els are increased two to eightfold, suggesting an RNA toxicity mechanism underlying this condition. Although the precise mechanism for this overexpression is still unknown, it has been postulated that a longer tract of CGG repeats near the FMR1 promoter results in a more open chromatin state, thereby promoting access by transcription factors (Kenneson et al. 2001; Tassone et al. 2000). Deep-RACE Here we modiﬁed an existing protocol developed in 2009 to identify in a high-throughput manner the transcription start site of genes of interest using 50RACE (Olivarius et al. 2009). We applied the same strategy using 30RACE to detect the end of transcripts of interest. Vast genomic regions are transcribed but not translated and many of the resulting transcripts, known as noncoding RNAs (ncRNAs), are enriched in the brain (Banfai et al. 2012; Cheng et al. 2005; Djebali et al. 2012). Long ncRNAs (lncRNAs), which are ncRNAs longer than 200 nucleotides, perform a wide range of functions, including modulation of transcription or of the epigenetic landscape of their loci of origin. LncRNAs can be transcribed from the sense and antisense strands of protein-coding genes, and can arise Sense oriented 30RACE The 30RACE protocol is based on the concept that mes- senger RNAs are polyadenylated transcripts and can be converted, via the reverse transcription step, to cDNA using a 30RACE adapter primer that binds the polyadeny- lated tail (polyA) of the RNA. Certain noncoding RNAs are polyadenylated while others are not, and the 30RACE protocol (Ambion cat#AM1700) can only detect polyA transcripts. RNA that is not polyadenylated cannot be detected by this method. Collection of tissue samples 19 human brain tissue samples were obtained from the NICHD Brain and Tissue Bank for Developmental Disor- ders at the University of Maryland, Baltimore, MD (Online Resource 5). RNA was extracted from tissue using the Trizol-Chloroform protocol and DNAse treated. Sense oriented 50 RACE 50 RACE for sense-oriented novel transcripts was per- formed using the Invitrogen 50 RACE System according to 123 Hum Genet (2014) 133:59–67 61 Antisense oriented 30RACE manufacturer instructions (Invitrogen cat#18374-058). Brieﬂy, strand-speciﬁc cDNA was synthesized from 1 lg of total human brain RNA (Clontech, cat#636530) using gene-speciﬁc primers (GSP1, Online Resource 3) located 500 bp upstream the TSS of FMR1. After ﬁrst strand cDNA synthesis, a homopolymeric tail was added to the 30- end of the cDNA, using TdT enzyme and dCTP. Tailed cDNA was then ampliﬁed using gene-speciﬁc primer 2 (GSP2, Online Resource 3) and the Abridged Anchor Pri- mer (AAP) provided with the system. PCR products were re-ampliﬁed in a nested PCR using gene-speciﬁc primer 3 (GSP3, Online Resource 3) and the abridged universal ampliﬁcation primer (AUAP) provided by the kit. The ﬁnal PCR products were submitted for next generation sequencing. manufacturer instructions (Invitrogen cat#18374-058). Brieﬂy, strand-speciﬁc cDNA was synthesized from 1 lg of total human brain RNA (Clontech, cat#636530) using gene-speciﬁc primers (GSP1, Online Resource 3) located 500 bp upstream the TSS of FMR1. After ﬁrst strand cDNA synthesis, a homopolymeric tail was added to the 30- end of the cDNA, using TdT enzyme and dCTP. Tailed cDNA was then ampliﬁed using gene-speciﬁc primer 2 (GSP2, Online Resource 3) and the Abridged Anchor Pri- mer (AAP) provided with the system. PCR products were re-ampliﬁed in a nested PCR using gene-speciﬁc primer 3 (GSP3, Online Resource 3) and the abridged universal ampliﬁcation primer (AUAP) provided by the kit. The ﬁnal PCR products were submitted for next generation sequencing. Total Human Brain RNA was reverse transcribed to cDNA according to the manufacturer’s protocol as described above for 30RACE-sense (Ambion cat#AM1700). The ﬁrst round of PCR was performed using primers located in exon1, exon5 and exon17 in order to ensure coverage of the entire locus. Nested PCR was performed using inner gene speciﬁc primers located in the previously mentioned regions (Online Resource 4). PCR products from all regions of the locus were pooled and submitted for next generation sequencing. Antisense oriented 50RACE 50 RACE for novel antisense transcripts was performed using the 50 RACE System according to manufacturer instructions (Invitrogen cat#18374-058). Brieﬂy, strand- speciﬁc cDNA was synthesized from 1 lg of total human brain RNA (Clontech) using gene-speciﬁc primers (GSP1, Online Resource 3) located in exon 1, exon 5 and exon 17, thereby spanning the entire locus of interest. cDNA was tailed with TdT enzyme and ampliﬁed using gene-speciﬁc primer 2 (GSP2, Online Resource 3) and AAP. PCR pro- ducts were re-ampliﬁed with gene-speciﬁc primer 3 (GSP3, Online Resource 3) and AUAP. The ﬁnal PCR products obtained from RACE experiments in the aforementioned regions of the locus were pooled and submitted for next generation sequencing. Next generation sequencing The reads coming from sequencing of 50- and 30RACE PCR products from Hiseq2000 sequencer were prepared for Alignment by trimming the adapters from the beginning and the end of the reads using PERL pro- grams. Mapping of the reads coming from the sequenc- ing of 50- and 30RACE PCR products was conducted using version 2.0.1 of TopHat, using default settings for Illumina reads. All reads were aligned to the hg19 assembly version (GRCh37) of the human genome and the prebuilt index of the hg19 genome assembly (To- pHat) was acquired from the TopHat homepage (http:// tophat.cbcb.umd.edu/). CDNA synthesis and quantitative PCR combining rapid ampliﬁcation of cDNA ends (RACE) with next generation sequencing to determine the 50 and 30ends of novel transcripts. This technique, also called Deep- RACE, was also applied to search for sense-oriented lncRNAs upstream of the transcription start site (TSS) of FMR1. Sense oriented lncRNAs overlapping gene pro- moters (Han et al. 2007; Kurokawa 2011; Martianov et al. 2007; Song et al. 2012) have been shown to regulate transcription initiation (Martianov et al. 2007; Song et al. 2012) and may therefore contribute to FMR1 gene dys- regulation in FXS/FXTAS. The two novel transcripts, FMR5 and FMR6, were vali- dated in several human brain regions. Strand speciﬁc reverse transcription (RT) was performed on 200 ng of commercial RNA (Clontech cat #636530, #636593, #636535, #636563, #636564, #636526, #636570, #636561) to make cDNA speciﬁc for FMR5 and FMR6. To rule out DNA contamination in the RNA samples, we included a ‘‘No RT’’ condition, in which the reverse transcriptase enzyme was omitted from the reaction. The primers used in the RT are reported in Online Resource 6. We interrogated the entire FMR1 locus by performing 50- and 30RACE spanning four different regions: the FMR1 promoter (up to 1 kb from the TSS), exon 1, exon 5 and exon 17. Our choice to explore these regions was based on reported expressed sequence tags antisense to the FMR1 locus. We conﬁrmed RACE-PCR ampliﬁcation of novel transcripts by capillary electrophoresis (Online Resource 1). These RACE PCR products were subjected to paired- end sequencing on Illumina’s HiSeq2000 sequencer. This procedure identiﬁed two novel noncoding RNAs, FMR5 (GenBank KC894604) and FMR6 (GenBank KC894603). FMR5 is a sense-oriented, unspliced 800 nt long transcript whose 50-end maps approximately 1 kb upstream of the TSS of FMR1 (Fig. 1a, Online Resource 2). We were unable to obtain 30RACE products for FMR5. One possible reason is that the high GC content in the promoter and 50UTR of FMR1 interfered with the RACE reaction. FMR6 is a spliced 600 nt long antisense transcript whose sequence is entirely complementary to the 30 region of FMR1. It begins in the 30UTR and ends in exon 15 of FMR1 (Fig. 1b, Online Resource 2). Interestingly, sequencing reads for FMR6 align only to the exons of FMR1, indicating that the transcript is spliced and that FMR6 and FMR1 share the same splice junctions. CDNA synthesis and quantitative PCR Quantitative PCR (qPCR) was used to compare the expression of FMR5 and FMR6 in commercially available RNA from several brain regions (Clontech), between human brain specimens and lymphocytes from control, premutation and full mutation individuals. FMR5 was measured using a custom TaqMan probe while FMR4 and FMR6 were quantiﬁed using SYBR Green, and the primers used were validated by melting curve. Glucose-6-Phos- phate Dehydrogenase and cyclophilin were used as housekeeping genes for expression normalization. QPCR data were analyzed by Delta Delta Ct method. Primers are listed in Online Resource 6. RNA from patient brain tissue and RNA from patient blood RNA from the cerebellum of 4 full mutation patients (MIND1031-09LZ, MIND1031-08GP, MIND1033-08WS, MINDJS-03) was provided courtesy of Dr. Tassone, UC Davis, MIND Institute, CA. Detailed information about patient’s samples can be found in Online Resource 5. Following reverse transcription, the cDNA is used in the following ampliﬁcation steps: a ﬁrst round of PCR was performed using the 30RACE Outer primer (provided by the kit) and a gene-speciﬁc primer for the sense noncoding RNA located 1 kb upstream of the TSS (Online Resource 4). An additional PCR step was performed using the 30RACE Inner primer and another gene speciﬁc primer. Nested PCR DNA products were submitted for next gen- eration sequencing. Blood from 2 control (616-11-ST, 378-11-JM), 2 pre- mutation (288-12-JC, 453-12-EG) and 2 full mutation (22- 12-FD, 294-12-LP) patients was provided courtesy of Dr. Tassone and it was processed to extract RNA (Tempus tubes, Applied Biosystems) according to University of California, Davis, Institutional Review Board-approved human subject protocols. 12 3 Hum Genet (2014) 133:59–67 62 Results The previous work reporting the presence of the antisense lncRNAs called ASFMR1 and FMR4 was performed in neuroblastoma (Khalil et al. 2008) and lymphoblastoid cell lines (Ladd et al. 2007), which display different epigenetic signatures than normal human brain potentially occluding discovery of additional transcripts. Here we attempted to identify novel transcripts derived from the FMR1 locus in human brain from unaffected, FXS and FXTAS patients. We screened the FMR1 locus for antisense transcripts by By deﬁnition, a noncoding RNA is a transcript that lacks an open reading frame (ORF) and is therefore not translated. Fig. 1 Alignment of Deep-RACE results to the FMR1 locus. The reads obtained by Deep-RACE were visualized using the IGV (Integrative Genomic Viewer) program after their alignment to the human genome, and appear as grey bars above the schematic of the FMR1 gene. a Those located in the promoter of FMR1 are the result of the 50RACE, and represent a partial sequence of FMR5, the sense transcript. b The reads that map to exon 15, exon 16, exon 17 and the 30UTR of FMR1 represent the antisense transcript, FMR6, and result from 50 and 30 RACE deep sequencing of the 50RACE, and represent a partial sequence of FMR5, the sense transcript. b The reads that map to exon 15, exon 16, exon 17 and the 30UTR of FMR1 represent the antisense transcript, FMR6, and result from 50 and 30 RACE deep sequencing of the 50RACE, and represent a partial sequence of FMR5, the sense transcript. b The reads that map to exon 15, exon 16, exon 17 and the 30UTR of FMR1 represent the antisense transcript, FMR6, and result from 50 and 30 RACE deep sequencing Fig. 1 Alignment of Deep-RACE results to the FMR1 locus. The reads obtained by Deep-RACE were visualized using the IGV (Integrative Genomic Viewer) program after their alignment to the human genome, and appear as grey bars above the schematic of the FMR1 gene. a Those located in the promoter of FMR1 are the result Fig. 1 Alignment of Deep-RACE results to the FMR1 locus. The reads obtained by Deep-RACE were visualized using the IGV (Integrative Genomic Viewer) program after their alignment to the human genome, and appear as grey bars above the schematic of the FMR1 gene. a Those located in the promoter of FMR1 are the result 123 Hum Genet (2014) 133:59–67 63 Fig. Results 2 FMR5 and FMR6 are widely expressed in the human brain. Expression of FMR5 (a) and FMR6 (b) was measured in RNA from human cerebellum, cerebral cortex, occipital pole, frontal lobe, temporal lobe, hippocampus and total fetal brain with qPCR. Data were normalized to expression of the housekeeping gene, glucose-6- phosphate dehydrogenase (G6PD). Error bars represent the standard deviation of four technical replicates transcripts. FMR6 was found to contain a few short ORFs (130 nt) (Online Resource 3). FMR5 contained a short ORF of 114nt and one of 459 nt potentially encoding a protein of 153 amino acids (Online Resource 3). To explore the possibility that this hypothetical protein is functional, we performed homology searches using the NCBI tool BLASTP. This query detected no putative conserved domains in any of the available databases (nr, refseq_protein, swissprot, pat, pdb and env_nr). Lack of homologous proteins and protein domains suggests that this sequence is unlikely to encode a functional protein. As a complementary strategy, we analyzed its potential domain proﬁle using the Conserved Domain Architecture Retrieval Tool (Geer et al. 2002). This search also resulted in no hits, further supporting the idea that the 459aa ORF does not encode a functional protein. Fig. 2 FMR5 and FMR6 are widely expressed in the human brain. Expression of FMR5 (a) and FMR6 (b) was measured in RNA from human cerebellum, cerebral cortex, occipital pole, frontal lobe, temporal lobe, hippocampus and total fetal brain with qPCR. Data were normalized to expression of the housekeeping gene, glucose-6- phosphate dehydrogenase (G6PD). Error bars represent the standard deviation of four technical replicates We next assessed FMR5 and FMR6 expression in vari- ous human brain regions. FMR5 and FMR6 expression was detected at low levels in comparison to housekeeping genes in adult human cerebellum, frontal and temporal lobes, occipital and cerebral cortices and hippocampus, suggest- ing their presence in most adult brain regions (Fig. 2). The two transcripts were also expressed in fetal total brain RNA (Fig. 2); however, we do not yet have evidence for a developmental function of these transcripts. In eukaryotes, protein-coding transcripts commonly contain an ORF [300 nucleotides (100 amino acids). We used the National Center for Biotechnology Information’s (NCBI’s) ‘‘ORF Finder’’ to determine ORFs in our two novel (G6PD), the housekeeping gene. In each panel, error bars for individual patients represent the standard deviation of four technical replicates. Results For mean expression values, error bars represent the standard deviation within control, FM and PM groups. p \ 0.05, *p \ 0.01, NS not signiﬁcant as determined by Student’s t test Fig. 3 Expression of FMR5, FMR6, FMR1 and FMR4 in the brain tissue of control, full mutation and premutation individuals. Expres- sion of FMR5 (a), FMR6 (b), FMR1 (c) and FMR4 (d) was measured in control, full mutation and premutation brain tissue by qPCR. Data were normalized to expression of glucose-6-phosphate dehydrogenase (G6PD), the housekeeping gene. In each panel, error bars for individual patients represent the standard deviation of four technical replicates. For mean expression values, error bars represent the standard deviation within control, FM and PM groups. p \ 0.05, *p \ 0.01, NS not signiﬁcant as determined by Student’s t test Fig. 3 Expression of FMR5, FMR6, FMR1 and FMR4 in the brain tissue of control, full mutation and premutation individuals. Expres- sion of FMR5 (a), FMR6 (b), FMR1 (c) and FMR4 (d) was measured in control, full mutation and premutation brain tissue by qPCR. Data were normalized to expression of glucose-6-phosphate dehydrogenase (G6PD), the housekeeping gene. In each panel, error bars for individual patients represent the standard deviation of four technical replicates. For mean expression values, error bars represent the standard deviation within control, FM and PM groups. p \ 0.05, **p \ 0.01, NS not signiﬁcant as determined by Student’s t test 12 Hum Genet (2014) 133:59–67 64 To explore the possibility that these novel transcripts contribute to FXS pathogenesis, we assessed FMR5 and FMR6 expression in post mortem brain tissue from full and premutation individuals and unaffected controls. RNA expression was quantiﬁed in tissue from 11 controls of mixed gender, 7 male full mutation (FM) and 5 male premutation (PM) individuals. On average, FMR5 expres- sion did not differ signiﬁcantly between full mutation, premutation and control samples (Fig. 3a). However, FMR6 expression was signiﬁcantly decreased in both the full mutation and premutation groups compared to controls, with the exception of sample #NICHD1421, which expresses FMR1 at levels similar to controls (Fig. 3b). All other full mutation samples have, as expected, reduced expression of FMR1 (Fig. 3c). We also determined expression levels of FMR4, a lncRNA previously reported by us (Khalil et al. 2008), in patient brain tissue. Results FMR4 was not detectable in full mutation (FM) samples, and its expression was increased in premutation (PM) samples compared to controls (Fig. 3d). A no-RT negative control was included in the detection of all ncRNA transcripts to ensure that the signal was not accounted for by genomic DNA contamination. The levels of FMR1, FMR4, FMR5 and FMR6 were variable in FXS and premutation brain samples. It is likely that differences in the number of repeats, extent of DNA methylation or extent of histone modiﬁcations causes this variability. Variable expression levels of these transcripts may translate into different clinical outcomes. These lncRNAs may thus be useful as biomarkers for FXS and FXTAS. To determine whether lncRNA expression is linked to clinical phenotype, it is necessary to measure these transcripts in a large cohort of patients, and blood presents the most practical option for both experimental and potential prognostic purposes. leukocytes in a limited number of patients (control n = 2, premutation n = 2, full mutation n = 2) by strand-speciﬁc reverse transcription quantitative PCR (RT-qPCR). All control and PM samples demonstrated robust FMR1 expression; additionally, a moderate level of FMR1 mRNA was detected in one of two FM patient samples, likely due to mosaicism (Fig. 4a). Expression of FMR4, FMR5, and FMR6 was also detectable in the majority of patient leu- kocyte RNA samples albeit expression levels were lower than those of FMR1 (Fig. 4b–d). These results establish the feasibility of conducting studies correlating expression levels of FMR4, FMR5, and FMR6 with clinical outcomes in FXS and FXTAS patients. Discussion As discussed above, FMR5 and FMR6 have distinct expression patterns, and additional studies are necessary to clarify whether any potential regulatory function of each transcript may contribute to FXS/FXTAS phenotypes. FMR6 is complementary to the 30 region of FMR1 and may therefore bind to the FMR1 mRNA, thereby regulating FMR10s stability, splicing, subcellular localization and translational efﬁciency. These regulatory functions have been described for other lncRNAs (reviewed in (Faghihi and Wahlestedt 2009)). For instance, stability of BACE1 mRNA is positively regulated by an antisense lncRNA to BACE1 called BACE1AS (Faghihi et al. 2008). The possi- bility that FMR6 regulates FMR1 mRNA splicing may be relevant since extensive alternative splicing of FMR1 has been demonstrated (Huang et al. 1996; Sittler et al. 1996). Finally, as FMR6 overlaps two microRNA binding sites for miR-19a and miR-19b in the 30UTR of FMR1 (Edbauer et al. 2010), it is possible that this lncRNA can modulate stability or translational efﬁciency of FMR1 through interference with microRNA binding. FMR6 is a spliced antisense-oriented lncRNA that overlaps exons 15-17 as well as the 30UTR of FMR1. Unexpectedly, the splicing sites in FMR6 correspond exactly to those of FMR1. Although very little is known about the consensus sequences for splicing of noncoding RNAs, it is possible that the reverse complement of the canonical sites in FMR1 are being recognized as non- canonical consensus sequences by the splicing machinery. Programs such as ‘‘Human Splicing Finder’’ (http://www. umd.be/HSF) can be used to predict non-canonical splicing sites by incorporating matrices for auxiliary sequences (Desmet et al. 2009). Further studies are required to address this possibility. The fact that FMR1-derived lncRNAs are differentially expressed in FXS and FXTAS suggests their usefulness as biomarkers for these diseases. The use of lncRNAs as biomarkers for human disease is a rather novel concept. LncRNAs have emerged as novel diagnostic/prognostic biomarkers in bodily ﬂuid samples of cancer patients. One example is the lncRNA prostate cancer antigen 3, which can be detected in urine samples and has been shown to improve diagnosis of prostate cancer (de Kok et al. 2002; Reis and Verjovski-Almeida 2012). Investigating the relationship between differential lncRNA expression and clinical outcomes requires screening a large number of patients with various degrees of deﬁned FXS or FXTAS symptoms. Discussion Graphical representation of the previously described and newly reported lncRNAs expressed from the FMR1 locus. FMR5 is transcribed from the FMR1 promoter in the sense direction, beginning around 1 kb upstream from the FMR1 transcription start site. FMR6 is an antisense-oriented lncRNA produced from the 30UTR of FMR1 unaffected by the repressive chromatin modiﬁcations dis- cussed above. Therefore it is possible that the observed reduction in FMR6 expression is a consequence of histone changes associated with the FM, rather than the DNA methylation responsible for FMR1 silencing. An unanticipated result is that FMR6 expression is reduced in premutation-range samples; however, the chromatin marks associated with the 30 end of FMR1 in premutation carriers are yet to be described. As mentioned previously, premutation-range expansions in the CGG repeat region are reported to result in an open chromatin state and increased FMR1 transcription (Tan et al. 2009). Our data suggest that in addition, the premutation-range CGG expansion somehow inﬂuences transcription or chromatin state near the far-distal 30 end of the gene at the premutation stage. Finally, we found that FMR4, similarly to FMR1, is downregulated in brain from FM patients and upregulated in PM carriers as we previously reported in blood leukocytes (Khalil et al. 2008). Fig. 5 The transcriptional landscape of the FMR1 gene locus is complex. Graphical representation of the previously described and newly reported lncRNAs expressed from the FMR1 locus. FMR5 is transcribed from the FMR1 promoter in the sense direction, beginning around 1 kb upstream from the FMR1 transcription start site. FMR6 is an antisense-oriented lncRNA produced from the 30UTR of FMR1 trimethylation of histone H3 at lysine 9 (H3K9me3) and trimethylation of histone H4 at lysine 20 (H4K20me3) associate with exon 1 of FMR1, which contains the CGG repeats, but do not associate with the promoter region (Kumari and Usdin 2010). On the other hand, levels of three active chromatin marks, H3 acetylation (H3Ac), H4 acetylation (H4Ac) and H3K4 dimethylation (H3K4me2), were reportedly lowered at the FMR1 promoter in FXS (Gheldof et al. 2006). Furthermore, Kumari et al. (2010) reported the presence of uncharacterized antisense lncR- NAs in the FMR1 promoter of both normal and full mutation cells, suggesting that the presence of repressive histone marks in the FMR1 locus would not necessarily inhibit the transcription of low abundance transcripts such as FMR5. Discussion In recent years, the importance of lncRNAs in many aspects of cell biology has received increasing attention from the scientiﬁc community. For decades, these tran- scripts were regarded as transcriptional noise, and thus their role in many disease processes was overlooked. Growing evidence points to a multitude of functions per- formed by lncRNA, including regulation of transcription and chromatin remodeling (Lee 2012). Here we report the discovery of two novel transcripts that originate from the FMR1 locus, adding complexity to a locus already known to produce other lncRNAs (Khalil et al. 2008; Kumari and Usdin 2010; Ladd et al. 2007) (Fig. 5). The ﬁrst lncRNA we report, FMR5, is a sense-oriented transcript that overlaps the FMR1 promoter. The FMR5 transcription start site (TSS) is located 1 kb upstream of the FMR1 TSS. FMR5 showed similar expression levels in control, FM and PM brain tissue, suggesting that FMR5 transcription remains independent of chromatin modiﬁca- tions in FM and PM carriers. This is consistent with the ﬁnding that in FXS repressive chromatin marks such as To determine whether we can detect these novel lncR- NAs in blood samples, we measured FMR4, FMR5 and FMR6 expression in RNA extracted from peripheral blood Fig. 4 FMR1, FMR4, FMR5 and FMR6 are detectable in peripheral blood leukocytes. Expression of FMR1 (a), FMR4 (b), FMR5 (c) and FMR6 (d) RNA was measured in leukocytes from the peripheral blood of control, full mutation and premutation individuals. Data were normalized to the housekeeping gene cyclophilin (CYC). Error bars represent the standard deviation of four technical replicates Fig. 4 FMR1, FMR4, FMR5 and FMR6 are detectable in peripheral blood leukocytes. Expression of FMR1 (a), FMR4 (b), FMR5 (c) and FMR6 (d) RNA was measured in leukocytes from the peripheral blood of control, full mutation and premutation individuals. Data were normalized to the housekeeping gene cyclophilin (CYC). Error bars represent the standard deviation of four technical replicates blood of control, full mutation and premutation individuals. Data were normalized to the housekeeping gene cyclophilin (CYC). Error bars represent the standard deviation of four technical replicates Fig. 4 FMR1, FMR4, FMR5 and FMR6 are detectable in peripheral blood leukocytes. Expression of FMR1 (a), FMR4 (b), FMR5 (c) and FMR6 (d) RNA was measured in leukocytes from the peripheral 3 Hum Genet (2014) 133:59–67 65 Fig. 5 The transcriptional landscape of the FMR1 gene locus is complex. Discussion We have demonstrated through our six-patient Our data show that FMR6 expression is signiﬁcantly downregulated in FXS brain samples, as is expected due to the reported decrease in H4Ac and H3K4me2 and increase in H3K9me2 throughout the FMR1 locus, including the 30 region of FMR1 (Gheldof et al. 2006). However, DNA methylation is restricted to the CGG repeat region at 50 end of the gene. One full mutation case in our study, NICHD#1421, displayed robust FMR1 expression in addition to higher expression of FMR6 compared to other FM patients. It is likely that in this case the 30UTR is 12 3 Hum Genet (2014) 133:59–67 66 RNAs are rarely translated in two human cell lines. Genome Res 22:1646–1657 pilot study that such a screening can be performed, as FMR4, FMR5 and FMR6 are detectable in peripheral blood leukocytes. If variability in expression of these transcripts in FM and PM individuals correlates with clinical vari- ability, it may be feasible to stratify FXS or FXTAS patients for early intervention and improve clinical outcomes. Brown V, Small K, Lakkis L, Feng Y, Gunter C, Wilkinson KD, Warren ST (1998) Puriﬁed recombinant FMRp exhibits selective RNA binding as an intrinsic property of the fragile X mental retardation protein. J Biol Chem 273:15521–15527 Chen L, Toth M (2001) Fragile X mice develop sensory hyperreac- tivity to auditory stimuli. Neuroscience 103:1043–1050 Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G et al (2005) Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308:1149–1154 In this study we used an innovative approach called Deep-RACE, which combines RACE and next generation sequencing (Olivarius et al. 2009) to identify novel tran- scripts in high-throughput manner. This technique is highly sensitive and enables detection of very low abundance transcripts. Although our study uncovered two new lncR- NAs, it is possible that we might have missed additional FMR1-derived transcripts because the RACE reaction is blocked by DNA sequences with high GC content, such as found in the promoter and 50UTR of the FMR1 gene. While there may well be other transcripts that are yet to be identiﬁed, this report of two novel lncRNAs, FMR5 and FMR6, further highlights the complexity of the FMR1 transcriptional landscape (Fig. 5). The functional proper- ties of these lncRNAs remain to be explored. 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https://openalex.org/W1964061173	http://file.scirp.org/pdf/JEMAA_2014092510183058.pdf	English	null	Some Consequences of Zero Point Energy	Journal of electromagnetic analysis and applications	2,014	cc-by	7,148	Abstract Both theory and experiments indicate that the vacuum is not a state of empty space, but is popu- lated by electromagnetic fluctuations at a lowest nonzero level, the Zero Point Energy (ZPE). This debouches into considerable changes of fundamental physics, as shown by a revised quantum electrodynamic theory (RQED) applied to elementary particles, and by a revised ZPE frequency spectrum applied to the expanding universe. The Standard Model based on a vacuum state of empty space is thus replaced by RQED, thereby resulting in massive elementary particles from the beginning, independently of the theory by Higgs. Also the basic properties of the Higgs-like par- ticle detected at CERN can be reproduced by RQED. It further leads to new fundamental results beyond the theories by Dirac and Higgs, such as to a deduced value of the elementary net charge, magnetic confinement of charged particle configurations, intrinsic local particle charges, photon spin with a very small but nonzero photon rest mass, and needle-like particle-wave properties which contribute to the understanding of the photoelectric effect and two-slit experiments. The real macroscopic pressure due to the revised ZPE frequency distribution further influences the dynamics of the expanding universe, by the ZPE photon pressure gradient acting as dark energy, and the ZPE photon energy density acting as dark matter. This results in a model being consistent with the observed scale, the rate of expansion, and the stability of a flat expanding observable un- iverse. Keywords Bo Lehnert Alfvén Laboratory, Royal Institute of Technology, Stockholm, Sweden Email: bo.lehnert@ee.kth.se Received 12 June 2014; revised 8 July 2014; accepted 1 August 2014 Copyright © 2014 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Copyright © 2014 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY http://creativecommons.org/licenses/by/4.0/ Journal of Electromagnetic Analysis and Applications, 2014, 6, 319-327 Published Online September 2014 in SciRes. http://www.scirp.org/journal/jemaa http://dx.doi.org/10.4236/jemaa.2014.610032 Journal of Electromagnetic Analysis and Applications, 2014, 6, 319-327 Published Online September 2014 in SciRes. http://www.scirp.org/journal/jemaa http://dx.doi.org/10.4236/jemaa.2014.610032 Journal of Electromagnetic Analysis and Applications, 2014, 6, 319-327 Published Online September 2014 in SciRes. http://www.scirp.org/journal/jemaa http://dx.doi.org/10.4236/jemaa.2014.610032 How to cite this paper: Lehnert, B. (2014) Some Consequences of Zero Point Energy. Journal of Electromagnetic Analysis and Applications, 6, 319-327. http://dx.doi.org/10.4236/jemaa.2014.610032 1. Introduction As shown in reviews of quantum mechanics by Pauling and Wilson [1] and Schiff [2] among others, the solu- tions of the one-dimensional harmonic oscillator do not only lead to the Planck electromagnetic wave energy but also to a lowest nonzero energy level of one-half quantum per state, i.e. the Zero Point Energy (ZPE). Conse- How to cite this paper: Lehnert, B. (2014) Some Consequences of Zero Point Energy. Journal of Electrom and Applications, 6, 319-327. http://dx.doi.org/10.4236/jemaa.2014.610032 B. Lehnert quently, the vacuum state is not merely that of an empty space. It includes quantum fluctuations part of which also carry electric charges, as pointed out by Abbot [3]. An example of these fluctuations was given by Casimir [4] who predicted that two metal plates will attract each other when being sufficiently close together. This is due to the fact that only small wavelengths of the fluctuations can exist in the spacing between the plates, whereas the full spectrum exerts a net force on the outsides of the plates. The same force was first demonstrated experi- mentally by Lamoreaux [5], in using a sensitive torsional pendulum. Its relation to the ZPE was already realized by Casimir [4], and further elucidated by Milonni [6] in a review. This important and experimentally confirmed result thus reveals the existence of a real macroscopic Casimir pressure and energy density originating from the ZPE. At his time, Casimir made the modest conclusion that his discovered “zero point pressure of electromagnetic waves might be of a certain interest”. Nevertheless later investigations indicate that the consequences of ZPE will debouch into considerable changes of fundamental physics, thereby removing severe limitations and weak points of the Standard Model of elementary particles. The present review demonstrates some of the consequences which originate from the Zero Point Energy, as outlined in Figure 1 and based on recent investigations by the author. The latter deal with the physics of the smallest elementary particles being treated in terms of a revised quantum electrodynamic theory (RQED) [7] [8], as well as the physics of the largest cosmical phenomena treated by means of a revised frequency distribution of the ZPE vacuum fluctuations [9]. For detailed deductions reference is made to the publications mentioned in this review. 2. Revised Quantum Electrodynamics 2.1. General Features of the Field Equations 2.1. 1. Introduction General Features of the Field Equations The present field equations are of the general four-dimensional Lorentz invariant form [10] The present field equations are of the general four-dimensional Lorentz invariant form [10] Figure 1. Some consequences of Zero Point Energy on the fundamental physics of the smallest elementary particles and on the largest cosmical phenomena. Figure 1. Some consequences of Zero Point Energy on the fundamental physics of the smallest elementary particles and on the largest cosmical phenomena. Figure 1. Some consequences of Zero Point Energy on the fundamental physics of the smallest elementary particles and on the largest cosmical phenomena. 320 B. Lehnert 2 2 0 2 2 1 c t µ   ∂ ∇− = −   ∂   V J (1) 2 2 0 2 2 1 c t µ   ∂ ∇− = −   ∂   V J 2 0 2 2 1 t µ  ∂ = −  ∂  V J (1) (1) here ( ) ,i c φ = V A is a four-potential with A as a three-dimensional magnetic vector potential, φ as an elec- trostatic potential, c standing for the velocity constant of light with 2 0 0 1 c µ ε = , ( ) ,icρ = J j being a four- current density with j as a three-dimensional current density, and ρ as an electric charge density. SI units are being used. The special case 0 ≡ J leads to the d’Alembert equation of an empty vacuum state. Then there are no local sources of the electromagnetic field, and there is no scope for a local energy density in a steady state which is the condition for particle models with nonzero rest mass and with electric charge. This is supported by a state- ment by Quigg [11] which implies that the symmetry of the conventional field equations of the Standard Model makes them strongly restricted and does not permit masses for leptons and quarks. Under the more general conditions of a nonzero current density J, and an associated nonzero electric charge density 0div ρ ε = E , there is a substantial change and an increased scope of the solutions originating from the field Equation (1). 1. Introduction Since J has to become invariant in a transition from one inertial frame K to another frame K′, the form of J should satisfy the condition 2 2 2 2 2 2 . j c j c const ρ ρ ′ ′ − = − = (2) (2) Further, since J has to vanish when there is no charge density ρ , the final form of J becomes [7] Further, since J has to vanish when there is no charge density ρ , the final form of J becomes [7] ( ) 2 2 ,ic c ρ = = J C C (3) (3) where C is a velocity vector in three-space. This can be regarded as a generalization of the Lorentz invariance from one to more dimensions, such as from plane to cylindrical waves. In a three-dimensional representation the extended field Equation (1) in the vacuum then become where C is a velocity vector in three-space. This can be regarded as a generalization of the Lorentz invariance from one to more dimensions, such as from plane to cylindrical waves. In a three-dimensional representation the extended field Equation (1) in the vacuum then become ( ) 0 0 0 curl div t µ ε ε ∂ = + ∂ E B E C (4) curl t ∂ = −∂ B E (5) curl div 0 = = B A B (6) t φ ∂ = −∇−∂ A E (7) 0 div ρ ε = E (8) (4) (5) (6) (7) 0 div ρ ε = E (8) thus being characterized by a broken symmetry between the electric and magnetic field strengths E and B. As compared to the conventional equations, the new features of these equations are represented by the space-charge current density of the first term in the right-hand member of Equation (4), and of Equation (8). thus being characterized by a broken symmetry between the electric and magnetic field strengths E and B. As compared to the conventional equations, the new features of these equations are represented by the space-charge current density of the first term in the right-hand member of Equation (4), and of Equation (8). These equations are also gauge invariant, because the new contributions include the electric field strength E. As in conventional electromagnetic theory, they can be treated in an analogous way in a gauge transformation of the potentials A and φ . 2 2 0 2 2 1 c t µ   ∂ ∇− = −   ∂   V J 1. Introduction The general properties of the field Equations (4)-(8) can now elucidate the connections between a nonzero div E and a nonzero ZPE. The questions on a revised spectral distribution of ZPE are postponed to Section 3. Here it is observed that steady electromagnetic states become possible through the source term of the space-charge cur- rent density in Equation (4), i.e. also when 0 t ∂∂= . There is then a nonzero energy density of the alternative form ( ) 1 2 s w ρ φ = + ⋅ C A (9) (9) including the charge density ρ . All basic features of ZPE can thus be reproduced by a nonzero local div E as follows: including the charge density ρ . All basic features of ZPE can thus be reproduced by a nonzero local div E as follows: • Local electrically charged as well as neutral electromagnetic fluctuations generated by this source. N l l i h ll i hi h f i ll i d i l d l • Net total electric charges as well as vanishing net charges of spatially integrated particle models. 321 321 B. Lehnert B. Lehnert • Intrinsic local charges of both polarities arising within limited regions of the particle models. • A nonzero spatially integrated total field energy and rest mass of such models. As described by Schiff [2] among others, Maxwell’s equations are used as a guideline for proper interpreta- tions of the conventional quantum electrodynamic theory. This also applies to the present extended equations. Here a short-cut and simplification will be made, by first determining the general solutions of the same equa- tions, and then imposing relevant quantum conditions. This is at least justified by the fact that the quantized eq- uations become identical to the original equations in which the potential V and current density J are merely be- ing replaced by their expectations values, as shown by Heitler [12]. Such a way of handling the quantum condi- tions should therefore not be too far from the truth, by using the most probable trajectories and states in a first approximation. It may finally be mentioned that, in a speculation by the author about a generalized law of gravitation, the lat- ter is proposed to be extended to full symmetry. 2.2.1. Steady Electromagnetic States Steady axisymmetric states representing particle models in a frame ( ) , , r θ ϕ of spherical coordinates, with all relevant quantities being independent of the angle φ, lead to a current density ( ) 0,0,Cρ = j and a magnetic vector potential ( ) 0,0, A = A . Here C c = ± represents the two spin directions. The general solutions can then be expressed in terms of a generating function ( ) ( ) 0 , , F r CA G G θ φ ρ θ = − = (10) (10) where G0 stands for a characteristic amplitude, G is a normalized dimensionless part, and 0 r r ρ = with r0 as a characteristic radial dimension. This yields the general solutions ds for a characteristic amplitude, G is a normalized dimensionless part, and 0 r r ρ = with r0 as a radial dimension. This yields the general solutions ( ) 2 sin CA DF θ = − (11) ( ) 2 1 sin D F φ θ   = − +   (12) ( ) ( ) 2 2 2 0 0 1 sin r D D F ρ ε ρ θ   = − +   (13) (11) (12) (13) with the operator D D D ρ θ = + where 2 2 2 cos sin D D ρ θ θ ρ ρ ρ θ θ θ   ∂ ∂ ∂ ∂ = − = − − ×   ∂ ∂ ∂ ∂   . (14) (14) These solutions lead to charged and neutral particle models of both matter and antimatter, having spin and rest mass. This is in conformity with the equation by Dirac which includes the elementary charge “e” and the elec- tron mass “me”, but as given and assumed parameters [10]. The mass me is then associated with a nonzero spin. the special results which come out of the present RQED theory, the following can be mentioned: Among the special results which come out of the present RQED theory, the follow • The theory by Higgs is based on a spontaneous nonlinear mechanism of symmetry breaking in an empty va- cuum state, by which an unstable boson of unspecified but large rest mass is being formed. This particle then decays into a succession of massive elementary particles. 1. Introduction Such a law would include both polarities of corresponding charge and attractive as well as repulsive gravitational forces, in analogy with the Coulomb interaction [7]. An additional question would then follow, i.e. whether states of positive and negative mass could become associated with those of matter and antimatter. 2.2. New Fundamental Results of RQED At this point more detailed descriptions can be given on the results which come out of the basic field equations, in respect to steady as well as time-dependent states. 2.2.1. Steady Electromagnetic States In the present RQED theory the source due to the space-charge current density results in elementary particles with rest mass, already from the beginning [8] [13] [14]. It is characterized by intrinsic linear broken symmetry. This provides an alternative explanation of the Higgs-like particle observed at CERN [15]-[17]. 18 • The theory by Higgs is based on a spontaneous nonlinear mechanism of symmetry breaking in an empty va- cuum state, by which an unstable boson of unspecified but large rest mass is being formed. This particle then decays into a succession of massive elementary particles. In the present RQED theory the source due to the space-charge current density results in elementary particles with rest mass, already from the beginning [8] [13] [14]. It is characterized by intrinsic linear broken symmetry. This provides an alternative explanation of the Higgs-like particle observed at CERN [15]-[17]. • The present theory yields a model of the Z boson having an effective radius of about 10−18 m, in agreement with measurements [14]. The decay of a Higgs-like boson into two Z bosons further initiates the idea of su- perimposing two Z bosons to form a model of such a particle. The resulting composite particle solution is • The present theory yields a model of the Z boson having an effective radius of about 10−18 m, in agreement with measurements [14]. The decay of a Higgs-like boson into two Z bosons further initiates the idea of su- perimposing two Z bosons to form a model of such a particle. The resulting composite particle solution is 322 B. Lehnert consistent with the point made by Quigg [11] that the Higgs is perhaps not a truly elementary particle, but is built out of as yet unobserved constituents. Thereby the present theory on a Higgs-like boson satisfies the ba- sic properties of the particle observed at CERN [16] [17], in having vanishing charge, vanishing spin, an ef- fective radius of about 10−18 m corresponding to a rest mass of about 125 GeV, and an unstable behavior due to its purely electrostatic nature [13] [14]. consistent with the point made by Quigg [11] that the Higgs is perhaps not a truly elementary particle, but is built out of as yet unobserved constituents. 2.2.1. Steady Electromagnetic States • In a variational analysis the absolute value of the deduced net elementary charge is found to fall within a narrow parameter range, positioned around the experimental value “e” and having the width of only a few percent of “e”, as shown by Lehnert and Scheffel [21] and Lehnert and Höök [19] [20]. • In a variational analysis the absolute value of the deduced net elementary charge is found to fall within a narrow parameter range, positioned around the experimental value “e” and having the width of only a few percent of “e”, as shown by Lehnert and Scheffel [21] and Lehnert and Höök [19] [20]. • In the present theory a confining magnetic field prevents the charged leptons from “exploding” under the ac- tion of their electrostatic eigenforce [7] [20]. • In the RQED lepton models there also exist local intrinsic charges of both polarities, being an order of mag- nitude larger than the net elementary charge “e”. The resulting Coulomb interaction becomes about two or- ders of magnitude larger than that due to the net charge. If such conditions would also hold true for quarks, this total Coulomb force would become comparable and similar to the short-range strong force. This raises the question whether the intrinsic charge force will interfere with the strong force, or even become identical with it, as stated in a speculation by the author [22]. 2.2.1. Steady Electromagnetic States Thereby the present theory on a Higgs-like boson satisfies the ba- sic properties of the particle observed at CERN [16] [17], in having vanishing charge, vanishing spin, an ef- fective radius of about 10−18 m corresponding to a rest mass of about 125 GeV, and an unstable behavior due to its purely electrostatic nature [13] [14]. p y • With a separable generating function ( ) ( ) G R T ρ θ = ⋅ of Equation (10), the spatially integrated net electric charge q0 and magnetic moment M0 of a particle-shaped state become subject to a number of symmetry properties. These are due to the convergence or divergence of R(ρ) at the origin ρ = 0, and the top-bottom symmetry or antisymmetry of T(θ) in respect to the equatorial plane π 2 θ = . Thus a divergent R(ρ) in com- bination with top-bottom symmetry of T(θ) is the only case leading to a charged particle such as the electron with q0 ≠ 0 and M0 ≠ 0, whereas q0 = 0 and M0 = 0 in all other cases, such as for a model of the neutrino [7]. • As described by Ryder [18] among others, the conventional divergence problem of infinite self-energy in point-charge-like geometries is tackled in a process of renormalization, by adding extra ad hoc counter terms to the Lagrangian. In this way a finite result is obtained from the difference between two “infinities”. In the present theory, the model of a charged particle is on the other hand treated in terms of a revised renormaliza- tion procedure, applied to the radial part R(ρ). Thereby the “infinity” of the latter is outbalanced by the “zero” of a “counter-factor”. This results in finite values of the net charge q0, magnetic moment M0, rest mass m0, and spin s0, associated with a very small characteristic radius of the model. In this way the problem of infi- nite self-energy can thus be tackled, as shown in more detail by the author [7] [19] and by Lehnert and Höök [20]. This revised process then has the result that all quantum conditions and all relevant experimental values of charge, magnetic moment, mass, and spin of the leptons can be reproduced by the choice of only two sca- lar free parameters, the so called first and second counter-factors. 2.2.2. Time-Dependent Electromagnetic States With the field Equations (4)-(8) including an explicit time dependence, the wave-particle dualism will become an intrinsic property of the resulting solutions. A physically relevant photon model should have the form of a wave or wave packet of preserved and limited geometrical shape, propagating in an undamped way and in a de- fined direction. No artificial boundaries have to be imposed. The angular momentum in the direction of propa- gation, the spin, should have the constant value 2π h . This leads to the representation in a cylindrical frame ( ) , , r z ϕ with z in the direction of propagation. The velocity vector in Equation (4) then has the form ( ) 0,cos ,sin c α α = C (15) ( ) 0,cos ,sin c α α = C (15) (15) leading to the dispersion relation leading to the dispersion relation ( ) sin kv v c ω α = = (16) (16) for a normal wave of the form ( ) ( ) exp f r i t kz ω − +     being associated with the photon mode. Here cos sin α α  , and the spin is provided at the expense of a very small reduction of the phase and group veloci- ties v from the value c. for a normal wave of the form ( ) ( ) exp f r i t kz ω − +     being associated with the photon mode. Here cos sin α α  , and the spin is provided at the expense of a very small reduction of the phase and group veloci- ties v from the value c. Some specific results can here be mentioned as follows [7]: Some specific results can here be mentioned as follows [7]: • The Standard Model leads to a vanishing total (integrated) spin of the individual photon [23], in contradic- tion with experiments. This is shown in terms of the quantized solutions obtained from the d’Alembert equa- tion in an empty vacuum state. The present theory of a cylindrical wave-packet model results on the other • The Standard Model leads to a vanishing total (integrated) spin of the individual photon [23], in contradic- tion with experiments. This is shown in terms of the quantized solutions obtained from the d’Alembert equa- tion in an empty vacuum state. 2.2.2. Time-Dependent Electromagnetic States This contributes to the understanding of electron-positron pair formation. • Introducing the alternative form g g g p y vidual photon wave packet. This contributes to the understanding of electron-positron pair formation. • Introducing the alternative form vidual photon wave packet. This contributes to the understanding of electron positron pair formation. • Introducing the alternative form ( ) ( ) 0, sinh ,cosh 0, , z c i c C C φ α α = = C (17) (17) of the velocity vector with α > 0, the dispersion relation becomes ( ) cosh z kC ku u c c ω α = = = > (18) (18) ( ) cosh z kC ku u c c ω α = = = > for a superluminal normal tachyon mode [7]. This mode and resulting relations become identical with those of the earlier elaborated tachyon theory given in reviews by Recami [27] and Bilaniuk, Deshpande and Su- darshan [28]. for a superluminal normal tachyon mode [7]. This mode and resulting relations become identical with those of the earlier elaborated tachyon theory given in reviews by Recami [27] and Bilaniuk, Deshpande and Su- darshan [28]. for a superluminal normal tachyon mode [7]. This mode and resulting relations become identical with those of the earlier elaborated tachyon theory given in reviews by Recami [27] and Bilaniuk, Deshpande and Su- darshan [28]. • One surprising consequence of quantum mechanics has been the entanglement of two or more distant par- ticles. This has raised the alternative question of nonquantum teleportation as stated by Rabounski and Bo- rissova [29]. At a first sight, the superluminal tachyon mode of large α in Equation (18) may be considered as a candidate for this purpose. Since the basic field Equations (4)-(8) with C2 = c2 apply both to expressions (15) and (17) for the photon and tachyon modes, a transition between these modes could be imagined in terms of the Complementarity principle of the Copenhagen school by Bohr. However, this has to be consi- dered as an open question needing further analysis, because superluminal speeds of the tachyons cannot be registered by a regular observer as anything but a motion at the speed of light, as stated by Rabounski and Borissova [29]. 2.2.2. Time-Dependent Electromagnetic States The present theory of a cylindrical wave-packet model results on the other 323 B. Lehnert hand in a spin, as well as in an extremely small but nonzero rest mass. The phase and group velocities are then hardly distinguishable from the velocity constant c. As an example, for a photon wave packet of a rele- vant and small characteristic radial dimension, the reduction of the velocity v in Equation (16) with respect to c occurs first in the tenth decimal. The photon spin and photon rest mass are mutually related, due to the ge- neralized Lorentz invariance. hand in a spin, as well as in an extremely small but nonzero rest mass. The phase and group velocities are then hardly distinguishable from the velocity constant c. As an example, for a photon wave packet of a rele- vant and small characteristic radial dimension, the reduction of the velocity v in Equation (16) with respect to c occurs first in the tenth decimal. The photon spin and photon rest mass are mutually related, due to the ge- neralized Lorentz invariance. • For the individual photon both a particle behaviour in the form of needle-like radiation and a wave behaviour in the form of interference phenomena can simultaneously be realized [24]. This satisfies the necessary crite- ria for the observed behaviour of the photoelectric effect and that in two-slit experiments. • The present theory on screw-shaped wave modes is consistent with the observed hollow geometry of cork- screw-shaped light beams [25]. • The present theory on screw-shaped wave modes is consistent with the observed hollow geometry of cork- screw-shaped light beams [25]. • The detected angular momentum of a light beam having a spatially limited cross-section can be explained by spin contributions from its boundary layers [26]. • The detected angular momentum of a light beam having a spatially limited cross-section can be explained by spin contributions from its boundary layers [26]. • The nonzero electric field divergence leads to intrinsic electric charges of alternating polarity with vidual photon wave packet. This contributes to the understanding of electron-positron pair formati • The nonzero electric field divergence leads to intrinsic electric charges of alternating polarity with vidual photon wave packet. This contributes to the understanding of electron-positron pair formati • The nonzero electric field divergence leads to intrinsic electric charges of alternating polarity within an indi- vidual photon wave packet. 3.2. New Fundamental Results of the ZPE Frequency Distribution 10 2 • An observed acceleration of about 4 × 10−10 m/s2 at the present stage of expansion can be expl namic unbalance due to a dominating dark energy of about 75% [9]. ed acceleration of about 4 × 10−10 m/s2 at the present stage of expansion can be explained by a dy- alance due to a dominating dark energy of about 75% [9]. • The expanding cloud of ZPE photons has at present the character of a nearly flat Euclidian geometry. This is found to be consistent with a stable dynamic state being subject to expansive or compressive perturbations [37]. • In a proposed generalized theory of gravitation with both polarities of mass and of mutual forces, a mechan- ism for separation of matter from antimatter may be provided at an early stage of the expanding universe [7] [38]. This could also reveal itself as a “dark flow” in certain regions. • In a proposed generalized theory of gravitation with both polarities of mass and of mutual forces, a mechan- ism for separation of matter from antimatter may be provided at an early stage of the expanding universe [7] [38]. This could also reveal itself as a “dark flow” in certain regions. which results in a total and local energy density 4 3 24π u h c ν = . (20) 4 3 24π u h c ν = . (20) here the average frequency ν is a so far arbitrary quantity, in the same way as the mean energy kT of the Planck distribution, and ν can also depend on its local position in the universe. The energy density in Equation (20) thus yields a finite total ZPE vacuum pressure 3 u . This revised frequency distribution further leads to an extended analysis of the Casimir force, with the aim of finding a method for determining the so far unknown average frequency ν [35]. This can be done at the largest available Casimir force, i.e. that acting between two metal plates with zero spacing and where the electromag- netic skin depth plays the rôle of an equivalent gap between the plates. To avoid microscopic matching of the plate metal structures, different plate metals should be used, also having different electric resistivities. As a re- sult, the Casimir pressure ˆp is then obtained as a function of the average frequency ν , and the shape of this function differs for different metals, thus giving a possibility for determination of the local value of ν . This would hold, provided that the plate adhering forces remain small as compared to the maximum Casimir force. 3.1. A Self-Consistent Distribution On account of the Casimir effect, the low-frequency part of the ZPE distribution has to be accepted as an expe- rimental fact, but there exists a crucial problem with the high-frequency part. As demonstrated by Terletskii [30], Milonni [6] and Loudon [31] among others, conventional theory results in a spectrum having an infinite total (integrated) energy density. Such a result is unacceptable, both from the physical point and from the point that the analysis is underdetermined and treats the included infinite number of states with the same statistical proba- bility. Several attempts have been made to truncate the involved integral at cut-off frequencies corresponding either to the Planck length or to an arbitrarily high energy of 100 GeV. This still leads to an excessive vacuum energy density being about 10120 or 1055 times greater than that being expected. There are also several investigators such as Riess and Turner [32] and Heitler [12] who have thrown doubt upon the conventional theory. To overcome these difficulties, the author has proposed that the ensemble of ZPE energy modes (photons) re- quires a separate treatment, also at the temperature limit T = 0, which first become self-consistent when the sys- tem is defined to have a given and finite total energy density [9] [33]-[35]. Such an analysis can be conducted in the standard way of an ensemble of ZPE photons with the frequency ν and energy 2 hν , populating the available states in statistical equilibrium with a probability given by the Bolzmann factor ( ) exp ν ν − , where 2 hν is the average particle energy of the ensemble. Then the energy density within the frequency range dν becomes 324 B. Lehnert ( ) ( ) 3 3 4π exp du h c d ν ν ν ν = − . (19) ( ) ( ) 3 3 4π exp du h c d ν ν ν ν = − . (19) ( ) ( ) 3 3 4π exp du h c d ν ν ν ν = − . (19) which results in a total and local energy density 3.2. New Fundamental Results of the ZPE Frequency Distribution vised ZPE frequency distribution has given rise to a number of new fundamental results as follows: The revised ZPE frequency distribution has given rise to a number of new fundamental results as follows: • This distribution of photons constitutes a real macroscopic pressure having an application to the large-scale dynamics of the expanding universe [9] [33] [34]. It is thus proposed that the pressure gradient of the ZPE photon gas can play the rôle of an expanding dark energy force, at the same time as the energy density of the same gas plays the rôle of gravitating dark matter and a contracting force. This should in particular apply to the present and later stages of the expanding universe. Being already in the lowest energy state, the ZPE photons do not radiate in a statistical equilibrium of maximum entropy. • The coincidence problem concerning equal orders of vacuum energy density and mass densit flexible, and does not have to be related to a fixed cosmological constant. • The coincidence problem concerning equal orders of vacuum energy density and mass de flexible, and does not have to be related to a fixed cosmological constant. • The present dynamical model of the expanding universe is reconcilable with the cosmical dimensions and the radius R0 = 1026 m of the observable universe [9]. This comes out from an average density 10−26 kg/m3 of normal matter according to Linde [36], the mass contents of 21% and 4% of dark and normal matter, and an integrated amount of dark matter having the mass • The present dynamical model of the expanding universe is reconcilable with the cosmical dimensions and the radius R0 = 1026 m of the observable universe [9]. 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Physical Review, 145, 1156-1168. http://dx.doi.org/10.1103/PhysRev.145.1156 [16] Aad, G., Abajyan, T., Abbott, B., Abdallah, J., Khalek, S.A., Abdelalim, A.A., et al. (2012) Observation of a New Par- ticle in the Search for the Standard Model Higgs Boson with the ATLAS Detector at the LHC. Physics Letters B, 716, 1-29. http://dx.doi.org/10.1016/j.physletb.2012.08.020 [17] Chatrchyan, S., Khachatryan, V., Sirunyan, A.M., Tumasyan, A., Adam, W., Aguilo, E., et al. (2012) CMS Collabora- tion. Observation of a New Boson at a Mass of 125 GeV with the CMS Experiment at the LHC. 4. Conclusions The same theory further leads to a number of fundamental results beyond those by Dirac and Higgs, such as a deduced value of the elementary charge positioned in a nar- row range close to its experimental value, a confining magnetic field which prevents the charged leptons from “exploding” under the action of their electrostatic eigenforce, large intrinsic and local electric charges of both polarities within the body of a particle configuration, a photon spin being associated with a very small but non- zero photon rest mass, and photon wave packet solutions of a needle-like character which contribute to the un- derstanding of the photoelectric effect and of two-slit experiments. The real macroscopic pressure due to the revised ZPE frequency distribution should further influence the large-scale dynamics of the expanding universe, in particular at its present and later stages. This concerns the mechanisms of dark energy and dark matter which are proposed to originate from the pressure gradient and mass density of the ZPE photon gas. The resulting theory leads to an explanation of the coincidence problem being independent of the cosmological constant, to an agreement between the deduced and observed scales of the un- iverse, to an accelerated expansion being consistent with observations, and to the stability of such a nearly flat universe. Also a “fine structure” with local variations and concentrations of ZPE dark matter would become im- aginable. g The future will indicate what is relevant and not relevant in these efforts and theories. 4. Conclusions According to both theory and experiments, the vacuum is not a state of empty space but is populated by elec- tromagnetic fluctuations at a lowest nonzero energy level, the Zero Point Energy. This review describes how the ZPE is included in a revised quantum electrodynamical theory (RQED) being applied on the scale of elementary particles. The ZPE also applies to the physics of large-scale cosmical phenomena such as the expanding universe, and in terms of a revised frequency distribution. The Standard Model of elementary particles, which is based on a vacuum state of empty space, is here re- placed by RQED theory. Thereby the ZPE is shown to be represented by a nonzero electric field divergence. This leads to massive elementary particles from the beginning, independently of and distinguished from the 325 B. Lehnert theory by Higgs. The basic features of the unstable Higgs-like particle recently detected in the experiments at CERN, such as zero charge, zero spin, and an effective radius of about 10−18 m being consistent with a rest mass of 125 GeV, are also reproduced by RQED theory. The same theory further leads to a number of fundamental results beyond those by Dirac and Higgs, such as a deduced value of the elementary charge positioned in a nar- row range close to its experimental value, a confining magnetic field which prevents the charged leptons from “exploding” under the action of their electrostatic eigenforce, large intrinsic and local electric charges of both polarities within the body of a particle configuration, a photon spin being associated with a very small but non- zero photon rest mass, and photon wave packet solutions of a needle-like character which contribute to the un- derstanding of the photoelectric effect and of two-slit experiments. theory by Higgs. The basic features of the unstable Higgs-like particle recently detected in the experiments at CERN, such as zero charge, zero spin, and an effective radius of about 10−18 m being consistent with a rest mass of 125 GeV, are also reproduced by RQED theory. References Physics Letters B, 716, 30-61. http://dx.doi.org/10.1016/j.physletb.2012.08.021 326 B. Lehnert [18] Ryder, L.H. (1966) Quantum Field Theory. 2nd Edition, Chapter 9, Cambridge University Press, Cambridge. [19] Lehnert, B. (2010) Deduced Fundamental Properties of the Electron. International Review of Physics (IRE 6. [20] Lehnert, B. and Höök, J. (2010) An Electron Model with Elementary Charge. Journal of Plasma Physics, 76, 419-428. [21] Lehnert, B. and Scheffel, J. (2002) On the Minimum Elementary Charge of an Extended Electromagnetic Theory. Phy- sica Scripta, 65, 200-207. http://dx.doi.org/10.1238/Physica.Regular.065a00200 [22] Lehnert, B. (2013) Intrinsic Charges and the Strong Force. Progress in Physics, 4, 17-20. [23] Lehnert, B. (2013) On the Angular Momentum and Rest Mass of the Photon. Journal of Plasma Ph 1135. [24] Lehnert, B. (2011) The Individual Photon in Two-slit Experiments. International Review of Physics (IREPHY), 5, 15- 18. [25] Lehnert, B. (2005) Screw-Shaped Light in Extended Electromagnetics. Physica Scripta, 72, 359-365. http://dx.doi.org/10.1238/Physica.Regular.072a00359 [26] Lehnert, B. (2006) Boundary Conditions and Spin of a Dense Light Beam. Physica Scripta, 74, 139-144. [27] Recami, E. (1986) Classical Tachyons and Possible Applications. La Rivista Del Nuovo Cimento, 9, 1-78. http://dx.doi.org/10.1007/BF02724327 [28] Bilaniuk, O.M, Deshpande, V.K. and Sudarshan, E.C.G (1962) “Meta” Relativity. American Journal of 718-723. [29] Rabounski, D. and Borissova, L. (2014) General Relativity Theory Explains the Sholl Effect and Makes Possible Fore- casting Earthquakes and Weather Cataclysms. Progress in Physics, 10, 63-70. [30] Terletskii, Y.D. (1971) Statistical Physics. North-Holland Publishing Company, Amsterdam, London. [31] Loudon, R. (2000) The Quantum Theory of Light. 3rd Edition, Oxford University Press, Oxford. [32] Riess, A.G. and Turner, M.S. (2004) From Slowdown to Speedup. Scientific American, 50-55. [33] Lehnert, B. (2009) Dark Energy and Matter of the Expanding Universe. Progress in Physics, 2, 77-82. [34] Lehnert, B. (2011) A Zero Point Distribution of Finite Density. International Review of Physics (IREPHY), 3, 304-308. [34] Lehnert, B. (2011) A Zero Point Distribution of Finite Density. International Review of Physics (IREPH [35] Lehnert, B. (2013) Extended Analysis of the Casimir Force. Progress in Physics, 10, 74-76. [36] Linde, A. (1994) The Self-Reproducing Inflatory Universe. Scientific American, 32-39. [37] Lehnert, B. (2013) On a Flat Expanding Universe. Advanced Studies in Theoretical Physics, 7, 191-197 [38] Lehnert, B. (2011) The Point Mass Concept. Progress in Physics, 2, 15-19.
https://openalex.org/W2075386319	https://bmcvetres.biomedcentral.com/counter/pdf/10.1186/1746-6148-10-98	English	null	A simplified evaluation system of surface-related lung lesions of pigs for official meat inspection under industrial slaughter conditions in Germany	BMC veterinary research	2,014	cc-by	8,665	* Correspondence: diana.meemken@tiho-hannover.de 2Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173 Hannover, Germany Full list of author information is available at the end of the article METHODOLOGY ARTICLE Open Access © 2014 Steinmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 A simplified evaluation system of surface-related lung lesions of pigs for official meat inspection under industrial slaughter conditions in Germany Thorsten Steinmann1, Thomas Blaha1 and Diana Meemken2* Thorsten Steinmann1, Thomas Blaha1 and Diana Meemken2* * Correspondence: diana.meemken@tiho-hannover.de 2Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173 Hannover, Germany Full list of author information is available at the end of the article © 2014 Steinmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Abstract Background: European and national administrative legislation require objective evaluation systems for organ lesions at pig slaughter. These results can be used as basis for herd health improvement programs by farmers and their consulting veterinarians. Various studies have shown that the current evaluation and recording of lesions by authorized meat inspectors are not reliable and produce significant inter-rater disagreement especially for lung lesions in pigs. The objectives of this study were to increase the usability of official meat inspection data by a developed and validated scheme and to analyze potential improvements in the reliability of the proposed system under industrialized slaughter conditions. Results: A simplified evaluation scheme for surface-related lung lesions was developed based on morphometric evaluations of unaffected lungs with quantitative relationships of each lobe to the whole lung (“Rule of Tens”). Furthermore, a theoretical as well as a hands-on training program for meat inspectors was developed and applied. Based on 5,183 lungs, the authors established a baseline of the inter-rater reliability of current routine assessments of lung lesions as documented by meat inspectors compared with the assessments of an independent veterinarian using the developed simplified evaluation scheme. Most frequent inter-rater disagreements greater than 75% were found for moderate pneumonia. Sources of the deviations most frequently included misinterpretations of technical artifacts, which were erroneously assessed by the meat inspectors as pneumonic lung lesions. Results of the post-training investigation based on 4,646 lungs showed a significantly improved reliability of lung lesion evaluation and the inter-rater agreement increased in all respects. Especially the disagreement of recording moderate cases of pneumonia decreased in total to 15% deviations from reference. Conclusions: The presented simplified lung evaluation scheme showed its capability to standardize the evaluation of lung lesions according to administrative legislation under industrialial slaughter conditions. The reliability of official meat inspections can be significantly increased with the help of the presented scheme to assess surface-related lung lesions of slaughter pigs. Continuous standardization and optimization can be achieved by personalized training programs in the framework of quality assurance systems for meat inspectors. Keywords: Risk-based meat inspection, Pneumonia, Reliability, Standardization, Training program, Slaughterhouse, Herd health, Swine, Porcine, Quality assurance Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 2 of 12 The score developed by Blaha [2] assesses pathological- anatomical lung lesions according to the estimating lung alteration. Background Official controls on products of animal origin intended for human consumption are important for protecting public health as well as animal health and animal welfare. The European Commission regulation calls for continuous improvement of all mentioned aspects, which have to be based on the most recent and relevant information avail- able. This information should constantly adjust current meat inspection practice [1]. Slaughter check results, which are obtained regularly during the slaughter process by official meat inspectors, have increasingly gained significance not only to protect the consumer but also to assess herd health of livestock [2-5]. After implementation of the risk-based meat inspection in the European Union, slaughter check results became important for farmers and veterinarians for several reasons. Firstly, feedback of slaughter check results to the farmers became mandatory in Europe [6]. Secondly, results impact the intensity of the meat inspection method [7-9]. Thirdly, slaughter check results are increasingly accepted as valuable indicators of herd health by farmers and their veterinarians [10-12,4]. Therefore, we report on a quantitative study to increase the reliability of official meat inspection based on a vali- dated, simplified and standardized scheme to assess surface-related lung lesions of slaughter pigs. According to a national administrative regulation in Germany, an objective evaluation system for organ le- sions has to be used for official meat inspections in abattoirs [13]. Outside Germany, diverse evaluation systems for organ lesions are in use. Particularly for lung lesions, various methods and standards are currently in place [14-17,2,18,19]. Godwin et al. [14] developed a 55- point lung lesion scheme, which is especially suitable for quantifying lesions caused by enzootic pneumonia in the cranial and medial lobes as well as cranial parts of the cau- dal lobes. The lung lesion scheme of Madec and Kobisch [15] divides each lobe into quarters, and scores each af- fected quarter with one point. The minimum score is zero point, i.e. the lung is completely unaltered, and the max- imum is 28 points (four points per lobe), i.e. all lobes are completely altered. Straw et al. [17] proposed a simple evaluation scheme for surface areas according to a “Rule of Ten”. However, underlying data in support of this scheme have not been published. Another more sophisti- cated data-based scheme was published by Christensen et al. [18]. Background Their scheme evaluates lung lesions depending on the weight of the altered lung lobe and reports the re- sult on a 100-points scale. Any alteration of pars cranialis of lobus cranialis pulmonis sinistri is denoted with five points, of pars caudalis of lobus cranialis pulmonis sinistri with six points, and of the lobus caudalis pulmonis sinistri with 29 points. In the right lung, the lobus cranialis scores eleven points, the lobus medius ten, the lobus caudalis 34, and the lobus accessorius five points, respectively. Abstract It scores alterations due to pneumonia from low (surface-related extent of lesion ≤10%) over moderate (surface-related extent of lesion 11-30%) to high (surface- related extent of lesion > 30%), denoted as Pneumonia 1 to 3 (Pn1, Pn2, Pn3). The German AVVLmH (2009) [12] proposes a modified evaluation scheme adopting the de- fined grades for inflammatory lung lesions from Blaha [2]. In addition, completely healthy lungs are defined as lungs without lesions (without any observable finding, result key o.b.B.) and merged with lungs having lesions extending to less than 10% of the total surface area (result key PN1) in result category 0. Lungs with lesions from 10 to 30% (result key PN2) are classified in category 1 and lungs with lesions with a surface extent more than 30% (result key PN3) in category 2. Although the German scoring systems are, at first glance, short and easy to handle, various stud- ies have shown that the current evaluation of lung lesions in identical organs by official meat inspectors (specialized authorized veterinarians and qualified assistants) is not reliable, produces significant inter-rater disagreement and unexplainable variation between different abattoirs [4,5]. Variance partitioning coefficients of a recent logistic multi- level analysis with cross-classified random effects of 20 post-mortem findings of official meat inspectors at an Austrian slaughterhouse suggest that especially meat inspection of scalding water lungs can be deemed as not sufficiently standardized [20]. The objective of this study was to increase the reliability of official meat inspection data on the basis of a validated, simplified and standard- ized scheme to evaluate surface-related lung lesions of slaughter pigs and to analyze potential improvements in the reliability of the proposed under field conditions. Morphometric quantification and definition of a simplified surface-related evaluation system for lung lesions For this purpose, the surfaces of macroscopically unaltered lungs of pigs at slaughter were measured and the quantita- tive proportion of each lobe to the whole lung was calcu- lated as a percentage of the total lung surface. In March 2011, five unaltered lungs, i.e. all over aerated lungs with a typical light salmon color (Figure 1), from pigs with an average of 100 to 110 kg slaughter weight were selected on a sample basis from a slaughterhouse in Lower Saxony, Germany. Both sides of the lungs were photographed with a digital camera. The distance between the working top and the tripod-fixed camera was 40 cm. The photographs of each lung were printed on cross-section paper (Max Bringmann, Wendelstein, Germany) with the smallest areal sensitivity of 1 mm2. Due to the anatomical condi- tions of the cranial lobes of the lung on both sides, lobes Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 3 of 12 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Figure 1 Lung surface morphometry, dorsal and ventral view on cross-section paper. Figure 1 Lung surface morphometry, dorsal and ventral view on cross-section paper. lobus accessorius pulmonis dextri [21], respectively. Each of the seven lobes was consecutively numbered on the ventral and dorsal side, and the surface proportion in relation to the total lung surface was calculated. After analyzing the results of the surface calculations of the five unaltered lungs, medians and arithmetic means per lung lobe were calculated. Based on the calculated means, simplifications of the proportion of each lobe were defined as a simplified surface-related evaluation scheme for lung lesions in order to improve operability of the assessments. were subdivided along a simulated line with 45° angle based on a horizontal line drawn at the bifurcatio trachea- lis (Figure 2). Subsequently, the lungs were schematically divided into lung lobes according to their anatomical nomenclature in lobus cranialis pulmonis sinistri pars cranialis and pars caudalis, lobus caudalis pulmonis sinistri, lobus cranialis pulmonis dextri, lobus medius pulmonis dextri, lobus caudalis pulmonalis dextri and Figure 2 Simplified lung scheme (dorsal) as standardized evaluating base for lung lesions (“Rule of Tens”). Status-quo investigation In order to establish a baseline of the inter-rater reliabil- ity of current routine assessments, results of routine assessments of lung lesions documented by Authorized Qualified Assistants (AQAs) were compared with the assessment results of an independent observer using the surface-related evaluation scheme. Between July and August 2011, the status-quo investigation of the actual evaluation of lung lesion according to the regulation of the German AVVLmH was performed in an abattoir in North Rhine Westphalia, Germany. The abattoir proc- essed 550 pigs per hour. Pigs were stunned by an auto- matic electro perfusion, and scalding was performed in a scalding tank. The official slaughter check included scoring of the lung lesions according to the system of the German AVVLmH (2009) [13] and was performed by 15 AQAs, who assessed one lung per five seconds. AQAs were professional butchers or farmers trained according to EC Regulation No 854/2004 [22] which is a non-academic training in Germany. All AQAs were subsequently certified and authorized for meat inspection by the State Government of North Rhine Westphalia, Figure 2 Simplified lung scheme (dorsal) as standardized evaluating base for lung lesions (“Rule of Tens”). Figure 2 Simplified lung scheme (dorsal) as standardized evaluating base for lung lesions (“Rule of Tens”). Page 4 of 12 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Germany after additional meat inspection seminars of up to 6 months’ duration. AQAs rotated between different positions and workstations every 20 minutes. Lung find- ings were entered via the touch screen at the slaughter line immediately after the macroscopical examination of each lung. The result keys PN1, 2, or 3 could be selected on the touch screen. As allowed by the German AVVLmH [13], Pneumonia 0 was not scored by AQAs. For this inves- tigation, PN 0 was subsequently calculated (total of examined lungs minus lungs with findings in result keys 1–3). The Observing Veterinarian (OV = T. Steinmann) examined the lungs immediately after the AQAs by using the simplified lung lesion evaluation scheme in combination with the AVVLmH scoring system [13] including a score for PN0. The OV documented the result key manually for predefined time periods in order to synchronize assessment series identical with series assessed by AQAs. The OV remained blinded regarding the entered results of the assistants throughout the period of data collection. Morphometric evaluation of ventral and dorsal lung surface areas Figure 1 shows the morphometric measurement method applied for lung no. 1 (L1) to determine the surface of this lung per lobe and on both sides. 2 A red square =1 mm2. Morphometric results of a sample of normal healthy lungs In Table 1, the morphometric data of the surfaces of each lobe were determined for the ventral and dorsal lobe and both surfaces were combined by cumulation. From five exemplary unaffected lungs, their medians and arithmetic means of the whole surfaces as well as the defined simplified surface proportion as basis for the development of a simplified lung lesion evaluation sys- tem for lung lesions are shown. Medians and arithmetic means of sample lobes dif- fered not more than 0.79% per site. The greatest dif- ference of 0.79% was observed for the accessory lobe (cf. Table 1). By using medians, rounding up or down to 10% or 50% for the small lobes or both caudal lobes, respectively, resulted in rounding differences below 0.75% (both caudal lobes taken together). Only the right cranial lobe showed a rounding difference of −3.17% (cf. Table 1). Status-quo investigation The absolute and relative differences in total and the deviations from the reference in total, which were col- lected in two weeks (cf. Table 2), as well as in predefined daily assessment periods (cf. Table 3) were observed. As shown in Tables 2 and 3, the scoring of lung lesions according to the AVVLmH criteria [13] by AQAs differed significantly compared with the assessments of the OV. In the total status-quo observation, the largest overall difference of 5.5% was observed for key PN2 resulting in an over-scoring of + 75.3% deviation from the observer reference. Key PN1 was associated with an under-scoring of - 20.4% in total, whereas key PN3 was associated with an under-scoring of - 11.7% (cf. Table 2). Status-quo investigation During the investigation period of eight slaughter days, lungs of 5,183 randomly selected pigs were scored by AQAs and by the inde- pendent OV in parallel. For the comparison, the daily and total sums per result key were used and the devia- tions were calculated. pigs scored parallel and independently by 15 AQAs and by the OV as described for the status-quo investigation (see above). The results were compared and the inter-rater reliability was determined based on absolute and relative agreement per result key between AQAs and OV. Development and conduct of a specific training program for AQAs In order to disclose the sources of the calculated inter- rater deviations within the status-quo inquiry, personal discussions between OV and AQAs specific evaluations were performed at the slaughter line. The most fre- quently analyzed sources of deviations were included in a specific training program for AQAs. The training was divided into two parts: (a) a theoretical training in two sessions lasting four hours each and (b) a practical on-site training of individual AQAs during operations at the slaughter line. The training took place during two weeks in August and September 2012. The core element of the training was the simplified surface-related lung lesion evaluation scheme as an easy-to-use and quick method to quantitatively evaluate pathologically-ana- tomically lung lesions. This was presented to the AQAs and discussed in detail. Each AQA received a laminated sheet with the scheme to be used at the slaughter line for the training period. Figure 2 shows the resulting simplified lung scheme dorsal view with the defined surface proportions of all lung lobes to be used as a standardized method applied to quickly assess how much of a lung surface area is affected by pathological alterations. By summarizing both caudal lobes of the lung (2×25 = 5×10), the simplification follows a “Rule of Tens” (5 × 10 + 10 + 10 + 10 + 10 + 10). Investigation of improvements in reliability of evaluation of lung lesions Especially the ability to differentiate between extensional areal hemorrhages caused by slaughter technique or by disease was remarkably restricted on the part of the AQAs. The deviations in the daily assessment periods were much larger as shown below (cf. Table 3, Figures 3 and 4). The extent of deviations was largest on the very first day of observation (Monday, 25.07). Subsequently, deviations ranged from an over-scoring of PN2 by 348.6% on the first observation day of the status-quo investigation to an under-scoring of PN3 by 50.0% on the last observation day. Within the keys, PN2 was consistently over-scored ranging from + 7.3% to + 348.6% on all observation days, whereas PN3 was under-scored during the last 6 obser- vation periods ranging from - 18.2% to - 50.0% (cf. Table 3 and Figure 4). For key PN0, Mondays seemed to be generally more burdened with deviations than other days of the week. 3. The individual sensitivity of judging lesions and the power of judgment, which was exhibited by AQAs during the status-quo investigation, was essentially heterogeneous and may depend on psychological and socio-economic factors, including a Hawthorne effect, motivation to work (Monday Blues), cultural differences and lack of understanding to fulfill an essential function in public health and animal welfare. 3. The individual sensitivity of judging lesions and the power of judgment, which was exhibited by AQAs during the status-quo investigation, was essentially heterogeneous and may depend on psychological and socio-economic factors, including a Hawthorne effect, motivation to work (Monday Blues), cultural differences and lack of understanding to fulfill an essential function in public health and animal welfare. The training program consisted of a theoretical and a practical part. The theoretical part of the training focused on the components: Investigation of improvements in reliability of evaluation of lung lesions In this second investigation, the results of post-training assessments of lung lesions by AQAs were compared with the assessment results of the OV, both using the simplified lung lesion evaluation scheme. During four slaughtering days in September 2012, lungs of 4,646 randomly selected Table 1 Total and relative surface areas of porcine pulmonary lobes Lung sample Total surface area Lobus cranialis pulmonis sinistri, pars cranialis Lobus. cranialis pulmonis sinistri, pars caudalis Lobus caudalis pulmonis sinistri Lobus caudalis pulmonis dextri Lobus medius pulmonis dextri Lobus cranialis pulmonis dextri Lobus accessorius pulmonis dextri cm2 cm2 (%) cm2 (%) cm2 (%) cm2 (%) cm2 (%) cm2 (%) cm2 (%) L1 462.59 31.70 (6.85) 44.72 (9.67) 111.70 (24.15) 111.32 (24.06) 41.76 (9.03) 60.94 (13.17) 60.45 (13.07) L2 412.93 39.34 (9.53) 45.57 (11.04) 105.99 (25.67) 86.26 (20.89) 39.34 (9.53) 59.93 (14.51) 36.48 (8.83) L3 349.10 37.90 (10.86) 30.67 (8.79) 98.72 (28.27) 96.61 (27.67) 38.47 (11.02) 46.77 (13.40) 37.49 (10.74) L4 538.55 56.95 (10.57) 67.04 (12.45) 146.48 (27.20) 105.25 (19.54) 50.17 (9.32) 56.32 (10.46) 56.34 (10.46) L5 625.89 46.43 (7.42) 43.49 (6.95) 139.40 (22.27) 168.34 (26.90) 63.00 (10.07) 74.23 (11.86) 91.00 (14.54) Arithmetic mean of relative surface (%) 9.05 9.78 25.51 23.81 9.79 12.68 11.53 Standard Deviation o f Arithmetic mean of relative surface (%) 1.63 1.88 2.14 3.2 0.7 1.39 2.02 Median of relative surface (%) 9.53 9.67 25.67 24.06 9.53 13.17 10.74 Simplified surface proportions (%) 10 10 25 + 25 = 50 10 10 10 Rounding error to median/mean (%) +0.47 + 0.95 +0.33 + 0.22 −0.46 −1.02 +0.94 + 1.19 +0.47 + 0.21 −3.17 −2.68 −0.74 −1.53 Table 1 Total and relative surface areas of porcine pulmonary lobes Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 6 of 12 Table 2 Status-quo investigation: evaluations of N = 5183 lungs, total results Result key Authorized qualified assistants n (%) Observing veterinarian (reference) n (%) Difference n (%) Deviation from observer (reference = 100) (%) PN0 3380 (65.2)* 3402 (65.6) - 22 (0.4) - 0.6 PN1 917 (17.7) 1152 (22.2) - 235 (2.5) - 20.4 PN2 666 (12.9) 380 (7.3) + 286 (5.5) + 75.3 PN3 220 (4.2) 249 (4.8) - 29 (0.6) - 11.7 Calculated value, cf. Methods. Table 2 Status-quo investigation: evaluations of N = 5183 lungs, total results technical artifacts, which were erroneously assessed as pneumonic lung lesions. Calculated value, cf. Methods. Sources for Under- or Over-scoring and training program for authorized qualified assistants In preparation of the specific training program, possible objective and subjective sources of status-quo deviations were identified: 1. Describing the importance for standardized lung lesion evaluation by showing the aims of informing farmers of the frequencies of lung lesions and the positive effects on herd health caused by a standardized scoring system. 1. The reason for the under-scoring of PN1 by AQAs might not be due to their inability to identify low grade pneumonia but due to a misunderstanding resulting in an underestimation of the importance of data collection even for low grade pneumonia and the impact on herd health. 2. Illustrating the anatomical, physiological, histological, immunological and pathological basis of the porcine lung as well as the differentiation of lung alterations caused by slaughter techniques and by diseases. ble 3 Status-quo investigation: evaluations of N = 5183 lungs, deviation from the observer (referen y Status-quo 2011 - deviation from observing veterinarian (reference) per predefined daily period, n (%) Result key Monday 07.25. Tuesday 07.26. Wednesday 07.27. Thursday 07.28. Monday 08.22. Tuesday 08.23. Wednesday 08.24. Thursday 08.25. PN0 −197 −19 −29 18 49 35 87 16 (−43.8) (−4.5) (−6.3) (5.0) (15.0) (7.3) (20.4) (3.4) PN1 66 −53 −2 −4 −48 37 −100 −58 (43.4) (−31.9) (−1.7) (−3.7) (−31.6) (28.0) (−58.5) (−37.4) PN2 122 49 40 18 12 4 4 37 (348.6) (87.5) (121.2) (30.0) (20.0) (7.3) (8.7) (105.7) PN3 9 23 −9 −33 −13 −2 −9 −5 (32.1) (63.9) (−25) (−47.8) (−40.6) (−18.2) (−33.3) (−50.0) Calculated value, cf. Methods. Status-quo 2011 - deviation from observing veterinarian (reference) per predefined daily period, n (%) Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 7 of 12 Page 7 of 12 -50% -40% -30% -20% -10% 0% 10% 20% 30% 40% 50% 6-Sep-12 7-Sep-12 10-Sep-12 11-Sep-12 Thu Fri Mon Tue PN0 Post-training 2012, n= 4,646 -50% -40% -30% -20% -10% 0% 10% 20% 30% 40% 50% 25-Jul-11 26-Jul-11 27-Jul-11 28-Jul-11 22-Aug-11 23-Aug-11 24-Aug-11 25-Aug-11 Mon Tue Wen Thu Mon Tue Wen Thu PN0 Status-quo inquiry 2011, n= 5,183 9:00-10:30 9:00-10:30 9:00-10:30 9:00-10:30 9:00-11:00 9:30-11:00 10:00-11:30 9:30-11:00 11:00-13:30 10:00-12:30 8:00-10:30 8:00-10:30 Figure 3 Status-quo and Post-training deviations of AQAs from the OV (Reference) per day in PN0. Figure 3 Status-quo and Post-training deviations of AQAs* from the OV (Reference) per day in PN0. 3. Revising the legal requirements of lung evaluation [7,13]. 3. Revising the legal requirements of lung evaluation [7,13]. 3. Sources for Under- or Over-scoring and training program for authorized qualified assistants Revising the legal requirements of lung evaluation [7,13]. daily assessment periods (cf. Table 3) were observed. As shown in Tables 4 and 5, the scoring of lung lesions according to the AVVLmH criteria [13] by AQAs im- proved dramatically and differed at a distinctly smaller degree compared with the assessments of the OV. In the entire post-training observation, an overall differ- ence of 1.5% was observed for key PN2 resulting in an over-scoring of + 15.4% deviation from the observer ref- erence. Key PN1 was associated with an under-scoring of – 12.9% in total, whereas key PN3 was associated with an under-scoring of - 11.5% (cf. Table 4). 4. Presenting the developed simplified surface-related lung lesion evaluation scheme to evaluate lung lesions according to its surface as a tool for homogeneous evaluations. 5. Showing affected lungs via photographs and abstracting the relevant lesions by means of a scheme with colorization of the affected lung areas to describe the extent (cf. Figure 5). The practical part included an individual one-to-one training of each AQA assistant conducted by the OV for 20 hours during the slaughter process at the line. The dif- ferences between technical artifacts and pathological le- sions were demonstrated by inspection and in doubtful cases by additional palpation and incision of the lung. The deviations within the daily assessment periods decreased as shown below (cf. Table 5, Figures 3 and 4). The extent of deviations did not exceed 50% and were largest on weekend-related days (Friday, 07.09 and Monday, 10.09.). Deviations ranged from an over-scoring of PN2 by + 42.3% to an under-scoring of PN1 by - 41.5%. Among the keys, PN2 was again consistently over-scored ranging from + 2.1% to + 42.3% on all observation days, whereas PN3 was slightly, but consistently under-scored during the whole post-training observation periods ran- ging from – 2.6% to - 25.0% (cf. Table 5 and Figure 4). Post-training investigation The absolute and relative differences in total and the deviations from the reference in total, which were col- lected in two weeks (cf. Table 2), as well as predefined Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 8 of 12 -400% -350% -300% -250% -200% -150% -100% -50% 0% 50% 100% 150% 200% 250% 300% 350% 400% 25-Jul-11 26-Jul-11 27-Jul-11 28-Jul-11 22-Aug-1123-Aug-1124-Aug-1125-Aug-11 Mon Tue Wen Thu Mon Tue Wen Thu PN1 PN2 PN3 -400% -350% -300% -250% -200% -150% -100% -50% 0% 50% 100% 150% 200% 250% 300% 350% 400% 6-Sep-12 7-Sep-12 10-Sep-12 11-Sep-12 Thu Fri Mon Tue PN1 PN2 PN3 Status-quo inquiry 2011, n= 5,183 Post-training inquiry 2012, n= 4,646 9:30-11:00 10:00-11:30 9:30-11:00 9:00-11:00 9:00-10:30 9:00-10:30 9:00-10:30 9:00-10:30 8:00-10:30 8:00-10:30 10:00-12:30 11:00-13:30 Figure 4 Status-quo and post-training deviations of AQAs to OV (Reference) per day in PN1-3. -400% -350% -300% -250% -200% -150% -100% -50% 0% 50% 100% 150% 200% 250% 300% 350% 400% 25-Jul-11 26-Jul-11 27-Jul-11 28-Jul-11 22-Aug-1123-Aug-1124-Aug-1125-Aug-11 Mon Tue Wen Thu Mon Tue Wen Thu PN1 PN2 PN3 -400% -350% -300% -250% -200% -150% -100% -50% 0% 50% 100% 150% 200% 250% 300% 350% 400% 6-Sep-12 7-Sep-12 10-Sep-12 11-Sep-12 Thu Fri Mon Tue PN1 PN2 PN3 Status-quo inquiry 2011, n= 5,183 Post-training inquiry 2012, n= 4,646 9:30-11:00 10:00-11:30 9:30-11:00 9:00-11:00 9:00-10:30 9:00-10:30 9:00-10:30 9:00-10:30 8:00-10:30 8:00-10:30 10:00-12:30 11:00-13:30 Figure 4 Status-quo and post-training deviations of AQAs to OV (Reference) per day in PN1-3. For key PN0, a Monday seemed to be generally more burdened with deviations (over-scoring by + 24.5%) than other days of the week. small porcine lung lobes down to 10%. However, mor- phometric data in support of this approximation and the method how to estimate surfaces (from ventral, dorsal or both) could not be found in the literature. Here, the authors provide data supporting this concept and extend it to an overall “Rule of Tens” for pig lungs. The advantage of this standardized estimation method for lung surfaces is that it is rapidly realizable and feas- ible to apply without expensive or sophisticated tech- nical tools or instruments. Furthermore, due to the determination that the cranial lobes are separated from each other by an imaginary line with an approximate 45° angle, this method was proven to have high practic- ability and usefulness during this study. Discussion h h The authors developed and validated a simplified and standardized scheme in order to assess surface-related lung lesions of slaughter pigs according to the German AVVLmH administrative regulation. Morphometric data of a sample of five lungs showed that medians and arithmetic means did not differ signifi- cantly from each other suggesting that their surfaces were nearly symmetrically distributed and that both, mean and median could be used for quantitative simplifications. In order to achieve an easy-to-use numerical aggregation rule for evaluating lung lesions the “Rule of Tens” was devel- oped. Estimation rules are regarded as highly effective for approximations under time pressure, e.g. in medical emer- gencies such as burns. A “Rule of Nines” is used in human medicine to determine severely burned body surface areas in adults using multiples of 9 and to guide treatment deci- sions [23]. Already Straw et al. [17] proposed breaking the A limitation of the method might be that the used two- dimensional photograph does not represent the three- dimensional lung tissue precisely. Nonetheless, according to Hill et al. [24] and Davies et al. [25], who quantified dif- ferences between two-dimensional and three-dimensional measuring methods of lungs, the agreement is remarkably high. In addition, the lack of precision is negligible due to the purpose to develop a standard scheme for measuring Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 9 of 12 Figure 5 Example of lung photographs and corresponding lung lesions abstracted by colorized schemes (dorsal and ventral). Figure 5 Example of lung photographs and corresponding lung lesions abstracted by colorized scheme ample of lung photographs and corresponding lung lesions abstracted by colorized schemes (dorsal and ventral). lung lesions in industrial abattoirs. Godwin et al. [14] developed a lung lesion evaluation scheme focused on lesions typically caused by enzootic pneumonia. The main deficits of this evaluation system are that lesions located in the diaphragmatic lobes as well as chronic lesions are not recorded, so that this system may over- estimate the respiratory herd health status. The benefit of the scheme by Madec and Kobisch [15] is that for application an extra surface standard is not necessary. This is due to the imaginary subdivision of each lobe into quarters which is adequately accessible by inspec- tion. Calculated value, cf. Methods. Discussion h h Nonetheless incor- rect scoring by the Observing Veterinarian cannot be precluded but the frequency of wrong lung assessments ought to be much lesser compared to AQAs. base their proportions on precise volume measurements of the different lung lobes. This leads to a distinctive lung dimorphism between the left and right lung, whereas the right lung is larger than the left lung. Espe- cially the right cranial and medial lung lobes are nearly double as large as the left cranial lobes. The most sub- stantial disadvantage of that scheme is reasoned in its difficult applicability at slaughter line especially under field conditions. In addition, the evaluation of lung le- sions in volume is impossible without incising the tissue. base their proportions on precise volume measurements of the different lung lobes. This leads to a distinctive lung dimorphism between the left and right lung, whereas the right lung is larger than the left lung. Espe- cially the right cranial and medial lung lobes are nearly double as large as the left cranial lobes. The most sub- stantial disadvantage of that scheme is reasoned in its difficult applicability at slaughter line especially under field conditions. In addition, the evaluation of lung le- sions in volume is impossible without incising the tissue. The subsequent status-quo investigation clearly re- vealed room for improvement. It is well known that par- ticipant observation can only do so much for the research as the sole presence of the observer in the field influences the participants' behavior. For this reason, we avoided any intervention or change in the working process of the AQAs as far as it was technically possible. We achieved a type of data collection characterized by a passive participation of only a single Observing Veterin- arian in the bystander role. To overcome limitations re- garding the ability to establish rapport, a separate and blinded documentation system was implemented for the Observing Veterinarian, and the assessment compari- sons were conducted based on predefined observation time-frames with incomplete overlapping of large lung samples rather than predefined small sizes of lungs sam- ples with incomplete overlapping of observation times. The design of our observational methods implied com- promises that did not support a rationale for calculating intra-observer repeatability and inter-observer reprodu- cibility based on correlation statistics. Descriptive statis- tics were regarded as being more appropriate for the approach of this study. Discussion h h The limitation of their system is that the relationship between the total number of the score points and the extent of the affected lung surface does not represent the realistic lung proportion, i.e. a totally affected cranial or medial lobe and a totally affected caudal lobe results in equal score points although the proportions are different. Furthermore, the evaluation as well as the documentation of the findings for each of the seven lung lobes are too time-consuming so that the scheme by Madec and Kobisch [15] is more suitable for a laboratory setting. An advantage of the scheme by Christensen et al. [18] is that it seems to be the most realistic one because they Table 4 Post-training investigation: evaluations of N = 4646 lungs, total results Result key Authorized qualified assistants Observing veterinarian (reference) Difference Deviation from observer (reference = 100) n (%) n (%) n (%) (%) PN0 2705 (58.2) 2568 (55,3) + 137 (3.0) + 5.3 PN1 1254 (27.0) 1440 (31.0) - 186 (4.0) - 12.9 PN2 526 (11.3) 456 (9.8) + 70 (1.5) + 15.4 PN3 161 (3.5) 182 (3.9) - 21 (0.5) - 11.5 *Calculated value, cf. Methods. Table 4 Post-training investigation: evaluations of N = 4646 lungs, total results Steinmann et al. BMC Veterinary Research 2014, 10:98 Page 10 of 12 http://www.biomedcentral.com/1746-6148/10/98 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 10 of 12 Table 5 Post-training investigation: evaluations of N = 4646 lungs, deviation from the observer (reference) per day Post-training 2012 - deviation from observing veterinarian (reference) per predefined daily periods, n (%) Result key Thursday Friday Monday Tuesday 06.09. 07.09. 10.09. 11.09. Discussion h h PN0 29 (4.2) −48 (−8.1) 157 (24.5) −1 (−0.2) PN1 −21 (−6.7) 4 (1.0) −162 (−41.5) −7 (−2.0) PN2 2 (2.1) 47 (42.3) 6 (4.7) 15 (12.4) PN3 −10 (−25.0) −3 (−6.1) −1 (−2.6) −7 (−13) ng investigation: evaluations of N = 4646 lungs, deviation from the observer (reference) per day training 2012 - deviation from observing veterinarian (reference) per predefined daily periods, n (%) estigation: evaluations of N = 4646 lungs, deviation from the observer (reference) per day Table 5 Post-training investigation: evaluations of N = 4646 lungs, deviation from the observer (re Post-training 2012 - deviation from observing veterinarian (reference) per predefined daily perio Table 5 Post-training investigation: evaluations of N = 4646 lungs, deviation from the observer (re purpose of the status-quo investigation and seemed to changed their behavior to fit that interpretation. A pos- sible reason for these characteristics is the expectation of AQAs that they will be evaluated and thus they fig- ured out how to control the observation and to attain good scores. The Hawthorne effect might have been fa- cilitated to appear in the status-quo investigation as the German AVVLmH administrative regulation allows low grade pneumonia to be scored in result category 0 im- plying an irrelevant finding. This was the routine at the investigated slaughter line, but this routine obviously re- duces scoring sensitivity for pathological alterations. It is important to note that the presumed strong Hawthorne effect at the beginning of the status-quo investigation became smaller in the course of the investigation. This effect can be seen in relation to ostensibly objective causes like misinterpretations of technical artifacts, which were erroneously assessed as pneumonic lung le- sions, and the limited ability to differentiate between ex- tensional pre- and post-mortal hemorrhages. It is conceivable that latter causes might play a greater role in the course of the status-quo investigation. The fact that these significant disagreements occurred mainly within the critical discrimination of keys PN0 and PN2 is seen as a matter of concern by the authors and, retro- spectively, justified a specific and efficient training of the AQAs. Although the described misinterpretations of ar- tifacts by the AQAs were at least subjective appraisals of the Observing Veterinarian, but he as a veterinarian has profound theoretical knowledge and practical skills in veterinary pathology backed up with knowledge about lung lesions and its histopathology. References E 1. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:83. 2. Blaha T: Die Erfassung pathologisch-anatomischer Organbefunde am Schlachthof. 1. Ansatz zu neuen Wegen bei der Wahrnehmung der Verantwortung für Verbraucherschutz und Tiergesundheit. Fleischwirtschaft 1993, 73:877–881. 1. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:83. 1. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:83. Nevertheless, results of the post-training investigation were far from ideal. Deviations from the observer contin- ued to revealed differences of greater than 10% in several periods for the critical keys PN2 and 3, and deviations peaked again slightly on working days close to the week- end. Further investigations of these remaining deficits, possibly based on unannounced spot checks, and data- derived trainings of AQAs seem to be an option to fur- ther increase the reliability of the current practice in meat inspections at industrial slaughter lines. g p 2. Blaha T: Die Erfassung pathologisch-anatomischer Organbefunde am Schlachthof. 1. Ansatz zu neuen Wegen bei der Wahrnehmung der Verantwortung für Verbraucherschutz und Tiergesundheit. Fl i h i t h ft 1993 73 877 881 g p g g Schlachthof. 1. Ansatz zu neuen Wegen bei der Wahrnehmung der Verantwortung für Verbraucherschutz und Tiergesundheit. Fleischwirtschaft 1993, 73:877–881. Verantwortung für Verbraucherschutz und Tiergesundheit. Fleischwirtschaft 1993, 73:877–881. Fleischwirtschaft 1993, 73:877–881. 3. Blaha T: Recording of lesions in organs of slaughter animals for quality assurance systems in meat production. Dtsch Tierärztl Wochenschrift 1994 101:264–267 [Article in German]. 4. Eckhardt P, Fuchs K, Kornberger B, Köfer J: Slaughter findings feedback systems-its use for farms of origin? Berl Munch Tierarztl Wochenschr 2010, 11–12:468–476 [Article in German]. 5. Hoischen-Taubner S, Blaha T, Werner C, Sundrum A: Repeatability of anatomical-pathological findings at the abattoir for characteristics of animal health. Journal for Food Safety and Food Quality 2011, 3:82–87 [Article in German] Discussion h h By using this means, an impact of the observer involvement in terms of a distinct effect on the result key PN0 and PN2 was seen on day 1 of the status-quo investigation (cf. Figures 3 and 4). This effect bears a resemblance to the Hawthorne effect as a form of reactivity whereby observed persons improve or mod- ify their behavior in response to the fact that they know they are being observed [26]. It may be assumed that participating AQAs acted differently under observation with the aim of achieving a similar result to that of the Observing Veterinarian. This observational artifact obvi- ously led the AQAs to an own interpretation of the The specific training followed established didactical principles and separated a theoretical part from a practical one. A thorough theoretical revision of specific patho- anatomical details of the lung and its lesions as an import- ant prerequisite for evaluating pneumonia was strongly appreciated by the AQAs. The colorized abstractions of pneumonic lesions which were put in context with corresponding photographs (cf. Figure 5) supported the detectability of lesions and their extensions caused by Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Page 11 of 12 pneumonia. It was confirmed by the AQAs that the sim- plified lung lesion evaluation scheme strongly improved the evaluation of the affected lung surface in the sense of an easy-to-use tool with the potential to enormously sim- plify the graduation of lung lesions into low, medium and high even under high time pressure and stress. Questions during the course about differentiation of alterations either caused by disease or by slaughter techniques were discussed directly at the slaughter line with the AQAs on a personal case-by-case basis. In doubtful cases, lungs were palpated and incised as proposed by Nathues et al. [27]. The authors were aware that this positive feedback to the training given by the AQAs could have been partially the result of polite responses. Thus, it became also an objective of the post-training investigation to check this particular question. Acknowledgments This investigation was supported by a research grant of the European Commission, the European Regional development Fund (ERDF) and the state government of North-Rhine Westphalia in the context of “Ziel2-Programm NRW 2007-2013” the stage comprehensive cluster project “gesunde Tiere - gesunde Lebensmittel”. In general, the post-training investigational data sug- gested a significant improvement potential compared with the status-quo investigation. Particularly, the critical keys PN1, 2 and 3 exhibited a major increase in reliability as judged by the AQAs. It seemed that the previously sug- gested Hawthorne effect, which was apparently associated with keys PN0 and PN2 in the status-quo investigation, had nearly vanished. This change might be one of the spe- cific positive results of the personalized practical training triggered by and due to the sympathy and interest of the Observing Veterinarian. Further discussion of this possible correlation may lead to an improved understanding of a management effect at real-world industrial slaughter lines with the important question how management can make AQAs perform better because they feel better. The authors wish to thank management, official veterinarians and authorized qualified assistants of the Meat Hygiene Department and Westfleisch Slaughter Plant, Hamm, Germany, for their unrestricted and continuous support of this study in all its steps. We also wish to thank Prof. Dr. G.G. Steinmann, Boehringer Ingelheim, Germany for his critical review of the manuscript. Authors’ contributions TS participated in the study design and carried out the data collection, developed the simplified standard surface area scheme, conducted the training program and drafted the manuscript. TB and DM planned and designed the study, coordinated the research project and participated in drafting the manuscript. TS, TB and DM performed the statistical analysis and were involved in the interpretation of data. All authors read and approved the final manuscript. Received: 21 August 2013 Accepted: 16 April 2014 Published: 27 April 2014 Received: 21 August 2013 Accepted: 16 April 2014 Published: 27 April 2014 Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Author details 1 ld S f 1Field Station for Epidemiology, University of Veterinary Medicine Hannover, Foundation, Buescheler Str. 9, D-49456 Bakum, Germany. 2Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173 Hannover, Germany. Conclusion It may be concluded that the reliability of official meat inspections in a real-world industrialized setting can be in- creased with the help of i) a validated, simplified and stan- dardized scheme to assess surface-related lung lesions of slaughter pigs (“The Rule of Tens”) and ii) an accom- panying theoretical and practical training of authorized qualified assistants (AQA) in evaluating pathological lung lesions. A continuative standardization and optimization can be realized by repetitive and individual training pro- grams based on controls within the framework of quality assurance. 6. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:98. 7. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:111. 8. Commission E: Regulation (EC) No 1244/2007 of the European Parliament of 24 October 2007amending Regulation (EC) No 2074/2005 as regards implementing measures for certain products of animal origin intended for human consumption and laying down specific rules on official controls for the inspection of meat (Text with EEA relevance). Off J Eur Union 2007, L281:12–17. 6. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:98. 7. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:111. 8. Commission E: Regulation (EC) No 1244/2007 of the European Parliament of 24 October 2007amending Regulation (EC) No 2074/2005 as regards implementing measures for certain products of animal origin intended for human consumption and laying down specific rules on official controls for the inspection of meat (Text with EEA relevance). Off J Eur Union 2007, L281:12–17. 6. Abbreviations AQAs: Authorized qualified assistants = synonymous to official auxiliaries in Reg. (EC) No. 854/2004; OV: Observing veterinarian (Reference). Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 9. Blaha T: One world-one health: the threat of emerging diseases. A European Perspective. Transbound Emerg Dis 2012, 59(Suppl 1):3–8. 10. Baeckstroem L, Bremer H: Disease registrations on pigs at slaughter as a method of preventive and therapeutic veterinary medicine in swine production. Sven Veterinaertidsskr 1976, 28:312–336. production. Sven Veterinaertidsskr 1976, 28:312–336. 11. Blaha T, Blaha ML: VET special:Qualitätssicherung in der Schweinefleischerzeugung. Stuttgart: Gustav Fischer Verlag, Jena; 1 12. Dickhaus CP, Meemken D, Blaha T: Attempts to quantify the health status of pig herds: developing and validating a Herd Health Score (HHS). In Sustainable Animal Production the Challenges and Potential Developments for Professional Farming. Edited by Aland A, Madec F. Wageningen: Wageningen Academic Publishers; 2009:191–201. 13. AVV Lebensmittelhygiene(AVV LmH) (9.November 2009) Anlage 3. access date:16 June 2013 from Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV): [http://www.verwaltungsvorschriften-im- internet.de/BMELV-329-20091109-KF-002-A008.htm] 14. Godwin R, Hodgeson RG, Whittlestone P, Woodhams RL: Some experiments relating to artificial immunity in enzootic pneumonia of pigs. J Hyg 1969, 67:465–476. 15. Madec F, Kobisch M: Bilan lésionnel des poumons des porcs charcutier à l’abattoir. In 14esJournées de la Recherche Porcine en France: 3 et 4 Février 1982; Paris. Edited by ITP, INRA. 1982:405–412. y 16. Morrison RB, Hilley HD, Leman AD: Comparison of methods for assessing the prevalence and extent of pneumonia in market weight swine. Can Vet J 1985, 26:381–384. 17. Straw BE, Backstrom L, Leman AD: Examination of swine at slaughter. Part II. Findings at slaughter and their significance. Compend Contin Educ Pract Vet 1986, 8:106–111. 18. Christensen G, Sorensen V, Mousing J: Diseases of the respiratory system. In Diseases of Swine. 8th edition. Edited by Straw B, D'Allaire S, Mengeling WL, Taylor DJ. Ames, Iowa: Iowa State University Press; 1999:913–940. 19. Maes DG, Deluyker H, Verdonck M, Castryck F, Miry C, Vrijens B, Ducatelle R, De Kruif A: Non-infectious factors associated with macroscopic and microscopic lung lesions in slaughter pigs from farrow-to-finish herds. Vet Rec 2001, 148:41–46. 20. Schleicher S, Scheriau I, Kopacka S, Wanda J, Hofrichter J, Köfer: Analysis of the variation in meat inspection of pigs using variance partitioning. Prev Vet Med 2013, Available online 26 June 2013, In Press, Corrected Proof, http://dx.doi.org/10.1016/j.prevetmed.2013.05.018. 21. Waibl H: 5 Atmungsapparat (H. Waibl). Lungen. In Nickel, Schummer, Seiferle: Lehrbuch der Anatomie der Haustiere, Band II Eingeweide. 9th edition. Conclusion European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:98. 7. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:111. 8. Commission E: Regulation (EC) No 1244/2007 of the European Parliament of 24 October 2007amending Regulation (EC) No 2074/2005 as regards implementing measures for certain products of animal origin intended for human consumption and laying down specific rules on official controls for the inspection of meat (Text with EEA relevance). Off J Eur Union 2007, L281:12–17. 6. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:98. Page 12 of 12 Page 12 of 12 Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Edited by Frewein J, Gasse H, Leiser R, Heider R, Thomé H, Vollmerhaus B, Waibl H. Stuttgart: Parey; 2004:259–284. 22. European Commission: Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union 2004, L226:105. 23. Knaysi GA, Crikelair GF, Cosman B: The rule of nines: its history and accuracy. Plast Reconstr Surg 1968, 41:560–563. 23. Knaysi GA, Crikelair GF, Cosman B: The rule of nines: its history and accuracy. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit y 27. Nathues H, Hewicker-Trautwein M, Grosse Beilage E: Differenzierung schlachtungsbedingeter Artefakte von pneumonischen Veränderungen beim Lungencheck an Schlachtschweinen (Differentiation between pneumonic lung lesions and technical artefacts during scoring of lungs from pigs at the abattoir). Tierärztliche Praxis Grosstiere 2008, 4:258–262. y 27. Steinmann et al. BMC Veterinary Research 2014, 10:98 http://www.biomedcentral.com/1746-6148/10/98 Nathues H, Hewicker-Trautwein M, Grosse Beilage E: Differenzierung schlachtungsbedingeter Artefakte von pneumonischen Veränderungen beim Lungencheck an Schlachtschweinen (Differentiation between pneumonic lung lesions and technical artefacts during scoring of lungs from pigs at the abattoir). Tierärztliche Praxis Grosstiere 2008, 4:258–262. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission Cite this article as: Steinmann et al.: A simplified evaluation system of surface-related lung lesions of pigs for official meat inspection under industrial slaughter conditions in Germany. BMC Veterinary Research 2014 10:98.
https://openalex.org/W2962952254	http://cds.cern.ch/record/2244882/files/fulltext.pdf	English	null	Supergravity at 40: Reflections and Perspectives	arXiv (Cornell University)	2,017	cc-by	9,988	2017 2017 Vol. 40, N. 6 DOI 10.1393/ncr/i2017-10136-6 RIVISTA DEL NUOVO CIMENTO c⃝Societ`a Italiana di Fisica S. Ferrara(1)(2)(3) and A. Sagnotti(4) (1) Theoretical Physics Department, CERN CH - 1211 Geneva 23, Switzerland (2) INFN - Laboratori Nazionali di Frascati - Via Enrico Fermi 40 I-00044 Frascati (RM), Italy (3) Department of Physics and Astronomy, Mani L. Bhaumik Institute for Theoretical Physics U.C.L.A., Los Angeles CA 90095-1547, USA (4) Scuola Normale Superiore e INFN - Piazza dei Cavalieri 7, I-56126 Pisa, Italy (1) Supersymmetry was inspired by “dual resonance models”, an early version of String Theory pioneered by Gabriele Veneziano [6] and extended by Andr´e Neveu, Pierre Ramond and John Schwarz [7]. Earlier work done in France by Jean-Loup Gervais and Benji Sakita [8], and in the Soviet Union by Yuri Golfand and Evgeny Likhtman [9] and by Dmitry Volkov and Vladimir Akulov [10], had anticipated some salient features. Supergravity at 40: Reﬂections and perspectives(∗) S. Ferrara(1)(2)(3) and A. Sagnotti(4) (1) Theoretical Physics Department, CERN CH - 1211 Geneva 23, Switzerland (2) INFN - Laboratori Nazionali di Frascati - Via Enrico Fermi 40 I-00044 Frascati (RM), Italy (3) Department of Physics and Astronomy, Mani L. Bhaumik Institute for Theoretical Physics U.C.L.A., Los Angeles CA 90095-1547, USA (4) Scuola Normale Superiore e INFN - Piazza dei Cavalieri 7, I-56126 Pisa, Italy S. Ferrara(1)(2)(3) and A. Sagnotti(4) (1) Theoretical Physics Department, CERN CH - 1211 Geneva 23, Switzerland (2) INFN - Laboratori Nazionali di Frascati - Via Enrico Fermi 40 I-00044 Frascati (RM), Italy (3) Department of Physics and Astronomy, Mani L. Bhaumik Institute for Theoretical Physics U.C.L.A., Los Angeles CA 90095-1547, USA (4) Scuola Normale Superiore e INFN - Piazza dei Cavalieri 7, I-56126 Pisa, Italy received 15 February 2017 received 15 February 2017 received 15 February 2017 Dedicated to John H. Schwarz on the occasion of his 75th birthday Dedicated to John H. Schwarz on the occasion of his 75th birthday Summary. — The fortieth anniversary of the original construction of Supergravity provides an opportunity to combine some reminiscences of its early days with an assessment of its impact on the quest for a quantum theory of gravity. Summary. — The fortieth anniversary of the original construction of Supergravity provides an opportunity to combine some reminiscences of its early days with an assessment of its impact on the quest for a quantum theory of gravity. 280 1. Introduction 280 2. The early times 282 3. The golden age 283 4. Supergravity and particle physics 284 5. Supergravity and string theory 286 6. Branes and M-theory 287 7. Supergravity and the AdS/CFT correspondence 288 8. Conclusions and perspectives (∗) Based in part on the talk delivered by S.F. at the “Special Session of the DISCRETE2016 Symposium and the Leopold Infeld Colloquium”, in Warsaw, on December 1 2016, and on a joint CERN Courier article. 279 280 S. FERRARA and A. SAGNOTTI 1. – Introduction The year 2016 marked the fortieth anniversary of the discovery of Supergravity (SGR) [1], an extension of Einstein’s General Relativity [2] (GR) where Supersymme- try, promoted to a gauge symmetry, accompanies general coordinate transformations. Supersymmetry, whose ﬁrst realization in four-dimensional Quantum Field Theory was introduced by Julius Wess and Bruno Zumino in [3,4], extends the very notion of space- time, adjoining to the Poincar´e group of translations and Lorentz rotations new symme- tries that change the Statistics (Bose-Einstein vs. Fermi-Dirac) of particles and ﬁelds. A peculiar mathematical structure, called “super-algebra”, achieves this goal while cir- cumventing classic no-go theorems that constrain attempts to unify space-time symme- tries (connected to mass and spin) with internal ones (connected to charges of various types) [5](1). Supergravity implies the existence of a new type of elementary quantum of gravitational origin, a spin 3/2 particle called gravitino. An exact Supersymmetry would require the existence of super-partners in the Stan- dard Model of Electroweak and Strong interactions and for the gravitational ﬁeld, but it would also imply mass degeneracies between the known particles and their super- partners. This option has been ruled out, over the years, by several experiments, and therefore Supersymmetry can be at best broken, with super-partner masses that seem to lie beyond the TeV energy region currently explored at the CERN LHC. In Supergrav- ity one would expect that the breaking be spontaneous, as in the Brout-Englert-Higgs (BEH) mechanism of the standard Model [11], which was remarkably conﬁrmed by the 2012 discovery of a Higgs particle [12]. Supersymmetry would have dramatic consequences. It would aﬀect the subatomic world, via supersymmetric extensions of the Standard Model (MSSM [13] and alike), but also large-scale phenomena and the cosmological evolution of our Universe [14]. Supergravity has the potential to provide important clues for dark matter, dark energy and inﬂation [15], and for the links between the corresponding breaking scale and the one that ought to have superseded it and presumably still characterizes the present epoch. The recent discovery of gravitational waves from black-hole (BH) mergers is a stunning conﬁrmation of GR [16], the gauge theory of the gravitational ﬁeld, and one can dream of future revelations of its spin-two quantum, the graviton. The gravitino ought to acquire mass via a supersymmetric version of the BEH mechanism, whose details would also control other mass splittings of crucial importance for super-particle searches (squarks, gluinos, sleptons). 1. – Introduction Let us now describe some key steps in the development of Supergravity, with an eye to achievements and diﬃculties of this endeavor and to its impact on diﬀerent ﬁelds. 2. – The early times The ﬁrst instance of Supergravity was built in the spring of 1976 by Daniel Freedman, Peter van Nieuwenhuizen and one of us (S.F.) [17], in a collaboration that had started in the fall of 1975 in Paris, at ´Ecole Normale Sup´erieure. The construction relied on 281 SUPERGRAVITY AT 40: REFLECTIONS AND PERSPECTIVES the vierbein formulation of General Relativity and on the Noether method, an iterative procedure that would result in the non-linear Yang-Mills or Einstein-Hilbert action prin- ciples if applied to gauge theories or gravity. Inconsistencies, if present, would have led to obstructions that no further modiﬁcations could have overcome. Shortly thereafter, Stanley Deser and Bruno Zumino recovered the result in a simpler and elegant way [18], extending the ﬁrst-order (Palatini) formalism of General Relativity(2). In their work the authors of [18] focussed on supergravity as a way to bypass inconsistencies of the Velo- Zwanziger type [19], which generally aﬀect theories with higher spin ﬁelds (for recent reviews see [20]). the vierbein formulation of General Relativity and on the Noether method, an iterative procedure that would result in the non-linear Yang-Mills or Einstein-Hilbert action prin- ciples if applied to gauge theories or gravity. Inconsistencies, if present, would have led to obstructions that no further modiﬁcations could have overcome. Shortly thereafter, Stanley Deser and Bruno Zumino recovered the result in a simpler and elegant way [18], extending the ﬁrst-order (Palatini) formalism of General Relativity(2). In their work the authors of [18] focussed on supergravity as a way to bypass inconsistencies of the Velo- Zwanziger type [19], which generally aﬀect theories with higher spin ﬁelds (for recent reviews see [20]). These original developments are well captured by eq. (2.1) below, where we display the Lagrangian of N = 1 Supergravity in four dimensions in the “1.5 order” formalism [1], the torsion equation for the gravitino ﬁeld ψμ and the supersymmetry transformations for the vierbein ﬁeld eμ a and ψμ (in “mostly plus” signature, as in other examples below)(3): S = 1 2 k2 d4x e eμ a eν b Rμν ab(ω) −ψμ γμνρ Dν(ω) ψρ , δ S δ ω = 0 −→Dμ ea ν −Dν ea μ = 1 2 ψμ γa ψν, (2.1) δ ea μ = 1 2 ϵ γa ψμ, δ ψμ = Dμ ϵ. (2.1) Further simpliﬁcations of the procedure emerged once its full signiﬁcance was better appreciated. (2) Alternative approaches were soon developed, including Supergravity as the gauge theory of the anti-de Sitter group [21], Supergravity on a group manifold [22] and Supergravity from broken superconformal symmetry [23]. (3) For convenience, we use in all equations the conventions set out by Daniel Freedman and Antoine Van Proeyen in [1]. Notice that their deﬁnition of ψμ, say, diﬀers from the usual one, since it includes an imaginary factor i. (3) For convenience, we use in all equations the conventions set out by Daniel Freedman and Antoine Van Proeyen in [1]. Notice that their deﬁnition of ψμ, say, diﬀers from the usual one, since it includes an imaginary factor i. (2) Alternative approaches were soon developed, including Supergravity as the gauge theory of the anti-de Sitter group [21], Supergravity on a group manifold [22] and Supergravity from broken superconformal symmetry [23]. 3 2. – The early times A mixed formalism was eventually adopted, where it became far simpler to track unwanted terms, and during a Summer Institute held at ´Ecole Normale in August 1976 the Noether procedure led to the ﬁrst matter couplings [24], which opened the way to a host of more complicated examples. Moreover, the “spinning string” [7], or String Theory as it is now called [25], was connected to space-time Supersymmetry via a Gliozzi-Scherk-Olive (or GSO) projection [26]. A ﬁrst extended version of four- dimensional Supergravity, involving two gravitinos, came to light shortly thereafter [27]. The low-energy spectra that emerged from the GSO projection pointed to yet un- known ten-dimensional versions of Supergravity, including the counterparts of several gravitinos [28], and to a four-dimensional supersymmetric Yang-Mills theory (SYM) in- variant under four distinct supersymmetries [26,29]. When S.F. visited Caltech in the Fall of 1976, he became aware that Murray Gell- Mann had worked out many consequences of Supersymmetry, including upper bounds on the number of gravitinos and on the gauge symmetries allowed, in principle, in all instances of “pure” Supergravity, where all particles would be connected to the gravi- ton [30] (see table I). Gell-Mann had realized, in particular, that the largest theory would include eight gravitinos, and would allow for a maximal gauge group, SO(8), which would not suﬃce to accommodate the SU(3) × SU(2) × U(1) gauge symmetry of the Standard Model. S. FERRARA and A. SAGNOTTI 282 Table I. – The helicity content of supergravity multiplets in four dimensions. The factors indicate the multiplicities, and the overall multiplets for N = 7 and N = 8 coincide. Table I. – The helicity content of supergravity multiplets in four dimensions. The factors indicate the multiplicities, and the overall multiplets for N = 7 and N = 8 coincide. Table I. – The helicity content of supergravity multiplets in four dimensions. The factors indicate the multiplicities, and the overall multiplets for N = 7 and N = 8 coincide. 2. – The early times Helicity multiplets of D = 4 supergravities N Helicity content 1 ˆ (2), ` 3 2 ´˜ 2 ˆ (2), 2 ` 3 2 ´ , (1) ˜ 3 ˆ (2), 3 ` 3 2 ´ , 3(1), ` 1 2 ´˜ 4 ˆ (2), 4 ` 3 2 ´ , 6(1), 4 ` 1 2 ´ , 2(0) ˜ 5 ˆ (2), 5 ` 3 2 ´ , 10(1), 11 ` 1 2 ´ , 10(0) ˜ 6 ˆ (2), 6 ` 3 2 ´ , 16(1), 26 ` 1 2 ´ , 30(0) ˜ 8 ˆ (2), 8 ` 3 2 ´ , 28(1), 56 ` 1 2 ´ , 70(0) ˜ 3. – The golden age The following years, 1977 and 1978, were most performing, and a widespread en- thusiasm drew into the ﬁeld many new adepts. Important developments followed read- ily, including the discovery of “minimal” formulations where N = 1 Supersymmetry is manifest [31, 32]. These involve extra (non-propagating) auxiliary degrees of freedom, which result in equal numbers of Bose and Fermi ﬁelds and particles. Finding these “oﬀ-shell” formulations has proved very hard beyond N = 1, but in this case they have allowed systematic investigations of the spontaneous breaking of local Supersymmetry and precise characterizations of scalar geometries. This is fortunate, since only N = 1 Supersymmetry is directly compatible with the chiral (parity-violating) interactions of the Standard Model. Auxiliary ﬁeld formulations proved important also to understand higher-derivative extensions of Supergravity [33], including the supersymmetric exten- sion [34] of Starobinsky’s model of inﬂation [35]. More recently, they were instrumental in supersymmetric localization techniques, where curved backgrounds providing infrared regulators are captured by diverse auxiliary ﬁeld conﬁgurations [36]. Complete matter couplings for N = 1, 2 in four dimensions were thus constructed by the early 1980s [37,38], and some key features of the general N = 1 case can be neatly summarized as in eq. (3.1) below S = d4x e 1 2 k2 eμ a eν b Rμν ab(ω) −∂i ∂¯j G Dμzi Dν ¯z ¯j gμν −V (z, ¯z) + . . . , (3.1) V = eG Gi G¯j G−1i¯j −3 , G = K + log \|W\|2. The main ingredients of the construction are the Kahler potential K(zi, zi) and the superpotential W(zi), which depend on the scalar ﬁelds and enter the theory via the invariant combination G. The main ingredients of the construction are the Kahler potential K(zi, zi) and the superpotential W(zi), which depend on the scalar ﬁelds and enter the theory via the invariant combination G. 283 SUPERGRAVITY AT 40: REFLECTIONS AND PERSPECTIVES A key step in the development of the theory had to do with the maximal model promptly identiﬁed by Gell-Mann. The maximal N = 8 Supergravity was derived in 1978 by Eugene Cremmer and Bernard Julia [39] from their previous, remarkable construction with Joel Scherk, of the unique Supergravity in eleven dimensions [40]. 3. – The golden age Its key features are summarized in S = 1 2 k2 d11x e eμ a eν b Rμν ab(ω) −ψμ γμνρ Dν ω + ˆω 2 ψρ (3.2) −1 24 Fμνρσ F μνρσ − √ 2 192 ψμ γμναβγδ ψν + 12 ψ α γγδ ψβ Fαβγδ + Fαβγδ −2 √ 2 (144)2 ϵα1...α4β1...β4μνρ F α1...α4 F β1...β4 Aμνρ , ωμ ab = ωμ ab + 1 8 ψα γμab αβ ψβ, Fμνρσ = Fμνρσ + 3 √ 2 2 ψ[μ γνρ ψσ], where ω solves its ﬁeld equation and “hats” denote supercovariant quantities. Curiously, at most seven additional spatial dimensions are indeed allowed in Super- gravity [41], in contrast with General Relativity. In general, extra dimensions beyond our space-time could exist, and yet be inaccessible to our senses, if they were curled into tiny internal spaces. This is the spirit of the Kaluza-Klein (KK) scenario that ﬁrst linked, in the 1920s, higher-dimensional GR and Electromagnetism [42]. The ﬁrst key ingredient of the N = 8 construction was the geometrical nature of the scalar interactions, which result from an analogue of the pion model involving the 70 diﬀerent ﬁelds present in the maximal theory, associated to the E7(7)/SU(8) coset. The second was a set of generalized electric-magnetic dualities, which extend the man- ifest symmetry of the vacuum Maxwell equations under the interchange of electric and magnetic ﬁelds and had already surfaced in simpler models [43]. For instance, in the N = 8 model, the E7(7) group acts on the 56 “electric” and “magnetic” ﬁeld strengths as a generalized electric-magnetic duality [39]. Hidden (inﬁnite-dimensional) symmetries extending it have been widely explored in recent years, following [44]. 4. – Supergravity and particle physics The MSSM [13] and other supersymmetric extensions of the Standard Model were introduced and widely investigated by Pierre Fayet, Savas Dimopoulos, Howard Georgi and others [13], relying heavily on the soft-breaking terms proposed by Luciano Girardello and Marc Grisaru in [45]. These low-dimensional couplings are not supersymmetric, and were introduced to overcome restrictions accompanying the spontaneous breaking of rigid Supersymmetry. A glimpse of the modiﬁcations induced by Supergravity is captured by the super-trace formula, here restricted for simplicity to chiral multiplets [37,46] (4.1) 1 2 Str M 2 Vzi=0,V =0 ≡1 2 J (−1)2 J (2 J + 1) m2 J = eG N −1 + Gi G ¯j Ri¯j , (4.1) where N is the number of scalar multiplets, which emerges in the m3/2/mP l →0 limit. For the sake of comparison, in renormalizable models of rigid Supersymmetry the r.h.s. would vanish, reﬂecting patterns where scalars pair, in mass, around fermions. 284 S. FERRARA and A. SAGNOTTI Table II. – Standard ﬁeld multiplets in D = 10, 11. The IIA model is directly related via KK to eleven dimensions. The IIB model contains doublets of gravitini, spinors and two-forms, and a chiral four-form, A+, with self-dual ﬁeld strength, together with two scalars parametrizing the SL(2, R)/U(1) coset. The ﬁeld content of the I model is a truncation of the others. The L, R suﬃxes indicate the chirality of Fermi ﬁelds. Field content of D = 11 and D = 10 supergravities and super Yang-Mills Model Field content D = 11 SGR ` ea μ, ψμ, Cμνρ ´ D = 10 IIA, or (1, 1) SGR ` ea μ, ψμ L, ψμ R, λL, λR, Cμνρ, Bμν, Aμ, φ ´ D = 10 IIB, or (2, 0) SGR ` ea μ, ψi μ L, λi R, D+ μνρσ, Bi μν, φ, φ′´ (i = 1, 2) D = 10, (1, 0) SGR ` ea μ, ψμ L, λR, Bμν, φ ´ D = 10, (1, 0) SYM (Aμ, λL) Supergravity provided a rationale for the emergence of soft-breaking terms as low- energy relics of the super-Higgs mechanism. This was shown by Riccardo Barbieri, Carlos Savoy and one of us (S.F.), and independently by Lawrence Hall, Joseph Lykken and Steven Weinberg, and by Richard Arnowitt, Ali Chamseddine and Pran Nath [47]. 4. – Supergravity and particle physics [ ] No-scale Supergravity, a theory with a naturally vanishing cosmological constant, was built in [48], and was readily applied to Physics beyond the Standard Model. All these developments made it possible to derive parameter spaces of masses and couplings for supersymmetric extensions of the Standard Model, which can be explored in experimental searches for new physics, as was done at LEP, at Tevatron and elsewhere in the past and is currently done extensively at the LHC collider [4]. “Split Supersymmetry” [49] was advocated in recent years to obtain large mass sepa- rations among MSSM super partners while maintaining a number of attractive features of Supersymmetry, at the price of a ﬁne-tuned cosmological constant [50], and was further developed by the same authors and many others. 5. – Supergravity and string theory The ultraviolet behavior of Supergravity theories was vigorously investigated soon after the original discovery. As in gravity [51], no divergences were found in the one-loop S-matrix of “pure” models involving solely the gravitational multiplet [52]. Symmetry arguments soon pushed them at least to the three-loop order [53, 54], and thus beyond GR, where divergences begin to show up at two loops [55]. In subsequent years reﬁned symmetry arguments and explicit computations relying on novel methods spurred by these studies have proceeded hand-in-hand, under the drive of Zvi Bern, Lance Dixon, David Kosower and others, revealing further, unexpected cancelations of divergences [56]. The case of N = 8 Supergravity remains unsettled, and some authors still envisage the possibility that this maximal theory be ﬁnite to all orders. In particular, the double- copy structure relating N = 4 Yang-Mills to N = 8 Supergravity might provide a clue to its actual divergence structure [57], and recently played a role in the identiﬁcation of SUPERGRAVITY AT 40: REFLECTIONS AND PERSPECTIVES 285 an unexpected symmetry [58], the dual superconformal symmetry, whose lessons could reverberate on Supergravity. Moreover, some subtleties that were well appreciated only recently might lead to deeper insights into the whole scenario [59]. an unexpected symmetry [58], the dual superconformal symmetry, whose lessons could reverberate on Supergravity. Moreover, some subtleties that were well appreciated only recently might lead to deeper insights into the whole scenario [59]. Supergravity allows in general continuous deformations that combine gauged inter- nal symmetries and the emergence of scalar potentials. Explicit constructions soon clariﬁed [60, 61], however, that these bring along negative vacuum energies, and thus maximally (super)symmetric anti-de Sitter, or AdS, vacua. This is the case, in particu- lar, for the N = 8 model of Bernard de Wit and Hermann Nicolai [62], which realized the full SO(8) gauge symmetry that we have already mentioned but does not admit a Minkowski vacuum. In a KK compactiﬁcation from eleven dimensions, a gauged SO(8) symmetry would be inherited from a seven-dimensional internal sphere [63]. A number of enticing variants were readily explored [64]. The GSO projection opened the way to connecting String Theory to gravity along lines that were foreseen, in the mid 1970s, by Scherk and Schwarz [65], and indepen- dently by Tamiaki Yoneya [66]. 5. – Supergravity and string theory All ten-dimensional versions of Supergravity (and cor- responding strings) were constructed by the 1980s [67, 68], but a widespread activity in this direction only started in 1984, when Michael Green and Schwarz discovered that gauge and gravitational [69] anomalies cancel, unexpectedly, in all versions of ten-dimensional supersymmetric String Theory (or Superstring Theory, for brief) [70]. Anomalies are quantum violations of classical symmetries that are very troublesome when they aﬀect gauge interactions. Their cancellation is a fundamental consistency condition, which is automatically granted in the Standard Model by its known particle content [71]. The allowed gauge groups in ten dimensions are SO(32) and E8 × E8 in closed Het- erotic superstrings [72], whose discovery followed closely the anomaly cancellation, and SO(32) in the Type-I theory involving both open and closed superstrings [73], akin re- spectively to segments and circles. Here E8 ×E8 denotes two copies of the largest excep- tional Lie algebra in the Cartan classiﬁcation, and both SO(32) and E8 ×E8 contain the SU(3) × SU(2) × U(1) gauge symmetry of the Standard Model as a small subgroup. At low energies, all these theories reduce to minimal Supergravity coupled to a Yang-Mills multiplet in ten dimensions. Unprecedented avenues thus opened up for linking ten-dimensional strings to the chiral interactions of Particle Physics, in scenarios that are free, by construction, of the ultraviolet problems of gravity. One might well say that Supergravity started oﬃcially, with this 1984 “ﬁrst superstring revolution”, a second life as a low-energy manifestation of String Theory. It actually led to reconsider the very notion of anomaly, since in the Green-Schwarz mechanism some of its gauge potentials remove part of it. The original construction involved a single antisymmetric tensor potential Bμν (to be contrasted with the electro-magnetic vector potential Aμ), but more complicated cases that have emerged later rest on several ﬁelds of this and similar types. The anomaly cancellation mechanism quickly resulted in deﬁnite KK scenarios grant- ing String Theory a four-dimensional interpretation [74]. It pointed to a speciﬁc class of six-dimensional internal manifolds leading to chiral spectra with N = 1 Supersymmetry in four dimensions that were widely studied in Mathematics, Calabi-Yau spaces. These lead naturally, in this context, to a grand-uniﬁcation gauge group E6, which was known to connect to the Standard Model with right-handed neutrinos. 6. – Branes and M-theory The early 1990s were marked by many detailed studies of classical solutions of Su- pergravity [82]. These generalize BHs, and form ﬁelds are their key new ingredient. In four dimensions, electric charges source a vector potential Aμ, whose Lorentz index reﬂects somehow their “world lines”. In a similar fashion, strings source antisymmet- ric potentials Bμν, while other potentials present in ten-dimensional Supergravity are related to extended objects of higher dimensionality, generically dubbed “p-branes”. Here p is the number of spatial dimensions of these extended objects (p = 0 for par- ticles, p = 1 for strings, p = 2 for membranes, and so on). Supergravity cannot forego the presence of a wide class of extended objects, with which String Theory would ap- pear at odds, insofar as it focuses solely on strings. This was strongly advocated, over the years, by a number of scientists, and most notably by Michael Duﬀ[83] and Paul Townsend [84]. The “second superstring revolution” is associated to the mid-1990s, and to the reso- lution of this apparent dichotomy when it became widely recognized that String Theory, after all, cannot be merely a theory of strings. Behind it, as we now concede, lies indeed a far more complicated soup of strings and more general p-branes. The novel ingredient of this wider picture was a class of p-branes that show up in string perturbation theory in the presence of boundaries, the D-branes whose role was clariﬁed by Joseph Polchin- ski [85], but the (electric-magnetic) dualities of the low-energy Supergravity were again a main tool. Strong-weak coupling dualities in String Theory were actually ﬁrst advocated by Anna-Maria Font, Dieter Lust, Luis Ibanez and Fernando Quevedo in [86], and were inspired by continuous symmetries of the low-energy Supergravity. The end result is an awesome uniﬁed picture, which was largely due to Edward Witten [87] and is usually referred to as M-theory. When strings combine with other extended objects, even the very notion of space-time is blurred, to the extent that ten and eleven dimensions emerge from singular corners of this correspondence. One of these is the eleven-dimensional Supergravity. These results are usually summarized via the hexagon-like diagram of ﬁg. 1, whose sides reﬂect diﬀerent duality links, three of which were deeply inspired by Supergravity [87-89]. 5. – Supergravity and string theory All thus converged into an intriguing dictionary between the resulting low-energy dynamics and the topology of these complicated manifolds, or if you will between four-dimensional Supergravity and Algebraic Geometry. A remarkable property of these spaces, with deep physical 286 S. FERRARA and A. SAGNOTTI implications, is the so-called “mirror symmetry” [75], which exchanges manifolds while swapping their Hodge numbers. This symmetry allows one to compute couplings that are non-perturbative from the string vantage point in terms of classical ones. A rationale is found in the C-map of the four-dimensional low-energy Supergravity [76], where N = 2 vector multiplets and hypermultiplets are interchanged. The structure of these multiplets rests on “special geometry”, whose mathematical signiﬁcance was ﬁrst stressed in [77]. No-scale Supergravity [48] was linked to Calabi-Yau compactiﬁcations in [78,79]. Actu- ally, strings behave properly also on singular (orbifold) limits that compare to smooth Calabi-Yau spaces like tetrahedra to spheres [80]. These limits aﬀord more complete descriptions, and have led to deep insights into the structure of String Theory, opening the way to early direct constructions of four-dimensional heterotic string spectra [81]. [ ] Other (type II) theories with maximal Supersymmetry exist in ten dimensions, are also free of anomalies and allow similar compactiﬁcations, but for a while they seemed totally unrelated to Particle Physics. (4) Recently, double ﬁeld theory [92] has emerged as an enticing intermediate framework to accommodate T-duality within Field Theory. (5) There were a number of notable anticipations of aspects of the correspondence, including some contributions in [68,94]. 6. – Branes and M-theory The others had already surfaced in the late Eighties: they are beyond its reach but ﬁnd their rationale in T-duality, a peculiar string correspondence between large and small KK radii [90], SUPERGRAVITY AT 40: REFLECTIONS AND PERSPECTIVES 287 Fig. 1. – Duality links among the ﬁve ten-dimensional supersymmetric versions of String Theory and eleven-dimensional Supergravity. Fig. 1. – Duality links among the ﬁve ten-dimensional supersymmetric versions of String Theory and eleven-dimensional Supergravity. and in the Ω orientifold link between type-IIB and type-I strings proposed by one of us (A.S.) [91]. Supergravity thus started oﬃcially a third, parallel life, as a probe into the elusive inner workings of String Theory(4). 8. – Conclusions and perspectives The last two decades have witnessed a multitude of applications of AdS/CFT outside its original realm. These have touched upon ﬂuid dynamics, the quark-gluon plasma, and more recently Condensed Matter Physics, providing a number of useful insights on strongly coupled matter systems. Perhaps more unexpectedly, AdS/CFT duality has stimulated work related to scattering amplitudes, which may also shed light on the old issue of the ultraviolet behavior of supergravity, but the reverse program of gaining information about gravity from gauge dynamics has proved harder. Above all, however, there is a pressing need to shed light on the geometrical principles and the deep symmetries underlying String Theory, which have proved elusive over the years. The interplay between Particle Physics and Cosmology is a natural arena to explore consequences of Supergravity. Recent experiments probing the Cosmic Microwave Back- ground, and in particular the results of the Planck mission [99], have lent some deﬁnite support to inﬂationary models of the Early Universe. An elusive particle, the inﬂaton, should have driven this primordial acceleration, and while our current grasp of String Theory does not allow a detailed analysis of the problem, Supergravity can provide fun- damental clues on this and the subsequent particle physics epochs. Applications of Supergravity to Cosmology have attracted an increasing interest in recent years [34, 100]. Supersymmetry is broken in a de Sitter-like inﬂationary phase, where one typically encounters more ﬁelds than would be needed for the early Universe, and moreover some familiar scenarios tend to be plagued by instabilities. The novel ingredient that appears to get around these problems is non-linear supersymmetry [101], whose foundations lie in the prescient 1973 work of Volkov and Akulov [10] and on the technique of constrained superﬁelds [102]. Non-linear supersymmetry arises when some super-partners are exceedingly massive, seems to play an intriguing role in String Theory [103] and connects naturally to the KKLT scenario [104]. The current lack of signals for supersymmetry at the LHC makes one wonder whether it might also hold a prominent place in an eventual picture of Particle Physics. This resonates with the idea of “split supersymmetry” [49], which allows for large mass separations among superpartners and can be accommodated in Supergravity at the price of reconsidering hierarchy issues. Crossing the current frontiers appears to require a deeper understanding of broken Supersymmetry in Supergravity and in String Theory. 7. – Supergravity and the AdS/CFT correspondence The late 1990s witnessed the emergence of a peculiar duality that has had a huge impact on the literature. This is the AdS/CFT correspondence [93], which links “holo- graphically” gravity and gauge theories(5). It was originally conjectured by Juan Mal- dacena, and its most explored form concerns String Theory compactiﬁed on a special KK manifold, AdS5 × S5, the direct product of a ﬁve-dimensional anti-de Sitter space and a ﬁve-dimensional internal sphere. AdS5 has a boundary at spatial inﬁnity, which is identiﬁed with a four-dimensional Minkowski space, and the correspondence posits the equivalence between a weakly coupled String Theory in the bulk and a strongly coupled ﬁeld theory, the N = 4 supersymmetric Yang-Mills theory that we have already met in a ten-dimensional guise, on this boundary. This surprising correspondence, which brought AdS Supergravity to the forefront, also vindicates some intriguing ideas that had been around since Jacob Bekenstein connected the black-hole entropy to its area [95]. Super- gravity thus started oﬃcially, with this “third superstring revolution”, one more parallel life as an unprecedented tool for exploring non-perturbative features of gauge theories. In N = 2 Supergravity supersymmetric black holes give rise to universal (duality- invariant) area formulas for their Bekenstein-Hawking Entropy, which generalize the Reissner-Nordstrom entropy formula for charged dyons and rest on the (square modu- 288 S. FERRARA and A. SAGNOTTI lus) of the central charge computed at its extremum in moduli space [96]. The name “attractors” was coined since, independently of the initial conditions at large distances, the scalar trajectories in moduli space approach the same extremal points, which coincide with the values at the BH Horizon. This explains, in particular, why the ADM mass is a continuous moduli-dependent function while the entropy is quantized in terms of BH charges. In the large-charge limit, Andrew Strominger and Cumrun Vafa associated to these types of results for extremal BH’s a microscopic counting inspired by String Theory [97]. The actual nature of the microstates, however, is still a debated issue [98]. [4] For a review of early results see: Fayet P. and Ferrara S., Phys. Rep., 32 (1977) 249. For a collection of early papers see: Ferrara S., Supersymmetry (North- Holland, Amsterdam, The Netherlands) 1987. More recent reviews are: West P. C., Introduction to supersymmetry and supergravity (World Scientiﬁc, Singapore) 1990; Wess J. and Bagger J., Supersymmetry and supergravity (Princeton University Press) 1992; Weinberg S., The quantum theory of ﬁelds. Vol. 3: Supersymmetry (Cambridge University Press) 2005. [5] Coleman S. R. and Mandula J., Phys. Rev., 159 (1967) 1251; Haag R., Lopuszanski J. T. and Sohnius M., Nucl. Phys. B, 88 (1975) 257. ∗∗∗ This overview was based in part on the talk delivered by S.F. at the “Infeld Collo- quium and Discrete”, in Warsaw, on December 1 2016, and on a joint CERN Courier article [115]. It is focussed on topics that are closer to the research interests of the au- thors, who apologize in advance if they have missed some important references. SF was supported in part by the CERN TH-Department and by INFN (IS CSN4-GSS-PI). AS was supported in part by Scuola Normale, by INFN (IS CSN4-GSS-PI) and by APC- U. Paris VII, and would like to thank APC-U. Paris VII for the kind hospitality. We are grateful to Carlo Angelantonj, Bianca Letizia Cerchiai, Emilian Dudas and Marine Samsonyan, who read the manuscript and oﬀered constructive suggestions. ( ) [6] Veneziano G., Nuovo Cimento A, 57 (1968) 190. [3] Wess J. and Zumino B., Phys. Lett., 49B (1974) 52; Nucl. Phys. B, 70 (1974) 39; Salam A. and Strathdee J. A., Nucl. Phys. 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https://openalex.org/W2291574278	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-016-2439-2	English	null	Erratum to: ‘Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq’	BMC genomics	2,016	cc-by	722	© 2016 Ayers et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: craig.smith@monash.edu 3Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3168, Australia Full list of author information is available at the end of the article ERRATUM ERRATUM Open Access Erratum to: ‘Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq’ Katie L. Ayers1,2, Luke S. Lambeth1, Nadia M. Davidson1, Andrew H. Sinclair1,2, Alicia Oshlack1 and Craig A. Smith3* * Correspondence: craig.smith@monash.edu 3Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3168, Australia Full list of author information is available at the end of the article © 2016 Ayers et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ayers et al. BMC Genomics (2016) 17:169 DOI 10.1186/s12864-016-2439-2 Ayers et al. BMC Genomics (2016) 17:169 DOI 10.1186/s12864-016-2439-2 Erratum to: ‘Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq’ Unfortunately, the original version of this article [1] contained an error in Figure 2. The correct figure is included below: F2 F2 Ayers et al. BMC Genomics (2016) 17:169 Page 2 of 3 g. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Ayers et al. BMC Genomics (2016) 17:169 Page 3 of 3 (See figure on previous page.) Fig. 2 Male candidate gene expression in vivo. Whole mount in situ hybridisation for 4 male-biased candidate genes, on E6 UGS (UGS) from males and females. BMPR2 is more highly expressed in males (a, e) than in females (i, m). In over-stained sections, BMPR2 appears to be expressed in the testis cords (e). ZNF385b shows greater expression in males (b, f) than females (j, n), consistent with RNA-seq. In males it is expressed in the cords (f). NZP, a novel Z-protein, is expressed in males more highly than females (c, g versus k, o). It is also expressed in the cords of males (g). LAMA1 is also higher in males (d) than females (l) and is expressed in testis cords in males (h), with some weak expression in the juxta-cortical medulla in females (p). These results are consistent with RNA-seq data. Typically, 3 UGS from each sex were used for each probe, and these images are representative. A sense control probe did not show any staining for any of the candidate genes (data not shown) 1. Ayers KL, Lambeth LS, Davidson NM. Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq’. BMC Genomics. 2015;16:704. Author details 1M d h Child 1Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, 3052 Parkville, VIC, Australia. 2Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia. 3Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3168, Australia. 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https://openalex.org/W2583373587	https://www.clinmedkaz.org/download/the-method-of-dynamic-proprioceptive-correction-in-the-rehabilitation-of-patients-with-childrens-8815.pdf	Russian	null	The method of dynamic proprioceptive correction in the rehabilitation of patients with children’s cerebral palsy	Ķazaķstannyṇ klinikalyķ medicinasy	2,016	cc-by	3,405	Озық мақала / Оригинальная статья / Original аrticle Озық мақала / Оригинальная статья / Original аrticle Материал поступил в редакцию: 01-09-2015 Материал принят к печати: 13-06-2016 УДК: 616.831-009.11-036.86-053.2 Тұжырымдама Тұжырымдама ұ р д Зерттеу мақсаты. Балалардың церебральды сал ауруымен ауыратын балаларды оңалтудағы динамикалық проприоцепциялық түзету әдісінің тиімділігін бағалау. Әдістері. Барлығы GMFCS II-IV деңгей жүйесі бойынша аурудың ауырлық дәрежесімен балалардың церебральды сал ауруы диагно- зы, диплегиялық түрімен ауыратын 4-тен 7- жасқа дейінгі 80 бала зерттелді. Негізгі топты динамикалық проприоцептивті коррекция әдісімен кешенді оңалту емін алушы 55 бала құрайды, ал динамикалық проприоцептивті коррекция әдісін қолданбайтын оңалту курсынан өткен 25 пациент бақылау тобына кіреді. Барлық пациенттердің емдеу алдында және емдеуден кейін Ашуорт шкаласы бойынша бұлшықет тонусының жағдайы, (GMAE) баллдарын компьютерлік бағдарламамен есептеуді пайдаланумен (GMFM-88) үлкен қозғалыс қызметінің шкаласы бой- ынша үлкен қозғалыс қызметінің динамикасы бағаланды және бас миының Nihon-Kohden ЭЭГ-1100К компьютерлік кешендегі биоэлектрлік көрсеткішімен таныстырылды. р р Нәтижесі. Алынған деректердің талдауы динамикалық проприоцептивті коррекция әдісін қолданумен ем қабылдаған пациенттердің бақылау тобындағы пациенттерге қарағанда жаңа қозғалыс дағдысын нақты тезірек меңгеретіндігін куәландырады. у р р р р у р ЭЭГ көрсеткішімен танысу патологиялық кері афферентацияны түзету бас миының қабыршағына белсенді әсер ететінін және қыртыстық электрогенездің қыртысастылық-таламистикалық құрылымына патологиялық әсер етуін төмендететінін көрсетті. қ р д ң қ р қ қ құр қ р у д р Қорытынды. Өткізілген зерттеу және алынған деректер балалардың церебральды сал ауруымен ауыратын амикалық проприоцепциялық түзету әдісінің тиімділігін куәландырады. кізілген зерттеу және алынған деректер балалардың церебральды сал ауруымен ауыратын балаларды емдеуд епциялық түзету әдісінің тиімділігін куәландырады. Маңызды сөздер: оңалту - балалардың церебральды сал ауруы - динамикалық проприоцепциялық түзету. метод динАмической проприоцептивной коррекции в реАБилитАции пАциентов с детским цере- БрАльным пАрАличом лисовский е.в., кусаинова к.к. АО Р б й й б й О б А К Abstract Research objective was the evaluation of effectiveness of the dynamic proprioceptive correction in complex rehabilitation of patients with Children Cerebral Palsy. Methods. The results of diagnostics and treatment of 80 patients at age from 4 to 7 years with Children’s Cerebral Palsy, diplegical form, level of severity II-IV according to GMFCS, were analyzed. 55 children of study group were treated with complex rehabilitation program included dynamic proprioceptive correction; 25 children of control group were treated with standard complex rehabilitation program. The state of muscular tone were estimate in patient of both groups according to Ashworth scale, dynamics of motor function according to GMFCS with calculation program GMAE, indicators of bioelectrical brain activity with computer complex Nihon-Kohden EEG-1100К. Results. The analysis of our date demonstrates that patients treated with rehabilitation program included dynamic propriocep- tive correction have statistically significant advantage over patients of control group in the gaining of new motor skills. Investigation of EEG-indicators shows that the correction of pathological afferentation makes activate influence on the brain cortex and decrease pathological influence of subcortical and thalamus structures on the cortical electrogenesis. i Conclusions. The data of conducted investigation confirm the effectiveness of the method of dynamic proprioceptive correc- tion in the treating of patients with Children Cerebral Palsy. Keywords: rehabilitation - children’s cerebral palsy - dynamic proprioceptive correction. J Clin Med Kaz 2016; 2(40):31 35 Автор для корреспонденции: Лисовский Евгений Владимирович, АО «Республиканский детский реабилитационный центр». Адрес: 36 проспект Туран, Астана. 010000. Тел.: +7(702) 584-17-41. E-mail: lisovski67@gmail.com Yevgen Lisovskyy, Kenzhe Kussainova «Republican Children’s Rehabilitation Centre» JSC, Scien «Republican Children’s Rehabilitation Centre» JSC, Science and Education Department, Аstana, Kazakhstan Abstract БАлАлАрдың цереБрАльды сАл Ауруымен АуырАтын БАлАлАрды оңАлтудАғы динАмикАлық проприоцепциялық түзету әдісі лисовский е.в., құсаинова к.к. лисовский е.в., құсаинова к.к. «Республикалық балаларды оңалту орталығы» АҚ, ғылым мен білім бөлімі, Астана, Қазақстан J Clin Med Kaz 2016; 2(40):31-35 The method of dynamic proprioceptive correction in the rehabilitation of patients with children’s cerebral palsy Yevgen Lisovskyy, Kenzhe Kussainova R bl Ch ld ’ R h b l C JSC S Yevgen Lisovskyy, Kenzhe Kussainova bl h ld h b l Введение В 1991 г. в Институте педиатрии РАН с целью коррекции аномально функционирующей системы ФСА в комплексном лечении детского церебрального паралича был впервые использован лечебный костюм (ЛК) «Адели». Главный принцип действия этого костюма состоит в исправлении (коррекции) афферентного проприоцептивного потока; исходя из этого, данный метод лечения был назван «динамической проприоцептивной коррекцией» (ДПК). В 1991 г. в Институте педиатрии РАН с целью коррекции аномально функционирующей системы ФСА в комплексном лечении детского церебрального паралича был впервые использован лечебный костюм (ЛК) «Адели». Главный принцип действия этого костюма состоит в исправлении (коррекции) афферентного проприоцептивного потока; исходя из этого, данный метод лечения был назван «динамической проприоцептивной коррекцией» (ДПК). В настоящее время детский церебральный паралич (ДЦП) принято определять, как «совокупность нарушений двигательной функции, движений и положения тела, при этом нарушения имеют постоянный, но не неизменный характер и вызваны непрогрессирующим повреждением или аномалиями головного мозга, возникающими в развивающемся/незрелом мозге» (SCPE – Европейское сообщество контроля детского церебрального паралича, 2000) [1]. В настоящее время метод ДПК с применением лечебных костюмов широко применяется в центрах реабилитации больных ДЦП в России, реже – за рубежом, появились новые модификации ЛК – «Гравистат», «Гравитон», «Регент», «Спираль» (Украина), «Терасьют» (TheraSuit, США). Согласно К.А. Семеновой, «существующие в настоящее время рутинные методы лечения, включающие многочисленные методики лечебной физкультуры, массажа, физиотерапии, фармакотерапии, иглорефлексотерапии, ортопедической коррекции, могут снизить выраженность двигательного дефекта, но не в состоянии устранить его полностью, при этом вероятность рецидива приближается к 100% («сизифова» реабилитация)» [2,3]. Такой парадоксальный вывод никак не может устроить врачей и родителей больных детей с ДЦП. Поэтому продолжение изучения методов и способов реабилитации и абилитации, направленных на улучшение состояния больных с ДЦП, продолжает оставаться актуальной проблемой современной реабилитологии [4]. Все вышеуказанные костюмы представляют собой систему эластичных тяг – аксиальных, которые обеспечивают компрессионную нагрузку, и ротационных, с помощью которых осуществляетсякоррекция различных патологических установок и патологического двигательного стереотипа в целом (ротационно-корригирующие тяги). В 2006 г. профессором Исановой В.А.был предложен ЛК «Атлант», который представляет собой комбинезон со смонтированным на нем пневматическим корсетом и пневматическим фиксатором конечностей. Таким образом обеспечивается формирование пневматического корсета для туловища и пневмоортезов для суставов конечностей, что обеспечивает удержание вертикальной позы больными ДЦП с минимальным расходом энергии. Устойчивость характерной для резидуальной стадии ДЦП патологической программы двигательного развития и отсутствие лекарственных средств ее эффективной коррекции привели к тому, что в последние десятилетия акцент в восстановительном лечении больных делается на поиск патогенетически целесообразных интегративных немедикаментозных воздействий [5]. Введение В основе современных нефармакологических технологий развития двигательных навыков и коррекции нарушений произвольной моторики при церебральном параличе лежат представления о нейропластичности как об адаптационном свойстве человеческого мозга модифицировать взаимодействие нейронов под влиянием приобретенного опыта [6]. Пластичность мозга проявляется устойчивыми целенаправленными изменениями реакций нейрона, преобразованиями его внутриклеточных структурв процессе обучения, что приводит к изменению межнейронных взаимодействий. В конечном итоге, именно эти изменения являются основанием для компенсации утраченной функции при органических неврологических заболеваниях. Целью работы явился анализ эффективности метода динамической проприоцептивной коррекцией в комплексной реабилитации детей с детским церебральным параличом. лисовский е.в., кусаинова к.к. лисовский е.в., кусаинова к.к. , у АО «Республиканский детский реабилитационный центр», Отдел науки и образования, Астана, Казахстан Резюме Цель исследования. Оценка эффективности метода динамической проприоцептивной коррекции в комплексной реабилитации паци- ентов с детским церебральным параличом. Цель исследования. Оценка эффективности метода динамической проприоцептивной коррекции в компл ентов с детским церебральным параличом. Методы. Обследовано 80 детей с диагнозом «детский церебральный паралич, диплегическая форма» в возрасте от 4-х до 7-ми лет со степенью тяжести заболевания по системе GMFCS II-IV уровень. Основную группу составили 55 детей, получавших в составе комплексной №2 (40) 2016 Clinical Medicine of Kazakhstan 31 реабилитации лечение методом динамической проприоцептивной коррекции; в контрольную группу вошли 25 пациентов, которые проходи- ли курс реабилитации без применения метода динамической проприоцептивной коррекции. У всех пациентов до и после лечения оценивали состояние мышечного тонуса по шкале Ашуорт, динамику больших моторных функций по шкале больших моторных функций (GMFM-88) с использованием компьютерной программы подсчета баллов (GMAE) и изучали показатели биоэлектрической активности головного мозга на компьютерном комплексе Nihon-Kohden ЭЭГ-1100К. р Результаты. Анализ полученных данных свидетельствует о том, что пациенты, получавшие лечение с использованием метода дина- мической проприоцептивной коррекции статистически достоверно быстрее осваивают новые двигательные навыки, чем пациенты контроль- ной группы. Изучение показателей ЭЭГ показывает, что коррекция патологической афферентации оказывает активирующее влияние на кору головного мозга и снижает патологическое влияние подкорково-таламических структур на корковый электрогенез. Выводы. Проведенное исследование и полученные данные свидетельствуют об эффективности метода динамической проприоцеп- тивной коррекции в лечении детей с дестким церебральным параличом. р Результаты. Анализ полученных данных свидетельствует о том, что пациенты, получавшие лечение с использованием метода дина- мической проприоцептивной коррекции статистически достоверно быстрее осваивают новые двигательные навыки, чем пациенты контроль- ной группы. Изучение показателей ЭЭГ показывает, что коррекция патологической афферентации оказывает активирующее влияние на кору головного мозга и снижает патологическое влияние подкорково-таламических структур на корковый электрогенез. Выводы. Проведенное исследование и полученные данные свидетельствуют об эффекти тивной коррекции в лечении детей с дестким церебральным параличом. е исследование и полученные данные свидетельствуют об эффективности метода динамической проприоцеп- детей с дестким церебральным параличом. рр р р р Ключевые слова: реабилитация - детский церебральный паралич - динамическая проприоцептивная коррекция. Материалы и методы исследования Исследование выполнено на базе АО «Республиканский детский реабилитационный центр» (Астана). Всего обследовано 80 детей с диагнозом «ДЦП, диплегическая форма». Исследование выполнено на базе АО «Республиканский детский реабилитационный центр» (Астана). Всего обследовано 80 детей с диагнозом «ДЦП, диплегическая форма». Критериями для включения пациентов в исследование являлись: клинически установленный диагноз диплегической формы ДЦП; возраст пациента от 4-х до 7-ми лет (включительно); степень тяжести заболевания по системе классификации больших моторных функций (GMFCS) – уровень II-IV; согласие родителей пациента на участие в исследование. Из исследования исключались дети с симптоматической эпилепсией, в том числе – в состоянии клинической ремиссии, но с высоким индексом представленности эпилептиформной активности на ЭЭГ. В основе двигательных нарушений у бальных ДЦП лежит патология именно функциональной системы антигравитации (ФСА) [4,7]. При этом в горизонтальном положении тела активность ФСА минимальна и на-рушения тонуса мышц больного так же минимальны; как только больной принимает вертикальное положение происходит резкое повышение тонусаантигравитационных мышц В основную (первую) группу вошли 55 детей, получавших в составе комплексной реабилитации лечение методом ДПК (ЛК «Атлант»). Средний возраст детей №2 (40) 2016 Clinical Medicine of Kazakhstan 32 в этой группе составил 5,3+0,9 года. Распределение по полу произошло следующим образом: мальчики – 30 (54,5%), девочки – 25 (45,5%). В контрольную (вторую) группу вошли 25 пациентов, которые проходили курсы комплексной реабилитации без применения метода ДПК. В этой группе распределение пациентов по полу произошло следующим образом: мальчики – 14 (56,0%), девочки – 11 (44,0%). Средний возраст детей контрольной группы оказался равен 5,2+1,0года. Таким образом, по возрастно- половому признаку, диагнозу и степени тяжести заболевания (по системе GMFCS, см. табл. 1) обе группы являются репрезентативными. Распределение детей основной и контрольной групп по системе GMFCS Таблица 1 Распределение детей основной и контрольной групп по системе GMFCS Таблица 1 Группа GMFCS II III IV абс % абс % абс % Основная 15 27,3 31 56,3 9 16,4 Контрольная 6 24,0 15 60,0 4 16,0 освоение этих моторных навыков имеет смысл направить усилия методиста ЛФК и родителей пациента. Занятия в ЛК «Атлант» проводились 1 раз в день, длительность занятия – 45 минут, количество процедур на курс лечения – 15. Во время проведения занятий ЛФК акценты выбирались в зависимости от этапа моторного развития ребенка: подавление патологических тонических рефлексов и совершенствование позно-тонического контроля, тренировка переворота, контроль позвоночника в положении сидя, тренировка опорной функции рук, способностьначинать и завершать движение, тренировка шагового рефлекса и опорной функции ног и др. Материалы и методы исследования У детей основной и контрольной групп с целью изучения влияния метода ДПК на показатели биоэлектрической активности головного мозга проводилась электроэнцефалография. Исследование проводилось на аппаратно-программном компьютерном комплексе Nihon-KohdenЭЭГ-1100К (Япония) с использованием международной схемы наложения электродов по Джасперу 10-20. На полученных электроэнцефалограммах проводили визуальный анализ кривой, оценку амплитуд и индексов основных ритмов биоэлектрической активности (БЭА) мозга. Необходимо отметить, что пациенты обеих групп в комплексной реабилитации получали ботулинотерапию препаратом Диспорт. Средняя доза препарата составила в основной группе 300+40,8 Ед, в контрольной – 312+46,3 Ед. Все исследования проводили при поступлении пациента и после окончания курса реабилитации. Клиническое обследование детей включало изучение динамики степени выраженности спастического синдрома по шкале Ашуорт и уровень моторного развития ребенка по шкале больших моторных функций в варианте GMFM- 88и GMFM-66 с использованием компьютерной программы подсчета баллов – «Gross Motor Ability Estimator» (GMAE). Последняя на основании полученных результатов позволяет построитьграфическую диаграмму, в которойоцениваемые двигательные актыраспределяются по степени сложности (рис.1). В этом случаеварианты ответной реакциислева от вертикальной линии (95% доверительный интервал обследованного пациента) представляют собой акты, которыми пациент овладел, а баллы справа – это двигательные акты, которыми пациент, вероятно, овладеет в будущем. Те двигательные акты, которые находятся ближе к линии справа, являются более легкими для пациента и должны быть освоены раньше остальных. Именно на На проведение исследования получено разрешение Локальной этической комиссии АО «Республиканский детский реабилитационный центр». Обследование пациентов проведено с соблюдением принципов медицинской этики; от родителей пациентов получено информированное согласие на участие детей в исследовании. Полученные результаты анализировали с помощью методов биостатистики в программном пакете Exсel 2016, с расчетом параметрического критерия достоверности Стьюдента. Результаты Общими особенностями БЭА мозга у обследованных детей (ка основной так и контрольной групп) являлись: сме- щение частоты альфа-ритма в сторону низких частот (8-10 Гц); некоторое снижение амплитуды альфа-ритма; значи- тельное повышение индекса представленности в структуре ЭЭГ медленных ритмов – волн дельта и тета диапазонов. Эти изменения являются отражением нарушения созрева- Динамика показателей основных показателей ЭЭГ у детей основной и контрольной групп в процессе лечения Таблица 4 * - разница между показателями основной и контрольной групп статистически достоверна (p<0,01) Основная группа Контрольная группа До лечения После лечения До лечения После лечения Средняя частота альфа- ритма 9,1+1,2 11,5+1,7* 9,0+1,1 9,8+1,2* Средняя амплитуда альфа- ритма 45,8+4,7 53,4+4,1* 47,1+5,8 48,6+5,1* Средняя амплитуда дельта- ритма 149,3+6,3 140,3+8,4 147,5+6,3 141,8+7,8 Индекс представленности дельта-ритма 51,6+8,2 36,9+7,9* 48,4+7,4 43,8+9,2* Средняя амплитуда тета- ритма 136,6+3,1 127,4+6,5 138,7+6,9 133,3+6,8 Индекс представленности тета-ритма 44,5+9,9 25,3+7,6* 45,8+8,5 41,7+7,5* Динамика показателей основных показателей ЭЭГ у детей основной и контрольной групп в процессе лечения * - разница между показателями основной и контрольной групп статистически достоверна (p<0,01) тонических рефлексов и патологических синергий, возник- ших на их основе), корригируют патологическую пропри- оцептивную афферентацию и, следовательно, способствуют формированию вестибулярных реакций, а в дальнейшем на их основе – обеспечивают адекватную работу функциональ- ной системы антигравитации. тонических рефлексов и патологических синергий, возник- ших на их основе), корригируют патологическую пропри- оцептивную афферентацию и, следовательно, способствуют формированию вестибулярных реакций, а в дальнейшем на их основе – обеспечивают адекватную работу функциональ- ной системы антигравитации. В процессе лечения у пациентов наблюдались однона- правленные изменения БЭА мозга: увеличение амплитуды и частоты базового ритма, уменьшение индекса представ- ленности в структуре ЭЭГ медленноволновой активности и снижение амплитуды дельта - и тета-волн. В группе де- тей, получавших в составе комплексной терапии лечение методом ДПК эти изменения более выражены в сравнении с пациентами контрольной группы и эта разница является статистически достоверной (p<0,01; табл. 4). Подобные из- менения отражают активирующее влияние на кору головно- го мозга афферентации, поступающей от периферических проприорецепторов в процессе коррекции патологического двигательного стереотипа в лечебном костюме и свидетель- ствуют о снижении патологических влияний подкорково-та- ламических структур на кору головного мозга. Анализ состояния и динамики биоэлектрической ак- тивности головного мозга больных с ДЦП, прошедших курс реабилитации с включением метода ДПК, показывает его- выраженное влияние на интегративную деятельность мозга, что выражается количественнымии качественными изме- нениями спектров биоэлектрической активности. Характер этих изменений демонстрирует уменьшение влиянияактив- ности подкорково-стволовыхструктурна кору головного мозга и совершенствование механизмов коркового тормо- жения. Результаты В таблице 2 представлена динамика показателей мышечного тонуса у обследованных детей по шкале Ашуорта. Показатели мышечного тонуса у пациентов основной и контрольной групп в процессе лечения Таблица 2 Группа Верхние конечности Нижние конечности До лечения После лечения p До лечения После лечения p Основная (n=55) 1,3+0,4 1,04+0,2 p> 0,05 3,8+0,6 2,8+0,6 p> 0,05 Контрольная (n=25) 1,2+0,5 1,0+0,3 3,8+0,6 3,0+0,6 Показатели мышечного тонуса у пациентов основной и контрольной групп в процессе лечения лечении детей с ДЦП. Как видно из представленных данных, несмотря на достигнутое в процессе лечения снижение тонуса мышц (преимущественно в нижних конечностях), статистиче- ски достоверной разницы между пациентами основной и контрольной групп нет. К тому же мы не можем отнести динамику мышечного тонуса исключительно за счет его коррекции методом ДПК. Скорее снижение тонуса мышц обусловлено применением ботулинотерапии в комплексном Анализ уровня и темпов моторного развития паци- ентов, полученных в процессе тестирования по шкале GMFM-88 (табл. 3), свидетельствует о том, что пациенты основной группы быстрее осваивают новые двигательные навыки, причем достоверность полученных результатов высокая(p<0,01). Мы отдаем себе отчет в том, что шкала GMFM не оценивает качество выполняемого движения, но №2 (40) 2016 Clinical Medicine of Kazakhstan 33 нованием для более быстрого перехода на новый уровень моторного развития. появление нового моторного акта с последующим его закре- плением в составе двигательного стереотипа и отработкой качественной составляющей этого движения является ос- появление нового моторного акта с последующим его закре- плением в составе двигательного стереотипа и отработкой качественной составляющей этого движения является ос- Динамика моторного развития пациентов по шкале GMFM-88 (в баллах) Таблица 3 * - разница между показателями основной и контрольной групп статистически достоверна (p< 0,01) Группа До лечения После лечения Степень прироста Основная (n=55) 58,2+8,5 65,3+8,8 7,0+2,2* Контрольная (n=25) 58,5+10,6 63,0+10,2 4,5+1,4* ния коркового электрогенеза, а увеличение индекса медлен- новолновой активности свидетельствует о функциональной- незрелости нервной ткани нафоне имеющихся структурных повреждений головного мозга у детей с ДЦП и о преобла- дании таламокортикальной составляющей в иерархической системе электрогенеза биоэлектрической активности голов- ного мозга. ния коркового электрогенеза, а увеличение индекса медлен- новолновой активности свидетельствует о функциональной- незрелости нервной ткани нафоне имеющихся структурных повреждений головного мозга у детей с ДЦП и о преобла- дании таламокортикальной составляющей в иерархической системе электрогенеза биоэлектрической активности голов- ного мозга. Результаты С современных позиций, ритмы головного мозга рас- сматриваются как иерархическая система, которая включает в себя три эволюционные подсистемы мозга – стволовую, лимбическую и корково-таламическую [8]. Согласно этой теории, медленные волны связаны с биологическими моти- вациямии эмоциональными процессами, а быстрые ритмы отражают процессы «восприятия и распознавания паттер- нов окружающей среды». Обсуждение Результаты проведенного исследования свидетель- ствуют о том, что применение метода ДПК с использова- нием ЛК «Атлант» позволяет оптимизировать программу реабилитации пациентов с ДЦП и значительно ускорить освоение новых двигательных навыков. Занятия в лечебном костюме приводят к снижению и нормализации мышечного тонуса (в том числе и за счет подавления патологических №2 (40) 2016 Clinical Medicine of Kazakhstan 34 №2 (40) 2016 Clinical Medicine of Kazakhstan y g ), y , , ( ), pp A.B. Lektsii po nevrologii razvitiya (Lectures on the development of neurology), M., 2012, 368 р. Выводы систем с качественно новыми функциональными возможностями формирования более адекватного двигательного стереотипа. Подобная динамика показателей ЭЭГ является косвенным признаком эффективности метода ДПК в лечении детей с ДЦП. Выводы Вышеописанные изменения в соотношении частотных диапазонов на ЭЭГ свидетельствуют не просто о новом уровне корково-подкорковых взаимоотношений, но являются отражением построения высокодифференцированных систем с качественно новыми функциональными возможностями формирования более адекватного двигательного стереотипа. Подобная динамика показателей ЭЭГ является косвенным признаком эффективности метода ДПК в лечении детей с ДЦП. p y g y ( p g y), g, , p 8. Klitochenko G.V., Tonkonozhenko N.L., Guyvan O.I. et al. Issledovanie faktorov razvitiya razlichnyih form detskogo tserebralnogo paralicha v Volgograde (Research of factors of various forms of cerebral palsy in Volgograd), Meditsinskie nauki, 2014, No.11, pp.25-28. p g y ( p gy) A.L. Eksperimentalnaya gravitatsiya (Experimental gravity), S-Peterburg, 2014, 112 p. Литература р ур 1. Aykardi Zhan. Zabolevaniya nervnoy sistemyi u detey (Diseases of the nervous system in children), M., 2013, T.1, 553 р. р ур 1. Aykardi Zhan. Zabolevaniya nervnoy sistemyi u detey (Diseases of the nervous system in children), M., 2013, T.1, 553 р. 1. Aykardi Zhan. Zabolevaniya nervnoy sistemyi u detey (Diseases of the nervous system in children), M., 2013, T.1, 553 р. 2. Semenov I.V. Referativnyiy obzor. Voprosyi etiopatogeneza detskogo tserebralnogo paralicha (Patent review. Questions etiopathogenesis of cerebral palsy), М., 2010, 557 р. 2. Semenov I.V. Referativnyiy obzor. Voprosyi etiopatogeneza detskogo tserebralnogo paralicha (Patent review. Questions etiopathogenesis of cerebral palsy), М., 2010, 557 р. 2. Semenov I.V. Referativnyiy obzor. Voprosyi etiopatogeneza detskogo tserebralnogo paralicha (Patent review. Questions etiopathogenesis of cerebral palsy), М., 2010, 557 р. 3. Semenova K.A. Problema vosstanovitelnogo lecheniya detskogo tserebralnogo paralicha (The problem of rehabilitation treatment of cerebral palsy), Zhurn. nevrologii i psihiatrii, 2012, No.7, pp.9-13. 3. Semenova K.A. Problema vosstanovitelnogo lecheniya detskogo tserebralnogo paralicha (The problem of rehabilitation treatment of cerebral palsy), Zhurn. nevrologii i psihiatrii, 2012, No.7, pp.9-13. 5. Dotsenko V.I. Ispolzovanie v sovremennoy neyroreabilitatsii priemov remodelirovaniya pravilnyih dvigatelnyih stereotipov i sensornogo obraza dvizheniy (The use of modern techniques of neurorehabilitation remodeling correct movement patterns and motion sensory image), Kurortnyie vedomosti, 2010, No.4 (61), pp.33-41. y g ) y ( ) pp 6. Palchik A.B. Lektsii po nevrologii razvitiya (Lectures on the development of neurology), M., 2012, 3 7. Dmitriev A.L. Eksperimentalnaya gravitatsiya (Experimental gravity), S-Peterburg, 2014, 112 p. 8. Klitochenko G.V., Tonkonozhenko N.L., Guyvan O.I. et al. Issledovanie faktorov razvitiya razlichnyih form detskogo tserebralnogo paralicha v Volgograde (Research of factors of various forms of cerebral palsy in Volgograd), Meditsinskie nauki, 2014, No.11, pp.25-28. №2 (40) 2016 Clinical Medicine of Kazakhstan 35
https://openalex.org/W2746097385	https://jomped.org/index.php/jomped/article/download/6/24	English	null	Protective effects of neem (<i>Azadirachta indica A. Juss</i>) seed oil on carbon tetrachloride-induced hepatotoxicity in Wistar rats	Journal of medicinal plants for economic development	2,017	cc-by	4,603	Journal of Medicinal Plants for Economic Development ISSN: (Print) 2519-559X Journal of Medicinal Plants for Economic Development ISSN: (Print) 2519-559X Page 1 of 5 Original Research Page 1 of 5 Original Research Page 1 of 5 Dates: Results: Treatment with neem seed oil lowered the aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels significantly (P < 0.05) in a dose-dependent manner compared to the control. The haematological parameters, organ weight index and animal body weight showed no significant difference (P > 0.05) when compared with the control. Histology assessment was in agreement with the biochemical result as tissues of CCl4 exhibited significant fatty tissue accumulation, as opposed to that of 0.25 mL/kg neem treatment, which showed only moderate accumulation of fatty tissues, while higher doses, 0.5 mL/kg and 1.0 mL/kg, showed a healthy liver as compared with the control. Received: 10 Jan. 2017 Accepted: 26 June 2017 Published: 18 Aug. 2017 Received: 10 Jan. 2017 Accepted: 26 June 2017 Published: 18 Aug. 2017 How to cite this article: Idu, M., Ovuakporie-Uvo, O. & Okojie, S.O., 2017, ‘Protective effects of neem (Azadirachta indica A. Juss) seed oil on carbon tetrachloride-induced hepatotoxicity in Wistar rats’, Journal of Medicinal Plants For Economic Development 1(1), a6. https://doi.org/ 10.4102/jomped.v1i1.6 Conclusion: The result of this study revealed that neem seed oil had a dose-dependent hepatoprotective effect on the experimental rats. Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Affiliations: Method: Hepatotoxicity was induced by the administration of 1.0 mL/kg of CCl4 subcutaneously to 72 healthy Wistar rats of both sexes (weight range: 145 g – 315 g). The seed oil of A. indica was orally administered daily in various doses of 0.25 mL/kg, 0.5 mL/kg and 1.0 mL/kg for 14 days. Animal body and organ weights were recorded, while blood and liver tissues were collected for biochemical, haematological and histological analyses. Introduction Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Plants serve various purposes in the world, one of which is treating various ailments. The neem tree (Azadirachta indica A. Juss) has been known as a wonder tree since time past in the Indian subcontinent. It is now known all over the world majorly because of its relevance in food production and its medicinal properties (Mugnai 2009). Neem plant is the most diverse and versatile tree with maximum useful non-wood products (leaves, barks, flowers, fruits, seeds, gum, oil and neem cake) (Uwimbabazi, Uwimana & Rutanga 2015). Neem plant popularly known as ‘village pharmacy’ contains limonoids in its seeds, bark and leaves which have been confirmed to exhibit antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer and antifungal properties (Nix 2007). Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Since time past, herbal medicine has been known as the oldest form of medicine because it is believed to be safe, gentle, accessible and cost friendly (Abebe 2002). However, a standard dose for these herbal drugs is not established and may result in its minimised potential (lesser dosage) or toxicity effect (excess dosage). The physiological activities of neem (A. indica) leaf meal and the responses of rabbits at graded doses have been reported to have mild to moderately toxic effects (Ogbuewu 2008). A 24-hLD50 of 14 mg/kg for acute toxicity in rats has been earlier reported for neem leaf meal. Gomase, Rangari and Verma (2011) reported that young stem bark extract of A. indica plant protected the liver of rats induced with carbon tetrachloride (CCL4) using silymarin as a positive control. Doses of 200 mg/kg and 500 mg/kg of A. indica stem bark extract were reported to steady the functional status of liver cells. Also, neem leaf extract has been observed to provide hepatoprotective effects against paracetamol-induced hepatic cell damage in rats (Gupta, Khosla & Singh 2008). However, the present study aims at investigating the hepatoprotective activity of neem seed oil in rats induced with liver damage using CCL4. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Protective effects of neem (Azadirachta indica A. Juss) seed oil on carbon tetrachloride-induced hepatotoxicity in Wistar rats Background: Azadirachta indica (neem) seed oil was evaluated for its hepatoprotective effect. Liver damage was induced using carbon tetrachloride (CCl4) while silymarin served as a positive control. Aim: This study is aimed at testing the hepatoprotective potentials of A. indica seed oil on Wistar rats. Introduction Open Access http://www.jomped.co.za http://www.jomped.co.za Page 2 of 5 Original Research Statistical analysis Data were expressed as mean ± standard error of mean. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Duncan multiple comparisons post- test to separate between the means. Data from the test groups were compared with their respective controls and the differences at P < 0.05 were regarded as significant. Group 4 animals were administered neem oil (0.25 mL/kg) after CCl4 treatment. Group 5 animals were administered neem oil (0.5 mL/kg) after being treated with CCl4. Group 5 animals were administered neem oil (0.5 mL/kg) after being treated with CCl4. Fatty liver tissues are evident on CCl4-treated animals only (Figure 1b), whereas moderate to mild fatty liver tissues were observed in silymarin (Figure 1c) and 0.25 mL/kg neem oil– treated rats (Figure 1d). Normal liver cells radially arranged around portal tracts and central veins were evident in 0.5 mL/kg and 1.0 mL/kg of neem oil–treated animals (Figure 1e and f), which are similar to control. Group 6 animals were administered neem oil (1 mL/kg) after being treated with CCl4. Group 6 animals were administered neem oil (1 mL/kg) after being treated with CCl4. At the end of the two-week experimental period, the animals were fasted overnight prior to the administration of CCl4 except for the control group. This was done for rapid absorption and metabolism of the administered CCl4. Then, animals were sacrificed under chloroform anaesthesia to collect their blood samples through cardiac puncture. The livers, heart, left and right kidneys, spleen and lungs were Materials and methods Source of oil collected and weighed. The livers were further placed in sterile bottles containing 40% formaldehyde for subsequent analysis. The blood sample collected from the sacrificed animal was transferred immediately into a lithium heparinised container to avoid clotting, and centrifuged at 4000 rpm for 5 min. After which the supernatant was transferred into a clean tube using a Pasteur pipette for subsequent liver function test. Histopathology was carried out on the harvested liver. The tissues were placed in molten paraffin wax and separated, stained with haematoxylin and eosin and were examined under a light microscope. A photomicroscope (Motic, Canada) was used to take pictures of the micrographs at magnification 100x. Fresh neem oil (cold pressed) was purchased from Kano State, Nigeria. The oil was authenticated by Professor MacDonald Idu at the herbarium of the Department of Plant Biology and Biotechnology, University of Benin. The oil was kept in a cool and dry place throughout the duration of the experiment. Experimental animals A total of 24 albino rats of both sexes, 4 animals per group with weight range of 145 g – 315 g, were used for the study. The animals were purchased from a local dealer. They were housed in clean, disinfected cages and allowed free access to feed during the period of the experiment. The animal quarter was a cool and dry place, and animals observed a light–dark cycle. The animals were allowed to acclimatise to the new environment (animal house) for two weeks before the experiment. The animals were fed on pelletised grower mash and clean water. For the purpose of the study, the animals were divided into six groups comprising four animals each. The animals were identified, stained and weighed every three days throughout the duration of the work. Results Neem seed oil at 0.25, 0.5 and 1.0 mL/kg significantly lowered the aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in a dose-dependent manner when compared with the control. AST, ALT and ALP levels of CCl4-treated rats increased significantly (P < 0.05) when compared with the control. All doses of neem oil, positive control and negative control had no significant impact on the total protein (TP) and albumin (ALB) levels when compared with the control (Table 1). No significant difference (P > 0.05) was observed in all haematological parameters for animals treated with all doses of neem oil, positive and negative control when compared with the control (Table 2). Experimental design The CCl4 solution was prepared by mixing 0.2 mL of CCL4 with olive oil (1:1). The prepared solution was administered to the animals using a dose of 1 mg/kg. All administration of neem oil and silymarin was done orally using an oral gastric tube. All administration of CCl4 was carried out subcutaneously using sterile disposable needles. Group 1 control animals received no form of administration. Group 2 animals were administered CCl4 only. Gross examination of animal organs, livers, hearts, spleens, left kidneys, right kidneys and lungs, shows that there was no significant difference (P < 0.05) in all animal groups when compared with the control (Table 3). Also, the recorded body weight of animals at various days had no significant difference across the various groups (Table 4). Group 3 animals were administered silymarin after CCl4 treatment. Group 4 animals were administered neem oil (0.25 mL/kg) after CCl4 treatment. Discussion Different superscripts across rows show the means are significant from others when compared with the control ted bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; GLOB, globulin. 4. *P < 0.01; highly significant, P < 0.05; significant compared to control. Different superscripts across rows show the means are significant from others w TB, total bilirubin; CB, conjugated bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; GLOB, globulin. Values are mean ± SEM, n = 4. *P < 0.01; highly significant, P < 0.05; significant compared to control. Different superscripts across rows show the means are significant from others when compared with the control. TABLE 2: Effect of neem seed oil on haematological parameters of Wistar rats pretreated with CCl4 (U/g of wet tissue). Biochemical parameters Control CCl4 Silymarin+CCl4 Neem oil (0.25mL/kg) Neem oil (0.5mL/kg) Neem oil (1.0mL/kg) P WBC × 103 (µL) 13.57 ± 2.05 11.07 ± 1.22 12.03 ± 2.63 18 ± 5.25 11.45 ± 3.45 10.13 ± 0.90 P > 0.05 LY (%) 60.97 ± 2.26 65.63 ± 6.27 63.87 ± 2.66 56.63 ± 8.56 70.33 ± 5.33 75.77 ± 6.50 P > 0.05 MO (%) 12.73 ± 1.73 13.77 ± 3.10 8.4 ± 0.81 7.88 ± 0.55 10.2 ± 2.25 11.07 ± 2.83 P > 0.05 GR (%) 26.3 ± 0.57 20.6 ± 3.35 27.73 ± 2.07 35.5 ± 9.08 19.48 ± 6.16 13.17 ± 3.70 P > 0.05 RBC × 106 (µL) 6.62 ± 0.16 6.7 ± 1.44 7.54 ± 0.53 7.14 ± 0.40 6.79 ± 0.61 7.15 ± 0.27 P > 0.05 HGB (g/dL) 12.3 ± 0.85 11.53 ± 2.44 13.43 ± 0.75 11.55 ± 0.6 12.05 ± 0.47 12.33 ± 0.58 P > 0.05 HCT (%) 34 ± 1.59 33.33 ± 6.11 38.63 ± 1.41 34.5 ± 1.47 44.53 ± 9.28 36.43 ± 2.03 P > 0.05 PLT × 103 (µL) 487.33 ± 26.56 582.33 ± 15.06 514.33 ± 81.17 917.25 ± 205.22 660.25 ± 181.65 509 ± 178.55 P > 0.05 Values are mean ± SEM, n = 4 animals. P > 0.05 = not significant. The experimental animals were compared with normal groups. WBC white blood cells; LY% lymphocytes; MO% monocytes; GR% granulocytes; RBC red blood cells; HGB haemoglobin; HCT haematocrit; PLT platelets The experimental animals were compared with normal groups. Discussion TABLE 3: Effects of neem seed oil on organ weight index (g) of Wistar rats pretreated with CCl4. Organs Control CCl4 Silymarin+CCl4 Neem oil (0.25 mL/kg) Neem oil (0.5 mL/kg) Neem oil (1.0 mL/kg) P Liver 2.86 ± 0.28 3.28 ± 0.27 3.12 ± 0.27 4.4 ± 0.46 4.55 ± 0.28 4.6 ± 0.63 P > 0.05 Heart 0.18 ± 0.05 0.17 ± 0.06 0.23 ± 0.02 0.13 ± 0.02 0.15 ± 0.01 0.48 ± 0.22 P > 0.05 Spleen 0.19 ± 0.09 0.14 ± 0.03 0.19 ± 0.03 0.11 ± 0.05 0.15 ± 0.06 0.19 ± 0.05 P > 0.05 Left kidney 0.19 ± 0.05 0.18 ± 0.06 0.23 ± 0.02 0.15 ± 0.06 0.13 ± 0.02 0.14 ± 0.01 P > 0.05 Right kidney 0.2 ± 0.07 0.21 ± 0.08 0.23 ± 0.02 0.15 ± 0.04 0.14 ± 0.03 0.16 ± 0.01 P > 0.05 Lung 0.69 ± 0.01 0.48 ± 0.06 0.54 ± 0.13 0.48 ± 0.08 0.48 ± 0.04 0.46 ± 0.06 P > 0.05 Values are mean ± SEM for 3–4 animals in each observation P > 0 05 = not significant The experimental animals were compared with normal groups TABLE 3: Effects of neem seed oil on organ weight index (g) of Wistar rats pretreated with CCl4. TABLE 4: Effects of neem seed oil on body weight of Wistar rats pretreated with CCl4. Treatment groups Day 0 Day 4 Day 9 Day 14 P Control 200.0 ± 5.00 205.0 ± 10.00 218.33 ± 11.67 215.0 ± 21.79 P > 0.05 CCl4 215.0 ± 23.63 226.67 ± 31.80 206.67 ± 10.14 236.67 ± 26.82 P > 0.05 Silymarin+CCl4 186.67 ± 24.55 191.67 ± 14.81 195.0 ± 15.00 205.0 ± 13.23 P > 0.05 Neem oil (0.25 mL/kg) 237.5 ± 8.54 243.75 ± 7.47 251.25 ± 12.97 248.75 ± 13.90 P > 0.05 Neem oil (0.5 mL/kg) 222.5 ± 12.67 226.25 ± 15.19 232.5 ± 11.27 232.5 ± 11.27 P > 0.05 Neem oil (1.0 mL/kg) 268.33 ± 23.33 265.0 ± 28.43 280.0 ± 25.17 286.67 ± 25.22 P > 0.05 P > 0.05 = not significant; there are no differences between each treatment group across the days. 4: Effects of neem seed oil on body weight of Wistar rats pretreated w P > 0.05 = not significant; there are no differences between each treatment group across the days. The experimental animals were compared with normal groups. WBC, white blood cells; LY%, lymphocytes; MO%, monocytes; GR%, granulocytes; RBC, red blood cells; HGB, haemoglobin; HCT, haematocrit; PLT, platelets. Discussion Carbon tetrachloride can cause liver damage at 0.5 mL/kg body weight (Hewawasam et al. 2004), which was less than the dose administered in this study. Some plants have been http://www.jomped.co.za Open Access Page 3 of 5 Original Research TABLE 1: Effects of neem seed oil on biochemical parameters of Wistar rats pretreated with CCl4 (U/g of wet tissue). Haematological parameters Control CCl4 Silymarin+CCl4 Neem oil (0.25 mL/kg) Neem oil (0.5 mL/kg) Neem oil (1.0 mL/kg) P TB (mg/dL) 0.97c ± 0.47 1.50b ± 0.59 1.97a ± 0.09 0.38c ± 0.05 0.45c ± 0.06 0.57c ± 0.09 *P < 0.01 CB (mg/dL) 0.20b ± 0.06 0.13b ± 0.03 0.17b ± 0.03 0.15b ± 0.03 0.25b ± 0.03 0.37a ± 0.09 P < 0.05 AST (IU/L) 108.33d ± 1.59 168.33a ± 1.59 118.33b ± 1.59 115.88c ± 1.21 115.33c ± 1.59 112.75d ± 1.27 P < 0.01 ALT (IU/L) 56.53d ± 1.30 116.53a ± 1.30 66.53b ± 1.30 65.15b ± 1.11 63.53c ± 1.30 60.65c ± 1.27 P < 0.01 ALP (IU/L) 45.30e ± 0.93 105.30a ± 0.93 55.30b ± 0.93 53.73c ± 0.78 52.30d ± 0.93 50.73d ± 0.78 P < 0.01 TP (g/dL) 4.97 ± 1.06 6.37 ± 1.13 7.33 ± 0.43 5.28 ± 0.26 5.33 ± 0.19 5.90 ± 0.26 P > 0.05 ALB (g/dL) 3.07 ± 0.39 3.20 ± 0.67 3.63 ± 0.12 3.18 ± 0.44 3.88 ± 0.45 2.53 ± 0.48 P > 0.05 GLOB (g/dL) 3.20b ± 0.06 2.97b ± 0.52 3.70a ± 0.36 2.10c ± 0.47 1.55c ± 0.24 3.37b ± 0.67 P < 0.01 TB, total bilirubin; CB, conjugated bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; GLOB, globulin. Values are mean ± SEM, n = 4. *P < 0.01; highly significant, P < 0.05; significant compared to control. Different superscripts across rows show the means are significant from others when compared with the control. TABLE 1: Effects of neem seed oil on biochemical parameters of Wistar rats pretreated with CCl4 (U/g of wet tissue). TB, total bilirubin; CB, conjugated bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; GLOB, globulin. Values are mean ± SEM, n = 4. *P < 0.01; highly significant, P < 0.05; significant compared to control. Discussion no significant difference (P > 0.05) in the conjugated bilirubin across the other groups. TP and ALB had no significant difference (P > 0.05) across the various groups, showing that CCl4, silymarin and neem oil had no effect on the animals’ TP and ALB in this study. However, globulin of animals with CCl4 treatment only and neem seed oil treatment (1.0 mL/kg) had no significant difference with that of the control group. Animals treated with 0.25 mL/kg and 0.5 mL/kg neem oil had a significantly different (P < 0.05) globulin level from that of the control. shown to have protective effects against organ damage, especially those caused by daily exposure to toxins (Amole & Ilori 2010). In the present study, the total bilirubin level increased significantly (P < 0.05) in serum of animals administered with CCl4 and those co-administered with CCl4 and silymarin as compared to the control. However, the lower the dose of the neem seed oil, the more the total bilirubin decreased significantly (P < 0.05) compared with the control. These observations are in line with the research findings of Prakash et al. (2008), suggesting that neem oil is more potent than the control drug in maintaining total bilirubin in animals. Conjugate bilirubin increased significantly (P < 0.05) in 1.0 mL/kg of neem oil treatment when compared with control animals. However, there was shown to have protective effects against organ damage, especially those caused by daily exposure to toxins (Amole & Ilori 2010). In the present study, the total bilirubin level increased significantly (P < 0.05) in serum of animals administered with CCl4 and those co-administered with CCl4 and silymarin as compared to the control. However, the lower the dose of the neem seed oil, the more the total bilirubin decreased significantly (P < 0.05) compared with the control. These observations are in line with the research findings of Prakash et al. (2008), suggesting that neem oil is more potent than the control drug in maintaining total bilirubin in animals. Conjugate bilirubin increased significantly (P < 0.05) in 1.0 mL/kg of neem oil treatment when compared with control animals. However, there was AST (Range: 108–168), ALT (Range: 56–116) and ALP (Range: 45–105) values significantly increased (P < 0.05) in the CCl4 group. This is in agreement with Kumar et al. Discussion http://www.jomped.co.za Page 4 of 5 Original Research Original Research Original Research Page 4 of 5 a b c d e f Source: Authors’ own work. FIGURE 1: (a) Section of rat liver from control rats showing control rat with normal liver cells radially arranged around portal tracts (blue arrows) and central veins (black arrows) (100x). (b) Rat liver from negative control (CCl4 administration alone) with accumulation of fat in the cytoplasm of liver cells (blue arrows) (100x). (c) Rat liver from positive control (CCl4 + silymarin) showing fatty change (blue arrow) (100x). (d) Liver section from rats treated with Azadirachta indica oil extract (0.25 mL/kg) and CCl4 showing fatty change (blue arrow) (100x). (e) Liver section from rats treated with Azadirachta indica oil extract (0.5 mL/kg) and CCl4 showing normal liver cells radially arranged around portal tracts (blue arrow) and central veins (black arrow) (100x). (f) Liver section from rats treated with Azadirachta indica oil extract (1.0 mL/kg) and CCl4 showing normal liver cells radially arranged around central veins (black arrows) (100x) b a c b a e f d f d e Source: Authors’ own work. Source: Authors’ own work. FIGURE 1: (a) Section of rat liver from control rats showing control rat with normal liver cells radially arranged around portal tracts (blue arrows) and central veins (black arrows) (100x). (b) Rat liver from negative control (CCl4 administration alone) with accumulation of fat in the cytoplasm of liver cells (blue arrows) (100x). (c) Rat liver from positive control (CCl4 + silymarin) showing fatty change (blue arrow) (100x). (d) Liver section from rats treated with Azadirachta indica oil extract (0.25 mL/kg) and CCl4 showing fatty change (blue arrow) (100x). (e) Liver section from rats treated with Azadirachta indica oil extract (0.5 mL/kg) and CCl4 showing normal liver cells radially arranged around portal tracts (blue arrow) and central veins (black arrow) (100x). (f) Liver section from rats treated with Azadirachta indica oil extract (1.0 mL/kg) and CCl4 showing normal liver cells radially arranged around central veins (black arrows) (100x) 1.0 mL/kg), CCl4 (1 mL/kg) and silymarin (1 mg/kg) had no effect on the haematological parameters. The organ weight index and animal body weight were insignificant (P > 0.05) across the various groups in this study. This observation suggests that neem seed oil, CCl4 and silymarin had no effect on organ weight and body weight. Conclusion Hewawasam, P., Chen, N., Ding, M., Natale, J.T., Boissard, C.G., Yeola, S. et al., 2004, ‘The synthesis and structure-activity relationships of 3-amino-4-benzylquinolin-2- ones; discovery of novel KCNQ2 channel openers’, Bioorganic & Medicinal Chemistry 14(7), 1615–1618. https://doi.org/10.1016/j.bmcl.2004.01.073 In conclusion, A. indica oil extract showed hepatoprotective activities. The activities were graded dose dependent. Furthermore, the hepatoprotective function of neem oil was comparable to the standard drug (silymarin). However, more work needs to be carried out on determining the toxicity profile (biosafety) of neem seed oil. Kaplowitz, N., 2004, ‘Drug-induced liver disorders: Implications for drug development and regulation’, Drug Safety 24, 483–490. https://doi.org/10.2165/00002018- 200124070-00001 Kelly, D. & Skidmore, S., 2002, ‘Hepatitis C-Z: Latest treatment options’, Archives of Disease in Childhood 86, 339–343. https://doi.org/10.1136/adc.86.5.339 Kumar, P.V., Sivaraj, A., Elumalai, E.K. & Senthil Kumar, B., 2009, ‘Carbon tetrachloride- induced hepatotoxicity in rats-protective role of aqueous leaf extracts of Coccinia grandis’, International Journal of PharmTech Research 1(4), 1612–1615. Discussion (2009), Kaplowitz (2004), Kelly and Skidmore (2002), Basu (2003), Manibusan, Odin and Eastmond (2007) and Planaguma et al. (2005) who reported significant increase in the enzyme activity on administration of CCl4. The reduction of the ALT, AST and ALP values decreased with increase in dose of neem oil when compared with the CCl4 group only. More so, silymarin-treated groups had reduced ALT, AST and ALP values when compared with the control. This suggests that neem oil is more active at a higher dose (1.0 mL/kg) and more effective than silymarin (a known hepatoprotective drug). 1.0 mL/kg), CCl4 (1 mL/kg) and silymarin (1 mg/kg) had no effect on the haematological parameters. The organ weight index and animal body weight were insignificant (P > 0.05) across the various groups in this study. This observation suggests that neem seed oil, CCl4 and silymarin had no effect on organ weight and body weight. Histological studies of liver tissues in each group are in agreement with the biochemical result in this study. High fat accumulation and massive necrosis were observed in the liver tissue of animal group administered with CCl4 only. However, groups treated with silymarin and 0.25 mg/kg neem oil had a moderate fatty liver. In addition, liver tissues of control and 1.0 mg/kg of neem oil–treated group had a normal liver. This suggests that the neem oil dose 1.0 mL/kg There was no significant difference (P > 0.05) in the haematological parameters in all groups. This observation suggests that neem oil (dose: 0.25 mL/kg, 0.5 mL/kg, http://www.jomped.co.za Open Access Original Research Page 5 of 5 Page 5 of 5 is more effective than the lower doses in protecting the liver against CCl4 damage. This might be because of the presence of some phytocompounds present in neem, which improve the ability of the liver to detoxify itself of chemical contamination (Ogbuewu et al. 2011). Competing interest The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. Ogbuewu, I.P., Odoemenam, V.U., Obikaonu, H.O., Opara, M.N., Emenalom, O.O., Uchegbu, M.C. et al., 2011, ‘The growing importance of neem (Azadirachta indica A. Juss) in agriculture, industry, medicine and environment: A review’, Research Journal of Medicinal Plant 5(3), 230–245. https://doi.org/10.3923/ rjmp.2011.230.245 Planaguma, A., Claria, J., Miquel, R., López-Parra, M., Titos, E., Masferrer, J.L. et al., 2005, ‘The selective cyclooxygenase-2 Inhibitor SC- 236 reduces liver fibrosis by mechanisms involving non-parenchyma cell apoptosis and PPAR gamma activation’, FASEB Journal 19, 1120–1122. References Abebe, W., 2002, ‘Mechanisms of liver cell injury’, Journal of Herpetology 32, 39–47. Amole, O.O. & Ilori, O.O., 2010, ‘Antimicrobial activity of the aqueous and ethanolic extracts of the stem bark of Alstonia boonei’, International Journal of Phytopharmacology 1, 119–123. Basu, S., 2003, ‘Carbon tetrachloride-induced lipid peroxidation: Eicosanoid formation and their regulation by antioxidant nutrients’, Toxicology 18(9), 113–127. https:// doi.org/10.1016/S0300-483X(03)00157-4 This study reveals that neem oil is hepatoprotective (0.25 mL/kg, 0.5 mL/kg, 1.0 mL/kg) but might become toxic at a comparatively higher dose. Neem seed oil has been reported to show acute toxicity in rats and rabbits with LD50 of 14 mg/kg and 24 mg/kg, respectively, possibly targeting organs for toxic effects, especially the central nervous system and the lungs (Gandhi et al. 1988). Gandhi, M., Lal, R., Sankaranarayanan, A., Banerjee, C.K. & Sharma, P.L., 1988, ‘Acute toxicity study of the oil from Azadirachta indica seed (neem oil)’, Journal of Ethnopharmacology 23, 39–51. https://doi.org/10.1016/0378- 8741(88)90113-4 Gomase, P.V., Rangari, V.D. & Verma, P.R., 2011, ‘Phytochemical evaluation and hepatoprotective activity of fresh juice of young stem (tender) bark of Azadirachta indica A. Juss’, International Journal of Pharmacy and Pharmaceutical Sciences 3(2), 55–59. Gupta, A., Khosla, P. & Singh, T.K., 2008, ‘Effect of neem leaf extract on isolated perfused preparation’, Indian Journal of Pharmacology 32, 132–175. Acknowledgements Manibusan, M.K., Odin, M. & Eastmond, D.A., 2007, ‘Postulated carbon tetrachloride mode of action: A review’, Journal of Environmental Science and Health 25, 185–209. https://doi.org/10.1080/10590500701569398 The authors wish to thank Mr Dickson for his assistance in the lab. They also appreciate Alhaji Rano for his assistance in helping them purchase locally extracted neem oil from Kano State, Nigeria. Mugnai, E., 2009, Azadirachta indica: Neem tree, the ‘village pharmacy’, ASAT- Associazione Scienze Agrarie Tropicali, Firenze. Nix, S., 2007, ‘Neem tree – “The Village Pharmacy”’, viewed from www.forestry. about.com. Ogbuewu, I.P., 2008, ‘Physiological responses of rabbits fed graded levels of neem (Azadirachta indica) leaf meal’, MSc thesis, Federal University of Technology, Owerri. Authors’ contributions M.I. designed this study and gave directions for its implementation. O.U-.O. helped to organise the results, proofread the final draft of the article and put it in the journal format. S.O.O. carried out the bench work with laboratory assistants and wrote the first draft of this article. Prakash, T., Fadadu, S.D., Sharma, U.R. & Dupadahalli, K., 2008, ‘Hepatoprotective activity of leaves of Rhododendron arboreum in CCl4 induced hepatotoxicity in rats’, Journal of Medicinal Plants Research 2(11), 315–320. Uwimbabazi, F., Uwimana, J. & Rutanga, J.P., 2015, ‘Assessment of antibacterial activity of neem plant (Azadirachta indica) on Staphylococcus aureus and Escherichia coli’, Journal of Medicinal Plants Studies 3(4), 85–91. http://www.jomped.co.za Open Access
https://openalex.org/W4297980216	https://cdr.lib.unc.edu/downloads/zp38wn935	English	null	Mice Lacking Phosphatidylinositol Transfer Protein-α Exhibit Spinocerebellar Degeneration, Intestinal and Hepatic Steatosis, and Hypoglycemia	Carolina Digital Repository (University of North Carolina at Chapel Hill)	2,003	cc-by	18,208	Mice Lacking Phosphatidylinositol Transfer Protein- Exhibit Spinocerebellar Degeneration, Intestinal and Hepatic Steatosis, and Hypoglycemia* PITP and PITP share 77% pri- mary sequence identity, are encoded by distinct genes, and exhibit biochemical differences. Yet both PITP and PITP (and even yeast PITPs) function as soluble factors that stimulate various reconstitutions of PIP-dependent functions in permeabi- lized mammalian cells. These functions include regulated and constitutive membrane trafficking and phospholipase C- dependent signaling through G-protein-coupled receptors (12– 14). Given the lack of PITP specificity in these assays, it re- mains unclear how faithful such reconstitutions are in reporting physiological functions for mammalian PITPs. Phosphatidylinositol transfer proteins (PITPs) regu- late the interface between lipid metabolism and cellular functions. We now report that ablation of PITP func- tion leads to aponecrotic spinocerebellar disease, hypo- glycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associ- ated with reduced proglucagon gene expression and gly- cogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intes- tinal and hepatic lipid metabolism and defective proglu- cagon gene expression contribute to hypoglycemia in PITP/ mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar dis- ease. Taken together, the data suggest an unanticipated role for PITP in with glucose homeostasis and in mam- malian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes. Genetic studies are providing initial clues regarding PITP function in metazoans. An inherited form of light-enhanced retinal degeneration in Drosophila results from inactivation of a membrane-bound PITP (15). In mice, reduction of PITP to 18% of wild-type levels is the basis for the vibrator neurode- generative disorder (16, 26). Gene ablation approaches suggest PITP plays an essential housekeeping function, whereas PITP is nonessential for ES cell viability and is not a quanti- tatively significant factor in membrane trafficking, PIP metab- olism, or growth factor signaling in ES cells (17). In this report, we describe the consequences associated with ablation of PITP function in the mouse. We find that PITP, although dispensable for prenatal development, is required for neonatal survival. PITP/ neonates suffer from a severe spinocerebellar neurodegenerative disease and exhibit defects in dietary fat and -tocopherol transport across the small in- testine. * This work was supported in part by National Institutes of Health Grant NS37723 (to V. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ Recipient of a National Institutes of Health grant. ‡‡ To whom correspondence should be addressed. Tel.: 919-962-9870; Fax: 919-966-1856; E-mail: vytas@med.unc.edu. 1 The abbreviations used are: PITPs, phosphatidylinositol transfer proteins; CHOP, CCAAT/enhancer-binding protein homology protein; CL, cardiolipin; CRD, chylomicron retention disease; DAG, diacylglyc- erol; ER, endoplasmic reticulum; ES cells, embryonic stem cells; FFA, free fatty acids; GFAP, glial fibrillary acidic protein; GM, gray matter; Glc-6-Pase, glucose-6-phosphatase; MEFs, murine embryonic fibro- blasts; PIP, phosphoinositide; PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; TG, triglyceride; TUNEL, terminal deoxynucleo- tidyltransferase-mediated dUTP nick-end-labeling; UPR, unfolded pro- tein response; WM, white matter; CE, cholesteryl ester. THE JOURNAL OF BIOLOGICAL CHEMISTRY THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 35, Issue of August 29, pp. 33501–33518, 2003 Printed in U.S.A. Mice Lacking Phosphatidylinositol Transfer Protein- Exhibit Spinocerebellar Degeneration, Intestinal and Hepatic Steatosis, and Hypoglycemia* This intestinal steatosis in some respects resembles CRD, a human disorder of unknown molecular etiology (18, 19). Liver steatosis is also prominent in the mutant mice, suggest- ing the possibility that PITP nullizygosity also compromises lipoprotein assembly and/or neutral lipid secretion in hepato- cytes. Finally, PITP/ mice are severely hypoglycemic. Our results suggest a novel and unanticipated role for PITP in regulating cargo-specific lipid transport from the enterocyte and hepatocyte ER, endocrine pancreas function, and glycogen metabolism. PITPs1 mobilize PtdIns or PtdCho between membrane bilay- ers in vitro (1, 2). In vivo studies demonstrate that PITPs control the interface between membrane trafficking and lipid metabolic pathways in yeast (3–6). By contrast, the physiolog- ical functions for mammalian PITPs, which are structurally unrelated to yeast PITPs (7, 8), are not understood at either the cellular or organismal levels. Mammals express at least three soluble PITPs: PITP, This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Mice Lacking Phosphatidylinositol Transfer Protein- Exhibit Spinocerebellar Degeneration, Intestinal and Hepatic Steatosis, and Hypoglycemia* Received for publication, April 7, 2003, and in revised form, May 30, 2003 Published, JBC Papers in Press, June 4, 2003, DOI 10.1074/jbc.M303591200 James G. Alb, Jr.‡, Jorge D. Cortese‡, Scott E. Phillips‡, Roger L. Albin§¶, Tim R. Nagy, Bruce A. Hamilton¶, and Vytas A. Bankaitis‡ ‡‡ From the ‡Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7090, the §Department of Neurology, University of Michigan School of Medicine, Ann Arbor Veterans Affairs Medical Center GRECC, Ann Arbor, Michigan 48104-0520, the Department of Nutrition, University of Alabama at Birmingham, Birmingham, Alabama 35294, and University of California School of Medicine, San Diego, La Jolla, California 92093-0644 James G. Alb, Jr.‡, Jorge D. Cortese‡, Scott E. Phillips‡, Roger L. Albin§¶, Tim R. Nagy, Bruce A. Hamilton¶, and Vytas A. Bankaitis‡ ‡‡ From the ‡Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7090, the §Department of Neurology, University of Michigan School of Medicine, Ann Arbor Veterans Affairs Medical Center GRECC, Ann Arbor, Michigan 48104-0520, the Department of Nutrition, University of Alabama at Birmingham, Birmingham, Alabama 35294, and University of California School of Medicine, San Diego, La Jolla, California 92093-0644 James G. Alb, Jr.‡, Jorge D. Cortese‡, Scott E. Phillips‡, Roger L. Albin§¶, Tim R. Nagy, Bruce A. Hamilton¶, and Vytas A. Bankaitis‡ ‡‡ From the ‡Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7090, the §Department of Neurology, University of Michigan School of Medicine, Ann Arbor Veterans Affairs Medical Center GRECC, Ann Arbor, Michigan 48104-0520, the Department of Nutrition, University of Alabama at Birmingham, Birmingham, Alabama 35294, and University of California School of Medicine, San Diego, La Jolla, California 92093-0644 PITP, and rdgB (9–11). PITP and PITP share 77% pri- mary sequence identity, are encoded by distinct genes, and exhibit biochemical differences. Yet both PITP and PITP (and even yeast PITPs) function as soluble factors that stimulate various reconstitutions of PIP-dependent functions in permeabi- lized mammalian cells. These functions include regulated and constitutive membrane trafficking and phospholipase C- dependent signaling through G-protein-coupled receptors (12– 14). Given the lack of PITP specificity in these assays, it re- mains unclear how faithful such reconstitutions are in reporting physiological functions for mammalian PITPs. PITP, and rdgB (9–11). RESULTS PITP/ Mice Develop to Term—Ablation of PITP func- tion in the mouse was achieved using two independent null alleles. First, a homologous recombination vector was con- structed where exons 8–10 of the PITP gene are replaced with a neo cassette (Fig. 1A). This mutation (PITP::neo) deletes PITP residues 162–257, a region critical for PITP function (17). Second, survey of the Lexicon Genetics OmniBankTM gene trap library (see “Experimental Procedures”) identified an inser- tion mutation in the PITP structural gene (PITP::neo/puro). This allele is genetically similar (although not identical) to PITP::neo as it also truncates PITP after residue 162 (Fig. 1B). Mice were derived from each targeted ES cell line. PITP/ offspring are phenotypically normal and fertile, and mice homozygous for either of these two mutations exhi- bit indistinguishable phenotypes. The phenotypic data pre- sented below were obtained from both PITP1::neo* and PITP::neo/puro homozygous animals. Histological Analysis—Mice were anesthetized with 1.25% Avertin and perfused with phosphate-buffered saline, 4% paraformaldehyde. Duodenum, ileum, cerebellum, pancreas, and spinal cord were har- vested, flushed with fixative (duodenum and ileum only), dissected, and infused with fixative for 24 h. Samples were mounted in paraffin, and 5-m-thick sections were stained and mounted. These sections were rehydrated by serial transfer from xylene to 50% EtOH, stained with hematoxylin/eosin, and mounted in Permount (Fisher). Where osmium staining was employed, duodenal and liver sections were stained in 5% potassium dichromate, 2% osmium tetroxide for 8 h prior to paraffin embedding (22) and counterstained with toluidine blue O. Histological Analysis—Mice were anesthetized with 1.25% Avertin and perfused with phosphate-buffered saline, 4% paraformaldehyde. Duodenum, ileum, cerebellum, pancreas, and spinal cord were har- vested, flushed with fixative (duodenum and ileum only), dissected, and infused with fixative for 24 h. Samples were mounted in paraffin, and 5-m-thick sections were stained and mounted. These sections were rehydrated by serial transfer from xylene to 50% EtOH, stained with hematoxylin/eosin, and mounted in Permount (Fisher). Where osmium staining was employed, duodenal and liver sections were stained in 5% potassium dichromate, 2% osmium tetroxide for 8 h prior to paraffin embedding (22) and counterstained with toluidine blue O. Whole brains were extracted from mice perfused with 4% paraform- aldehyde and 2.5% glutaraldehyde, washed for several days in phos- phate-buffered saline, and mounted in paraffin. RESULTS Brains were sliced in half along the sagittal plane; each half was embedded in paraffin; and 5- or 8-m-thick slices were mounted onto treated slides. Sections were rehydrated to 50% EtOH. For visualization of Purkinje cells, slices were incubated with goat anti-calbindin antibodies (Santa Cruz Biotechnol- ogy; Santa Cruz, CA) and developed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). After incubation in 2% osmium fumes for 10 min, slices were counterstained with toluidine blue O. Intercrosses with heterozygous mice carrying either PITP1::neo* or the PITP::neo/puro allele yielded genotypes consistent with a fully penetrant autosomal recessive muta- tion. From a dedicated pool of 408 live births, 89 PITP/ progeny were recovered (Fig. 1C). The genotypic distribution of 106 PITP/, 213 PITP/, 89 PITP/ corresponds to a 1.000:2.009:0.840 ratio that approximates closely the 1:2:1 ra- tio predicted by Mendel’s rules. Correct gene targeting in the progeny was verified by PCR genotyping and immunoblotting of brain extracts with a specific PITP antiserum (Fig. 1D). Antibodies directed against the PITP N terminus failed to detect a truncation product in mice homozygous for PITP1::neo* or PITP::neo/puro, suggesting that both al- leles represent null mutations. Finally, we find that relative PITP levels are unchanged in PITP/ and PITP/ brain (Fig. 1D), indicating that PITP/ mice do not activate com- pensatory processes that increase PITP expression. For Oil Red O staining, livers were extracted from mice perfused with 4% paraformaldehyde, washed with phosphate-buffered saline, and frozen at 20 °C. Frozen livers were mounted, sectioned (8 M), fixed to a histological slide, and placed in absolute propylene glycol (2 min). Slides were moved into Oil Red O solution (Newcomer Supply, Middleton, WI, catalog number 12722) for 1 h, differentiated in 85% propylene glycol (1 min), rinsed 2 in distilled water, counterstained with hematoxylin (10 s), and mounted in glycerin. Electron Microscopy—Mice were perfused with 4% formaldehyde, 2.5% glutaraldehyde. Biopsies from intestine, liver, and spinal cord were post-fixed with 1% OsO4, dehydrated with acetone, embedded in epoxy (23), sectioned (65 nm-thick), and stained with 4% uranyl acetate and Sato’s lead mixture (24). Samples were viewed at 80 kV in a Phillips Tecnai 12 microscope (FEI Co., Eindhoven, The Netherlands) and im- aged with a Gatan MultiScan model 794 digital camera (Gatan, Pleas- anton, CA). Epoxy-embedded samples were sectioned for histological analysis (2-m thickness) and stained with 1% toluidine blue O in 1% sodium borate. Ablation of PITP Function in Mice Care was exercised to separate islets from patches of connecting ducts and intervening con- nective tissue, and not to re-score the same islet in successive sections. Islet-like, encapsulated structures larger than 100 m that were de- tected in at least three consecutive sections were scored as islets. The total pancreatic area studied was similar in all sections. Pancreatic Histology—Whole pancreas from mice perfused with 4% paraformaldehyde was embedded in paraffin and serially sectioned (thickness 5 m). Islet numbers were assessed by sequential obser- vation of hematoxylin/eosin-stained sections. Care was exercised to separate islets from patches of connecting ducts and intervening con- nective tissue, and not to re-score the same islet in successive sections. Islet-like, encapsulated structures larger than 100 m that were de- tected in at least three consecutive sections were scored as islets. The total pancreatic area studied was similar in all sections. Carcass Analyses—Carcass analyses were as described previously (21). Gastrointestinal tracts were removed (stomach, small and large intestine, and cecum) and carcasses weighed. Body water content of eviscerated carcasses was determined by drying to constant weight in a 60 °C oven and measuring differences between the pre- and post-drying carcass mass. Dried carcasses were minced, ground to a homogeneous mixture, and extracted with petroleum ether in a Soxhlet apparatus to determine fat mass and fat-free dry mass. Fat-free dry mass was burned overnight at 600 °C (8 h) to determine eviscerated carcass ash. Carcass Analyses—Carcass analyses were as described previously (21). Gastrointestinal tracts were removed (stomach, small and large intestine, and cecum) and carcasses weighed. Body water content of eviscerated carcasses was determined by drying to constant weight in a 60 °C oven and measuring differences between the pre- and post-drying carcass mass. Dried carcasses were minced, ground to a homogeneous mixture, and extracted with petroleum ether in a Soxhlet apparatus to determine fat mass and fat-free dry mass. Fat-free dry mass was burned overnight at 600 °C (8 h) to determine eviscerated carcass ash. Ablation of PITP Function in Mice 33502 PerkinElmer Life Sciences model LC200 gradient pump, an AS 200 Autosampler, and an LC 295 programmable UV-visible light detector set at 292 nm. PITP/ genotype (AB-2; 5-GCGAGGCATCACTCTTCCCCTC-3), the heterozygous PITP/ genotype (AB-1B; 5-CACCATCCCCCAC- GGTGACTG-3), and the PITP/ genotype (PG-1; 5-GAATGTGTG- CGAGGCCAGAGG-3) in a 33-cycle reaction (53 °C annealing temper- ature). Genotypes for PITP::neo/puro mice (L1 line) were determined in two steps. First, a two primer assay that monitored Neo distinguishes PITP/ from PITP/ and PITP/ mice. Primers GE-UP (5- GGGCGCCCGGTTCTTT TTGTGA-3) and GE-DO (5-TTGGTGGTC- GAATGGGCAGGTAGC-3) were used in a 28-cycle reaction (60 °C an- nealing temperature). To distinguish PITP/ from PITP/ L1 mice, we resorted to immunoblot analyses of mouse brain using PITP- specific serum (17). Liver Glycogen Analysis—Quantification of liver glycogen was by the method of Passonneau and Lauderdale (26). Glycogen was extracted from acidified liver homogenates and hydrolyzed to glucose with amylo- glucosidase (Sigma). Glucose was determined by a glucose oxidase- coupled Trinder assay (Sigma). Adenylate Nucleotide Analysis—ATP and ATP/ADP ratios were measured using the ApoGlowTM kit (BioWhittaker Molecular Applica- tions; Rockville, MD). Samples (0.5 mg tissue) were collected and rap- idly frozen. Tissues were thawed and homogenized in the presence of a nucleotide-releasing mixture supplied by the manufacturer. Acid-ex- tracted ATP was assayed with a luciferase-based system with picomolar sensitivity. ADP was converted to ATP and measured after a 5-min incubation at 22 °C. Adenylate Nucleotide Analysis—ATP and ATP/ADP ratios were measured using the ApoGlowTM kit (BioWhittaker Molecular Applica- tions; Rockville, MD). Samples (0.5 mg tissue) were collected and rap- idly frozen. Tissues were thawed and homogenized in the presence of a nucleotide-releasing mixture supplied by the manufacturer. Acid-ex- tracted ATP was assayed with a luciferase-based system with picomolar sensitivity. ADP was converted to ATP and measured after a 5-min incubation at 22 °C. Serum Analyses—Blood was collected from mice immediately after heart puncture and clotted, and serum was clarified by centrifugation. Serum glucose was determined by using either the Trinder assay (Sig- ma) or was measured by Antech Diagnostics (Farmingdale, NY). Insu- lin and -hydroxybutyrate were determined using the Immunoassay System (Crystal Chem Inc.) and the -hydroxybutyrate assay kit (Sig- ma), respectively. All other serum analyses were performed by Antech Diagnostics (Farmingdale, NY). Pancreatic Histology—Whole pancreas from mice perfused with 4% paraformaldehyde was embedded in paraffin and serially sectioned (thickness 5 m). Islet numbers were assessed by sequential obser- vation of hematoxylin/eosin-stained sections. EXPERIMENTAL PROCEDURES Generation and Genotyping of PITP/ Mice—AB1-derived /PITP1::neo* ES cells have been described previously (17). The /PITP::neo/puro ES cells corresponded to the OST 1152 line (Lexicon Genetics OmniBankTM library (20)). Mice were generated by injection of ES cells into C57BL/6 blastocysts, implantation of blastocysts into pseudopregnant foster mothers, and identification of male chimeric mice competent for germ line transmission of each allele. Genotypes for PITP1::neo* mice (JG line) were determined by using a three-primer PCR assay. We employed a primer specific for the homozygous 33501 33501 Ablation of PITP Function in Mice RESULTS Digital images were collected with a SPOT RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI), using Plan Fluor Nikon objectives mounted in a Nikon Eclipse E400 microscope (Nikon Inc., Melville, NY). Morphometric analysis of spinal cord sections was carried out with Scion Image software (Scion Corp., Frederick, MD). Neonatal Mortality of PITP/ Mice—PITP/ mice failed to thrive and died at a very young age. In a sample pool of 57 PITP/ mice, 40% died within 48 h after birth (Fig. 2A). These early P0 and P1 deaths were not characterized by obvious external abnormalities. Moreover, postmortem analy- ses revealed that the stomachs of the expired PITP/ mice contained copious quantities of milk, indicating that mutant animals had nursed. Of the PITP/ progeny that survived past P1, a steady incidence of mortality was observed between -Tocopherol Analyses—Brain -tocopherol was extracted as de- scribed (25), injected onto a 250 4.6 mm Phenomenex ODS 20 re- versed phase C18 column (5-m particle diameter), and eluted with methanolic 0.05% ammonium acetate using a flow rate of 1.5 ml/min. The high pressure liquid chromatography system consisted of a Ablation of PITP Function in Mice 33503 FIG. 1. Generation and characterization of PITP-deficient mice. A, targeted replacement of the wild-type PITP locus with PITP::neo. Organization of the targeting vector is shown. Probe 1 represents a 500-bp DNA fragment that resides outside the bounds of the targeting vector and is employed for diagnosis of targeting events. Exons 7–10 of the PITP structural gene are indicated as closed bars and are numbered accordingly. Restriction enzyme sites: E, EcoRI; X, XbaI; K, KpnI; S, SacI. B, OmniBankTM gene trap library at the PITP locus. Retroviral construct VICTR20 is depicted (20). The PITP::neo/puro mutation represents integration of VICTR20 between PITP exons 7 and 8 and truncates PITP after residue 162. The abbreviations used are as follows: LTR, long terminal repeat; PGK, phosphoglycerate kinase-1 promoter; puro, puromycin N-acetyltransferase gene; SD, spice donor sequence; SA, splice acceptor sequence; IRES, internal ribosome entry site; geo, galactosidase/neomycin phosphotransferase fusion gene; pA, polyadenylation sequence. C, distribution of PITP genotypes in the F1. The number of live births obtained for each PITP genotype (indicated at top), from a dedicated set of 408 F1 progeny of PITP/ intercrosses, is given above the corresponding bar. D, confirmation of viable PITP/ progeny. RESULTS Restriction enzyme sites: E, EcoRI; X, XbaI; K, KpnI; S, SacI. B, OmniBankTM gene trap library at the PITP locus. Retroviral construct VICTR20 is depicted (20). The PITP::neo/puro mutation represents integration of VICTR20 between PITP exons 7 and 8 and truncates PITP after residue 162. The abbreviations used are as follows: LTR, long terminal repeat; PGK, phosphoglycerate kinase-1 promoter; puro, puromycin N-acetyltransferase gene; SD, spice donor sequence; SA, splice acceptor sequence; IRES, internal ribosome entry site; geo, galactosidase/neomycin phosphotransferase fusion gene; pA, polyadenylation sequence. C, distribution of PITP genotypes in the F1. The number of live births obtained for each PITP genotype (indicated at top), from a dedicated set of 408 F1 progeny of PITP/ intercrosses, is given above the corresponding bar. D, confirmation of viable PITP/ progeny. Upper left panel, diagnostic PCR profiles of PITP/, PITP/, and PITP/ progeny derived from a PITP/ intercross. Upper right panel, total brain lysates (20 g) harvested from each of five sibling pups (genotypes indicated) derived from a PITP/ intercross were resolved by SDS-PAGE and developed by immunoblotting with PITP-specific antibodies. Lower panel, immunoblot of PITP in brain lysates (20 g) from neonates of indicated genotype. FIG. 1. Generation and characterization of PITP-deficient mice. A, targeted replacement of the wild-type PITP locus with PITP::neo. Organization of the targeting vector is shown. Probe 1 represents a 500-bp DNA fragment that resides outside the bounds of the targeting vector and is employed for diagnosis of targeting events. Exons 7–10 of the PITP structural gene are indicated as closed bars and are numbered accordingly. Restriction enzyme sites: E, EcoRI; X, XbaI; K, KpnI; S, SacI. B, OmniBankTM gene trap library at the PITP locus. Retroviral construct VICTR20 is depicted (20). The PITP::neo/puro mutation represents integration of VICTR20 between PITP exons 7 and 8 and truncates PITP after residue 162. The abbreviations used are as follows: LTR, long terminal repeat; PGK, phosphoglycerate kinase-1 promoter; puro, puromycin N-acetyltransferase gene; SD, spice donor sequence; SA, splice acceptor sequence; IRES, internal ribosome entry site; geo, galactosidase/neomycin phosphotransferase fusion gene; pA, polyadenylation sequence. C, distribution of PITP genotypes in the F1. The number of live births obtained for each PITP genotype (indicated at top), from a dedicated set of 408 F1 progeny of PITP/ intercrosses, is given above the corresponding bar. D, confirmation of viable PITP/ progeny. RESULTS Upper left panel, diagnostic PCR profiles of PITP/, PITP/, and PITP/ progeny derived from a PITP/ intercross. Upper right panel, total brain lysates (20 g) harvested from each of five sibling pups (genotypes indicated) derived from a PITP/ intercross were resolved by SDS-PAGE and developed by immunoblotting with PITP-specific antibodies. Lower panel, immunoblot of PITP in brain lysates (20 g) from neonates of indicated genotype. f FIG. 1. Generation and characterization of PITP-deficient mice. A, targeted replacement of the wild-type PITP locus with PITP::neo. Organization of the targeting vector is shown. Probe 1 represents a 500-bp DNA fragment that resides outside the bounds of the targeting vector and is employed for diagnosis of targeting events. Exons 7–10 of the PITP structural gene are indicated as closed bars and are numbered accordingly. Restriction enzyme sites: E, EcoRI; X, XbaI; K, KpnI; S, SacI. B, OmniBankTM gene trap library at the PITP locus. Retroviral construct VICTR20 is depicted (20). The PITP::neo/puro mutation represents integration of VICTR20 between PITP exons 7 and 8 and truncates PITP after residue 162. The abbreviations used are as follows: LTR, long terminal repeat; PGK, phosphoglycerate kinase-1 promoter; puro, puromycin N-acetyltransferase gene; SD, spice donor sequence; SA, splice acceptor sequence; IRES, internal ribosome entry site; geo, galactosidase/neomycin phosphotransferase fusion gene; pA, polyadenylation sequence. C, distribution of PITP genotypes in the F1. The number of live births obtained for each PITP genotype (indicated at top), from a dedicated set of 408 F1 progeny of PITP/ intercrosses, is given above the corresponding bar. D, confirmation of viable PITP/ progeny. Upper left panel, diagnostic PCR profiles of PITP/, PITP/, and PITP/ progeny derived from a PITP/ intercross. Upper right panel, total brain lysates (20 g) harvested from each of five sibling pups (genotypes indicated) derived from a PITP/ intercross were resolved by SDS-PAGE and developed by immunoblotting with PITP-specific tib di L l i bl t f PITP i b i l t (20 ) f t f i di t d t FIG. 1. Generation and characterization of PITP-deficient mice. A, targeted replacement of the wild-type PITP locus with PITP::neo. Organization of the targeting vector is shown. Probe 1 represents a 500-bp DNA fragment that resides outside the bounds of the targeting vector and is employed for diagnosis of targeting events. Exons 7–10 of the PITP structural gene are indicated as closed bars and are numbered accordingly. RESULTS Upper left panel, diagnostic PCR profiles of PITP/, PITP/, and PITP/ progeny derived from a PITP/ intercross. Upper right panel, total brain lysates (20 g) harvested from each of five sibling pups (genotypes indicated) derived from a PITP/ intercross were resolved by SDS-PAGE and developed by immunoblotting with PITP-specific antibodies. Lower panel, immunoblot of PITP in brain lysates (20 g) from neonates of indicated genotype. Ablation of PITP Function in Mice FIG. 2. Overt phenotypes of PITP/ mice. A, mortality profile derived from a pool of 57 PITP/ mice generated from 5 mating pairs over a period of 5 mating cycles. Data are presented as percentage of surviving progeny (/, open circles; /, closed circles) as a function of time (postnatal day). B, rates of body mass increase of PITP/ and PITP/ littermates. Values represent averages for two sibling mice of each genotype (/, open circles; /, closed circles). The data are representative. C, visual comparison of PITP/ and PITP/ littermates. Genotypes are at bottom. D, stomachs (S) of age-matched PITP/ and PITP/ mice (P7) are filled with milk. Subcutaneous axillary (AF) and inguinal (IF) fat pads are indicated. E, comprehensive chemical analysis of eviscerated carcasses. Measurements represent the averages from six P6 and three P8 PITP/ animals (solid bars) and six P6 and three P8 PITP/ mice (hatched bars). The abbreviations used are as follows: total, eviscerated carcass mass; FFDM, fat-free dry mass; and ASH, non-combustible carcass bone ash. FIG. 2. Overt phenotypes of PITP/ mice. A, mortality profile derived from a pool of 57 PITP/ mice generated from 5 mating pairs over a period of 5 mating cycles. Data are presented as percentage of surviving progeny (/, open circles; /, closed circles) as a function of time (postnatal day). B, rates of body mass increase of PITP/ and PITP/ littermates. Values represent averages for two sibling mice of each genotype (/, open circles; /, closed circles). The data are representative. C, visual comparison of PITP/ and PITP/ littermates. Genotypes are at bottom. D, stomachs (S) of age-matched PITP/ and PITP/ mice (P7) are filled with milk. Subcutaneous axillary (AF) and inguinal (IF) fat pads are indicated. E, comprehensive chemical analysis of eviscerated carcasses. Measurements represent the averages from six P6 and three P8 PITP/ animals (solid bars) and six P6 and three P8 PITP/ mice (hatched bars). RESULTS The abbreviations used are as follows: total, eviscerated carcass mass; FFDM, fat-free dry mass; and ASH, non-combustible carcass bone ash. FIG. 2. Overt phenotypes of PITP/ mice. A, mortality profile derived from a pool of 57 PITP/ mice generated from 5 mating pairs over a period of 5 mating cycles. Data are presented as percentage of surviving progeny (/, open circles; /, closed circles) as a function of time (postnatal day). B, rates of body mass increase of PITP/ and PITP/ littermates. Values represent averages for two sibling mice of each genotype (/, open circles; /, closed circles). The data are representative. C, visual comparison of PITP/ and PITP/ littermates. Genotypes are at bottom. D, stomachs (S) of age-matched PITP/ and PITP/ mice (P7) are filled with milk. Subcutaneous axillary (AF) and inguinal (IF) fat pads are indicated. E, comprehensive chemical analysis of eviscerated carcasses. Measurements represent the averages from six P6 and three P8 PITP/ animals (solid bars) and six P6 and three P8 PITP/ mice (hatched bars). The abbreviations used are as follows: total, eviscerated carcass mass; FFDM, fat-free dry mass; and ASH, non-combustible carcass bone ash. Ablation of PITP Function in Mice 33505 P2 and P11. Almost all mice expired by P11, and only one PITP/ mouse lived to P14 (Fig. 2A). Degenerative Disease in PITP/ Cerebellum—PITP/ cerebellum suffered major degenerative insult as judged by the extent of reactive gliosis. Wild-type cerebellum shows low lev- els of GFAP, a specific marker for activated astrocytes (Fig. 3C). By contrast, PITP/ cerebellum exhibits a dramatic staining for GFAP, and reactive gliosis is especially prominent in the white matter trunk of this organ (Fig. 3C). Even in these relatively less affected areas, GFAP immunohistochemistry suggests some 10% of the cells represent activated astrocytes that are frequently seen to be enveloping neuron cell bodies (Fig. 3D). Reactive gliosis is not observed throughout the mu- tant brain. Whereas PITP/ cerebellum and brain stem are heavily infiltrated with activated glial cells, and the subtha- lamic region is also involved, significant gliosis is not observed in the neocortex, striatum, hippocampal formation, and other forebrain regions (not shown). g PITP/ progeny that survived past P1 were initially in- distinguishable from PITP/ and PITP/ littermates in size, external morphology, and behavior. By P4, however, two phenotypes rapidly asserted themselves. First, most PITP/ mice were moribund and exhibited little spontaneous move- ment. RESULTS Analyses from six PITP/ mice indicate that 18 1% of the total motor neuron cell bodies in the ventral horn exhibit properties of aponecrosis. These include pericyto- plasmic vacuolation, reduced cytoplasmic contents, and cyto- plasmic proliferation of irregular electron-translucent vesicles (Fig. 4B). Elevated Apoptosis and Purkinje Cell Defects in PITP/ Cerebellum—Nissl staining does not reveal obvious defects in development or morphology of PITP/ cerebrum, thalamus, hippocampus, or cerebellum. Normal cellularity was observed in all regions examined (data not shown). However, PITP insufficiencies evoked functional defects in the cerebellum. These defects were apparent at several levels. Whereas TUNEL staining showed a sparse and random distribution of apoptotic foci in wild-type brain, apoptosis was more prevalent throughout PITP/ cerebellum. This is particularly evident in the external granule layer of mutant cerebellum (Fig. 3A). We did not observe aponecrotic motor neurons in the ventral horn of PITP/ spinal cord. Extensive vacuolation and mem- brane blebbing is prevalent in cells that line the vasculature of PITP/ spinal cord (not shown). Axons surrounding capil- laries and small vessels in the GM are enlarged and often damaged. This is unlikely to represent a perfusion artifact, as this phenotype was not recorded in any of the spinal cord sections analyzed from 14 PITP/ mice. Second, we find Purkinje cell defects in PITP/ animals. Purkinje cells normally align themselves into a sharply defined monolayer that lies between the molecular and external gran- ular layers of the cerebellum (Fig. 3B). These cells elaborate well developed apical dendritic stalks which arborize into lux- urious branches that penetrate into the molecular layer of the cerebellum. PITP/ Purkinje cells, while retaining normal flask-shaped cell body morphologies, exhibit either abbreviated apical dendritic stalks or no obvious stalks at all (Fig. 3B). Moreover, the dendritic branches emanating from the abbrevi- ated apical stalks are less arborized than those of PITP/ Purkinje cells. We also observed cases where these cells exhibit defects in spatial alignment, reside off of the defined layer, or are otherwise misoriented. Toluidine blue O-staining reveals the presence of cells con- taining an abundance of purple intracellular granules in the perivascular matrix (Fig. 4C, left panel). This obvious purple metachromasia is diagnostic of inflammatory mast cells (29, 30). Accordingly, we find extratissular macrophages in perivascular tissue or even in the vessels themselves (Fig. 4C, right panel), suggesting the blood/brain barrier of PITP/ mice is itself com- promised. RESULTS These pathologies encompass a spec- trum of cell death events. These range from occasional apop- totic neurons (condensed nuclei, fragmented cytoplasm; not shown) to large numbers of neurons undergoing aponecrotic processes (Fig. 4B). Aponecrosis is a form of cell death associ- ated with reductions in cellular ATP and increases in ADP levels (27, 28). Analyses from six PITP/ mice indicate that 18 1% of the total motor neuron cell bodies in the ventral horn exhibit properties of aponecrosis. These include pericyto- plasmic vacuolation, reduced cytoplasmic contents, and cyto- plasmic proliferation of irregular electron-translucent vesicles (Fig. 4B). Neurodegeneration and Inflammation in PITP/ Spinal Cord—We consistently observed WM deficits in PITP/ cer- vical, thoracic, and lumbar spinal cord. Whereas ventral WM is well developed in PITP/ cervical spinal cord, the corre- sponding WM regions are thin and sparse in PITP/ ani- mals (Fig. 4A). WM to GM area ratios in cervical spinal cord are 0.45 0.1 for PITP/ and 0.32 0.02 for PITP/ mice, respectively (p 0.01; n 6). In addition to the WM deficits, evidence of inflammation pervades all regions of the PITPa/ Cord—We consistently observed WM deficits in PITP / cer- vical, thoracic, and lumbar spinal cord. Whereas ventral WM is well developed in PITP/ cervical spinal cord, the corre- sponding WM regions are thin and sparse in PITP/ ani- mals (Fig. 4A). WM to GM area ratios in cervical spinal cord are 0.45 0.1 for PITP/ and 0.32 0.02 for PITP/ mice, respectively (p 0.01; n 6). In addition to the WM deficits, evidence of inflammation pervades all regions of the PITPa/ spinal cord. Toluidine blue O-staining reveals areas of abnor- mally heavy staining in the ventral horn where motor neuron cell bodies reside, suggesting cell damage in these areas (Fig. 4A). Moreover, whereas the WM/GM interface of PITP/ cervical spinal cord presents normal vascular structures, neu- rons, and accessory cells, corresponding regions of the PITP/ spinal cord exhibit densely stained cells and dam- aged neurons (Fig. 4B). These pathologies encompass a spec- trum of cell death events. These range from occasional apop- totic neurons (condensed nuclei, fragmented cytoplasm; not shown) to large numbers of neurons undergoing aponecrotic processes (Fig. 4B). Aponecrosis is a form of cell death associ- ated with reductions in cellular ATP and increases in ADP levels (27, 28). RESULTS This reduction is obvious when the subcutaneous fat pads of PITP/ and PITP/ mice are compared. Whereas PITP/ controls exhibit large axillary and inguinal fat pads, these structures are absent from PITP/ animals (Fig. 2D). Chemical analyses of eviscerated carcasses quantified these differences; PITP/ and PITP/ mice exhibited total body fat contents of 15.0 1.1 and 4.0 0.5% of total body mass, respectively (Fig. 2E). Significantly, the relative water contents of PITP/ and PITP/ carcasses are similar (Fig. 2E), indicating that PITP/ mice are not dehydrated, as would be expected if there were substantial suckling defects. The rela- tive fat-free dry mass contents and carcass bone ash contents are also comparable (Fig. 2E). Consistent with those measure- ments, we find that organ/total body mass ratios in PITP/ mice for brain, liver, and other major organs are also propor- tional to total body mass (not shown). Finally, electron microscopy reveals ER defects in cerebellar neurons of PITP/ mice. These defects manifest themselves as significant vacuolations of the smooth ER region, even though adjacent regions of rough ER retain normal morphology (not shown). Such vacuolations are not observed in smooth ER of PITP/ cerebellar neurons. Finally, electron microscopy reveals ER defects in cerebellar neurons of PITP/ mice. These defects manifest themselves as significant vacuolations of the smooth ER region, even though adjacent regions of rough ER retain normal morphology (not shown). Such vacuolations are not observed in smooth ER of PITP/ cerebellar neurons. Neurodegeneration and Inflammation in PITP/ Spinal Cord—We consistently observed WM deficits in PITP/ cer- vical, thoracic, and lumbar spinal cord. Whereas ventral WM is well developed in PITP/ cervical spinal cord, the corre- sponding WM regions are thin and sparse in PITP/ ani- mals (Fig. 4A). WM to GM area ratios in cervical spinal cord are 0.45 0.1 for PITP/ and 0.32 0.02 for PITP/ mice, respectively (p 0.01; n 6). In addition to the WM deficits, evidence of inflammation pervades all regions of the PITPa/ spinal cord. Toluidine blue O-staining reveals areas of abnor- mally heavy staining in the ventral horn where motor neuron cell bodies reside, suggesting cell damage in these areas (Fig. 4A). Moreover, whereas the WM/GM interface of PITP/ cervical spinal cord presents normal vascular structures, neu- rons, and accessory cells, corresponding regions of the PITP/ spinal cord exhibit densely stained cells and dam- aged neurons (Fig. 4B). RESULTS These mice did respond to touch, however. By contrast, 10% of the PITP/ mice experienced spontaneous seizures. All PITP/ mice were severely ataxic and were generally incapable of maintaining themselves upright. We also observed coarse action tremors upon limb extension in PITP/ mice. Second, these mutant mice failed to thrive. Although the PITP/ mice gained body mass, they did so slowly (Fig. 2B). By P10, surviving PITP/ mice were 2.5-fold less massive than their PITP/ and PITP/ littermates (Fig. 2C). PITP/ Mice Suckle Effectively—Several lines of evidence indicate that the failure of PITP/ mice to thrive is not the simple consequence of neurological defects. We observed PITP/ mice in the act of suckling, and postmortem analy- ses indicated both copious quantities of milk in stomachs of these mice (Fig. 2D) and substantial amounts of digested mat- ter throughout the PITP/ intestinal tract (not shown). The suckling competence of PITP/ mice notwithstanding, the mutant animals exhibited pathologically low body fat levels. This reduction is obvious when the subcutaneous fat pads of PITP/ and PITP/ mice are compared. Whereas PITP/ controls exhibit large axillary and inguinal fat pads, these structures are absent from PITP/ animals (Fig. 2D). Chemical analyses of eviscerated carcasses quantified these differences; PITP/ and PITP/ mice exhibited total body fat contents of 15.0 1.1 and 4.0 0.5% of total body mass, respectively (Fig. 2E). Significantly, the relative water contents of PITP/ and PITP/ carcasses are similar (Fig. 2E), indicating that PITP/ mice are not dehydrated, as would be expected if there were substantial suckling defects. The rela- tive fat-free dry mass contents and carcass bone ash contents are also comparable (Fig. 2E). Consistent with those measure- ments, we find that organ/total body mass ratios in PITP/ mice for brain, liver, and other major organs are also propor- tional to total body mass (not shown). PITP/ Mice Suckle Effectively—Several lines of evidence indicate that the failure of PITP/ mice to thrive is not the simple consequence of neurological defects. We observed PITP/ mice in the act of suckling, and postmortem analy- ses indicated both copious quantities of milk in stomachs of these mice (Fig. 2D) and substantial amounts of digested mat- ter throughout the PITP/ intestinal tract (not shown). The suckling competence of PITP/ mice notwithstanding, the mutant animals exhibited pathologically low body fat levels. RESULTS The non- neuronal area (neuropil) comprises 54 3% in PITP/ spinal cord (n 6; p 0.01) and 70 6% of total area in PITP/ spinal cord, respectively. Degenerative processes in neuropil, typified by swollen axons lacking organelles and cytoskeletal filaments, are also obvious (not shown). Second, demyelination is scored in both WM and GM areas of PITP/ mice and especially in the dorsal spinal columns. Supporting cells that may be remodeling myelin are also observed (Fig. 4D). PITP/ mice to thrive suggests a malabsorption disorder. Histological analyses of duodenum indicate the architecture of this organ and its muscle layers are similar in PITP/ and PITP/ mice. Moreover, intestinal motility also appears nor- mal (not shown). There are obvious anomalies in PITP/ intestinal epithelium, however. Most strikingly, PITP/ en- terocytes exhibit an obviously heavy impregnation with the lipophilic agent osmium relative to PITP/ enterocytes (Fig. 5A). These histological properties are consistent with an abnor- mal accumulation of intracellular lipid in mutant enterocytes. If the accumulated lipid is derived from dietary fat, rather than manifestation of some other cellular pathology, active feeding is expected to be required for the loading of mutant enterocytes with lipid. Indeed, enterocytes harvested from PITP/ mice subjected to a 10-h fast are clear of accumulated lipid (not shown). includes processes that resemble those of other myelin-related cen- tral nervous system inflammatory disorders (31). First, macro- scopic swelling in the dorsal spinal columns is apparent. The non- neuronal area (neuropil) comprises 54 3% in PITP/ spinal cord (n 6; p 0.01) and 70 6% of total area in PITP/ spinal cord, respectively. Degenerative processes in neuropil, typified by swollen axons lacking organelles and cytoskeletal filaments, are also obvious (not shown). Second, demyelination is scored in both WM and GM areas of PITP/ mice and especially in the dorsal spinal columns. Supporting cells that may be remodeling myelin are also observed (Fig. 4D). The fragility of the mutant central nervous system notwith- standing, a variety of PITP/ neurons are amenable to pri- mary culture. Cultured cortical neurons, cerebellar granule cells, and spinal cord dorsal root ganglion neurons from PITP/ mice are not more fragile than their PITP/ counterparts in any obvious way. RESULTS Finally, spinocerebellar injury in PITP/ mice also Ablation of PITP Function in Mice 33506 FIG. 3. Cerebellar injury in PITP-deficient mice. A, TUNEL staining of cerebellum. Genotypes are indicated. The ApopTag Fluorescein in Situ Detection Kit (Intergen Co.) was used as instructed by the manufacturer. B, Purkinje cell defects in PITP/ cerebellum. The Purkinje cell layer was revealed by staining with calbindin antibodies and counterstaining with toluidine blue O. C, reactive gliosis in PITP/ cerebellum. Sagittal sections of PITP/ and PITP/ cerebellum (as indicated) were stained with anti-GFAP antibodies with or without counterstaining with toluidine blue O (right and left columns, respectively). D, higher magnification images of PITP/ and PITP/ cerebellum stained with anti-GFAP antibodies and counterstained with toluidine blue O. Genotypes are at top. Ablation of PITP Function in Mice 33506 FIG. 3. Cerebellar injury in PITP-deficient mice. A, TUNEL staining of cerebellum. Genotypes are indicated. The ApopTag Fluorescein in Situ Detection Kit (Intergen Co.) was used as instructed by the manufacturer. B, Purkinje cell defects in PITP/ cerebellum. The Purkinje cell layer was revealed by staining with calbindin antibodies and counterstaining with toluidine blue O. C, reactive gliosis in PITP/ cerebellum. Sagittal sections of PITP/ and PITP/ cerebellum (as indicated) were stained with anti-GFAP antibodies with or without counterstaining with toluidine blue O (right and left columns, respectively). D, higher magnification images of PITP/ and PITP/ cerebellum stained with anti-GFAP antibodies and counterstained with toluidine blue O. Genotypes are at top. FIG. 3. Cerebellar injury in PITP-deficient mice. A, TUNEL staining of cerebellum. Genotypes are indicated. The ApopTag Fluorescein in Situ Detection Kit (Intergen Co.) was used as instructed by the manufacturer. B, Purkinje cell defects in PITP/ cerebellum. The Purkinje cell layer was revealed by staining with calbindin antibodies and counterstaining with toluidine blue O. C, reactive gliosis in PITP/ cerebellum. Sagittal sections of PITP/ and PITP/ cerebellum (as indicated) were stained with anti-GFAP antibodies with or without counterstaining with toluidine blue O (right and left columns, respectively). D, higher magnification images of PITP/ and PITP/ cerebellum stained with anti-GFAP antibodies and counterstained with toluidine blue O. Genotypes are at top. includes processes that resemble those of other myelin-related cen- tral nervous system inflammatory disorders (31). First, macro- scopic swelling in the dorsal spinal columns is apparent. RESULTS Indeed, titration of nerve growth factor concentrations from 50 to 5 ng/ml revealed no differences in the thresholds of trophic factor required to sus- tain viability of PITP/ versus PITP/ dorsal root gan- glion neurons in culture (not shown). PITP/ cerebellar granule cells are similarly robust. These data suggest a signif- icant cause of neuronal injury in PITP/ mice is a hostile physiological environment, rather than some overriding cell autonomous defect. Closer inspection of PITP/ duodenal enterocytes by elec- tron microscopy demonstrates that these cells contain a heter- ogeneous population of lipid bodies (Fig. 5B). Even at this high resolution, these structures stain with osmium in a manner consistent with a lipid interior, and these vesicles exhibit an average diameter of 300–700 nm. Inspection of the borders of these lipid bodies at very high magnifications reveals a classi- cal trilaminar membrane surrounding these structures (not shown). PITP/ enterocytes, on the other hand, are packed with vesicles that are also membrane-enclosed but are much Lipid Dysregulation in PITP/ Enterocytes—Failure of 33507 Ablation of PITP Function in Mice FIG. 4. Neurodegenerative and inflammatory changes in PITP/ spinal cord. A, complete spinal cord sections were generated by reconstructing images from 2-m-thick, epoxy resin-embedded sections of PITP/ stained with toluidine blue O (left, number of images n 30) and PITP/ spinal cord (right, n 22). Bars are 0.15 mm. B, images from the ventral interface between gray and white matter (GM/WM interface) of cervical spinal cord sections. Top panels, representative images of PITP/ motor neuron cell bodies (left, open arrows) and abnormally dark staining PITP/ motor neuron cell bodies (right, dark arrows) are indicated. Bars are as follows: left, 50 m; right, 100 m. WM regions are indicated. Bottom panels, additional images from GM/WM interface of PITP/ cervical spinal cord are shown. Characteristic neurodegenerative symptoms include highly vacuolated cells (center panel, solid arrow), dark cells (right panel, solid arrows), and abnormally lightly stained cells without discrete intracellular structure (right panel, ). Bars are as follows: left, 100 m; center, 50 m; right, 20 m. C, inflammatory cells in PITP/ thoracic spinal cord. Left panel, mast cell (MC) and capillary lumen (C) are indicated. Right panel, macrophage (MF) and capillary lumen (C) are indicated. Bars are as follows: left, 20 m; right, 5 m. D, electron micrographs of myelin from the WM/GM interface of PITP/ (left) and PITP/ spinal cord (center). RESULTS Examples of myelinated axons are indicated by arrows. Right panel, cells that may be remodeling myelin are present in large numbers in PITP/ mice and an example is indicated (). Bars are as follows: left, 15 m; center, 10 m; right, 6 m. FIG. 4. Neurodegenerative and inflammatory changes in PITP/ spinal cord. A, complete spinal cord sections were generated by reconstructing images from 2-m-thick, epoxy resin-embedded sections of PITP/ stained with toluidine blue O (left, number of images n 30) and PITP/ spinal cord (right, n 22). Bars are 0.15 mm. B, images from the ventral interface between gray and white matter (GM/WM interface) of cervical spinal cord sections. Top panels, representative images of PITP/ motor neuron cell bodies (left, open arrows) and abnormally dark staining PITP/ motor neuron cell bodies (right, dark arrows) are indicated. Bars are as follows: left, 50 m; right, 100 m. WM regions are indicated. Bottom panels, additional images from GM/WM interface of PITP/ cervical spinal cord are shown. Characteristic neurodegenerative symptoms include highly vacuolated cells (center panel, solid arrow), dark cells (right panel, solid arrows), and abnormally lightly stained cells without discrete intracellular structure (right panel, ). Bars are as follows: left, 100 m; center, 50 m; right, 20 m. C, inflammatory cells in PITP/ thoracic spinal cord. Left panel, mast cell (MC) and capillary lumen (C) are indicated. Right panel, macrophage (MF) and capillary lumen (C) are indicated. Bars are as follows: left, 20 m; right, 5 m. D, electron micrographs of myelin from the WM/GM interface of PITP/ (left) and PITP/ spinal cord (center). Examples of myelinated axons are indicated by arrows. Right panel, cells that may be remodeling myelin are present in large numbers in PITP/ mice and an example is indicated (). Bars are as follows: left, 15 m; center, 10 m; right, 6 m. Ablation of PITP Function in Mice 33508 . 5. Accumulation of lipid in PITP/ duodenal epithelium. A, intracellular lipid in enterocytes of PITP/ duodenum. hick) of duodenum from PITP/ and PITP/ P5 siblings were stained with osmium and counterstained with toluidine blu les identify lipid. Relevant genotypes are given. B, electron micrographs of duodenal epithelium from PITP/ (left) and PITP Ablation of PITP Function in Mice 08 Ablation of PITP Function in Mice 508 FIG. 5. Accumulation of lipid in PITP/ duodenal epithelium. A, intracellular lipid in enterocytes of PITP/ duodenum. Ablation of PITP Function in Mice 33509 TABLE I Serum analyses larger than those observed in PITP/ enterocytes. Moreover, the structures that accumulate in PITP/ enterocytes often appear multilobed (Fig. 5C). Representative profiles of such structures from PITP/ and PITP/ enterocytes are shown at higher magnification in Fig. 5D. The size differences of the structures in wild-type and nullizygous enterocytes not- withstanding, these share many structural features. It is there- fore likely that the biogenesis of the lipid bodies initially fol- lows a common course in PITP/ and PITP/ enterocytes. Morphometric measurements indicate a rather unimodal fre- quency distribution of vesicle perimeters from PITP/ mice (average is 2 m; Fig. 5E). By contrast, the accumulated structures in PITP/ enterocytes are much larger and have multimodal size distributions. Perimeters of 20 m are often observed, and perimeters in excess of 50 m are also encoun- tered (Fig. 5E). Finally, our results indicate that the lipid structures in PITP/ enterocytes reside predominantly in dilated regions of smooth ER (Fig. 5F). larger than those observed in PITP/ enterocytes. Moreover, the structures that accumulate in PITP/ enterocytes often appear multilobed (Fig. 5C). Representative profiles of such structures from PITP/ and PITP/ enterocytes are shown at higher magnification in Fig. 5D. The size differences of the structures in wild-type and nullizygous enterocytes not- withstanding, these share many structural features. It is there- fore likely that the biogenesis of the lipid bodies initially fol- lows a common course in PITP/ and PITP/ enterocytes. Serum pooled from six PITP/ and PITP/ mice each, varying in age from P6 to P9, were subjected to a comprehensive mammalian chemistry panel performed by Antech Diagnostics (Farmingdale, NY). Values represent the averages of triplicate measurements obtained from three different sets of mice. Parameters that differ significantly between PITP/ and PITP/ mice are highlighted at top of the table. The abbreviations used are: APase, alkaline phosphatase; AST, aspartate aminotransferase; CPK, creatinine phosphokinase; ALT, al- anine aminotransferase. Morphometric measurements indicate a rather unimodal fre- quency distribution of vesicle perimeters from PITP/ mice (average is 2 m; Fig. 5E). By contrast, the accumulated structures in PITP/ enterocytes are much larger and have multimodal size distributions. Perimeters of 20 m are often observed, and perimeters in excess of 50 m are also encoun- tered (Fig. 5E). Finally, our results indicate that the lipid structures in PITP/ enterocytes reside predominantly in dilated regions of smooth ER (Fig. 5F). Ablation of PITP Function in Mice Immunoblot ex- periments indicate that apolipoprotein B48 and microsomal triglyceride transfer protein, two components required for chy- lomicron assembly, are expressed in PITP/ mice and that serum apoB levels are similar to those recorded for PITP/ mice (not shown). Quantitative Analyses of Lipids and Fatty Acids in PITP/ Liver and Brain—The lipid derangement in PITP/ duodenum and liver suggests increased neutral lipid mass. To test this interpretation directly, the mass of a variety of lipid species in PITP/ and PITP/ liver and brain was quantified. The intestine provides technical chal- lenges in this assay as the mice analyzed are neither fasted nor germ-free. We assume analysis of liver lipid mass is generally representative of the situation in intestine. PITP/ Mice Exhibit Microvesicular Steatosis in Liver— The pathways for lipid transport from the ER lumen to the cell surface are similar in enterocytes and hepatocytes. In that regard, PITP/ liver also exhibits obvious microvesicular steatosis, as evidenced by its dramatically enhanced staining with osmium (Fig. 6A) and with the lipophilic dye Oil Red O (Fig. 6B). Because Oil Red O exhibits specific affinity for neu- tral lipids (e.g. TG, DAG, and CE), we conclude that the accu- mulated lipid is largely of the neutral class. These histochem- ical data are extended by electron microscopy. Relative to wild- type hepatocytes, PITP/ cells exhibit an abundance of large intracellular lipid bodies. We often detect such structures in the nuclear matrix of mutant hepatocytes, a circumstance we have yet to encounter in wild-type cells (Fig. 6C). PITP/ Mice Exhibit Microvesicular Steatosis in Liver— The pathways for lipid transport from the ER lumen to the cell surface are similar in enterocytes and hepatocytes. In that regard, PITP/ liver also exhibits obvious microvesicular steatosis, as evidenced by its dramatically enhanced staining with osmium (Fig. 6A) and with the lipophilic dye Oil Red O (Fig. 6B). Because Oil Red O exhibits specific affinity for neu- tral lipids (e.g. TG, DAG, and CE), we conclude that the accu- mulated lipid is largely of the neutral class. These histochem- ical data are extended by electron microscopy. Relative to wild- type hepatocytes, PITP/ cells exhibit an abundance of large intracellular lipid bodies. We often detect such structures in the nuclear matrix of mutant hepatocytes, a circumstance we have yet to encounter in wild-type cells (Fig. 6C). As shown in Fig. duodenal enterocytes as revealed by electron microscopy. Lipid-engorged regions are identified by arrows. These smooth ER luminal regions are contiguous with the lumen of adjacent rough ER that is easily recognized by the associated ribosomes (not shown). Bar is 0.4 m. G, PITP/ mice exhibit reduced brain -tocopherol and post-prandial TG levels. Parameters are indicated. Measurements were made from nine PITP/ and nine PITP/ mice. Averages S.D. are given. Ablation of PITP Function in Mice Parameter PITP/ PITP/ Bilirubin 0.84 0.16 mg/dl 1.54 0.10 mg/dl APase 809 160 units/liter 1497 179 units/liter AST 135 9 units/liter 250 18 units/liter CPK 4892 42 units/liter 6439 147 units/liter Creatinine 0.44 0.08 mg/dl 0.44 0.08 mg/dl Total protein 3.10 0.34 g/dl 3.10 1.33 g/dl ALT 52.4 18.3 units/liter 46.1 13.2 units/liter Albumin 1.70 0.18 g/dl 1.60 0.17 g/dl Cholesterol 134 14 mg/dl 137 11 mg/dl Calcium 7.04 0.24 mg/dl 8.28 1.70 mg/dl Phosphorus 11.4 1.5 mg/dl 11.3 1.3 mg/dl Sodium 115 14 Meq/liter 113 8 Meq/liter Potassium 7.2 1.2 Meq/liter 7.3 0.9 Meq/liter Chloride 50.6 11.9 Meq/liter 45.3 3.0 Meq/liter Globulin 1.20 0.25 g/dl 1.50 32 g/dl Urea N2 22.0 5.0 mg/dl 25.0 7.5 mg/dl -Tocopherol and Plasma TG Deficiency in PITP/ Mice— The failure of PITP/ mice to thrive, when coupled with their retention of lipid in the enterocyte smooth ER, predicts a malabsorption-mediated deficit in fat-soluble vitamins, partic- ularly -tocopherol (vitamin E), and reduced levels of post- prandial plasma TGs. We find PITP/ mice are deficient in -tocopherol. Whereas PITP/ brain exhibits 19.48 3.41 g of -tocopherol/gm tissue, values for PITP / brain are 10-fold reduced (2.11 0.04 g of -tocopherol/g of tissue; Fig. 5G). Post-prandial plasma TG levels in PITP/ mice are also 3-fold reduced relative to PITP/ mice (55 13 versus 183 15 mg/dl). enterocytes. Although we cannot yet exclude the possibility that allocation of fatty acids and lipids between storage pools and lipoprotein pools is deranged in PITP/ liver, at least some key fatty acid and lipid biosynthetic enzymes (acetyl-CoA carboxylase, stearyl-CoA reductase, and fatty-acid synthase) are not transcriptionally up-regulated in this mutant tissue (not shown). Moreover, the hepatic microvesicular steatosis, although qualitatively resistant to a short 6-h fast, is reversed by fasting mutant mice for 17 h (not shown). By contrast, PITP/ and PITP/ plasma cholesterol levels are similar. Indeed, comprehensive blood chemistry analyses demonstrate many (although not all) parameters are largely normal for PITP/ mice (Table I). Immunoblot ex- periments indicate that apolipoprotein B48 and microsomal triglyceride transfer protein, two components required for chy- lomicron assembly, are expressed in PITP/ mice and that serum apoB levels are similar to those recorded for PITP/ mice (not shown). By contrast, PITP/ and PITP/ plasma cholesterol levels are similar. Indeed, comprehensive blood chemistry analyses demonstrate many (although not all) parameters are largely normal for PITP/ mice (Table I). RESULTS Sections (5 m thick) of duodenum from PITP/ and PITP/ P5 siblings were stained with osmium and counterstained with toluidine blue O. Black granules identify lipid. Relevant genotypes are given. B, electron micrographs of duodenal epithelium from PITP/ (left) and PITP/ (right) P8 mice are shown. Bars are 5 m. C, electron micrographs of enterocytes from PITP/ and PITP/ mice as indicated. Bars are 2 m. D, electron micrographs of lipid bodies from PITP/ (left panels) and PITP/ enterocytes (right panels) as indicated. Dimensions of the lipid bodies aside, the general morphologies of these structures exhibit many similarities in wild-type versus mutant enterocytes, and most are membrane-enclosed. Boundary membranes are indicated by arrows. A cytoplasmic lipid droplet (L) with a fuzzy border is shown for contrast. Bars are 0.2 m. E, distribution histogram of vesicle perimeters in PITP/ (solid symbols) and PITP/ mice (open symbols). Perimeter measure- ments were made for 428 and 398 lipid bodies from PITP/ and PITP/ enterocytes, respectively. F, enlargement of smooth ER in PITP/ Ablation of PITP Function in Mice Ablation of PITP Function in Mice Bars (from left) are 2 and 0.4 m, respectively. Ablation of PITP Function in Mice 33510 Ablation of PITP Function in Mice 33510 FIG. 6. Microvesicular steatosis in PITP/ hepatocytes. A, liver sections from PITP/ and PITP/ P5 siblings were stained with osmium and counterstained with toluidine blue O. Black granules identify lipid. Relevant genotypes are given at left. B, liver sections from mice of the indicated genotype were stained with Oil Red O and counterstained with hematoxylin/eosin. The red globules prevalent in sections of PITP nullizygous liver identify neutral lipid bodies. C, electron micrographs of PITP/ and PITP/ liver sections are shown. Nuclei (N) and examples of lipid bodies are highlighted by arrows. Bars (clockwise from upper left) are 2, 2, 3, and 2 m, respectively. D, electron micrograph of lipid bodies that accumulate in PITP nullizygous hepatocytes. The right panel is a magnification of the region identified by the box in the left panel. One lipid body is membrane-enclosed (arrow), whereas the other lacks a distinct border and is interpreted to represent a large cytoplasmic lipid droplet. Bars (from left) are 2 and 0.4 m, respectively. FIG. 6. Microvesicular steatosis in PITP/ hepatocytes. A, liver sections from PITP/ and PITP/ P5 siblings were stained with osmium and counterstained with toluidine blue O. Black granules identify lipid. Relevant genotypes are given at left. B, liver sections from mice of the indicated genotype were stained with Oil Red O and counterstained with hematoxylin/eosin. The red globules prevalent in sections of PITP nullizygous liver identify neutral lipid bodies. C, electron micrographs of PITP/ and PITP/ liver sections are shown. Nuclei (N) and examples of lipid bodies are highlighted by arrows. Bars (clockwise from upper left) are 2, 2, 3, and 2 m, respectively. D, electron micrograph of lipid bodies that accumulate in PITP nullizygous hepatocytes. The right panel is a magnification of the region identified by the box in the left panel. One lipid body is membrane-enclosed (arrow), whereas the other lacks a distinct border and is interpreted to represent a large cytoplasmic lipid droplet. Bars (from left) are 2 and 0.4 m, respectively. CL levels are altered in both mutant brain and liver relative to matched wild-type tissues (Fig. 7C). increased proliferation of electron-dense autophagic bodies throughout the cytoplasm (cytolysosomes) and regions of di- lated smooth ER (not shown). Ablation of PITP Function in Mice 7A, although the relative TG, DAG, and CE contents are very similar in matched wild-type and PITP nullizygous brain tissue, bulk neutral lipid mass is dramati- cally elevated in PITP nullizygous liver. Bulk TG and FFA mass is 2.0- and 2.6-fold increased relative to the respective values recorded for wild-type liver. Bulk CE and DAG mass is also increased in PITP/ liver. Unexpectedly, PITP/ brain exhibits a 3.3-fold increase in FFA mass relative to PITP/ brain (Fig. 7A). / The lipid bodies are either membrane-bound or represent large cytoplasmic lipid droplets (Fig. 6D). Unlike in mutant enterocytes, where accumulated lipid is predominantly found in membrane-enclosed compartments, 60% of the lipid bodies in PITP/ hepatocytes represent what we interpret to be cytosolic lipid droplets due to lack of identifiable boundary membrane (Fig. 6D). This raises the possibility that the mech- anism of hepatic steatosis differs from that of the PITP/ The derangements in neutral lipid/FFA content in PITP/ tissues do not reflect a universal derangement of lipid homeo- stasis. PtdCho, phosphatidylethanolamine, and phosphatidyl- serine mass values are similar in matched wild-type and PITP nullizygous brain and liver tissue (Fig. 7B). We do note, how- ever, that mass of specific polar lipids such as sphingomyelin and lyso-PtdCho are elevated in PITP nullizygous brain, and CL levels are altered in both mutant brain and liver relative to matched wild type tissues (Fig 7C) increased proliferation of electron-dense autophagic bodies throughout the cytoplasm (cytolysosomes) and regions of di FIG. 6. Microvesicular steatosis in PITP/ hepatocytes. A, liver sections from PITP/ and PITP/ P5 siblings were stained with osmium and counterstained with toluidine blue O. Black granules identify lipid. Relevant genotypes are given at left. B, liver sections from mice of the indicated genotype were stained with Oil Red O and counterstained with hematoxylin/eosin. The red globules prevalent in sections of PITP nullizygous liver identify neutral lipid bodies. C, electron micrographs of PITP/ and PITP/ liver sections are shown. Nuclei (N) and examples of lipid bodies are highlighted by arrows. Bars (clockwise from upper left) are 2, 2, 3, and 2 m, respectively. D, electron micrograph of lipid bodies that accumulate in PITP nullizygous hepatocytes. The right panel is a magnification of the region identified by the box in the left panel. One lipid body is membrane-enclosed (arrow), whereas the other lacks a distinct border and is interpreted to represent a large cytoplasmic lipid droplet. Ablation of PITP Function in Mice patic injury with reduced cellular ATP (Fig. 8, A and B). The significant decrease in mass of the mitochondrial lipid CL in PITP/ liver is consistent with energetic compromise (Fig. 7C). The mass increase of CL in PITP/ brain may reflect compensatory attempts by this tissue to enhance mitochondrial activity in response to an unfavorable energy status (Fig. 7C). Hypoglycemia in PITP/ Mice—The aponecrotic cell death observed in the PITP/ spinocerebellar system and liver diagnoses a systemic problem. In this regard, we find PITP/ neonates are severely hypoglycemic. We measure 9-fold reductions in serum glucose levels of PITP/ mice (9.6 3.6 mg/dl) relative to PITP/ siblings (88.0 6.6 mg/dl serum; Fig. 9A). This reduction in serum glucose is of similar magnitude to that measured for PITP/ mice that are fasted overnight, despite the fact that stomachs of all PITP/ mice analyzed were filled with milk at the time of analysis (not shown). Reduction in serum glucose is not secondary to hyperinsu- linemia. Circulating insulin levels in PITP/ mice are 9-fold lower than those of PITP/ siblings (0.5 0.2 versus 4.5 0.2 ng/ml, respectively; Fig. 9A). Given the hypoglycemia of PITP/ mice, these low insulin levels are appropriate (see below). We also measured an increase in circulating -hydroxy- butyrate in PITP/ versus PITP/ serum (19.1 1.2 and 11.2 1.8 mg/dl, respectively; Fig. 9A), and this increase is recapitulated in serum of PITP/ mice that were fasted overnight (20.2 0.1 mg/dl). -Hydroxybutyrate is generated by mitochondrial oxidation of fatty acids and serves as an alternative energy source for hypoglycemic tissue. Modest ele- vations are scored for bilirubin and alkaline phosphatase in PITP/ mice (consistent with modest hepatic stress), and a 2-fold increase in the gluconeogenic enzyme aspartate ami- notransferase is also measured (Table I). Reduced Proglucagon Gene Expression in PITP/ Mice— Mice normally effect dramatic changes in circulating insulin, corticosteroid, and glucagon levels to activate hepatic glucone- ogenesis late in gestation. This reprogramming of hormonal signaling serves as a protective mechanism for the impending loss of the glucose source provided by the maternal circulatory system in utero (33). Mutant animals are appropriately re- sponding to hypoglycemia as evidenced by elevated levels of circulating corticosteroids, a hormonal stimulus for gluconeo- genesis. Levels of plasma corticosteroid are highly elevated in PITP/ mice relative to PITP/ littermates (51.64 versus 19.58 ng/ml, respectively). and hatched bars, respectively. Numerical values for each bar are given. A, neutral lipid mass measurements. B, polar lipids whose mass is unaltered in PITP/ versus PITP/ tissues. C, polar lipids whose mass is altered in PITP/ versus PITP/ tissues. SM, sphingomy- elin; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine. Ablation of PITP Function in Mice Aponecrosis in the spinocerebel- lar system and in liver suggests a fundamental defect in energy metabolism in PITP nullizygous mice. Indeed, ATP levels are diminished in PITP/ relative to PITP/ liver (40.4 8.6 versus 98.1 9.3 pmol of ATP/mg of tissue; Fig. 8A). ATP/ADP ratios further emphasize this reduction (2.8 0.5 for PITP/ and 9.1 3.1 for PITP/ liver; Fig. 8B). Simi- larly, PITP/ and PITP/ animals exhibit 83.3 5.3 and 44.9 5.9 pmol of ATP/mg of cerebellum, respectively (Fig. 8A). Reduced cerebellar ATP/ADP ratios are measured as well (7.7 1.0 for wild-type versus 4.2 0.5 for mutant; Fig. 8B). Interestingly, PITP/ and PITP/ brain ATP levels (40 59 and 343 65 pmol of ATP/mg of brain, respectively) and ATP/ADP ratios (13.6 2.3 versus 9.1 0.7, respectively) are comparable, thereby regionally correlating neuronal and he- Fatty acid compositions of the CE, DAG, TG, FFA, sphingomy- elin, and CL pools of PITP/ and PITP/ brain (Table II) and liver (Table III) were also compared. The most striking qualitative and quantitative difference is the reduced content of 18:2n6 (9,12-octadecadienoic acid) in mutant brain TG, DAG, and CL pools (Table II), and an increase in 18:1n9 (9-octadenoic acid) in CE, DAG, and FFA pools in nullizygous liver (Table III). Fatty acid profiles of brain and liver lyso-PtdCho were determined for PITP/ and PITP/ mice, and no differences were scored (not shown). Reduced ATP Content in PITP/ Cerebellum and Liver— Electron microscopic analyses reveal not only lipid accumula- tion but also indications of aponecrosis in mutant hepatocytes. These indications resemble those observed in the PITP/ spinocerebellar system (see above) and include the significantly Ablation of PITP Function in Mice 33511 patic injury with reduced cellular ATP (Fig. 8, A and B). The significant decrease in mass of the mitochondrial lipid CL in PITP/ liver is consistent with energetic compromise (Fig. 7C). The mass increase of CL in PITP/ brain may reflect compensatory attempts by this tissue to enhance mitochondrial activity in response to an unfavorable energy status (Fig. 7C). patic injury with reduced cellular ATP (Fig. 8, A and B). The significant decrease in mass of the mitochondrial lipid CL in PITP/ liver is consistent with energetic compromise (Fig. 7C). The mass increase of CL in PITP/ brain may reflect compensatory attempts by this tissue to enhance mitochondrial activity in response to an unfavorable energy status (Fig. 7C). Ablation of PITP Function in Mice TABLE II Acyl chain compositions of bulk brain lipids Acyl chain compositions of bulk brain lipids Fatty acid compositions of the lipid classes analyzed from pooled brain tissue samples depicted in Fig. 7 were determined by Lipomics TrueMass™technology. Profiles of the indicated fatty acid species (at left) for the indicated lipid species (at top) are presented in nanomoles of fatty acid/total moles of fatty acid in each lipid class. The data are presented as PITP/ value/PITP/ value. Some differences between correspond- ing PITP/ and PITP/ values are indicated in boldfase. Omega fatty acid nomenclature is employed. The t prefix indicates trans-double bond, and the dm prefix indicates plasmalogen-linked fatty acid. SM indicates sphingomyelin. A precise molecular description of each fatty acid species analyzed is provided at www.lipomics.com/Fatty%20Acids/fatty_acid.htm. analyzed is provided at www.lipomics.com/Fatty%20Acids/fatty_acid.htm. Ablation of PITP Function in Mice We also compared expression of the gluconeogenic enzymes Glc-6-Pase and phosphoenolpyruvate carboxykinase in PITP/ and PITP/ mice. Expression of both genes in PITP / liver compares favorably to PITP/ control as their corresponding mRNA levels in PITP/ liver are 1.3 0.1- and 1.1 0.2-fold increased relative to those of PITP/ liver (Fig. 9B). Thus, transcriptional control of key gluconeogenic enzymes also appears intact in the mutant mice. Elevated serum levels of the gluconeogenic enzyme aspartate aminotransferase in PITP/ further support this conclusion (Table I). By contrast, proglucagon gene expression is inappropriately depressed in PITP/ animals (Fig. 9B). By using -actin mRNA as a normalizing factor, proglucagon gene expression in pancreas is reduced 11.7 0.1-fold in PITP/ mice relative to PITP/ siblings. In independent agreement with the Northern data, we record a 5-fold reduction in circulating glu- FIG. 7. Quantification of lipid mass in PITP/ and PITP/ brain and liver. Brains and livers were collected from each of four freshly euthanized non-fasted PITP/ and PITP/ mice and com- bined to generate defined tissue pools for each genotype. Pools were frozen and quantified for individual lipid species and FFAs via the Lipomics Technologies, Inc., TrueMassTM protocol. Individual lipid spe- cies are given at the top, and brain and liver values for each lipid species are given in a vertical column below each heading. Brain values are represented in the top panels, and the liver values are in the bottom panels as indicated. All lipid species are quantified as nanomoles per g of tissue. PITP/ and PITP/ values are indicated by solid bars FIG. 7. Quantification of lipid mass in PITP/ and PITP/ brain and liver. Brains and livers were collected from each of four freshly euthanized non-fasted PITP/ and PITP/ mice and com- bined to generate defined tissue pools for each genotype. Pools were frozen and quantified for individual lipid species and FFAs via the Lipomics Technologies, Inc., TrueMassTM protocol. Individual lipid spe- cies are given at the top, and brain and liver values for each lipid species are given in a vertical column below each heading. Brain values are represented in the top panels, and the liver values are in the bottom panels as indicated. All lipid species are quantified as nanomoles per g of tissue. PITP/ and PITP/ values are indicated by solid bars 33512 Ablation of PITP Function in Mice Acyl species CE TG DAG FFA SM CL 14:0 5.4/6.0 3.3/0.0 4.1/5.4 4.1/4.2 2.6/3.5 1.9/2.0 14:1n5 0.0/0.0 0.7/1.0 0.0/0.0 0.0/0.0 0.1/0.2 0.0/0.0 15:0 1.0/1.6 1.6/2.8 0.8/1.1 1.3/1.0 0.5/1.1 1.2/1.7 dm16:0 1.7/3.3 0.7/1.3 0.0/0.4 0.0/0.3 0.2/0.3 2.6/3.1 16:0 23.2/21.4 27.2/28.5 28.9/36.0 33.4/33.9 17.1/16.8 16.1/16.9 t16:1n7 0.9/1.8 0.7/1.3 3.1/3.3 5.4/5.5 1.6/2.6 0.5/2.2 16:1n7 9.1/13.2 3.7/5.6 1.3/1.4 1.2/0.9 1.3/1.8 3.3/1.8 dm18:0 0.0/0.0 0.9/0.7 0.0/0.0 0.4/0.7 0.2/0.1 1.8/2.1 dm18:1n9 1.9/2.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.1/0.2 dm18:1n7 0.0/0.0 0.8/1.0 0.0/0.0 0.0/0.0 0.0/0.0 0.2/0.2 18:0 16.4/11.9 15.4/18.8 23.4/27.9 27.4/30.4 28.7/23.2 21.8/18.9 t18:1n9 0.0/0.0 0.0/0.0 0.0/0.4 0.0/0.0 0.3/0.9 0.0/0.0 18:1n9 12.3/11.5 17.3/18.2 15.2/8.7 12.2/10.7 18.4/25.0 6.0/6.0 18:1n7 0.6/0.0 1.5/1.2 1.6/1.0 1.5/1.3 1.7/1.7 2.1/1.5 t18:2n6 0.0/0.0 0.0/0.0 0.2/0.3 0.0/0.1 0.0/0.0 0.0/0.0 18:2n6 7.2/6.8 16.3/8.9 9.1/4.8 6.1/4.2 2.7/3.1 8.4/2.8 18:3n6 0.0/0.6 0.3/0.6 0.3/0.0 0.2/0.0 0.0/0.0 0.0/0.0 18:3n3 0.2/0.0 0.6/0.4 0.8/0.8 0.3/0.4 0.4/0.4 0.4/0.3 18:4n3 0.0/0.3 0.3/0.5 0.0/0.0 0.0/0.0 0.0/0.0 0.2/0.2 20:0 0.4/1.0 0.8/0.4 0.8/0.7 0.9/0.9 0.4/0.3 0.2/0.2 20:1n15 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 20:1n12 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 20:1n9 6.2/5.6 1.1/2.0 0.4/0.0 0.2/0.3 0.5/0.4 0.4/0.2 20:3n9 0.0/0.0 0.0/0.0 0.3/1.4 0.5/0.4 0.1/0.1 0.0/0.1 20:2n6 0.7/0.9 0.8/0.3 0.3/0.5 0.4/0.4 0.1/0.2 16.9/25.2 20:3n6 0.0/0.0 1.0/0.3 0.4/0.0 0.4/0.3 0.6/0.5 0.5/0.4 20:4n6 1.1/0.0 1.4/0.4 7.7/2.8 3.5/1.9 4.5/3.3 4.4/4.1 20:3n3 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.1 0.1/0.0 20:4n3 0.0/0.0 0.3/0.2 0.0/0.0 0.0/0.0 0.0/0.0 0.3/0.3 20:5n3 0.0/0.0 0.3/0.0 0.2/0.0 0.0/0.1 0.0/0.0 0.0/0.1 22:0 1.1/1.8 0.3/0.3 0.0/0.0 0.0/1.1 0.6/0.4 0.1/0.2 22:1n9 9.3/8.6 1.3/0.3 0.0/1.1 0.0/0.4 0.4/0.3 0.3/0.4 22:2n6 0.0/0.3 0.0/0.0 0.0/2.1 0.0/0.2 0.0/0.1 0.7/0.6 22:4n6 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.2/0.2 0.4/0.0 22:5n6 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 22:5n3 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.6/0.4 0.1/0.2 22:6n3 0.0/0.0 0.3/0.3 1.1/0.0 0.0/0.0 15.9/12.4 8.5/7.3 24:0 1.3/1.5 0.8/0.3 0.0/0.0 0.5/0.6 0.2/0.3 0.2/0.4 24:1n9 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.2/0.4 0.2/0.3 24:6n3 0.0/0.0 0.2/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 cagon levels in mutant animals (246.68 and 49.32 pg of gluca- gon/ml of plasma for PITP/ and PITP/ mice, respectively). complete depletion of glycogen from mutant liver (Fig. 9C). When levels of liver glycogen are measured as a function of duration of fast in PITP/ and PITP/ animals, we found the rate of glycogen depletion from fasted PITP/ liver is delayed. Whereas PITP/ mice deplete 50 and 70% of their liver glycogen stores by 2 and 4 h of fast, respectively, PITP/ mice effect only a modest 23 and 27% depletion in liver glycogen stores after 2 and 4 h of fast, respectively (Fig. 9D). Ablation of PITP Function in Mice Taken together, these results are not consistent with an absolute block in PITP/ liver glycogen catabolism. Rather, the data indicate PITP/ liver exhibits kinetic defects in the rate of glycogenolysis and additionally suggest an inappropri- ate channeling of glucose to glycogen synthesis in the hypogly- cemic mutant mouse. complete depletion of glycogen from mutant liver (Fig. 9C). When levels of liver glycogen are measured as a function of duration of fast in PITP/ and PITP/ animals, we found the rate of glycogen depletion from fasted PITP/ liver is delayed. Whereas PITP/ mice deplete 50 and 70% of their liver glycogen stores by 2 and 4 h of fast, respectively, PITP/ mice effect only a modest 23 and 27% depletion in liver glycogen stores after 2 and 4 h of fast, respectively (Fig. 9D). Taken together, these results are not consistent with an absolute block in PITP/ liver glycogen catabolism. Rather, the data indicate PITP/ liver exhibits kinetic defects in the rate of glycogenolysis and additionally suggest an inappropri- ate channeling of glucose to glycogen synthesis in the hypogly- cemic mutant mouse. Glycogen Metabolism in PITP/ Mice—Normally, liver glycogen is rapidly consumed in response to hypoglycemia. Reductions in plasma glucagon therefore predict defects in glycogenolysis in PITP/ mice. Periodate acid-Schiff stain- ing and visualization of glycogen rosettes by electron micros- copy indicate substantial and inappropriate glycogen stores in PITP/ liver (not shown). Quantitative analyses support these observations. As expected, PITPa/ liver contains 109.0 15.3 mol of glycogen-derived glucose/g of tissue, and liver glycogen stores are depleted by a 10-h fast (Fig. 9C). Strikingly, the hypoglycemia of the mutant mice notwithstand- ing, PITPa/ liver contains 107.0 12.2 mol of glycogen- derived glucose/g of tissue (Fig. 9C). This compares favorably to wild-type values. Morphological Alterations in PITP/ Pancreas—Reduced proglucagon gene expression could reflect specific derangement of proglucagon gene expression in the mutant animals or could arise indirectly from generalized pancreatic dysfunction. Al- though exocrine pancreas morphologies of PITP/ and PITP/ mice are similar, PITP/ pancreas exhibits obvi- To characterize further the derangement in glycogen metab- olism, we tested whether glycogen stores in PITP/ liver are resistant to depletion by fast. Ablation of PITP Function in Mice In addition to the significant reduction in the number of islets, the mor- phology of identifiable islets in PITP/ pancreas is also highly abnormal. Islet cells are sparse and shrunken, and large vacuolations are abundant in the islets themselves (Fig. 10B). The damage of pancreatic islets in PITP/ mice provides a direct rationale for reduced proglucagon expression and defec- tive gluconeogenesis in PITP/ animals. Moreover, the very low levels of circulating insulin measured for PITP/ mice (see above) may also result from islet -cell insufficiencies and not simply from the hypoglycemic state of these animals. / Second, 35S-labeled amino acid pulse-radiolabeling experi- ments demonstrate that treatment of PITP/ and isogenic PITP/ ES cells with agents that activate UPR (i.e. dithio- threitol) evoke reduced incorporation of 35S-labeled amino ac- ids into nascent protein in both wild-type and mutant ES cells. The rate of protein synthesis in the face of dithiothreitol is 7.2 0.7 and 5.2 0.7% of unchallenged control for PITP/ and PITP/ ES cells, respectively (Fig. 10D). This effect is a result of a UPR-mediated translational block (34). These find- ings are qualitatively reproduced in other experiments that employed thapsigargin challenge to induce UPR (Fig. 10D). Second, 35S-labeled amino acid pulse-radiolabeling experi- ments demonstrate that treatment of PITP/ and isogenic PITP/ ES cells with agents that activate UPR (i.e. dithio- threitol) evoke reduced incorporation of 35S-labeled amino ac- ids into nascent protein in both wild-type and mutant ES cells. The rate of protein synthesis in the face of dithiothreitol is 7.2 0.7 and 5.2 0.7% of unchallenged control for PITP/ and PITP/ ES cells, respectively (Fig. 10D). This effect is a result of a UPR-mediated translational block (34). These find- ings are qualitatively reproduced in other experiments that employed thapsigargin challenge to induce UPR (Fig. 10D). PITP/ Cells Are Not Generally Defective in UPR—A link- age between activation of UPR and survival of pancreatic se- cretory islet cells has been demonstrated (34, 35). The ER abnormalities in PITP-deficient enterocytes, hepatocytes, and neurons, when coupled with loss of functional pancreatic islets in mutant mice, suggested the possibility that PITP/ cells are intrinsically defective in UPR. PITP/ Cells Are Not Generally Defective in UPR—A link- age between activation of UPR and survival of pancreatic se- cretory islet cells has been demonstrated (34, 35). Ablation of PITP Function in Mice Serial sectioning of PITP/ and PITP/ pan- creas, and quantification of total islets in the organ, revealed a greater than 2-fold reduction in absolute islet number in PITP/ pancreas relative to wild type (Fig. 10A). In addition to the significant reduction in the number of islets, the mor- phology of identifiable islets in PITP/ pancreas is also highly abnormal. Islet cells are sparse and shrunken, and large vacuolations are abundant in the islets themselves (Fig. 10B). The damage of pancreatic islets in PITP/ mice provides a direct rationale for reduced proglucagon expression and defec- tive gluconeogenesis in PITP/ animals. Moreover, the very low levels of circulating insulin measured for PITP/ mice (see above) may also result from islet -cell insufficiencies and not simply from the hypoglycemic state of these animals. / ES cells and MEFs to glucose deprivation. Cells defective in UPR are sensitive to such a challenge (34). In neither case do we record an unusual sensitivity of PITP/ cells to glucose deprivation. PITP/ ES cells maintain levels of viability that are 71 5 and 55 8% of control after 42 and 72 h of glucose starvation, respectively. The corresponding viability levels for isogenic PITP/ ES cells are 71 4 and 54 10% (Fig. 10C). Similarly, PITP/ MEFs exhibit viabilities of 82 5 and 73 10% after glucose deprivations of 42 and 72 h, respec- tively. Isogenic PITP/ MEFs exhibit corresponding viabil- ities of 84 4 and 76 8% (Fig. 10C). ES cells and MEFs to glucose deprivation. Cells defective in UPR are sensitive to such a challenge (34). In neither case do we record an unusual sensitivity of PITP/ cells to glucose deprivation. PITP/ ES cells maintain levels of viability that are 71 5 and 55 8% of control after 42 and 72 h of glucose starvation, respectively. The corresponding viability levels for isogenic PITP/ ES cells are 71 4 and 54 10% (Fig. 10C). Similarly, PITP/ MEFs exhibit viabilities of 82 5 and 73 10% after glucose deprivations of 42 and 72 h, respec- tively. Isogenic PITP/ MEFs exhibit corresponding viabil- ities of 84 4 and 76 8% (Fig. 10C). ous structural defects in that pancreatic islets are not at all prominent. Serial sectioning of PITP/ and PITP/ pan- creas, and quantification of total islets in the organ, revealed a greater than 2-fold reduction in absolute islet number in PITP/ pancreas relative to wild type (Fig. 10A). Ablation of PITP Function in Mice We found that a 10-h fast evokes Ablation of PITP Function in Mice Ablation of PITP Function in Mice 33513 TABLE III Acyl chain compositions of bulk liver lipids Fatty acid compositions of the lipid classes analyzed from pooled liver tissue samples depicted in Fig. 7 were analyzed. See the legend to Table II for essential details. Acyl species CE TG DAG FFA SM CL 14:0 3.3/2.8 4.5/3.4 5.3/5.0 4.2/3.5 2.2/2.0 1.0/1.2 14:1n5 0.1/0.0 0.1/0.1 0.1/0.1 0.3/0.0 0.2/0.1 0.0/0.0 15:0 0.5/0.2 0.2/0.1 0.4/0.2 0.4/0.3 0.5/0.4 0.4/0.6 dm16:0 0.6/0.5 0.0/0.0 0.1/0.0 0.0/0.0 0.1/0.1 0.9/0.9 16:0 38.3/35.9 28.4/28.4 27.8/24.3 35.4/37.6 19.3/20.0 17.5/15.2 t16:1n7 1.0/0.5 0.4/0.5 1.0/0.8 1.4/1.2 1.0/0.5 0.6/0.6 16:1n7 5.3/5.8 2.1/1.8 1.6/1.6 1.7/1.2 1.7/1.4 0.7/0.9 dm18:0 0.0/0.1 0.1/0.1 0.0/0.0 0.1/0.1 0.0/0.0 0.3/0.3 dm18:1n9 0.8/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.1 0.1/0.1 dm18:1n7 0.1/0.4 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.1/0.1 18:0 9.5/5.8 4.1/3.3 9.0/6.2 9.4/10.5 22.5/22.3 15.9/14.5 t18:1n9 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.6/0.5 0.0/0.0 18:1n9 17.3/26.3 24.2/28.0 16.5/22.5 15.4/22.6 23.0/20.6 3.1/4.2 18:1n7 1.0/1.4 2.1/2.7 1.8/2.2 1.8/2.8 1.5/1.5 3.1/3.9 t18:2n6 0.1/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.1/0.2 0.0/0.0 18:2n6 11.5/11.8 21.8/23.4 18.9/23.2 15.8/12.1 5.0/4.5 29.5/36.9 18:3n6 0.3/0.3 0.4/0.4 0.4/0.5 0.2/0.1 0.1/0.1 0.1/0.1 18:3n3 0.7/0.6 0.6/0.4 0.8/0.7 0.5/0.1 0.5/0.4 0.5/0.4 18:4n3 0.0/0.0 0.1/0.1 0.0/0.1 0.0/0.0 0.0/0.0 0.0/0.0 20:0 0.2/0.1 0.1/0.0 0.3/0.2 0.2/0.2 0.3/0.5 0.1/0.2 20:1n15 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 20:1n12 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 20:1n9 0.3/0.2 0.6/0.5 0.2/0.4 0.3/0.6 0.4/0.3 0.1/0.3 20:3n9 0.0/0.0 0.2/0.2 0.3/0.3 0.2/0.4 0.0/0.0 0.0/0.1 20:2n6 0.4/0.2 0.7/0.6 0.7/0.6 0.9/1.0 0.2/0.2 6.6/3.6 20:3n6 0.8/0.6 1.0/0.7 1.0/1.0 1.2/0.7 0.8/0.9 0.6/0.6 20:4n6 3.2/2.7 2.4/1.5 4.5/3.7 7.3/4.0 9.8/11.2 5.6/5.2 20:3n3 0.0/0.0 0.0/0.0 0.1/0.1 0.1/0.0 0.1/0.1 0.1/0.1 20:4n3 0.2/0.0 0.1/0.0 0.1/0.1 0.1/0.0 0.0/0.0 0.1/0.1 20:5n3 0.4/0.3 0.3/0.2 0.4/0.3 0.5/0.1 0.4/0.4 0.3/0.2 22:0 0.3/0.3 0.0/0.0 0.1/0.0 0.0/0.0 0.6/0.4 0.1/0.1 22:1n9 1.3/0.9 0.2/0.1 0.0/0.1 0.1/0.2 0.4/0.2 0.6/0.4 22:2n6 0.0/0.0 0.1/0.0 0.0/0.2 0.1/0.1 0.0/0.0 0.0/0.0 22:4n6 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 22:5n6 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 22:5n3 0.2/0.1 1.0/0.4 1.0/0.7 0.0/0.4 0.7/0.7 0.3/0.3 22:6n3 1.8/1.5 4.1/1.7 7.6/5.1 1.6/0.2 6.4/7.2 11.4/8.7 24:0 0.6/0.5 0.1/0.1 0.0/0.0 0.3/0.1 0.3/0.6 0.1/0.1 24:1n9 0.1/0.0 0.0/1.0 0.0/0.0 0.0/0.0 1.3/2.6 0.1/0.1 24:6n3 0.0/0.0 0.2/0.2 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 Ablation of PITP Function in Mice 33513 TABLE III Acyl chain compositions of bulk liver lipids ous structural defects in that pancreatic islets are not at all prominent. Ablation of PITP Function in Mice PITP/ and PITP/ data are in solid and hatched bars, respectively. PITP Deficiency and Steatosis—The most energy-rich com- ponent of the neonatal diet is TG, and the evidence suggests PITP/ mice are unable to effectively avail themselves of this nutrient source. In mammals, long chain TGs of maternal milk are hydrolyzed to monoacylglycerols and fatty acids. These compounds are transported across the apical enterocyte plasma membrane and reassembled into TG cores in the smooth ER lumen. The TG cores are then co-assembled with other lipids, fat-soluble vitamins, and apolipoproteins into chy- lomicrons either in the ER or in the Golgi complex. Subse- quently, chylomicrons are transported across the enterocyte basolateral plasma membrane via the secretory pathway (37). Enterocytes and hepatocytes share analogous pathways for assembly and secretion of lipoproteins into the circulation. The suckling-dependent microvesicular steatosis of PITP/ in- testine and liver suggests that PITP plays a common role in both intestinal and hepatic pathways for secretion of dietary lipids. A failure in transport early in the secretory pathway is indicated as electron microscopy suggests neutral lipids accu- mulate in smooth ER of PITP nullizygous enterocytes and in hepatocytes. FIG. 8. Tissue-specific energy deficits in PITP/ mice. A, tis- sue ATP levels in PITP/ mice. Measurements were averaged for three determinations from each of three independent P4-P6 PITP/ (solid bars) and PITP/ animals (hatched bars). B, ATP/ADP ratios for the indicated tissues. ADP was measured indirectly by conversion to ATP and measurement of ADP-derived ATP. No significant differences in ATP or ATP/ADP ratio were observed between measurements of cerebellar hemispheres, or selected brain or liver regions, and those of whole-organ homogenates. PITP/ and PITP/ data are in solid and hatched bars, respectively. The intestinal steatosis of PITP/ mice exhibits similari- ties with human CRD, a family of autosomal recessive disor- ders. Human CRD patients are characterized by pediatric failure to thrive, accumulation of chylomicrons within villus- associated enterocytes of the duodenum, reductions in post- prandial plasma TG and -tocopherol levels, and spinocerebel- lar involvement (18, 19, 32, 38). Similarly, PITP/ mice gain body mass slowly and are deficient in body fat. Moreover, villus-associated PITP/ enterocytes accumulate intracellu- lar lipid in a suckling-dependent manner. Finally, PITP/ mice exhibit large reductions in both post-prandial plasma TG and in brain -tocopherol. Ablation of PITP Function in Mice These data suggest PITP/ mice hydrolyze dietary TG in the lumen of the duodenum, transport the products into the enterocyte, and properly reassemble die- tary TG in the ER lumen but are defective in transport of re-esterified TGs across the PITP/ enterocyte to the baso- lateral plasma membrane. The microvesicular hepatic steato- sis we observe in PITP/ mice is not a symptom associated with human CRD, however. PITP/ MEFs and ES cells with the UPR-activating agent tunicamycin leads to a differential response in expression of the ER stress-induced transcription factor CHOP and the chap- erone protein calnexin. Expression of these two polypeptides is potently enhanced by induction of the UPR. Thus, CHOP and calnexin serve as reliable reporters of the UPR status in cells. As shown in Fig. 10E, CHOP is normally expressed at very low levels in PITP/ and PITP/ MEFs, but challenge with tunicamycin evokes a robust induction of CHOP expression in MEFs of either genotype. Similarly, calnexin levels increase in both PITP/ and PITP/ MEFs upon tunicamycin chal- lenge (Fig. 10E). Congruent results are recorded with PITP/ and PITP/ ES cells (not shown). We conclude PITP/ cells are not only competent for induction of UPR but that the nullizygous cells are not subject to constitutive activation of the UPR. Ablation of PITP Function in Mice The ER abnormalities in PITP-deficient enterocytes, hepatocytes, and neurons, when coupled with loss of functional pancreatic islets in mutant mice, suggested the possibility that PITP/ cells are intrinsically defective in UPR. First, we compared the response of PITP/ and PITP/ Finally, we assessed whether challenge of PITP/ and First, we compared the response of PITP/ and PITP/ FIG. 8. Tissue-specific energy deficits in PITP/ mice. A, tis- sue ATP levels in PITP/ mice. Measurements were averaged for three determinations from each of three independent P4-P6 PITP/ (solid bars) and PITP/ animals (hatched bars). B, ATP/ADP ratios for the indicated tissues. ADP was measured indirectly by conversion to ATP and measurement of ADP-derived ATP. No significant differences in ATP or ATP/ADP ratio were observed between measurements of cerebellar hemispheres, or selected brain or liver regions, and those of whole-organ homogenates. PITP/ and PITP/ data are in solid and hatched bars, respectively. Ablation of PITP 33514 Ablation of PITP Function in Mice 33514 Ablation of PITP 33514 homeostasis in the liver. Finally, PITP/ mice are severely hypoglycemic. The hypoglycemia is associated with reduced proglucagon gene expression, compromise of pancreatic islet integrity, and defects in liver glycogen metabolism. These find- ings suggest unanticipated roles for PITP in signaling path- ways that interface with trafficking of luminal lipid cargo from the ER and glucose homeostasis in mammals. PITP and Neonatal Survival—The development of PITP/ mice to term supports our finding that PITP-defi- cient ES cells maintain pluripotency and competence for mem- brane trafficking and growth factor signaling (17). Rather, the neonatal mortality of PITP/ mice indicates a role for PITP in regulating physiological functions that are critical in the context of the whole mouse. We conclude PITP nullizygosity manifests itself primarily upon extinction (by birth) of the nutritional resource provided by maternal circulation in utero, and reliance of the neonate on assimilation of its own dietary intake. / FIG. 8. Tissue-specific energy deficits in PITP/ mice. A, tis- sue ATP levels in PITP/ mice. Measurements were averaged for three determinations from each of three independent P4-P6 PITP/ (solid bars) and PITP/ animals (hatched bars). B, ATP/ADP ratios for the indicated tissues. ADP was measured indirectly by conversion to ATP and measurement of ADP-derived ATP. No significant differences in ATP or ATP/ADP ratio were observed between measurements of cerebellar hemispheres, or selected brain or liver regions, and those of whole-organ homogenates. DISCUSSION Age-matched PITP/ (G) and PITP/ () mice with stomachs full of milk were fasted as indicated, sacrificed, and liver glycogen measured. Mean glycogen values are given for PITP/ and PITP/ liver as solid and open bars, respectively, and statistical significance of the mean differences is given above each data set. Ablation of PITP Function in Mice 33515 Ablation of PITP Function in Mice 33515 FIG. 9. Hypoglycemia in PITP/ mice. A, serum glucose, insulin, and -hydroxybutyrate levels. Serum was collected from age-matched PITP/ (closed bars) and PITP/ (hatched bars) mice. Glucose data are averaged from six age-matched PITP/ and PITP/ mice of each genotype. Serum insulin and -hydroxybutyrate values represent averages of triplicate measurements obtained from three mice of each genotype. Values from fasted PITP/ mice (18 h) are indicated by F. B, Glc-6-Pase, phosphoenolpyruvate carboxykinase (PEPCK), and -actin mRNA levels in PITP/ and PITP/ liver, and proglucagon and -actin mRNA levels in pancreas. Data are representative of at least three independent mRNA extractions and hybridizations RNA load was normalized to 10 g total mRNA per lane and the actin data serve as indicators of FIG. 9. Hypoglycemia in PITP/ mice. A, serum glucose, insulin, and -hydroxybutyrate levels. Serum was collected from age-matched PITP/ (closed bars) and PITP/ (hatched bars) mice. Glucose data are averaged from six age-matched PITP/ and PITP/ mice of each genotype. Serum insulin and -hydroxybutyrate values represent averages of triplicate measurements obtained from three mice of each genotype. Values from fasted PITP/ mice (18 h) are indicated by F. B, Glc-6-Pase, phosphoenolpyruvate carboxykinase (PEPCK), and -actin mRNA levels in PITP/ and PITP/ liver, and proglucagon and -actin mRNA levels in pancreas. Data are representative of at least three independent mRNA extractions and hybridizations. RNA load was normalized to 10 g total mRNA per lane, and the -actin data serve as indicators of normalization quality. C, glycogen was quantified for non-fasted and fasted PITP/ and PITP/ liver, as indicated. D, kinetics of liver glycogen depletion. Age-matched PITP/ (G) and PITP/ () mice with stomachs full of milk were fasted as indicated, sacrificed, and liver glycogen measured. Mean glycogen values are given for PITP/ and PITP/ liver as solid and open bars, respectively, and statistical significance of the mean differences is given above each data set. volvement of a nuclear PITP pool in regulating proglucagon promoter function. DISCUSSION Loss of PITP activity in mice manifests itself in spinocere- bellar disease characterized by reactive gliosis of the cerebel- lum and brain stem and WM and GM deficits in spinal cord. The spinal cord pathologies include infiltration by inflamma- tory cells, aponecrosis of motor neuron cell bodies in the ventral horn, and pervasive demyelination that is particularly appar- ent in the dorsal columns. PITP is also required for proper absorption of dietary fat and fat-soluble vitamins through du- odenal enterocytes into the circulation and for proper lipid PITP Deficiency and Glucose Homeostasis—PITP/ neo- nates are hypoglycemic, even though key gluconeogenic en- zymes appear to be expressed normally. However, gluconeogen- esis is an energetically expensive process as 4ATP, 2GTP, 2NADH, and 4H2O are consumed in conversion of 2 pyruvate units to 1 glucose molecule (39). Fatty acid oxidation is a potent engine that drives production of the ATP, GTP, and NADH utilized in glucose synthesis and is also involved in production PITP Deficiency and Glucose Homeostasis—PITP/ neo- nates are hypoglycemic, even though key gluconeogenic en- zymes appear to be expressed normally. However, gluconeogen- esis is an energetically expensive process as 4ATP, 2GTP, 2NADH, and 4H2O are consumed in conversion of 2 pyruvate units to 1 glucose molecule (39). Fatty acid oxidation is a potent engine that drives production of the ATP, GTP, and NADH utilized in glucose synthesis and is also involved in production of ketone bodies that serve as energy sources under conditions volvement of a nuclear PITP pool in regulating proglucagon FIG. 9. Hypoglycemia in PITP/ mice. A, serum glucose, insulin, and -hydroxybutyrate levels. Serum was collected from age-matched PITP/ (closed bars) and PITP/ (hatched bars) mice. Glucose data are averaged from six age-matched PITP/ and PITP/ mice of each genotype. Serum insulin and -hydroxybutyrate values represent averages of triplicate measurements obtained from three mice of each genotype. Values from fasted PITP/ mice (18 h) are indicated by F. B, Glc-6-Pase, phosphoenolpyruvate carboxykinase (PEPCK), and -actin mRNA levels in PITP/ and PITP/ liver, and proglucagon and -actin mRNA levels in pancreas. Data are representative of at least three independent mRNA extractions and hybridizations. RNA load was normalized to 10 g total mRNA per lane, and the -actin data serve as indicators of normalization quality. C, glycogen was quantified for non-fasted and fasted PITP/ and PITP/ liver, as indicated. D, kinetics of liver glycogen depletion. DISCUSSION Rather, we hypothesize that glucagon defi- cit in PITP/ mice more likely represents an indirect man- ifestation of the structurally compromised endocrine pancreas that characterizes such mutants. Yet, because the glycogen stores of PITP/ liver are consumed during a modest fast, we conclude that regulation of glucose allocation into the gly- cogen synthesis pathway must also be deranged in PITP/ mice. of ketone bodies that serve as energy sources under conditions of low glucose availability. We suggest that the inability to effectively deliver lipid from enterocytes and hepatocytes to peripheral tissues imposes a heavy metabolic burden on PITP/ neonates by compromising fatty acid oxidation as a pathway for powering both gluconeogenesis and ketone produc- tion. Reductions in PITP/ liver ATP are consistent with this interpretation of the data. In addition, PITP/ animals exhibit kinetic defects in glycogenolysis, and these maintain substantial liver glycogen in the face of hypoglycemia. These defects coincide with dra- matic reductions in proglucagon gene expression in PITP/ pancreas. Presently, we do not believe that dysregulation of proglucagon expression reflects a direct transcriptional in- PITP Deficiency and Integrity of the Spinocerebellar Sys- tem—PITP/ neonates develop a spinocerebellar neurode- generative disease characterized by aponecrotic cell death. In this regard, we find a correlation between site of neuronal injury and regional specificity of dramatic reduction in cellular FIG. 10. Pancreatic islet cells and the unfolded protein response in PITP/ mice. A, number of pancreatic islets in whole pancreas fro PITP/ and PITP/ mice. Whole pancreata were recovered from each of two PITP/ and PITP/ mice. The individual PITP pancreata were serially sectioned into 48 and 60 consecutive sections, respectively, whereas both PITP/ pancreata were each sectioned into serial sections. Islets were then comprehensively counted for every section of each individual pancreas so that the total number of islets per org was determined. The data represent the averages of the two reconstructions for PITP/ and PITP/ pancreas (solid and open ba respectively). B, histology of endocrine pancreas. Images from hematoxylin/eosin-stained sections of PITP/ and PITP/ pancreas. Bars a 50 m. C, viability of PITP/ (solid bars) and PITP/ (hatched bars) ES cells and MEFs as a function of period of glucose starvation indicated. Living cells were identified by a trypan blue exclusion assay. D, ER stress-induced inhibition of protein synthesis. DISCUSSION MEFs of appropria genotype (PITP/, solid bars; PITP/, hatched bars) were incubated in the presence or absence of 10 mM dithiothreitol (DTT; 15 min) or 1 thapsigargin (Tg; 30 min) prior to and during a 20-min pulse-radiolabeling with [35S]methionine (100 Ci/ml). Relative incorporation of radiolab into protein was quantified as trichloroacetic acid-precipitable radioactivity and is presented as an average percentage of that measured mock-treated control cells (100%). Values are derived from triplicate determinations from three independent experiments. PITP/ a PITP/ MEFs exhibit essentially indistinguishable rates of incorporation of [35S]methionine into protein. E, CHOP and calnexin expressio Cell-free extracts were prepared from PITP/ and PITP/ MEFs after an 18-h incubation with 0, 10, 20, and 50 g/ml tunicamycin bottom). Equivalent amounts of protein were loaded for each sample (20 g) and resolved by SDS-PAGE. Proteins were transferred to nitroc lulose, and the resulting blot was probed with anti-CHOP and anti-calnexin antibodies (Santa Cruz Biotechnology) and developed by enhanc chemiluminescence Ablation of PITP Function in Mice 33516 Ablation of PITP Function in Mice 33516 33516 FIG. 10. Pancreatic islet cells and the unfolded protein response in PITP/ mice. A, number of pancreatic islets in whole pancreas from PITP/ and PITP/ mice. Whole pancreata were recovered from each of two PITP/ and PITP/ mice. The individual PITP/ pancreata were serially sectioned into 48 and 60 consecutive sections, respectively, whereas both PITP/ pancreata were each sectioned into 48 serial sections. Islets were then comprehensively counted for every section of each individual pancreas so that the total number of islets per organ was determined. The data represent the averages of the two reconstructions for PITP/ and PITP/ pancreas (solid and open bars, respectively). B, histology of endocrine pancreas. Images from hematoxylin/eosin-stained sections of PITP/ and PITP/ pancreas. Bars are 50 m. C, viability of PITP/ (solid bars) and PITP/ (hatched bars) ES cells and MEFs as a function of period of glucose starvation is indicated. Living cells were identified by a trypan blue exclusion assay. D, ER stress-induced inhibition of protein synthesis. MEFs of appropriate genotype (PITP/, solid bars; PITP/, hatched bars) were incubated in the presence or absence of 10 mM dithiothreitol (DTT; 15 min) or 1 M thapsigargin (Tg; 30 min) prior to and during a 20-min pulse-radiolabeling with [35S]methionine (100 Ci/ml). DISCUSSION We are particularly grateful to Gail Grossman for expert assistance in histochemistry; Hal Mekeel for helpful advice on electron microscopy; Patricia Gallagher for expert assistance in -to- copherol measurements (University of North Carolina Clinical Nutri- tion Research Center supported by National Institutes of Health Grant DK56350); David Brenner and Joe Grisham for expert advice on liver histopathology; Lisa M. Bain for brain histology; and Heather Collins and Bryan Wolf (University of Pennsylvania Diabetes Center) for glu- cagon and corticosteroid measurements. Support for body composition assays were provided by grants from the National Institutes of Health to the University of Alabama at Birmingham Clinical Nutrition Research Center. Acknowledgments—We acknowledge Douglas Cyr, Andrew Morris, Patrick Brennwald, Peter Petrusz, John York, Tushar Patel, William Snider, Virginia Godfrey, Scott Traske, and Rosalind Coleman for help- ful discussions. We are particularly grateful to Gail Grossman for expert assistance in histochemistry; Hal Mekeel for helpful advice on electron microscopy; Patricia Gallagher for expert assistance in -to- copherol measurements (University of North Carolina Clinical Nutri- tion Research Center supported by National Institutes of Health Grant DK56350); David Brenner and Joe Grisham for expert advice on liver histopathology; Lisa M. Bain for brain histology; and Heather Collins and Bryan Wolf (University of Pennsylvania Diabetes Center) for glu- cagon and corticosteroid measurements. Support for body composition assays were provided by grants from the National Institutes of Health to the University of Alabama at Birmingham Clinical Nutrition Research Center. With regard to dysregulation of cellular homeostasis in PITP nullizygous mice, we find that derangement of ER func- tion is a common theme. 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A., Cavalcanti, F., Crescibene, L., Bagala, A., Bono, F., Oliveri, R. L., Valentino, P., Zappia, M., and Quattrone, A. (2000) Ann. Neurol. 47, 260–264 , 20. Zambrowicz, B. P., Friedrich, G. A., Buxton, E. C., Lilleberg, S. L., Person, C., and Sands, A. T. (1998) Nature 392, 608–611 21. Nagy, T. R., Gower, B. A., and Stetson, M. H. (1994) Can. J. Zool. 72, 1726–1734 22. Luna, L. (1968) Manual of Histological Staining Methods of the Armed Forces Institute of Pathology, pp. 143–145, Armed Forces Institute of Pathlogy, Washington, DC g , 23. Mascorro, J. DISCUSSION Alternatively, PITP may play unforeseen ER signaling functions that do not require PITP-mediated trans- fer of lipid between organelles. DISCUSSION Relative incorporation of radiolabel into protein was quantified as trichloroacetic acid-precipitable radioactivity and is presented as an average percentage of that measured for mock-treated control cells (100%). Values are derived from triplicate determinations from three independent experiments. PITP/ and PITP/ MEFs exhibit essentially indistinguishable rates of incorporation of [35S]methionine into protein. E, CHOP and calnexin expression. Cell-free extracts were prepared from PITP/ and PITP/ MEFs after an 18-h incubation with 0, 10, 20, and 50 g/ml tunicamycin (at bottom). Equivalent amounts of protein were loaded for each sample (20 g) and resolved by SDS-PAGE. Proteins were transferred to nitrocel- lulose, and the resulting blot was probed with anti-CHOP and anti-calnexin antibodies (Santa Cruz Biotechnology) and developed by enhanced chemiluminescence. ATP. Neonatal cerebellum may be particularly sensitive to injury in PITP/ mice because it attempts to sustain the extraordinary rates of cell proliferation that accompany the postnatal development of this organ in the face of a challenging energetic environment. Similarly, Purkinje cells and motor neurons are very large and metabolically active cells that are sensitive to a variety of insults. We suggest PITP/ Purkinje cell and motor neuron defects also represent manifestations of energy deficit in these cells, thereby raising the possibility that a significant component of the spinocerebellar disorder arises from the intrinsic hypoglycemia and/or the intestinal and he- patic steatosis of PITP/ mice. Cellular Mechanisms for PITP Function in the Mouse?— The lines of evidence linking PITP to potentiation of PIP Ablation of PITP Function in Mice 33517 GTPase-activating proteins required for biogenesis of secretory cargo-laden transport vesicles from trans-Golgi membranes in yeast (6, 42). synthesis suggest that the tissue defects of PITP/ mice result from deranged PIP metabolism. A demonstration that this is indeed the case remains elusive as such derangements are not apparent in bulk PtdIns, PtdIns-3-phosphate, PtdIns- 4-phosphate, or PtdIns-4,5-bisphosphate levels in PITP/ ES cells (17) or MEFs (this study, not shown). Our data indi- cate that PITP does not play a critical role in regulating dynamics of cell-essential phospholipid pools in most cell types. Acknowledgments—We acknowledge Douglas Cyr, Andrew Morris, Patrick Brennwald, Peter Petrusz, John York, Tushar Patel, William Snider, Virginia Godfrey, Scott Traske, and Rosalind Coleman for help- ful discussions. REFERENCES A., and Kirby, G. S. (1986) Proceedings of the 44th Annual Meeting of the Electron Microscopy Society of America, pp. 222–223 f py y f , 24. Sato, T. (1968) J. Electron Microsc. 17, 158–159 25. Jishage, K., Arita, M., Igarashi, K., Iwata, T., Watanabe, M., Ogawa, M., Ueda, O., Kamada, N., Inoue, K., Arai, H., and Suzuki, H. (2001) J. Biol. Chem. 276, 1669–1672 Is there independent supporting evidence for such a pro- posal? It was recently demonstrated that inactivation of the SARA2 isoform of the Sar1p GTPase forms a genetic basis for human CRD (40). Sar1p is required for the budding of COPII vesicles from ER membranes and for the concentration of cargo into such transport vesicles (41). Our data, when coupled with the demonstrations of a role for Sar1p in human CRD, suggest the attractive hypothesis that PITP regulates the Sar1p GTPase cycle in ER membranes. We propose that a dedicated PITP/Sar1p interface is required to optimize assembly and packaging of unusual luminal cargoes (such as large TG cores) into ER-derived transport vesicles. This model shares common features with previous proposals positing that the Sec14p PITP regulates the activity of specific ADP-ribosylation factor , 26. Passonneau, J., and Lauderdale, V. (1974) Anal. Biochem. 60, 405–412 27. Formigli, L., Papucci, L., Tani, A., Schiavone, N., Tempestini, A., Orlandini, G. E., Capaccioli, S., and Zecchi Orlandini, S. (2000) J. Cell. Physiol. 182, 41–49 28. Sperandio, S., de Belle, I., and Bredesen, D. E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 14376–14381 , 29. Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1984) Prog. Allergy 34, 1–141 29. Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1984) Prog. Allergy 34, 1–141 30. Bebo, B. F., Jr., Yong, T., Orr, E. L., and Linthicum, D. S. (1996) J. Neurosci. Res. 45, 340–348 30. Bebo, B. F., Jr., Yong, T., Orr, E. L., and Linthicum, D. S. (1996) J. Neurosci. Res. 45, 340–348 31. Pender, M. P., Stanley, G. P., Yoong, G., and Nguyen, K. B. (1990) Acta Neuropathol. 80, 172–183 32. Roy, C. C., Levy, E., Green, P. H. R., Sniderman, A., Letarte, J., Buts, J.-P., Orquin, J., Brochu, P., Weber, A. M., Morin, C. L., Marcel, Y., and Deckelbaum, R. J. (1987) Gastroenterology 92, 390–399 Orquin, J., Brochu, P., Weber, A. M., Morin, C. L., Marcel, Y., and Deckelbaum, R. J. , , , , 42. Li, X., Rivas, M. P., Fang, M., Marchena, J., Mehrotra, B., Chaudhary, A., Feng, L., Prestwich, G. D., and Bankaitis, V. A. (2002). J. Cell Biol. 157, 63–77 REFERENCES (1987) Gastroenterology 92, 390–399 33. Hanson, R. W., and Reshef, L. (1997) Annu. Rev. Biochem. 66, 581–611 34. Harding, H. P., Zeng, H., Zhang, Y., Jungries, R., Chung, P., Plesken, H., Sabatini, D. D., and Ron, D. (1983) Mol. Cell 7, 1153–1163 35. Scheuner, D., Song, B., McEwen, E., Liu, C., Laybutt, R., Gillespie, P., Saunders, T., Bonner-Weir, S., and Kaufman, R. J. (2001) Mol. Cell 7, 1165–1176 39. Nelson, D. L., and Cox, M. M. (2000) Lehninger Principles of Biochemistry, 3rd Ed., p. 729, Worth Publishers, New York , p , , 40. Jones, B., Jones, E. L., Bonney, S. A., et al. (2003) Nat. Genet. 34, 29–31 p y , 38. Gauthier, S., and Sniderman, A. (1983) Ann. Neurol. 14, 591 37. Hussain, M. M., Kanch, R. K., Zhou, Z., Luchmoon, J., Zu, H., and Bakillah, A. 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https://openalex.org/W2508222713	https://researchportal.bath.ac.uk/files/153878847/npj_Yates.pdf	English	null	Using approximate Bayesian computation to quantify cell-cell adhesion parameters in a cell migratory process	bioRxiv (Cold Spring Harbor Laboratory)	2,016	cc-by	11,978	DOI: 10.1038/s41540-017-0010-7 DOI: 10.1038/s41540-017-0010-7 DOI: 10.1038/s41540-017-0010-7 Publication date: 2017 Publication date: 2017 Publisher Rights CC BY University of Bath General rights General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Citation for published version: Ross, R, Baker, RE, Parker, A, Ford, M, Mort, R & Yates, C 2017, 'Using approximate Bayesian computation to quantify cell-cell adhesion parameters in a cell migratory process', npj Systems Biology and Applications, vol. 3, 9. https://doi.org/10.1038/s41540-017-0010-7 Alternative formats If you require this document in an alternative format, please contact: openaccess@bath.ac.uk General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version: Ross, R, Baker, RE, Parker, A, Ford, M, Mort, R & Yates, C 2017, 'Using approximate Bayesian computation to quantify cell-cell adhesion parameters in a cell migratory process', npj Systems Biology and Applications, vol. 3, 9. https://doi.org/10.1038/s41540-017-0010-7 INTRODUCTION representing biological processes, such as cell–cell interactions and volume exclusion, to be easily assigned to agents in the ABM. Cell–cell interactions are known to play an important role in several cell migration processes. For example, multiple different cell–cell interactions, such as cell–cell signalling and cell–cell adhesion,1 have been identiﬁed as promoting metastasis in breast cancer. Repulsive interactions mediated via ephrins on the surface of neural crest stem cells are known to coordinate the early stages of melanoblast migration away from the neural tube.2 More fundamentally, it is hypothesised that the emergence of cell–cell interactions over one billion years ago helped establish the necessary conditions for multicellular organisms.3 If an ABM is an effective (By an effective representation, we mean the ABM captures the salient features of the process of interest, and is, therefore, a viable research tool with which to study the process of interest.) representation of a cell migration process it can be used for a number of purposes. One such purpose for an ABM is to perform in silico experiments to test scientiﬁc hypotheses. For instance, a recent study used an ABM to demonstrate that a simple mechanism of undirected cell move- ment and proliferation could account for neural crest stem cell colonisation of the developing epidermis in the embryonic mouse.4 Other studies involving ABMs have tested hypotheses concerning the inﬂuence of matrix stiffness and matrix architec- ture on cell migration,17 and the mechanism by which cranial neural crest stem cells become ‘leaders’ or ‘followers’ in the embryonic chick to facilitate their collective migration.6–8 y g A well-established approach for studying cell migration is to construct an agent-based model (ABM) to simulate the cell migratory process of interest.4–8 Typically, this involves using a computational model to simulate a population of agents on a two- dimensional surface, or in a three-dimensional volume. The agents in the ABM represent cells, and each agent is able to move and interact with other agents in the ABM. In this work, we use an ABM to simulate a wound-healing assay (Wound-healing assays are also often referred to as scratch assays.), an experiment commonly used for studying cell motility.9–15 Other modelling approaches apart from ABMs have been employed to study wound-healing. For instance, a huge amount of research has been completed using continuum methods to model the wound-healing process (see Flegg et al.16 for a recent review of the ﬁeld). Download date: 24. Oct. 2024 www.nature.com/npjsba 1Wolfson Centre for Mathematical Biology, Mathematical Institute, University of Oxford, Radcliffe Observatory Quarter, Woodstock Road, Oxford OX2 6GG, UK; 2MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, UK; 3Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Furness Building, Lancaster University, Bailrigg, Lancaster LA1 4YG, UK and 4Department of Mathematical Sciences, Centre for Mathematical Biology, University of Bath, Claverton Down, Bath BA2 7AY, UK Correspondence: Robert J H Ross (ross@maths ox ac uk) ARTICLE OPEN Using approximate Bayesian computation to quantify cell–cell adhesion parameters in a cell migratory process Robert J. H. Ross1, R. E. Baker1, Andrew Parker1, M. J. Ford2, R. L. Mort3 and C. A. Yates4 In this work, we implement approximate Bayesian computational methods to improve the design of a wound-healing assay used to quantify cell–cell interactions. This is important as cell–cell interactions, such as adhesion and repulsion, have been shown to play a role in cell migration. Initially, we demonstrate with a model of an unrealistic experiment that we are able to identify model parameters that describe agent motility and adhesion, given we choose appropriate summary statistics for our model data. Following this, we replace our model of an unrealistic experiment with a model representative of a practically realisable experiment. We demonstrate that, given the current (and commonly used) experimental set-up, our model parameters cannot be accurately identiﬁed using approximate Bayesian computation methods. We compare new experimental designs through simulation, and show more accurate identiﬁcation of model parameters is possible by expanding the size of the domain upon which the experiment is performed, as opposed to increasing the number of experimental replicates. The results presented in this work, therefore, describe time and cost-saving alterations for a commonly performed experiment for identifying cell motility parameters. Moreover, this work will be of interest to those concerned with performing experiments that allow for the accurate identiﬁcation of parameters governing cell migratory processes, especially cell migratory processes in which cell–cell adhesion or repulsion are known to play a signiﬁcant role. npj Systems Biology and Applications (2017) 3:9 ; doi:10.1038/s41540-017-0010-7 npj Systems Biology and Applications (2017) 3:9 ; doi:10.1038/s41540-017-0010-7 Received: 8 August 2016 Revised: 23 November 2016 Accepted: 10 January 2017 Published in partnership with the Systems Biology Institute RESULTS We choose these times as they are the times (in minutes) we will later analyse for the simulations of the practically realisable experiment, and correspond to 4, 8 and 12 h into an experiment. 3. We repeat step 2 ten times and calculate the ensemble average for each summary statistic for each individual time- point. Other methods to identify parameters from experimental data using ABMs also exist. For instance, a standard approach is to generate point estimates of model parameters that best reproduce statistics of the experimental data in the ABM. For example, the generation of motility and proliferation rates for agents in an ABM representing a biological process.4 This approach, while applicable in some circumstances, often gives little insight into how much uncertainty exists in the parameters chosen, a factor that can be of importance when analysing biological systems. For example, relationships between parameter uncertainty and system robustness are thought to be connected in biological function at a systems level.25 This procedure generates synthetic data for which we will now attempt to identify the parameters. In this work, we present representative results using Pm = 0.5 and α = 0.1 for model A, and Pm = 0.5 and α = 0.25, and Pm = 0.5 and α = −0.1 for model B. Throughout this work, we sample Pm and α for our model from uniform priors. In the case of model A, Pm ∈[0, 1] and α ∈ [−0.2, 0.25], and for model B, Pm ∈[0, 1] and α ∈[−0.2, 1.0]. We stipulate these lower and upper bounds for α for both models A and B to make sure inequalities (2) and (4) are satisﬁed. q We begin by implementing an ABC rejection algorithm that proceeds as follows: 4 g y The outline of this work is as follows: in the Methods Section we introduce our ABM and deﬁne the cell–cell interactions we implement. We also outline the method of ABC, and the summary statistics we use to analyse the ABM output. In the Results Section we present results and demonstrate that, given an ABM representing an unrealistic experiment, we are able to identify ABM parameters for agent motility and adhesion. Following this, we replace our ABM representing an unrealistic experiment with an ABM that simulates a practically realisable experiment. RESULTS We begin by demonstrating that for an ABM representing an unrealistic experiment we are able to identify model parameters, given appropriate summary statistics. Unrealistic experiment To ascertain the effectiveness of the chosen summary statistics to identify model parameters, we attempt to identify Θ from data generated synthetically. Synthetic data is ABM data generated with ﬁxed parameter values, and so can be thought of as a simulation equivalent of experimental data. To generate the synthetic data using the ABM we proceed as follows: 1. We choose parameters Θ to identify. To help clarify this explanation let us make these parameters Θ = (Pm, α) = (0.5,0.1) in model A (A value of Pm = 0.5, given that the simulation time will later be deﬁned to be in minutes, and the length of a lattice site represents cell length (typically between 10–100 μm, means that the motility of the agents is biologically realistic. The parameter α is dimensionless. The experimental realism of these parameters will be expanded on when we address the simulation of a practically realisable experiment.). p p The focus of our study is to determine the experimental conditions, and experimental data, required for the accurate identiﬁcation of cell motility and adhesion parameters in an ABM of a wound-healing assay. To do so, we employ approximate Bayesian computation (ABC), a probabilistic approach whereby a probability distribution for the parameter(s) of interest is estimated, as opposed to a point estimate.10, 19, 20 Although ABC is well-established in some ﬁelds, for instance in population genetics,21 its applicability for ABMs representing cell migration is still an area of active research.9–11, 22–24 Recent studies combining ABC and ABMs have been able to identify motility and proliferation rates in cell migratory processes,10 and improve the experimental design of scratch assays.11 However, as far as we are aware no study to date has used ABC methods to examine the experimental conditions, and experimental data, required for the accurate identiﬁcation of cell motility and adhesion parameters in a wound-healing assay. 2. p ) 2. For model A we perform a simulation of the ABM with Θ = (0.5,0.1), generate data, D, and calculate summary statistics, S(D), from the simulation at our time-points of interest. These times are t = [240,480,720]. INTRODUCTION However, we employ an ABM in this work because they provide an intuitive representation of cells, and allow for complex behaviours y g ABMs can also be used to identify parameters in experimental data (with the caveat that the parameters are model-dependent). The reasoning behind using an ABM to identify parameters in experimental data is as follows: if an ABM is an effective representation of an experiment, then the parameter values the ABM requires to reproduce the experimental data may be representative of the parameter values in the biological process that is the focus of the experiment. For instance, the value of a parameter that describes cell proliferation rate. Even if the parameter values in the parameterised ABM are not representative Published in partnership with the Systems Biology Institute Published in partnership with the Systems Biology Institute Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al 2 the ﬁeld of view of the microscope used to collect the experimental data. For instance, generating ﬁve experimental replicates on a larger domain enables more accurate identiﬁcation of ABM parameters than generating 500 experimental replicates on a domain eight times smaller. Finally, we discuss the results presented in this work. of the parameter values in the biological process, the parame- terised ABM may still be used to make predictions about the process of interest by performing in silico experiments, as described above. These predictions can then be experimentally tested. Alternatively, if the ABM is an effective representation of an experiment (i.e., the experimental data can be reproduced), but the parameters of the ABM are not identiﬁable, this may suggest the experiment is not well-designed (that is, if the experiment has been designed to estimate parameters). By parameters not being identiﬁable we mean that different parameter values in the ABM can reproduce the same experimental data. If this is the case, the ABM can then be used to suggest improvements to the experiment’s design, namely by altering the ABM design such that the ABM parameters become identiﬁable. These alterations can then be applied to the experiment to improve parameter identiﬁability. For example, a recent study using an ABM has examined the time-points at which data should be collected from an experiment to maximise the identiﬁability of ABM para- meters.11 Other theoretical work has shown how to maximise the information content of an experiment by choosing an appropriate experimental set-up.18 Published in partnership with the Systems Biology Institute npj Systems Biology and Applications (2017) 9 RESULTS In doing so, we show that agent motility and adhesion parameters cannot be successfully identiﬁed using ABC given the current experi- mental design. To improve parameter identiﬁability we compare different experimental set-ups, and show that identiﬁcation of ABM parameters is made more accurate if the size of the domain upon which the experiment is performed is expanded, as opposed to the number of experimental replicates increased. Experimen- tally, expanding the size of the domain is equivalent to increasing 1. Run 104 ABM simulations, in each case using Θ sampled uniformly at random from the prior distribution. 1. Run 104 ABM simulations, in each case using Θ sampled uniformly at random from the prior distribution. 2. Compute the distance d as deﬁned in Eq. 13 for simulation times t = [240,480,720]. 3. Accept the 100 parameter values, Θ, that give the smallest values of d. In Fig. 1, the posteriors generated using each of the three summary statistics applied to data from simulations of an unrealistic experiment are displayed. The most effective summary statistic for identifying the synthetic data parameters is the PCF. This is evident in the location of the posterior distribution density relative to the red dot (the red dot represents the synthetic data parameter values), and the narrow spread of the posterior distribution density as indicated by the scale bar in Fig. 1c, f and i. The agent density proﬁle summary statistic performs less In Fig. 1, the posteriors generated using each of the three summary statistics applied to data from simulations of an unrealistic experiment are displayed. The most effective summary statistic for identifying the synthetic data parameters is the PCF. This is evident in the location of the posterior distribution density relative to the red dot (the red dot represents the synthetic data parameter values), and the narrow spread of the posterior distribution density as indicated by the scale bar in Fig. 1c, f and i. The agent density proﬁle summary statistic performs less Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al Fig. 1 a–c Posterior distributions for model A for an unrealistic experiment with different summary statistics: a average displacement of agents in the horizontal direction; b agent density proﬁle; c PCF. In all cases the red dot indicates the value of the parameters used to generate the synthetic data, Pm = 0.5, α = 0.1. RESULTS As indicated by the colour bar the yellow regions indicate areas of high relative density of the posterior distribution, while the blue regions indicate areas of low relative density of the posterior distribution. d–f Model B, Pm = 0.5, α = 0.25: d average displacement of agents in the horizontal direction; e agent density proﬁle; f PCF. g–i Model B, Pm = 0.5, α = −0.1; g average displacement of agents in the horizontal direction; h agent density proﬁle; i PCF Using Bayesian computation to quantify cell cell adhesion RJH Ross et al 3 3 Fig. 1 a–c Posterior distributions for model A for an unrealistic experiment with different summary statistics: a average displacement of agents in the horizontal direction; b agent density proﬁle; c PCF. In all cases the red dot indicates the value of the parameters used to generate the synthetic data, Pm = 0.5, α = 0.1. As indicated by the colour bar the yellow regions indicate areas of high relative density of the posterior distribution, while the blue regions indicate areas of low relative density of the posterior distribution. d–f Model B, Pm = 0.5, α = 0.25: d average displacement of agents in the horizontal direction; e agent density proﬁle; f PCF. g–i Model B, Pm = 0.5, α = −0.1; g average displacement of agents in the horizontal direction; h agent density proﬁle; i PCF well than the PCF for parameter identiﬁcation, especially for model A (Fig. 1b). In the case of the average agent displacement summary statistic many combinations of Pm and α lead to the same average agent displacement, which results in an extended region of possible parameter values. To some extent this is to be expected, as increasing either Pm or α will have opposing effects on the average agent displacement. This means that using agent displacement as a summary statistic results in parameter identiﬁability issues in this example. pairs (i.e., all combinations of Pm and α). A larger DKL(p\|π) value suggests that more information is obtained (the entropy of the distribution is reduced) when moving from the prior distribution to the posterior distribution. However, this does not necessarily mean the posterior distribution is a more accurate representation of the parameter distribution. Therefore, the KLD should not be seen as ubiquitously applicable to inference problems similar to those described in this work. Published in partnership with the Systems Biology Institute RESULTS Practically realisable experiment In the previous section, we demonstrated that for unrealistic experimental conditions the PCF summary statistic is best able to identify synthetic data parameters (for data generated from an ABM of an unrealistic experiment), and so moving forward we will only use the PCF summary statistic for parameter identiﬁcation. Previous work has combined summary statistics to improve parameter identiﬁcation, and how best to combine summary statistics has been the focus of a signiﬁcant amount of research, with a wide range of different methods examined.10, 27–30 However, in this case combining our summary statistics results in a negligible improvement to the posterior distribution (An example of a posterior distribution generated by combining all three summary statistics can be found in the supplementary material (Section S1)). y We also alter the ABM to have ﬂux (nonperiodic) boundary conditions at the left-hand and right-hand boundaries of the domain (i.e., for lattice sites with j = 1 or j = Ly). The left-most column is kept at or above a constant density throughout the simulation time course. That is, after any movement event from the left-most column in the simulation the column density of the left-most column is calculated, and if found to be below a certain density agents are added to empty sites in this column chosen uniformly at random until the required density is achieved. This mechanism ensures that the agent density proﬁle in the ABM replicates the evolution of the experimental data throughout the simulation. Further details regarding the implementation of this boundary condition are provided in the supplementary material (Section S3). The top and bottom boundaries of the ABM domain remain periodic as cells were seen to move in and out of the microscope ﬁeld at these boundaries in the experimental images, at an approximately equal rate. We now replace our ABM that represents an unrealistic experiment with an ABM that represents an actual experiment, and examine if synthetic data parameters can be identiﬁed in the ABM. That is, from this point on, we generate all synthetic data from an ABM based on a realistic experimental set-up. We provide brief details of the experiment here, however, a more detailed description can be found in the supplementary material (Section S2). In Fig. 2a typical initial frame of the experimental data can be seen. RESULTS To reduce computational time we now implement a Markov Chain Monte Carlo variant of ABC.19 Details of the implementation of the algorithm are given in the supplementary material (Section S4). As before we sample from uniform priors Pm ∈[0, 1] and α ∈ [−0.2, 0.25] for model A, and Pm ∈[0, 1] and α ∈[−0.2, 1.0] for model B, and collect simulation data at t = [240,480,720]. We collect simulation data at three time-points so that the computa- tional time is of practical length (our longest ABC Markov Chain Monte Carlo implementations took approximately 192 h). A value of Pm = 0.5, given that the simulation time is in minutes, and the length of a lattice site is 26 µm, means that the motility of the agents is biologically realistic. To be precise, the agents here are approximately ﬁve times faster than cell motility rates previously published (Using the relationship that the diffusion coefﬁcient is equal to PmΔ2.) (refs 4, 9). However, the cells considered in refs 4, 9 are not thought to exhibit cell–cell adhesion, and so a higher motility rate for the agents is sensible as agent movement is reduced by cell–cell adhesion in our ABM. In total, we have data from ﬁve replicates of the experiment. Therefore, we now generate our synthetic data from ﬁve replicates of the ABM, using the same procedure as described before. One key difference between the unrealistic and practically realisable experiments is the size of the domain and, because of this, the number of agents in a simulation. The experimental images were captured by a microscope with a ﬁeld of view of 597.24 µm by 597.24 µm. The cell size in the experimental images is consistent with each cell occupying a Fig. 2 Typical initial frame of the experimental data. The cells are positioned such that they will migrate primarily horizontally into the space without cells, this space represents a wound (the direction of migration is indicated by the white arrow) In Fig. 3 it can be seen that the synthetic data parameters cannot be accurately identiﬁed using ABC, with the PCF summary statistic, given the current ABM design. This is evident in the location of the red dots (indicating the parameter values used to generate the synthetic data) relative to the posterior distributions, and the wide spread of the posterior distributions (indicated by the scale bar in Fig. 3). RESULTS In particular, the KLD should be used with caution in scenarios in which an informative prior is used. In such scenarios, other methods to measure the improvement of an inference procedure have been examined and may be more suitable.27 To quantify the difference between the performance of the different summary statistics we use the Kullback-Leibler diver- gence (KLD), which is a measure of the information gained in moving from the prior distribution to the posterior distribution.26 The KLD for a discrete probability distribution is deﬁned as follows: To compute the KLD, we discretise our posterior distribution onto a lattice with 26 equally spaced values of Pm and 26 equally spaced values of α. Computing DKL(p\|π) for all nine plots in Fig. 1 gives: (a) 1.77; (b) 1.70; (c) 2.32; and (d) 2.15; (e) 2.57; (f) 3.35; and (g) 2.45; (h) 2.72; (i) 3.27. In tandem with the proximity of the peak of the posterior distribution densities to the red dots in Fig. 1c, f and i, compared to Fig. 1a, b, d, e, g and h, this is increase in the DKL pjπ ð Þ ¼ X l p ΘljD ð Þ log p ΘljD ð Þ π Θl ð Þ ; ð1Þ ð1Þ where the index l accounts for all possible discretised parameter where the index l accounts for all possible discretised parameter Published in partnership with the Systems Biology Institute npj Systems Biology and Applications (2017) 9 Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al 4 KLD suggests that the PCF summary statistic is more effective for parameter identiﬁcation than the average agent displacement and agent density proﬁle summary statistics. 26 µm by 26 µm square lattice site. Given the size of the microscope ﬁeld of view this means the ABM domain size is Lx = 23 by Ly = 23. We use the average initial conditions from the experiment to generate the initial conditions in the ABM of a realistic experiment. Exact details of how the initial condition is generated in the ABM, and how experimental data is mapped to a lattice, can be found in the supplementary material (Section S3). Published in partnership with the Systems Biology Institute npj Systems Biology and Applications (2017) 9 Improving the experimental design Speciﬁcally, the number of occupied lattice pairs for each horizontal pair distance that generates the PCF is proportional to (This is not quite correct as a distance of ‘0’ between agents, that is they share the same column, is not accounted for in Eq. 9. To make Eq. 2 exact is not trivial as the expected number of agents each agent shares a column with depends on both the column position and simulation time.) g In Fig. 6a–c, we plot the posterior distribution for synthetic data generated from 500 replicates of our ABM on a Lx = 23 by Ly = 23 sized domain, while in Fig. 6d–f we plot the posterior distribution generated from synthetic data generated from ﬁve replicates of our ABM on a Lx = 23 by Ly = 184 sized domain (A Markov chain Monte Carlo trace corresponding to Fig. 7e can be found in the supplementary material (Section S5)). As predicted, it is apparent that increasing the domain size is more effective for parameter identiﬁcation than increasing the number of replicates used to generate the synthetic data. This is evident in the location (and narrow spread) of the posterior distribution relative to the red dot, whereby the peak of the posterior distribution is closer to the red dot in the case of Fig. 6d–f compared to Fig. 6a–c. Despite this, the identiﬁcation of the parameters for repulsive interactions remains somewhat elusive (Fig. 6f). A possible reason for this is that the repulsive interaction we present here is a weak one, due to the constraint of Eqs. 4 and 6, and larger values of \|α\| are easier to identify as they have a more profound effect on the behaviour of the agent population. NðN 1Þ 2 : ð2Þ Computing DKL(p\|π) for all six plots in Fig. 6 gives: (a) 2.55; (b) 2.69; (c) 1.53; and (d) 3.69; (e) 2.97; (f) 3.54. In tandem with the proximity of the peak of the posterior distribution densities to the red dots in Fig. 6d–f compared to Fig. 6a–c, this increase in the KLD suggests that generating synthetic data on a larger domain is more effective for improving parameter identiﬁcation than increasing the number of replicates used to generate the synthetic data. RESULTS We have included the ABC Markov chain Monte Carlo traces corresponding to Fig. 3 in the supplementary material (Section S5). A possible reason why the synthetic data parameters cannot be identiﬁed is that the synthetic data does not accurately represent the parameter values used to generate it, making parameter identiﬁcation infeasible. To examine this possibility, we calculated the variance in the PCF synthetic data. In Fig. 4a–c the blue line indicates the variance in the PCF synthetic data for the current simulation design generated from ﬁve replicates of the ABM on a domain of dimension Lx = 23 by Ly = 23. y If the variance in the summary statistics of the synthetic data precludes accurate identiﬁcation of model parameters using ABC, a sensible strategy may be to examine methods to reduce the variance in the summary statistics of the synthetic data. Reducing the variance of the summary statistics may mean the synthetic data is a more accurate reﬂection of the parameters values used to generate it. This may also explain why parameter identiﬁcation for the unrealistic experiment was successful, as the variance in the Fig. 2 Typical initial frame of the experimental data. The cells are positioned such that they will migrate primarily horizontally into the space without cells, this space represents a wound (the direction of migration is indicated by the white arrow) npj Systems Biology and Applications (2017) 9 Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al 5 Fig. 3 Posterior distributions for simulations of the realistic experiment described using the PCF as a summary statistic for an ABM of dimension Lx = 23 and Ly = 23. The synthetic data is generated from ﬁve replicates of the ABM. a Model A: Pm = 0.5, α = 0.1, b model B: Pm = 0.5, α = 0.25, c model B: Pm = 0.5, α = −0.1. In all cases the red dot indicates the value of the parameters used to generate synthetic data 5 Fig. 3 Posterior distributions for simulations of the realistic experiment described using the PCF as a summary statistic for an ABM of dimension Lx = 23 and Ly = 23. The synthetic data is generated from ﬁve replicates of the ABM. a Model A: Pm = 0.5, α = 0.1, b model B: Pm = 0.5, α = 0.25, c model B: Pm = 0.5, α = −0.1. Published in partnership with the Systems Biology Institute Improving the experimental design Therefore, the identiﬁcation of parameters in experimental data using the PCF as a summary statistic may be best facilitated by increasing the size of the domain upon which the experiment is performed, rather than increasing the number of replicates of an experiment with a smaller domain. Further variance plots for models A and B for the PCF summary statistic can be found in the supplementary material (Section S6). It is important to note that it is also the case for the agent density proﬁle synthetic data, that increasing the size of the domain is more effective at reducing variance in the synthetic data than increasing the number of replicates. If generated from 500 replicates of our ABM on a Lx = 23 by Ly = 23 sized domain, the agent density proﬁle synthetic data will have greater variance than the agent density proﬁle synthetic data generated from ﬁve replicates of our ABM on a Lx = 23 by Ly = 184 sized domain (data not shown). In this case, the reduction in variance is an artefact of the lattice-based model. This is because the density of each Improving the experimental design Improving the experimental design We now conﬁrm that more accurate identiﬁcation of synthetic data parameters can be carried out by expanding the domain upon which the experiment is performed, as opposed to increasing the number of experimental replicates. p In Fig. 4, the variance in the PCF synthetic data for model B with Pm = 0.5 and α = 0.25 for different domain sizes and varying numbers of replicates can be seen. It is evident that the variance in the PCF calculated from 500 replicates of our ABM on a Lx = 23 by Ly = 23 sized domain (blue line in Fig. 4d–f) is greater than the variance in the PCF calculated from ﬁve replicates of our ABM on a Lx = 23 by Ly = 184 sized domain (purple line in Fig. 4a–c). This can be understood by considering Eq. 9: the number of occupied lattice pairs for each horizontal pair distance used to generate the PCF does not increase linearly with the number of agents. Speciﬁcally, the number of occupied lattice pairs for each horizontal pair distance that generates the PCF is proportional to (This is not quite correct as a distance of ‘0’ between agents, that is they share the same column, is not accounted for in Eq. 9. To make Eq. 2 exact is not trivial as the expected number of agents each agent shares a column with depends on both the column position and simulation time.) p In Fig. 4, the variance in the PCF synthetic data for model B with Pm = 0.5 and α = 0.25 for different domain sizes and varying numbers of replicates can be seen. It is evident that the variance in the PCF calculated from 500 replicates of our ABM on a Lx = 23 by Ly = 23 sized domain (blue line in Fig. 4d–f) is greater than the variance in the PCF calculated from ﬁve replicates of our ABM on a Lx = 23 by Ly = 184 sized domain (purple line in Fig. 4a–c). This can be understood by considering Eq. 9: the number of occupied lattice pairs for each horizontal pair distance used to generate the PCF does not increase linearly with the number of agents. npj Systems Biology and Applications (2017) 9 RESULTS In all cases the red dot indicates the value of the parameters used to generate synthetic data summary statistics of the synthetic data was much smaller than for the practically realisable experiment (data not shown). column in the ABM can take on a greater range of values between 0 and 1 as the column length is increased, leading to a reduction in variance in the agent density proﬁle synthetic data (especially in the initial conditions of the simulations used to generate the synthetic data). However, as we do not use the agent density proﬁle summary statistic to identify parameters in the current simulation design we do not pursue this matter further. We conjectured that the variance in the summary statistics of the synthetic data could be reduced in two ways: We conjectured that the variance in the summary statistics of the synthetic data could be reduced in two ways: 1. increasing the number of ABM replicates used to generate the synthetic data; 1. increasing the number of ABM replicates used to generate the synthetic data; 2. increasing the size of the ABM domain while keeping the column density of the initial conditions invariant. An example of this proposed initial condition is given in Fig. 5b, in which the domain is twice the size of that in Fig. 5a. Importantly, increasing the size of the ABM domain increases the number of agents in the simulation, and can be thought of as equivalent to increasing the ﬁeld of view of the microscope. DISCUSSION In this work, we have presented methods to identify motility and adhesion parameters in an ABM of a wound-healing assay. Our ﬁndings suggest that for a commonly performed experiment increasing the size of the experimental domain can be more effective in improving the accuracy of parameter identiﬁcation, when compared to increasing the number of replicates of the npj Systems Biology and Applications (2017) 9 Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al 6 Fig. 4 The variance in the PCF synthetic data for model B with Pm = 0.5, α = 0.25 and different ABM domain sizes. Panels a–c display synthetic data generated from ﬁve replicates of the ABM, panels d–f display synthetic data generated from 500 replicates of the ABM. The domain size is indicated in the legend Fig. 4 The variance in the PCF synthetic data for model B with Pm = 0.5, α = 0.25 and different ABM domain sizes. Panels a–c display synthetic data generated from ﬁve replicates of the ABM, panels d–f display synthetic data generated from 500 replicates of the ABM. The domain size is indicated in the legend synthetic data for model B with Pm = 0.5, α = 0.25 and different ABM domain sizes. Panels a–c display syntheti ates of the ABM, panels d–f display synthetic data generated from 500 replicates of the ABM. The domain siz experiment. This is because increasing the size of the domain, which is equivalent to increasing the number of cells in the experiment, more effectively reduces the variance in the summary statistics of the synthetic data from which the parameters are identiﬁed. The reason for this reduction in variance is explained by Eq. 9, where the number of agent pair counts that generate the PCF increases nonlinearly with the number of agents on the domain. In addition, increasing the size of the experimental domain may make the collection of experimental data less time- consuming, as potentially fewer replicates of the experiment will have to be conducted. For instance, ﬁve replicates of the experiment on a larger domain provides more information about parameters than 500 replicates of the experiment on a smaller domain (in the examples we have presented in this work). DISCUSSION Therefore, a comprehensive study of all summary statistics commonly used for analysing cell migration, to understand how their variance scales with the size of the experimental domain, is an interesting avenue for further research. behaviours to the ABM, such as the role of the cell cycle. This may allow us to better capture the behaviour of the cell populations we have studied here, and so produce more realistic models of cell migration. experiment. This is because increasing the size of the domain, which is equivalent to increasing the number of cells in the experiment, more effectively reduces the variance in the summary statistics of the synthetic data from which the parameters are identiﬁed. The reason for this reduction in variance is explained by Eq. 9, where the number of agent pair counts that generate the PCF increases nonlinearly with the number of agents on the domain. In addition, increasing the size of the experimental domain may make the collection of experimental data less time- consuming, as potentially fewer replicates of the experiment will have to be conducted. For instance, ﬁve replicates of the experiment on a larger domain provides more information about parameters than 500 replicates of the experiment on a smaller domain (in the examples we have presented in this work). Therefore, a comprehensive study of all summary statistics commonly used for analysing cell migration, to understand how their variance scales with the size of the experimental domain, is an interesting avenue for further research. To conclude, the ﬁndings presented in this work will be of particular interest to those concerned with performing experi- ments that enable the effective parameterisation of cell migratory processes. In particular, cell migratory processes in which cell–cell adhesion or repulsion are known to play an important role. More generally, we have also suggested time and cost-saving alterations to a commonly performed experiment for identifying cell motility parameters. Published in partnership with the Systems Biology Institute METHODS In this section, we ﬁrst introduce the ABM. We then deﬁne our summary statistics and explain ABC and its implementation. g We also studied using the average horizontal displacement of agents and the agent density proﬁle as summary statistics. These were found to be less effective than the PCF in parameter identiﬁcation. This was especially the case for the averaged agent displacement, whereby a range of adhesion and motility parameters could result in the same average agent displacement. This result suggests that agent displacement may not be a suitable summary statistic for identifying cell motility and adhesion parameters, due to parameter identiﬁability issues. npj Systems Biology and Applications (2017) 9 Agent-based model where α is the adhesion parameter. The subscript A on the transition probability in Eq. 3 indicates that this is the transition probability for model A. If α > 0 Eq. 3 models cell–cell adhesion, and if α < 0 Eq. 3 models cell–cell repulsion. The transition probabilities stated in Eq. 3 must satisfy where α is the adhesion parameter. The subscript A on the transition probability in Eq. 3 indicates that this is the transition probability for model A. If α > 0 Eq. 3 models cell–cell adhesion, and if α < 0 Eq. 3 models cell–cell repulsion. The transition probabilities stated in Eq. 3 must satisfy 0 X U v′2U TA v′ vj ð Þ 1: ð4Þ ð4Þ Inequality (4) ensures the probability of an agent, if selected to move, attempting to move to any of its unoccupied nearest neighbour sites never exceeds unity, and so constrains the value α can take. The transition probability in the second model, which we refer to as model B,13, 36 is deﬁned as TB v′ vj ð Þ ¼ 1 α ð Þn 4 ; ð5Þ TB v′ vj ð Þ ¼ 1 α ð Þn 4 ; ð5Þ For the ABM of an unrealistic experiment, all simulations have periodic boundary conditions at the top and bottom of the domain (i.e. for lattice sites indexed by j = 1 or j = Ly), and no-ﬂux boundary conditions at the left- hand and right-hand boundaries of the domain (i.e. for lattice sites indexed by i = 1 or i = Lx). and must satisfy and must satisfy 0 X U v′2U TB v′ vj ð Þ 1: 0 X U v′2U TB v′ vj ð Þ 1: ð6Þ ð6Þ y It is important to stress that throughout this work we assume that cellular processes such as migration have constant parameter values associated with them. Inference procedures do exist in which the parameter values associated with cell processes are not assumed to be constant, but are instead treated as a random variable sampled from a distribution. These methods are often important for sensitivity analysis, or if the data is sampled from a heterogeneous population.33–35 However, we do not implement these methods in this work as it would serve to prematurely complicate our research question. Agent-based model g An ABM is a computational model for simulating the behaviour of autonomous agents. The agents in the ABM represent cells, and each agent is able to move and interact with other agents. The ABM is simulated on a two-dimensional square lattice with lattice spacing31 and size Lx by Ly, where Lx is the number of lattice sites in each row, and Ly is the number of sites in each column. Each agent is initially assigned to a lattice site, from which it can move into adjacent sites. If an agent attempts to move into a site that is already occupied by another agent, the movement event is aborted. Processes such as this whereby one agent is allowed per site are often referred to as exclusion processes.31 In the ABM time evolves continuously, and as our ABM can be modelled as a continuous-time Markov process we use the Gillespie algorithm32 to generate sample paths. Attempted agent movement events occur with rate Pm per unit time. Pmδt, therefore, is the probability of an agent attempting to move in the next inﬁnitesimally small time interval δt. In our ABM, a lattice site is denoted by v = (i, j), where i indicates the column number and j the row number. Each lattice site has four adjacent lattice sites (except for those sites situated on nonperiodic boundaries), and so the number of nearest neighbour lattice The most obvious extension to the work presented here is to experimentally validate the ﬁndings. That is, expand the wound- healing experimental domain and demonstrate: (i) the cell migratory process can be effectively described by the model we have presented here; and (ii) the experimental parameters are identiﬁable given a larger experimental domain. If validated, evidence may be provided that demonstrates which adhesion model, A or B, is more applicable to the cell type under consideration. Subsequently, we could add further agent Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al j 45 40 35 30 25 20 15 10 5 0 i 0 5 10 15 20 i 20 15 10 5 0 j 0 5 10 15 20 (a) (b) Fig. 5 Increasing the size of the simulation domain while keeping the initial column densities the same. Agent-based model It is also important to acknowledge that in migrating cell populations there are often many more factors at play than simply cell motility and adhesion. For instance, the cell cycle and a cell’s response to environmental cues may be important factors in a cell’s behaviour. Again, however, we have purposely simpliﬁed our model to ﬁrst ascertain if we can accurately estimate parameters associated with cell motility and adhesion. As in model A if α > 0 Eq. 5 models cell–cell adhesion, and if α < 0 Eq. 5 models cell–cell repulsion. Models A and B simulate different types of cell–cell adhesion. In model A, the transition probability is a linear function of n. Meanwhile, in model B the transition probability is a nonlinear function of α. Not only may these different types of cell–cell adhesion be relevant for different cell types, but implementing two models of cell–cell adhesion allows us to test the robustness of the methods we present in this work for identifying cell–cell adhesion parameters. Summary statistics Summary statistics are lower-dimensional summaries of data that provide a tractable means to compare different sets of data. Summary statistics are important because experimental data is often of high dimensionality, and if we want to use experimental data to efﬁciently guide computational algorithms we require ways to accurately summarise it. We now deﬁne the summary statistics we apply to the ABM output and experimental data. Following this, we describe how we utilise these summary statistics to implement ABC. npj Systems Biology and Applications (2017) 9 Agent-based model The domain in b is twice the size of that in a, however, the average initial density of each column is the same in both a and b 7 j 45 40 35 30 25 20 15 10 5 0 i 0 5 10 15 20 (b) 0 5 10 15 20 i 20 15 10 5 0 j (a) (a) 20 j i Fig. 5 Increasing the size of the simulation domain while keeping the initial column densities the same. The domain in b is twice the size of that in a, however, the average initial density of each column is the same in both a and b Fig. 5 Increasing the size of the simulation domain while keeping the initial column densities the same. T that in a, however, the average initial density of each column is the same in both a and b probability in the ﬁrst model, which we refer to as model A, is deﬁned as sites that are occupied by an agent, denoted by n, is 0 ≤n ≤4. We denote the set of unoccupied nearest neighbour lattice sites by U. TA v′ vj ð Þ ¼ 1 nα 4 ; ð3Þ ð3Þ The ABM domain size for simulations representing unrealistic experi- ments is Lx = 100 by Ly = 100, and the lattice sites indexed by 1 ≤j ≤Ly and 1 ≤i ≤10, and1 ≤j ≤Ly and 91 ≤i ≤Lx are initially occupied by agents. In Fig. 7, the initial conditions in the ABM for the unrealistic experiment can be seen. The initial condition in Fig. 7 represents a ‘wound’, in that agents are positioned either side of a space, the ‘wound’, that they can migrate into. The agent migration into this space simulates one aspect of the wound-healing process. We refer to this simulation as unrealistic because the uniformity of the initial conditions would not be possible in a realistic experimental setting. The initial condition is also improved from our experimentally realisable simulation as it is ‘double-sided’, as opposed to the ‘single-sided’ experimental data that we will later simulate for our ABM of a realistic experiment. It has been shown that double-sided initial conditions can provide more information than single-sided initial conditions for some model parameters.11 For instance, double-sided initial conditions can improve parameter identiﬁability if increasing the number of agents in a simulation improves parameter identiﬁability. Published in partnership with the Systems Biology Institute Cell–cell adhesion models In the ABM cell–cell interactions are simulated by altering the probability of an agent attempting to move, depending on the number of nearest occupied neighbours, n, an agent has. We employ two models to simulate cell–cell interactions in the ABM, one of which has been published before.13, 36 We deﬁne T(v′\|v) as the transition probability that an agent situated at site v, having been selected to move, attempts to move to site v′, where v′ indicates one of the nearest neighbour sites of v. Therefore, T(v′\|v) is only non-zero if v and v′ are nearest neighbours. The transition We initially use three summary statistics to evaluate the ABM output, all of which have been considered previously.9, 36, 37 Our aim is to ascertain which summary statistic (or combination of summary statistics) is most effective for the identiﬁcation of agent motility and adhesion parameters in the ABM. npj Systems Biology and Applications (2017) 9 Published in partnership with the Systems Biology Institute Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al 8 Fig. 6 a–c Posterior distributions for simulations of the realistic experiment using the PCF as a summary statistic for an ABM simulated on a domain of dimension Lx = 23 by Ly = 23 with synthetic data generated from 500 replicates. a Model A: Pm = 0.5, α = 0.1, b model B: Pm = 0.5, α = 0.25, c model B: Pm = 0.5, α = −0.1. d–f Posterior distribution plots for simulations of the experiment using the PCF as a summary statistic for an ABM simulated on a domain of size Lx = 23 by Ly = 184 with synthetic data generated from ﬁve replicates. a Model A: Pm = 0.5, α = 0.1, b model B: Pm = 0.5, α = 0.25, c model B: Pm = 0.5, α = −0.1. Further ﬁgure information can be found in Fig. 1 Fig. 6 a–c Posterior distributions for simulations of the realistic experiment using the PCF as a summary statistic for an ABM simulated on a domain of dimension Lx = 23 by Ly = 23 with synthetic data generated from 500 replicates. a Model A: Pm = 0.5, α = 0.1, b model B: Pm = 0.5, α = 0.25, c model B: Pm = 0.5, α = −0.1. Agent density proﬁle The agent density proﬁle at time t in the ABM is calculated as The agent density proﬁle at time t in the ABM is calculated Ct ið Þ ¼ 1 Ly X Ly j¼1 1 v f g: ð8Þ Ct ið Þ ¼ 1 Ly X Ly j¼1 1 v f g: ð8Þ Here Ct(i) is the agent density proﬁle and 1 is the indicator function for the occupancy of a lattice site v (i.e., 1 if an agent occupies lattice site v, and 0 if it is not occupied by an agent). We have shown previously that different cell–cell interactions have different effects on the agent density proﬁle.36 For instance, repulsive interactions between agents can create a concave agent density proﬁle, whereas adhesive interactions between agents can create a convex agent density proﬁle. Therefore, the agent density proﬁle may be an effective summary statistic for distinguishing between repulsive and adhesive cell–cell interactions in the ABM. Fig. 7 The initial condition in the ABM for the unrealistic experiment. Yellow indicates a site occupied by an agent and blue indicates an empty lattice site Cell–cell adhesion models d–f Posterior distribution plots for simulations of the experiment using the PCF as a summary statistic for an ABM simulated on a domain of size Lx = 23 by Ly = 184 with synthetic data generated from ﬁve replicates. a Model A: Pm = 0.5, α = 0.1, b model B: Pm = 0.5, α = 0.25, c model B: Pm = 0.5, α = −0.1. Further ﬁgure information can be found in Fig. 1 0 20 40 60 80 100 i 100 80 60 40 20 0 j Fig. 7 The initial condition in the ABM for the unrealistic experiment. Yellow indicates a site occupied by an agent and blue indicates an empty lattice site time ti, and ik tf is the column position of agent k at time tf. We only look at the horizontal displacement of agents as this is the direction in which the majority of agent displacement occurs, due to the initial conditions of the ABM (Fig. 7). It has previously been shown that different cell–cell interactions have different effects on the average displacement of agents in an ABM.36 As may be expected, repulsive (adhesive) interactions between agents tend to increase (decrease) the average displacement of agents, and so the average displacement of agents may be a useful summary statistic for distinguishing between repulsive and adhesive cell–cell interactions in the ABM. 0 20 40 60 80 100 i 100 80 60 40 20 0 j Published in partnership with the Systems Biology Institute Average horizontal displacement of agents Average horizontal displacement of agents The ﬁnal summary statistic, we consider is the pairwise-correlation function (PCF). The PCF provides a measure of the spatial clustering between agents in an ABM, and has been used frequently in the analysis of cell migratory processes.4, 9, 38, 39 The PCF has also been successfully used as a summary statistic for the parameterisation of ABMs of cell migration.10 We use ik t to denote the column position of agent k at time t, il t to denote the column position of agent l at time t, and deﬁne ct(m) to be the number of occupied pairs of lattice sites for each nonperiodic (By nonperiodic it is meant the The average horizontal displacement of all agents, ī, in a given time interval, [ti, tf], in the ABM is calculated as i ¼ 1 N X N k¼1 ik ti ik tf ; ð7Þ i ¼ 1 N X N k¼1 ik ti ik tf ; ð7Þ p y y statistic for the parameterisation of ABMs of cell migration.10 We use ik t to denote the column position of agent k at time t, il t to denote the column position of agent l at time t, and deﬁne ct(m) to be the number of occupied pairs of lattice sites for each nonperiodic (By nonperiodic it is meant the Where ī is the average horizontal displacement of agents, N is the total number of agents in the simulation, ik ti is the column position of agent k at j Systems Biology and Applications (2017) 9 Published in partnership with th Published in partnership with the Systems Biology Institute Using Bayesian computation to quantify cell–cell adhesion RJH Ross et al 9 Table 1. The summary statistics we implement and the properties of the agent population they summarise Summary statistic Description Average horizontal displacement of agents Summarises the displacement of agents into the ‘wound’. This displacement is affected by the adhesion of agents and their motility rate. Mathematically the average horizontal displacement of agents is deﬁned as i ¼ 1 N PN k¼1 ik ti ik tf Agent density proﬁle Summarises the macroscopic shape of the population as it moves into the ‘wound’. Average horizontal displacement of agents 10 describes the expected number of pairs of occupied lattice sites, for each nonperiodic horizontal pair distance, m, in a population distributed uniformly at random on the domain. Combining Eqs. 9 and 10, the PCF is d ¼ X T t¼1 X R r¼1 SðDÞr;t S ~D r;t ;j ð13Þ ð13Þ Where R is the number of data points in S(D) and T is the number of sampling times. We repeat the above process many times, that is, sample Θ from π, produce ~D, calculate d with Eq. 13, and only accept Θ for which d is below a user deﬁned certain threshold (alternatively, a predeﬁned number of Θ that minimise d can be accepted). This enables us to generate a distribution for Θ that is an approximation of the posterior distribution, p (Θ\|D), given M.40 More speciﬁc details of the ABC algorithms we implement are introduced when necessary in the text. qtðmÞ ¼ ctðmÞ ^ctðmÞ ; ð11Þ qtðmÞ ¼ ctðmÞ ^ctðmÞ ; ð11Þ Where qt(m), the PCF, is a measure of how far ct(m) departs from describing the expected number of occupied lattice pairs for each horizontal distance of an agent population spatially distributed uniformly at random on the ABM domain. It is important to brieﬂy discuss why we chose these summary statistics and not others that have also been used to analyse cell migration.10, 22, 24 Other summary statistics were initially implemented in this study, such as the concavity of agent trajectories, the total distance travelled by agents and the leading edge of the agent population. However, these summary statistics were found not to be informative for the identiﬁcation of agent motility and adhesion parameters in our ABM, and so were excluded from this work. The three summary statistics we implement are encapsulated in Table 1 for the reader’s convenience, in addition to the properties each summary statistic summarises in the agent population. ACKNOWLEDGEMENTS R.J.H.R. would like to thank the UK’s Engineering and Physical Sciences Research Council (EPSRC, EP/G03706X/1) for funding through a studentship at the Systems Biology programme of The University of Oxford’s Doctoral Training Centre. R.L.M. was supported by a Medical Research Scotland Project Grant (436FRG). Approximate Bayesian computation R.J.H.R., R.E.B., A.P. and C.A.Y. conceived the work, and performed the mathematical and computational analysis. Data collection and analysis was performed by R.L.M. and M.J.F. R.J.H.R., R.E.B. and C.A.Y. drafted the manuscript. All authors agree with manuscript results and conclusions. All authors approved the ﬁnal version. Here, we introduce our ABC algorithm.19 We deﬁne M as a stochastic model that takes parameters Θ and produces data D. This relationship can be written as D~M(Θ). For the ABM presented in this work Θ = (Pm, α), where Θ is sampled from a prior distribution, π, and so this relationship can be written as Θ~π. The relationship between π and Θ is often written as ~π (Θ), which indicates that a new Θ sampled from the prior distribution may depend on the previous Θ. This relationship will be relevant later on in this work, however, initially each Θ sampled from π is independent of the previous Θ. COMPETING INTERESTS The authors declare no competing ﬁnancial interests. The authors declare no competing ﬁnancial interests. The identiﬁcation of ABM parameters in this work centres around the following problem: given a stochastic model, M, and data, D, what is the probability density function that describes Θ being the model parameters that produced data D? More formally, we seek to obtain a posterior distribution, p(Θ\|D), which is the conditional probability of Θ given D (and the model, M). Average horizontal displacement of agents Given the translational invariance of the initial conditions in the vertical direction of the ABM, the majority of important spatial information will be in the horizontal direction (This approach is in agreement with previous studies39, which showed the most relevant information from the PCF summary statistic is perpendicular to the wound axis in a wound-healing assay). Binder and Simpson39 demonstrated that is necessary to normalise Eq. 9 to account for volume exclusion. The normalisation term is Where 1 is the indicator function equal to unity if ik t il t = m, and is equal to zero otherwise. In Eq. 9 only the pair agent distances in the horizontal direction are counted. Given the translational invariance of the initial conditions in the vertical direction of the ABM, the majority of important spatial information will be in the horizontal direction (This approach is in agreement with previous studies39, which showed the most relevant information from the PCF summary statistic is perpendicular to the wound axis in a wound-healing assay). Binder and Simpson39 demonstrated that is necessary to normalise Eq. 9 to account for volume exclusion. The normalisation term is y In many cases for ABC, due to the high dimensionality of the data, D, it is necessary to utilise a summary statistic, S = S(D). The summary statistics we employ in this work are of varying dimension. For instance, the agent density proﬁle at time t has Lx data points, whereas the average agent displacement at time t has one data point. Therefore, we write S(D) as S(D)r,t, where S(D)r,t is the rth data point in the summary statistic at the tth sampling time. The ABC method proceeds in the following manner: we wish to estimate the posterior distribution of Θ given D. We now simulate model M with parameters Θ, sampled from π, and produce data ~D. We calculate the difference between a summary statistic applied to D and ~D with ^ct m ð Þ ¼ L2 y Lx m ð Þρ^ρ; 8 m ¼ 1; ¼ ; Lx 1; ð10Þ ^ct m ð Þ ¼ L2 y Lx m ð Þρ^ρ; 8 m ¼ 1; ¼ ; Lx 1; ð10Þ where = N/(LxLy) , and ^ρ = (N−1)/(LxLy−1). Eq. Published in partnership with the Systems Biology Institute Average horizontal displacement of agents We have previously shown this shape is partly determined by agent interactions and motility.36 Mathematically, the agent density proﬁle is deﬁned as Ct ið Þ ¼ 1 Ly PLy j¼1 1 v f g: Pair-wise correlation function Summarises the spatial correlations/structure established by agent movement and interactions. Mathematically the pair-wise correlation function is deﬁned as qt m ð Þ ¼ ct m ð Þ ^ct m ð Þ Table 1. The summary statistics we implement and the properties of the agent population they summarise Summary statistic Description Summarises the macroscopic shape of the population as it moves into the ‘wound’. We have previously shown this shape is partly determined by agent interactions and motility.36 Mathematically, the agent density proﬁle is deﬁned as Ct ið Þ ¼ 1 Ly PLy j¼1 1 v f g: y j Summarises the spatial correlations/structure established by agent movement and interactions. Mathematically the pair-wise correlation function is deﬁned as qt m ð Þ ¼ ct m ð Þ ^ct m ð Þ distance measured between two agents cannot cross the ABM boundary) horizontal pair distance m = 1,…,Lx−1 at time t. This means ct(m) is given by distribution are related in the following manner by Bayes’ theorem: distribution are related in the following manner by Bayes’ theorem: distribution are related in the following manner by Bayes’ theorem: p ΘjD ð Þ / L DjΘ ð Þπ Θ ð Þ: ð12Þ p ΘjD ð Þ / L DjΘ ð Þπ Θ ð Þ: ð12Þ ð12Þ That is, the posterior distribution is proportional to the product of the likelihood function and the prior distribution. That is, the posterior distribution is proportional to the product of the likelihood function and the prior distribution. ct m ð Þ ¼ X N k¼1 X N l¼kþ1 1 ik t il t ¼ m ; 8 m ¼ 1; ¼ ; Lx 1; ð9Þ ð9Þ ABC is a well-known method for estimating posterior distributions of model parameters in scenarios where the likelihood function is intractable i.e., it is impossible or computationally prohibitive to obtain.19 Where 1 is the indicator function equal to unity if ik t il t = m, and is equal to zero otherwise. 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https://openalex.org/W4287100448	https://zenodo.org/records/5070570/files/Camassa%20et%20al%202021.pdf	English	null	Metastability of Down states in the cerebral cortex: dynamics and modulation	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	12,491	Camassa A1., Galluzzi A.2, Mattia M.2, Sanchez-Vives M.V. 1,3 1 Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 2 Natl. Center for Radioprotection and Computational Physics, Istituto Superiore di Sanità, 00161 Rome, Italy 3 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 1 Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 2 Natl. Center for Radioprotection and Computational Physics, Istituto Superiore di Sanità, 00161 Rome, Italy 3 Institució Catalana de Recerca i Estudis Avançats (ICREA) Barcelona Spain * shared senior authorship. 1 1 Abstract Slow oscillations are an emergent activity of the cerebral cortex network consisting of alternating periods of activity or Up states and periods of silence or Down states. These silent periods are critical for cerebral cortex function since they act to reset the system and break casual interactions between cortical areas. The occurrence of Down states is almost invariably associated with unconsciousness. By analysing the instantaneous phase and synchronization (Kuramoto order parameter) of the local field potential and multiunit activity from multielectrode recordings, we identified metastability within Down states: a “deterministic” and a “stochastic” period, explored in in vivo, in vitro and in silico cortical networks. A large parameter space was investigated through different experimental manipulations to validate a frame that integrates a broad range of oscillatory frequencies. We propose a more detailed Up and Down state cycle dynamics that bridges cortical properties emerging at the mesoscale with their underlying mechanisms at the microscale. 2 2 Introduction Slow oscillations are a multiscale phenomenon that dominate cerebral cortex dynamics not only in slow wave sleep but also in a variety of situations including deep anesthesia, and disorders of consciousness and perilesional activity, while they also emerge in isolated gyri or slabs in vivo and cortical slices in vitro (for a review, see 1). This is a network emergent pattern that results from the integration of cellular and network properties, which shape this activity and its propagation as a slow wave 2,3. For this reason, the resulting pattern of slow wave activity can vary within a certain range, being highly informative about the physiological, pharmacological, or pathological state of the network 1. This is a network emergent pattern that results from the integration of cellular and network properties, which shape this activity and its propagation as a slow wave 2,3. For this reason, the resulting pattern of slow wave activity can vary within a certain range, being highly informative about the physiological, pharmacological, or pathological state of the network 1. Slow oscillations consist of the alternation of Up states, active periods with neuronal firing, and Down states or periods of silence. Up states share properties with wakefulness: persistent activity generated by recurrent networks with comparable firing rates 4,5, eventual synchronization in beta and gamma frequencies 6,7, or a balanced excitation and inhibition 8,9. Indeed, Up states have been referred to as “fragments of wakefulness” 10, since in a sleeping or anesthetized brain, this seemingly awake activity can be recorded at intervals. 3 So, what separates these “windows into wakefulness” from actual wakefulness, and thus, from conscious states? The answer is: the silent periods in between, or Down states. Intracellular recordings from cortical neurons transitioning between wakefulness and slow wave sleep 11 or between wakefulness and anesthesia 4 evidence that the main difference associated with the collapse of wakefulness is the appearance of quiescent periods or Down states. Down states are not only a temporal interruption in the information processing and a reset of the system that has been associated with different metabolic and recalibration functions 12, they also imply a spatial interruption of functional connectivity that breaks the causal interactions between areas 13. Results To investigate the cerebral cortex network dynamics and underlying mechanisms during Up and Down states, we studied in parallel three cortical networks: in vivo, in vitro and in silico, while expressing slow oscillations. We focused on the dynamic properties of Down states and their relationship with the micro- and mesoscale networks, both in control conditions and under different experimental manipulations, which allowed us a broader investigation of the parameter space. Introduction The role 3 of Down states—or its evoked counterpart, “off-periods” —on consciousness levels has been thoroughly studied, since evoking prominent “off-periods” using transcranial magnetic stimulation in humans, is a validated measure of loss of consciousness which is used in unresponsive patients 14. Furthermore, local injury in the brain also generates “off-periods” as a response to stimulation 15, which can then percolate cortical networks disrupting their physiological processing 16. Loss of consciousness associated with focal limbic seizures has also been associated with slow waves 17. Even in awake brains, the propagation of isolated sleep- like slow waves causes lapses of attention 18, evidencing the disruptive impact on processing. All this evidence suggests that a detailed understanding of Down states is important, since these silent periods are critical for cortical function and for the cancellation of conscious processing. In the current study, we have used novel analytical approaches for the analysis of Up and Down state transitions and found that Down states are not only silent periods, but have a dynamical structure including “deterministic” and “stochastic” stages. With this we define a cycle for slow oscillations that is compatible with different frequencies of oscillation and that provides a novel framework that connects network properties and cellular mechanisms. 4 4 Network dynamics revealed by input versus output synchronization First, to investigate the network dynamics occurring at different scales in vivo, in vitro and in silico (Fig. 1A), we computed the time evolution of the phase and the synchronization during the oscillatory cycles, i.e. during ongoing slow-wave activity. To determine the dominant phase fluctuations of the signals over time, we calculated the instantaneous phase at each network node via the analytic signal framework19,20 (Supp. Fig. 1) applying the Hilbert transform to broadband signals (multiunit activity (MUA) and local field potential (LFP)) 21. The instantaneous phase was used to quantify the network-level phase synchrony as a function of time 22,23. We considered each of our recording electrodes in the experimental preparations as a node of the network and estimated the instantaneous synchrony among the nodes computing the Kuramoto order parameter (KOP; Methods). To capture the multiscale processes underlying network activity, we studied the synchronization dynamics at two different levels: between neuronal populations in the LFP signal and the MUA, considered as signals mainly made of synaptic input currents and of the output action potentials generated by the neurons, respectively 24. We show the network phase time course with dynamic phase histograms (Fig. 1D). In it, the color 5 5 scale indicates the number of electrodes with a given phase (vertical axis) at a given time (horizontal axis). Phase bundles marked by dark pixels over time indicate many sites with the same phase (in-phase locking; 23). The dynamic phase histograms reveal differences in the synchronization dynamics of the MUA and LFP, supporting the idea that the two components of the the MUA and LFP, supporting the idea that the two components of the electrophysiological activity provide different information about network dynamics. The MUA signals exhibited patterns of synchronization characterized by in-phase locking during the Up states and thus high synchronization (high KOP) (Fig. 1 Da, Ea, Fa). This high synchronization was partially maintained during the early phases of the Down state, but progressively decayed, displaying asynchronous “stochastic” behavior in the later phases with a significant decrease of the KOP. This result was confirmed at a population level, where we computed the average network synchronization associated with the Up and Down states, respectively, as shown in Fig. 1, Dc, Ec and Fc. Network dynamics revealed by input versus output synchronization We found that, on average, the KOP of the MUA is high during the Up states, but significantly decreases on average during the Down states, all in vivo, in vitro and in silico (Fig. 1 Ac Wilcoxon signed-rank test in vivo: Up vs Down p=0.04; Bc in vitro Up vs Down: p=0.02; Cc in silico Up vs Down: p=0.04). To study the dynamics arising in the extracellular voltage resulting from the combination of synaptic activity and neuronal transmembrane processes (i.e. ionic channels), we also studied the network synchronization of the LFP signal. Here we found an intermediate level of synchronization associated with the Up states (KOP mean ± std: in vivo 0.65 ± 0.14, in vitro 0.64 ± 0.10 , in silico 0.62 ± 0.01). Interestingly, in the LFP, the absolute difference between the Up and Down synchronization was not as consistent as in the MUA: no significant difference was found in vivo (Fig. 1Dc), significantly higher synchronization in the Down states in vitro (Fig. 1Ec), and significantly lower synchronization during Down states in silico (Fig. 1Fc). 6 6 We found the explanation to this diversity when looking into the dynamics of Down states: as suggested by the MUA analysis, the LFP clearly expressed a metastable dynamics during these periods. Metastability in the brain is phenomenologically intended as the transient persistence of states different than the natural equilibrium state. In such circumstantial conditions, the elements of the neural system adjust their mode of interaction in order to generate adaptive behavior within a continuously changing environment 25. The metastability in the Down states was characterized by two phases: 1) a sustained period of high synchronization right after the Up-to-Down transition, where the collective phase behavior shows an exponential trend and the KOP is close to 1 (in orange in Fig. 1); and 2) a second period characterized by a loss in network synchronization, in which the collective phase randomly fluctuates between plus and minus pi (in grey in Fig. 1). According to some previously proposed models, a random fluctuation in the second period would be critical to trigger the subsequent Up state 26–31. Given the properties of these two periods, we decided to name these stages “deterministic” and “stochastic” respectively, and we will refer to them like this in the rest of the article. Network dynamics revealed by input versus output synchronization To investigate in depth the metastability of Down states as well as the differences in the synchronization dynamics in the MUA and LFP, we zoomed into single slow oscillation (SO) cycles. The 3D representation of a signal amplitude, phase and synchrony over time provides significant insights into the dynamics of the network activity in this framework. In Fig. 2 we show a comparison of the one-cycle dynamics of MUA and LFP. Independently of the network (in vivo, in vitro, in silico), high values of MUA (i.e. the Up states) correspond to periods of high synchronization. After the end of the Up state, and once the firing rate is collapsed, the synchrony is progressively lost (in orange), KOP decreases and transitions into the “stochastic” phase regime (Fig. 2 Aa, Ba, Ca). Ba, Ca). 7 7 Conversely, in the LFP, the period immediately after the Up-to-Down transition (in orange) is still characterized by a high synchronization with a KOP≈1, highlighting again the presence of the “deterministic” stage at the beginning of the Down state at the local population level. This “deterministic” state then transitions towards a “stochastic” one (in grey), which will eventually lead to the next Up state. Thus, we demonstrate that it is not possible to distinguish the different stages of the metastable quiescent states by looking at the MUA synchronization dynamics alone, stressing the importance of the simultaneous recording and analysis of the LFP and MUA signals,. Characteristic time scales of the metastable Down states The synchronization analysis at the level of neuronal populations in the LFP revealed the existence of a metastable dynamic during the Down states. A “deterministic” and a “stochastic” period were identified, having variable duration, and occurring at different time points of the silent periods, being the “deterministic” stage the one arising right after the Up to Down transition. During this stage, we observed a correlation between the synchronization dynamic and the strength and time course of the after- hyperpolarization mechanism dominating the network dynamic at the beginning of the Down states26. Accordingly, we propose here that it could be possible to infer microscopic phenomena; that is, the evolution of the after-hyperpolarization following Up states 32,33, through the study of macroscopic variables, such as the time constant of network synchronization. To quantitatively characterize these phenomena, we estimated the time constant of the exponential phase growth (τ, Fig. 3A orange area, see Methods) during the “deterministic” stage of the Down states. This estimation was done for cortical networks in vivo, in vitro and in silico. The synchronization analysis at the level of neuronal populations in the LFP revealed the existence of a metastable dynamic during the Down states. A “deterministic” and a “stochastic” period were identified, having variable duration, and occurring at different time points of the silent periods, being the “deterministic” stage the one arising right after the Up to Down transition. During this stage, we observed a correlation between the synchronization dynamic and the strength and time course of the after- the synchronization dynamic and the strength and time course of the after- hyperpolarization mechanism dominating the network dynamic at the beginning of the Down states26. Accordingly, we propose here that it could be possible to infer microscopic phenomena; that is, the evolution of the after-hyperpolarization following Up states 32,33, through the study of macroscopic variables, such as the time constant of network synchronization. To quantitatively characterize these phenomena, we estimated the time constant of the exponential phase growth (τ, Fig. 3A orange area, see Methods) during the “deterministic” stage of the Down states. This estimation was done for cortical networks in vivo, in vitro and in silico. 8 For the in silico one, we modeled a cortical slice using a network of spiking neurons (single neuron parameters and network connectivity as in 2). Briefly, the network model is composed of a two-dimensional 7×20 lattice of interacting cortical modules. Characteristic time scales of the metastable Down states Each 8 module is composed of excitatory (80%) and inhibitory (20%) neurons. Excitatory neurons incorporated activity-dependent after-hyperpolarizing currents underlying spike-frequency adaptation. In order to use the same analytical approach developed for in vitro and in vivo recordings, we transduced the simulated activity (i.e., emitted spikes) in an in silico representation of MUA and LFP (see Methods for details). We modulated the decay time τa of the adaptation current and in doing so, we fixed the mean adaptation current by changing the adaptation strength. The estimation of τ used in vivo, in vitro and in silico was validated by computing the Pearson correlation and the residual sum of squares (RSS) of the regression between the real τa set in the simulated neurons and the one estimated from the in silico LFP synchronization (Fig. 3B, bottom right panel, Pearson correlation=0.97, p<0.001, The estimation of τ used in vivo, in vitro and in silico was validated by computing the Pearson correlation and the residual sum of squares (RSS) of the regression between the real τa set in the simulated neurons and the one estimated from the in silico LFP synchronization (Fig. 3B, bottom right panel, Pearson correlation=0.97, p<0.001, RSS=2.35). The significant linear relationship between the single-neuron parameter τa and the synchronization decay constant τ of the whole network provided an unexpected opportunity to bridge the gap between experimental observations at the macroscale and the microscopic parameters of the system. unexpected opportunity to bridge the gap between experimental observations at the macroscale and the microscopic parameters of the system. Through a multiscale exploration of the in vitro cortical preparations, we verified that the τ estimated at the whole network level is linearly correlated with the one estimated in a single triod of the recording array (Supplementary Fig. 3Ad: Pearson correlation=0.64, p=0.04; Ba: Pearson correlation=0.59, p=0.003; Ca: Pearson correlation=0.56, p=0.001), that is in turn correlated with the exponential decay time constant directly estimated from the LFP trace of a single channel (Supplementary Fig.3Ad, Bd, Cd). Our results are represented here as continuous probability density curves (python seaborn.kdeplot) showing the distribution of Down state duration and τ obtained through a kernel density estimation (KDE).). We thus demonstrate that neuronal properties as the characteristic time constant of the adaptation and associated post-Up after-hyperpolarization currents, may be estimated at network 9 level, highlighting the scalability of such phenomena in both experimental and simulated environments. Characteristic time scales of the metastable Down states When comparing the in vivo, in vitro and in silico joint distributions of the Down state durations and time constants of the network synchronization, τ (Fig. 3C), we found a much larger range of Down state durations and τ in vivo under deep anesthesia (mean ± std: Down duration 18.82 ± 7.76s, τ duration 2.05±1.52s) with respect to the in vitro (mean ± std: Down duration 2.89 ± 1.28s, τ duration 0.95±0.61s) and in silico cases (Down duration 9.41 ± 3.94s, τ duration 1.35±0.1321s). To be able to explain the Down state metastability in different systems at various scales, we performed a linear regression analysis between the Down duration and τ. Our results show that a significant correlation between the two parameters is present when the metastable quiescent dynamics are dominated by the “deterministic” period, which is more common in the control conditions in vitro, as pointed out in Fig. 1Eb. For in vitro cortical networks, the relationship between τ and Down states (0.51–8.42 s) was of R2 = 0.34 (RSS = 681, 𝑃= 10−15; Fig. 3D). To be able to explain the Down state metastability in different systems at various scales, we performed a linear regression analysis between the Down duration and τ. The case of Down states in vivo included a much larger range of Down state durations (0.76–43.22 s) and the tau was significantly larger than in vitro, with an average value of 2.36 +/- 1.53 s. Even when in vivo Down states were characterized by both “deterministic” and “stochastic” stages (Fig. 1Db), the “deterministic” stage still ruled the Down states to some extent, although with a slightly lower correlation with respect to the in vitro case (R2=0.29, RSS=9e+03, p=0.01). This positive relationship was almost non-existent in the in silico cortical network model (R2=0.14, RSS=2e+03, p=0.11), where all Down states are truly metastable, suggesting that the correlation between Down state duration and τ may be used as a relevant variable in the study of the cellular and network mechanisms dominating the cortical off-period dynamics. 10 metastability We have shown that different cortical networks express different metastability in the Down states, conveyed by the synchronization dynamics of neuronal pools in the local field potential. By examining the characteristics of each network, we systematically observed that the linear regression between the mean values of KOP in each Down state and the Down state duration revealed a significant negative correlation (Fig. 4): shorter Down states are more synchronized than longer ones. Thus, the KOP is high when the Down states have short durations; that is, when the silent state is dominated by the afterhyperpolarization dynamics (“deterministic” stage), while the KOP decreases when the Down states have longer durations and manifest metastability; that is, when the network has the time to desynchronize and shows “stochastic” fluctuations (stochastic stage), as observed in all networks in vivo, in vitro and in silico under control conditions. Still, we will show below that there can be exceptions to this rule, with specific experimental manipulations that enhance afterhyperpolarization. In light of these results, we speculate that the metastability of the Down states is a state- dependent phenomena that varies with the dynamical conditions independently of the network structure and observation scale, which is reflected in the overall degree of network synchronization. Exogenous modulation of Down state’s metastability and tuning of system dynamics To explore a wider space of cortical dynamics and to assess metastability quantitatively, we introduced here some experimental manipulations that vary the duration of Down states and thus, the cortical dynamics. We did this in the three networks: in vivo, in vitro and in silico. 11 11 In vivo, we analyzed the emergent cortical dynamics under three different levels of anesthesia (Fig. 5), which provided us with a significant modulation of the SO frequency (Fig. 5C) and Down state duration, as we have described in 34. The three levels of anesthesia expressed different Down state durations (Down duration mean ± std Deep anesthesia: 18.82 ± 7.76s; Mid anesthesia level: 5.35 ± 1.02s; Light anesthesia level: 1.81 ± 1.01s). This experimental manipulation resulted in significantly different time constants of synchronization (Fig. 5B), such that deep anesthesia had the longest and light anesthesia the shortest time constant values and Down state durations. The correlation between τ and Down state duration significantly increased when moving from deep anesthesia to lighter anesthesia states, , the light levels of anesthesia expressing the highest correlation: shortest Down states of high regularity 35 correspond to a cycle sharply controlled by the “deterministic” stage of the Down state. In vitro we used two experimental manipulations of the Down state duration. First, the modulation of slow oscillatory activity by using constant electric fields of different polarities (+3V/m and −3V/m). Exogenous electric fields vary the neuronal membrane potential (0.5 mV per V/m, 36) causing a net depolarization/hyperpolarization in pyramidal neurons. The variation of direct current (DC) causes an exponential modulation of the slow oscillatory frequency in cortical slices that we have described in detail in 37,38 (Fig. 6A). With positive DC stimulation, we obtained a decrease in Down state duration and an increase in oscillatory frequency, while negative DC stimulation induced an increase in Down duration and a decrease in oscillatory frequency with respect to the control condition (Fig. 6B-C, Down duration mean ± std Control: 2.89 ± 1.28s; Positive DC: 1.44 ± 0.46s; Negative DC: 6.36 ± 3.11). The linear regression analysis reveals a lack of significance in the τ versus Down state duration for control and DC− (Fig. 6D). However, DC+, which corresponds to the highest frequency of slow oscillations, displayed an R2 of 0.39 (p=0.03), being a situation with short and mostly In vivo, we analyzed the emergent cortical dynamics under three different levels of anesthesia (Fig. Exogenous modulation of Down state’s metastability and tuning of system dynamics 5), which provided us with a significant modulation of the SO frequency (Fig. 5C) and Down state duration, as we have described in 34. The three levels of anesthesia expressed different Down state durations (Down duration mean ± std Deep anesthesia: 18.82 ± 7.76s; Mid anesthesia level: 5.35 ± 1.02s; Light anesthesia level: 1.81 ± 1.01s). This experimental manipulation resulted in significantly different time constants of synchronization (Fig. 5B), such that deep anesthesia had the longest and light anesthesia the shortest time constant values and Down state durations. The correlation between τ and Down state duration significantly increased when moving from deep anesthesia to lighter anesthesia states, , the light levels of anesthesia expressing the highest correlation: shortest Down states of high regularity 35 correspond to a cycle sharply controlled by the “deterministic” stage of the Down state. In vitro we used two experimental manipulations of the Down state duration. First, the modulation of slow oscillatory activity by using constant electric fields of different polarities (+3V/m and −3V/m). Exogenous electric fields vary the neuronal membrane potential (0.5 mV per V/m, 36) causing a net depolarization/hyperpolarization in pyramidal neurons. The variation of direct current (DC) causes an exponential modulation of the slow oscillatory frequency in cortical slices that we have described in detail in 37,38 (Fig. 6A). With positive DC stimulation, we obtained a decrease in Down state duration and an increase in oscillatory frequency, while negative DC stimulation induced an increase in Down duration and a decrease in oscillatory frequency with respect to the control condition (Fig. 6B-C, Down duration mean ± std Control: 2.89 ± 1.28s; Positive DC: 1.44 ± 0.46s; Negative DC: 6.36 ± 3.11). The linear regression analysis reveals a lack of significance in the τ versus Down state duration for control and DC− (Fig. 6D). However, DC+, which corresponds to the highest frequency of slow oscillations, displayed an R2 of 0.39 (p=0.03), being a situation with short and mostly 12 “deterministic” Down states that tightly control the triggering of the following Up state and reminiscent of light anesthesia. But, is it that simple? Are shorter Down states invariably and tightly bound to a highly “deterministic” interval? Our second manipulation in vitro shows that this physiological relationship, which dominates in control states, can be altered in non-physiological conditions. Exogenous modulation of Down state’s metastability and tuning of system dynamics In this case, departing from the control slow oscillation at a frequency of 0.36 +/−0.09 Hz, we blocked GABAA receptors by means of bicuculine methiodide (BMI). The blockade of fast inhibition led to highly synchronized Up states of lower frequency, which were followed by increasingly prominent afterhyperpolarizations (Fig. 6E), as we have previously described 33,39. The τ versus Down state regression analysis (Fig. 6H) revealed a highly significant relation for 1µM BMI, despite corresponding to the longest Down states. This finding reveals that it is not simply the duration of the Down states, but the mechanism underlying this duration that is critical: when inhibition is blocked, the afterhypolarization increases in amplitude (Fig. 6E), corresponding to a larger intracellular afterhyperpolarization attributed to potassium currents 33. This is highly suggestive of the mechanism bridging cellular and network properties during Down states. corresponding to a larger intracellular afterhyperpolarization attributed to potassium currents 33. This is highly suggestive of the mechanism bridging cellular and network properties during Down states. Finally, in silico we modulated the Down state duration simulating the positive and negative DC stimulation (Fig. 7A-B, Down duration mean ± std Control: 9.41 ± 3.94s; Positive DC: 3.06 ± 1.48s; Negative DC: 13.7 ± 8.79s). As shown for the experiments, the frequency of the oscillations increased with positive DC (and decreased with negative DC) (Fig. 7C). The relation between τ and Down state duration was absent for negative DC (Fig. 7D), with long Down states and thus longer “stochastic” periods. The strongest relationship was, as for the data, for positive DC (R2=0.64; p<0.001), thus for shorter Down states dominated by the “deterministic” period. 13 13 Discussion The mechanisms underlying slow oscillations, or the alternation between Up and Down states, have been a matter of debate since the seminal work of Steriade et al. 42 which characterized this activity pattern. That a relatively simple spontaneous rhythm is so difficult to fully understand is mainly due to two factors: recurrent networks and close- circle processes, which led to a causality dilemma. In the case of Up and Down states, the properties of the Up state (e.g. activity pattern, synchrony) determine the subsequent Down state, and vice versa. This being an emergent pattern from a complex network, where all sort of elements from ionic currents to excitation/inhibition or to synaptic properties have an impact on the resulting rhythm, there has been space for various experimental, computational and clinical studies investigating this intriguing and prevalent oscillation (for a review 1,43,44). In order to clarify this picture here we have focused on the less-studied silent period, the Down state, and found that it has more structure than a simple “absence of activity”. The mechanisms underlying slow oscillations, or the alternation between Up and Down states, have been a matter of debate since the seminal work of Steriade et al. 42 which characterized this activity pattern. That a relatively simple spontaneous rhythm is so difficult to fully understand is mainly due to two factors: recurrent networks and close- circle processes, which led to a causality dilemma. In the case of Up and Down states, the properties of the Up state (e.g. activity pattern, synchrony) determine the subsequent Down state, and vice versa. This being an emergent pattern from a complex network, where all sort of elements from ionic currents to excitation/inhibition or to synaptic properties have an impact on the resulting rhythm, there has been space for various experimental, computational and clinical studies investigating this intriguing and prevalent oscillation (for a review 1,43,44). In order to clarify this picture here we have focused on the less-studied silent period, the Down state, and found that it has more structure than a simple “absence of activity”. A global picture of slow oscillations and Down state metastability across states In this final section, we integrate the concepts presented along these results. Through the analysis of the cortical network dynamics by means of phase and synchronization of MUA and LFP, we found that Down states are more than silent periods in between Up states. They have a structure, which is defined by its metastability and that is at the base of the emergent oscillatory pattern. We have differentiated two phases in the Down states (Fig. 8A): a “deterministic” phase, with high synchronization (KOP) and a time course determined by the exponential growth of the phase (τ) and a “stochastic” one, with low synchronization (Figs 1 and 2). In sum, Fig. 8A represents the oscillatory cycle between Up and Down states that our data and analysis support: the end of the Up state is highly synchronous as commonly reported (e.g. 40,41), the network goes into a silent, synchronized and “deterministic” period, synchrony starts decreasing and enters a “stochastic” stage of low synchronization. The system does not always go through all these stages, and very short Down states are commonly dominated by the “deterministic” stage, thus the inverse relation between KOP and Down state duration (Fig. 4), while longer Down states spend more time in “stochastic” mode. Throughout this study we have used the linear regression of the τ versus Down states duration as a measure of how tightly driven was the duration of the Down states by the time constant of the phase and thus, by the synchronized or “deterministic” period. In order to cover a wide parameter space of slow oscillatory cycles, not only we have used three networks (in vivo, in vitro and in silico) but also explored experimental manipulations of these cycles. In Fig. 8B we integrate all these results in the same axes, namely the “deterministic” fraction of Down states (“deterministic”/“deterministic+stochastic”) against the regression values displayed along the figures, providing a final picture that depicts the state-dependence of the (“deterministic”/“deterministic+stochastic”) against the regression values displayed along the figures, providing a final picture that depicts the state-dependence of the 14 metastability of Down states and that connects the mechanisms with the network emergent properties. metastability of Down states and that connects the mechanisms with the network emergent properties. Metastability in the Down states The synchronization analysis at the level of neuronal pools in the local field potential signal (LFP) revealed the metastability of the cortical Down states. This metastability is characterized by two different periods: 1) a highly synchronized one following the Up- to-Down transition that we have called the “deterministic” state, with a time course that is defined by the exponential decay of the phase, and 2) a desynchronized period that we have referred to as “stochastic”. 15 The identification of these periods and the possibility of inferring local and global features of network dynamics through the analytical approach used here, illustrates the value of the combined recording and analysis of LFPs and spikes, which offer insights that cannot be obtained at present by examining spikes alone45 The identification of these periods and the possibility of inferring local and global features of network dynamics through the analytical approach used here, illustrates the value of the combined recording and analysis of LFPs and spikes, which offer insights that cannot be obtained at present by examining spikes alone45 A new proposal for an oscillatory cycle These newly identified periods within Down states give rise to a detailed Up and Down state cycle (Fig. 8A), closely connected to the underlying cellular and network mechanisms, revealing the possibility of inferring microscopic features (i.e. time scales of potassium currents causing adaptation) in network dynamics by studying macroscopic variables such as collective synchronization. Our computational model 2 has further proven this extreme, where the adaptation time constant has been used to validate the τ of the population phase during the “deterministic” period (Fig. 3B). Further, the newly proposed stages of the oscillatory cycle can be used to predict the responsiveness of the network in different time windows of the Down states, the period of refractoriness to the generation of a new Up state 32, or the probability of inducing a new Up state as a response to an input. 16 Moreover, this more detailed cycle conciliates existing models of the mechanisms underlying Up and Down states. We propose that there is an inescapable Down state that is the “deterministic” period, which is dependent on the activity on the precedent Up state 26,29,46, probably due to the same mechanism that terminated the Up state: slow hyperpolarizing potassium currents of a different kind (calcium and sodium dependent, 26,33 ATP-dependent K+ current 47 or GABAB receptor-mediated 48 are compatible in time course and properties). For example, intracellular recordings have demonstrated that the enhancement of this afterhyperpolarization elongates the Down states, while the larger membrane conductance due to channel opening contributes to 16 the refractoriness 49. Interestingly, with extracellular recordings, here we have demonstrated that for larger posthyperpolarizations, the “deterministic” period can also be elongated (Fig. 6E, F, H). Once this “deterministic” refractory period is terminated, the network can trigger a new Up state if there is enough excitability or inputs to the system. We have shown that this is the case under experimental manipulations such as light anesthesia (Fig. 5A) or positive DC injection in vitro (Fig. 6A) or in silico (Fig. 7A). Under those conditions, the Down state is mostly formed by the “deterministic” period, in association with the higher slow oscillatory frequency. However, under control conditions, or those with artificially decreased excitability (deep anesthesia, or negative DC) the Down state is longer than the “deterministic” period, and the system enters the “stochastic” period. A new proposal for an oscillatory cycle Computer models of Up and Down states have proposed the initiation of Up states by the stochastic summation of miniature synaptic potentials 50 or by spontaneous firing of layer 5 neurons 26, that build up the activity through recurrent connections and trigger a new Up state. The new cycle that we propose here (Fig. 8A) can explain the variety of slow oscillatory frequencies (and thus, of Down state durations) that can be expressed by the system, either spontaneously or under experimental manipulations 28,34,38,39,51, some of which have been represented with respect to the Down state periods in Fig. 8B. Down states versus “Off-periods” Down states are spontaneous silent periods, however, cortical stimulation evokes a response that consists of a limited activation followed by an “off-period” or period of silence. These “off-periods” are prominent in states of slow oscillatory activity 14, but also in pathological conditions such as unresponsive wakefulness syndrome and even in perilesional regions 16. When “off-periods” occur, they break the causal relations 17 17 across areas and decrease the complexity of the cortical network, a clinical validated measure of consciousness levels 52,53. “Off-periods” are periods of hyperpolarization and only they break down complexity in brain states when there is a state with spontaneous Down states (e.g., slow wave sleep, pathological slow waves), sharing properties with them. We speculate that the metastability of the cortical Down states is closely related to the phases observed in “off-periods”, with a “deterministic” stage associated with the peak of hyperpolarization breaking causality 14 and the “stochastic” one associated with the period recovery of network interactions, highlighting the multiscale implications of Down state metastability 54. Experimental methods Extracellular local field potential (LFP) recordings from different preparations were used. The in vivo data were recorded with a 32-channel electrode array placed on the cortical surface of deeply anesthetized mice (N=5, Fig. 1A) (see 34for details). The in vitro recordings were obtained from a 16-channel electrode array placed on the top of slices of ferret visual cortex (N=6 slices from two ferrets, Fig. 1B, details in2). All procedures were approved by the Ethics Committee at the Hospital Clinic of Barcelona and were carried out to the standards laid down in Spanish regulatory laws (BOE 34/11370-421, 2013) and in the European Union directive 2010/63/EU. Computation of the phase signal The instantaneous phase at each network node was computed via the analytic signal framework 22,55. This approach entails transforming a real-valued time series into a complex phasor, whose modulus (length) and argument (angle) in the complex plane 18 represent the signal instantaneous amplitude and phase, respectively (Fig. 1D, E). Given the time series 𝑥𝑖(𝑡), with 𝑖 corresponding to the recording channel, its analytic representation is given by: represent the signal instantaneous amplitude and phase, respectively (Fig. 1D, E). Given the time series 𝑥𝑖(𝑡), with 𝑖 corresponding to the recording channel, its analytic representation is given by: (𝑡) = 𝑥𝑖(𝑡) + 𝑗𝐻[𝑥𝑖(𝑡)] 𝑧𝑖(𝑡) = 𝑥𝑖(𝑡) + 𝑗𝐻[𝑥𝑖(𝑡)] (𝑡) = 𝑥𝑖(𝑡) + 𝑗𝐻[𝑥𝑖(𝑡)] where 𝐻[𝑥𝑖(𝑡)] denotes the Hilbert transform of the signal 𝑥𝑖(𝑡) and the instantaneous phase of the time series can be obtained as follows (Fig. 1D,1E): where 𝐻[𝑥𝑖(𝑡)] denotes the Hilbert transform of the signal 𝑥𝑖(𝑡) and the instantaneous phase of the time series can be obtained as follows (Fig. 1D,1E): 𝜑𝑖(𝑡) = tan−1 (Im[𝑧𝑖(𝑡)] Re[𝑧𝑖(𝑡)]) Similar to 21, in order to preserve the spectral content of the signals and obtain a representation that captures the dominant phase fluctuations of the signals over time, avoiding distortions and artifacts introduced by filtering, we applied the Hilbert transform to broadband signals (MUA and LFP). We did not resort to any narrow-band filtering of MUA and LFP on purpose. In this way we aimed at having an unbiased approach in determining the time scales associated to the adaptation-driven recovery phase, i.e., the one characterizing the determinist stage of the Down states. This comes with a caveat in considering as reliable the phase 𝜑𝑖(𝑡) of the unfiltered signals when they differ from smooth sinusoidal waveforms as in the case of the relatively fast MUA Up-Down transitions. Network synchronization The instantaneous phase computed as explained above was used to quantify the network-level phase synchrony as a function of time 19,23. According to Strogatz 56 and Acebrón et al.57, we can consider our networks as systems composed of weakly coupled, nearly identical, interacting limit-cycle oscillators, where all the oscillators 19 exert a phase-dependent influence on the others. When the frequencies of the oscillators are too diverse, they are not able to synchronize and the system behaves incoherently, whereas when the coupling is strong enough, the oscillators behave in a synchronized way 58. Kuramoto proposed a mathematical model to outline network synchronization59, which is suited to the mean field approach, where the dynamics of the neuronal population is measured by the macroscopic complex order parameter whose modulus measures the phase coherence of the population over time. The values 𝑟= 1 and 𝑟= 0 describe the limits in which all oscillators are either phase locked or move incoherently, respectively. We considered each of our recording electrodes in the experimental preparations as a node of the network, and estimated the instantaneous synchrony among the nodes computing the Kuramoto order parameter given by: 𝑟(𝑡) = 1 𝑛\|∑𝑒−𝑖𝜑𝑗(𝑡) 𝑛 𝑗=1 \| where 𝑛 is the number of recording channels and 𝜑𝑗(𝑡) is the instantaneous phase of each of them. The same was done in the simulated data, considering each module of the model as a node of the network, and estimating the instantaneous synchrony among them. Periods of high phase synchrony (i.e., low phase difference) corresponded to values of 𝑟 close to 1, while periods of low network synchronization corresponded to values of 𝑟 close to 0. To obtain a visual representation of network synchronization over time, we imaged the instantaneous phases versus time using dynamic phase histograms as in Yang et al.23 (Fig. 4). Dynamical parameters estimation We estimated the duration of the Down states in each SO cycle from the MUA as in 7. Then, we estimated the time constant of the LFP synchronization dynamics in all the experimental conditions and in the simulated data. To do so, we extracted from the 20 dynamic phase histograms (Figs 3Ab, Bb, Cb) the phase related to the maximum counts over time (Fig. 3A). Using this time series, we fitted an exponential curve 𝑓(𝑡) = 𝐴(1 −𝑒−𝑡 𝜏) + 𝑐 𝑓(𝑡) = 𝐴(1 −𝑒−𝑡 𝜏) + 𝑐 𝑓(𝑡) = 𝐴(1 −𝑒−𝑡 𝜏) + 𝑐 in the time periods right after each Up-to-Down transition (Fig. 3A, orange area) and extracted the variable 𝜏 which is the characteristic time constant of the exponential (computed in Python using scipy.optimize.curve_fit). Note that in the simulated data the 𝜏 parameter is known, therefore we used the simulation to validate our fitting method, comparing the estimated and the actual 𝜏 (Fig. 3B, bottom right). According to this validation, and in order to avoid high fitting errors, we included here only the estimations in which one standard deviation errors on the parameters’ estimation is lower than 1. in the time periods right after each Up-to-Down transition (Fig. 3A, orange area) and extracted the variable 𝜏 which is the characteristic time constant of the exponential (computed in Python using scipy.optimize.curve_fit). Note that in the simulated data the 𝜏 parameter is known, therefore we used the simulation to validate our fitting method, comparing the estimated and the actual 𝜏 (Fig. 3B, bottom right). According to this validation, and in order to avoid high fitting errors, we included here only the estimations in which one standard deviation errors on the parameters’ estimation is lower than 1. Computational methods: spiking neuron networks We modeled a cortical slice using a spiking neural network; details about single neuron parameters and network connectivity can be found in 2. Briefly, the network model is composed of a two-dimensional 7 × 20 lattice of interacting cortical modules. Each module is a subnetwork of two homogenous populations of 502 excitatory and 126 inhibitory leaky integrate-and-fire (LIF) neurons. The membrane potential 𝑉𝑖(𝑡) of the 𝑖-th neuron evolves according to 𝑉𝑖̇ = −𝑉𝑖/𝜏+ 𝐼𝑖−𝑎𝑖, where 𝐼𝑖(𝑡) is the input synaptic current and 𝑎(𝑡) is an activity-dependent hyperpolarizing current present only in excitatory neurons, responsible for spike-frequency adaptation. The decay constant 𝜏 of the membrane potential is 20 ms and 10 ms for excitatory and inhibitory neurons, respectively. The synaptic current is a linear combination of the spikes emitted by the presynaptic neurons: 𝐼𝑖(𝑡) = ∑𝐽_𝑖𝑘 𝑘 𝛿(𝑡−𝑡𝑘−𝑑𝑖𝑘) + 𝐼𝑒𝑥𝑡, with additional contribution 𝐼𝑒𝑥𝑡 given by the spikes emitted by external neurons, modeled as Poisson processes with spike rate 𝐶𝑒𝑥𝑡𝜈𝑒𝑥𝑡 that is 1466Hz for the “foreground”, for the “background” 1511 21 Hz and for the inhibitory 2100 Hz. Each spike emitted at time 𝑡𝑘 by the 𝑘-th presynaptic neuron contributes with an instantaneous current 𝐽𝑖𝑘 (synaptic efficacy). Spikes were delivered with a transmission delay 𝑑𝑖𝑘. The current 𝑎𝑖(𝑡) follows the first-order dynamics 𝜏𝑎𝑎𝑖̇ = −𝑎𝑖+ 𝑔𝑎𝜏𝑎∑𝛿(𝑡−𝑡𝑖) 𝑖 . In the absence of emitted spike by the 𝑖-th neuron, the adaptation current decay with a time constant 𝜏𝑎= 1 s. The adaptation strength 𝑔𝑎= 0.06 mV/ms determines the sudden increase in 𝑎𝑖 when a spike is emitted. We also modulated the decay time 𝜏𝑎 of the adaptation current by setting sequentially the values {1,2,2.5,3,3.5,4} s every 100 s. In doing so, we fixed the mean adaptation current by changing the adaptation strength while keeping constant the product 𝜏𝑎𝑔𝑎. Neurons emit a spike when 𝑉(𝑡) crosses the threshold 𝜃= 20 mV, after which the potential reset to 15 mV during an absolute refractory period of 2 ms and 1 ms for excitatory and inhibitory neurons respectively. The connectivity (i.e. the probability to have two neurons synaptically coupled) is determined by the position of the postsynaptic module in the lattice, and by the relative distance with the presynaptic population in the lattice 2. The connectivity is not homogeneous in space, aiming at modeling a strip of more excitable assemblies representing layer 5 neurons. Numerical simulations of the model network were performed in NEST 60. Acknowledgements We want to acknowledge former experimenters from the Sanchez-Vives laboratory that have contributed to the laboratory data collection, many of which are available along with previous publications. We also thank Tony Donegan for his help with languaje edition. This work has received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 945539 (Human Brain Project SGA3) to MVSV and MM. It has also been funded by by the Spanish Ministry of Science and Innovation through the MICINN under grant BFU2017-85048-R to MVSV. Computational methods: spiking neuron networks In order to use the same analytical approach developed for in vitro and in vivo recordings, we transduced the simulated activity (i.e., emitted spikes) in an in silico representation of MUA and LFP. The MUA is computed starting from the firing rate of the foreground excitatory neurons 𝜈𝑓𝑔(𝑡) and adding a proper white noise 𝜁(𝑡) with 〈𝜁(𝑡)〉= 0.3 and 〈𝜁2(𝑡)〉= 3 in order to reproduce the background fluctuations observed in the experiments. LFP is computed as a linear combination of the average (across neurons) incoming currents 𝑎𝑓𝑔= 〈𝑎𝑖(𝑡)〉 (adaptation-related) and 𝐼𝑓𝑔= 〈𝐼𝑖(𝑡)〉 to the foreground excitatory neurons in a cortical module, while adding a noise 𝜂(𝑡) 22 with zero mean and variance 〈𝜂2(𝑡)〉= 〈0.25(5𝑎𝑓𝑔(𝑡) −𝐼𝑓𝑔(𝑡))2〉, resulting in the following equation: 𝐿𝐹𝑃= 5𝑎𝑓𝑔−𝐼𝑓𝑔+ 𝜂. with zero mean and variance 〈𝜂2(𝑡)〉= 〈0.25(5𝑎𝑓𝑔(𝑡) −𝐼𝑓𝑔(𝑡))2〉, resulting in the following equation: 𝐿𝐹𝑃= 5𝑎𝑓𝑔−𝐼𝑓𝑔+ 𝜂. This network configuration is the one used to obtain in silico control simulations (N=5). We operated several changes in order to investigate how different microscopic and mesoscopic features of the system affect the properties of the spontaneous slow-wave activity. To model the effects of a stimulation by a homogeneous DC field applied to a cortical slice, the synaptic input current was modulated by adding an extra DC current 𝐼𝐷𝐶= {−1, −0.5, −0.25, +2.5, +5, +10, +20} 𝑝𝐴 to make the membrane potential more or less polarized (DC- and DC+ simulations37. Code availability Numerical simulations of the model network were performed in NEST (2.12.0. DOI: 10.5281/zenodo.259534) on the high-performance computing facility hosted by the Istituto Superiore di Sanità. Python code of the simulation is freely available in EBRAINS 23 Knowledge Graph where all the parameters and information of the network design are fully accessible. 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Orange and grey shades represent the “deterministic” and “stochastic” periods of the Down states, respectively. Boxplots of the average KOP during Up and Down states of the MUA in orange and the LFP in purple: in vivo (Dc N 5 mice n 75 Down states) in vitro (Ec Figure 1. Dynamics phase histograms and state related synchronization in in vivo, in vitro and in silico cortical networks. Schematic representation of the in vivo (A), in vitro (B) and in silico (C) cortical networks, the orange traces represent the extracellular Figure 1. Dynamics phase histograms and state related synchronization in in vivo, in vitro and in silico cortical networks. Schematic representation of the in vivo (A), in gure 1. Dynamics phase histograms and state related synchronization in in vivo, g y p g y in vitro and in silico cortical networks. Schematic representation of the in vivo (A), in vitro (B) and in silico (C) cortical networks, the orange traces represent the extracellular local field potential recorded by one electrode or one node of the network (orange circle). Representation of dynamic phase histograms, the corresponding Kuramoto order parameter (KOP) and cortical activity for MUA signals in vivo (Da), in vitro (Ea) and in silico (Fa), and LFP signals in vivo (Db), in vitro (Eb) and in silico (Fb). Orange and grey shades represent the “deterministic” and “stochastic” periods of the Down states, respectively. Boxplots of the average KOP during Up and Down states of the MUA in orange and the LFP in purple: in vivo (Dc, N=5 mice, n=75 Down states), in vitro (Ec, N=6 slices, n=478 Down states) and in silico (N=5 simulations, n=113 Down states). in vitro and in silico cortical networks. Neurophysiol. 104, 1314–1324 (2010). & Sejnowski, T. J. 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Representation of dynamic phase histograms, the corresponding Kuramoto order parameter (KOP) and cortical activity for MUA signals in vivo (Da), in vitro (Ea) and in silico (Fa), and LFP signals in vivo (Db), in vitro (Eb) and in silico (Fb). Orange and grey shades represent the “deterministic” and “stochastic” periods of the Down states, respectively. Boxplots of the average KOP during Up and Down states of the MUA in orange and the LFP in purple: in vivo (Dc, N=5 mice, n=75 Down states), in vitro (Ec, N=6 slices, n=478 Down states) and in silico (N=5 simulations, n=113 Down states). Figure 1. Neurophysiol. 104, 1314–1324 (2010). Schematic representation of the in vivo (A), in vitro (B) and in silico (C) cortical networks, the orange traces represent the extracellular local field potential recorded by one electrode or one node of the network (orange circle). Representation of dynamic phase histograms, the corresponding Kuramoto order parameter (KOP) and cortical activity for MUA signals in vivo (Da), in vitro (Ea) and in silico (Fa), and LFP signals in vivo (Db), in vitro (Eb) and in silico (Fb). Orange and grey shades represent the “deterministic” and “stochastic” periods of the Down states, respectively. Boxplots of the average KOP during Up and Down states of the MUA in orange and the LFP in purple: in vivo (Dc, N=5 mice, n=75 Down states), in vitro (Ec, N=6 slices, n=478 Down states) and in silico (N=5 simulations, n=113 Down states). 28 Figure 2. MUA vs LFP synchronization dynamics. A) In vivo 3D representation of the phase, amplitude, and Kuramoto order parameter (KOP) corresponding to the slow oscillation cycle depicted in the 2D insert in MUA (Aa) and LFP (Ab). B) In vitro 3D representation of the phase, amplitude, and KOP corresponding to the SO cycle depicted in the 2D insert in MUA (Ba) and LFP (Bb). C) In silico 3D representation of the phase, amplitude, and KOP corresponding to the slow oscillation cycle depicted in the 2D insert in MUA (Ca) and LFP (Cb). The points are colored according to time; grey shades represent the projections of the cloud on each plane. Figure 2. MUA vs LFP synchronization dynamics. A) In vivo 3D representation of the phase, amplitude, and Kuramoto order parameter (KOP) corresponding to the slow oscillation cycle depicted in the 2D insert in MUA (Aa) and LFP (Ab). B) In vitro 3D representation of the phase, amplitude, and KOP corresponding to the SO cycle depicted in the 2D insert in MUA (Ba) and LFP (Bb). C) In silico 3D representation of the phase, amplitude, and KOP corresponding to the slow oscillation cycle depicted in the 2D insert in MUA (Ca) and LFP (Cb). The points are colored according to time; grey shades represent the projections of the cloud on each plane. 29 Figure 3. Metastability of the Down state and its characteristic time scale. Neurophysiol. 104, 1314–1324 (2010). A) τ estimation method: bottom panel shows the LFP extracted from one module of a simulated network (in silico); upper panel shows the corresponding time evolution of the maximum of the LFP phase histogram; the grey area corresponds to the “deterministic” stage of the Down state where we estimated the τ, while the pink area corresponds to the “stochastic” stage. B) Example of exponential fitting for τ estimation in vivo (top left), in vitro (bottom left), and in silico (top right); regression plot of the real τ versus the estimated τ in silico with residual sum of squares (RSS) of 1.46. C) kernel density estimation (KDE) of Down durations vs τ in vivo, in vitro and in silico. D) Linear correlation between Down duration and τ in vivo (RSS=9e+03), in vitro (RSS=681) and in silico (RSS=2e+03). Figure 3. Metastability of the Down state and its characteristic time scale. A) τ g y ) estimation method: bottom panel shows the LFP extracted from one module of a simulated network (in silico); upper panel shows the corresponding time evolution of the maximum of the LFP phase histogram; the grey area corresponds to the “deterministic” stage of the Down state where we estimated the τ, while the pink area corresponds to the “stochastic” stage. B) Example of exponential fitting for τ estimation in vivo (top left), in vitro (bottom left), and in silico (top right); regression plot of the real τ versus the estimated τ in silico with residual sum of squares (RSS) of 1.46. C) kernel density estimation (KDE) of Down durations vs τ in vivo, in vitro and in silico. D) Linear correlation between Down duration and τ in vivo (RSS=9e+03), in vitro (RSS=681) and in silico (RSS=2e+03). g y estimation method: bottom panel shows the LFP extracted from one module of a simulated network (in silico); upper panel shows the corresponding time evolution of the maximum of the LFP phase histogram; the grey area corresponds to the “deterministic” stage of the Down state where we estimated the τ, while the pink area corresponds to the “stochastic” stage. B) Example of exponential fitting for τ estimation in vivo (top left), in vitro (bottom left), and in silico (top right); regression plot of the real τ versus the estimated τ in silico with residual sum of squares (RSS) of 1.46. Neurophysiol. 104, 1314–1324 (2010). C) kernel density estimation (KDE) of Down durations vs τ in vivo, in vitro and in silico. D) Linear correlation between Down duration and τ in vivo (RSS=9e+03), in vitro (RSS=681) and in silico (RSS=2e+03). 30 Figure 4. Activity modulation effect on Down states synchronization. Linear correlation between the mean Kuramoto order parameter (KOP) of the LFP during the Down states, and the Down state duration A) in vivo (R2=-0.45, p=1e-29), B) in vitro (R2=-0.1, p=0.02) and C) in silico (R2=-0.9, p=1e-93). Figure 4. Activity modulation effect on Down states synchronization. Linear correlation between the mean Kuramoto order parameter (KOP) of the LFP during the Down states, and the Down state duration A) in vivo (R2=-0.45, p=1e-29), B) in vitro (R2=-0.1, p=0.02) and C) in silico (R2=-0.9, p=1e-93). 31 Figure 5 Activity modulation effect on network dynamics. A) Traces representing the LFP recording from one electrode in vivo at Deep, Mid and Light anesthesia level (adapted from Dasilva et al.34). B) kernel density estimation (KDE) of Down durations vs τ in vivo under Deep, Mid and Light anesthesia levels. C) boxplot of the average frequency of the slow oscillations for each subject (N=5) under Light, Mid and Deep anesthesia (Friedman test p=0.002, Wilcoxon signed-rank test: p<0.05). D) Linear correlation between τ and Down duration in vivo under Deep (RSS=9e+03), Mid (RSS=1e+03) and Light anesthesia (RSS=114). Figure 5 Activity modulation effect on network dynamics. A) Traces representing the LFP recording from one electrode in vivo at Deep, Mid and Light anesthesia level (adapted from Dasilva et al.34). B) kernel density estimation (KDE) of Down durations vs τ in vivo under Deep, Mid and Light anesthesia levels. C) boxplot of the average frequency of the slow oscillations for each subject (N=5) under Light, Mid and Deep anesthesia (Friedman test p=0.002, Wilcoxon signed-rank test: p<0.05). D) Linear correlation between τ and Down duration in vivo under Deep (RSS=9e+03), Mid (RSS=1e+03) and Light anesthesia (RSS=114). 32 Figure 6. In vitro activity modulation effect on network dynamics. A) Traces representing the LFP recording from one electrode in vitro under negative DC stimulation, control conditions and positive DC stimulation. B) Kernel density estimation (KDE) of Down durations vs τ in vitro with negative DC, control conditions and positive DC. C) boxplot of the average frequency of slow oscillations for each slice (N=6) under negative DC stimulation, control conditions and positive DC stimulation (Friedman test p=0.014, p<0.05). Neurophysiol. 104, 1314–1324 (2010). D) Linear correlation between Down duration and τ in vitro under negative DC stimulation (RSS=201), control conditions (RSS=19.9) and positive DC stimulation (RSS=3.6). E) Traces representing the LFP recording from one electrode in vitro under control conditions and with bath application of bicuculline (BMI) at two different concentrations: 0.6uM and 1uM. F) KDE of Down durations vs τ in vitro under control conditions and with BMI at 0.6uM and 1uM. G) boxplot of the average frequency of slow oscillations for each slice (N=6) under control conditions and with BMI at 0.6uM and 1uM (Friedman test p=0.014, p<0.05). H) Linear correlation b t D d ti d i it d t l diti (RSS 88 6) ith BMI Figure 6. In vitro activity modulation effect on network dynamics. A) Traces representing the LFP recording from one electrode in vitro under negative DC stimulation, control conditions and positive DC stimulation. B) Kernel density estimation (KDE) of Down durations vs τ in vitro with negative DC, control conditions and positive DC. C) boxplot of the average frequency of slow oscillations for each slice (N=6) under negative DC stimulation, control conditions and positive DC stimulation (Friedman test p=0.014, p<0.05). D) Linear correlation between Down duration and τ in vitro under negative DC stimulation (RSS=201), control conditions (RSS=19.9) and positive DC stimulation (RSS=3.6). E) Traces representing the LFP recording from one electrode in vitro under control conditions and with bath application of bicuculline (BMI) at two different concentrations: 0.6uM and 1uM. F) KDE of Down durations vs τ in vitro under control conditions and with BMI at 0.6uM and 1uM. G) boxplot of the average frequency of slow oscillations for each slice (N=6) under control conditions and with BMI at 0.6uM and 1uM (Friedman test p=0.014, p<0.05). H) Linear correlation between Down duration and τ in vitro under control conditions (RSS=88.6), with BMI 0.6uM (RSS=1e+03), and BMI 1uM (RSS=721). 33 Figure 7. In silico activity modulation effect on network dynamics. A) Traces representing the LFP reconstructed from one network module in silico under negative DC stimulation, control conditions and positive DC stimulation. B) kernel density estimation (KDE) of Down durations vs τ in silico under negative DC stimulation, control conditions and positive DC stimulation. C) boxplot of the average frequency of slow oscillations for each simulation (N=5) under negative DC stimulation, control conditions and positive DC stimulation (Friedman test p=ns). Neurophysiol. 104, 1314–1324 (2010). D, Linear correlation between Down duration and τ in silico under negative DC stimulation (RSS=696), control conditions (RSS=2e+03) and positive DC stimulation (RSS=50.6). Figure 7. In silico activity modulation effect on network dynamics. A) Traces representing the LFP reconstructed from one network module in silico under negative DC stimulation, control conditions and positive DC stimulation. B) kernel density estimation (KDE) of Down durations vs τ in silico under negative DC stimulation, control conditions and positive DC stimulation. C) boxplot of the average frequency of slow oscillations for each simulation (N=5) under negative DC stimulation, control conditions and positive DC stimulation (Friedman test p=ns). D, Linear correlation between Down duration and τ in silico under negative DC stimulation (RSS=696), control conditions (RSS=2e+03) and positive DC stimulation (RSS=50.6). Figure 7. In silico activity modulation effect on network dynamics. A) Traces Figure 7. In silico activity modulation effect on network dynamics. A) Traces representing the LFP reconstructed from one network module in silico under negative DC stimulation, control conditions and positive DC stimulation. B) kernel density estimation (KDE) of Down durations vs τ in silico under negative DC stimulation, control conditions and positive DC stimulation. C) boxplot of the average frequency of slow oscillations for each simulation (N=5) under negative DC stimulation, control conditions and positive DC stimulation (Friedman test p=ns). D, Linear correlation between Down duration and τ in silico under negative DC stimulation (RSS=696), control conditions (RSS=2e+03) and positive DC stimulation (RSS=50.6). 34 Figure 8. The slow oscillatory cycle mechanisms and its effect on the dynamics of different cortical networks. A) Schematic representation of the periods composing an oscillatory cycle in relation to network synchronization dynamics of MUA and LFP activity (upper panels). B) Representation of the state-dependent metastability in different cortical networks in the space defined by the deterministic fraction of the Down states and the correlation between the duration of the entire Down states and the time constant of the “deterministic” period (τ). gure 8. The slow oscillatory cycle mechanisms and its effect on the dynamics of Figure 8. The slow oscillatory cycle mechanisms and its effect on the dynamics of different cortical networks. A) Schematic representation of the periods composing an oscillatory cycle in relation to network synchronization dynamics of MUA and LFP activity (upper panels). Neurophysiol. 104, 1314–1324 (2010). B) Representation of the state-dependent metastability in different cortical networks in the space defined by the deterministic fraction of the Down states and the correlation between the duration of the entire Down states and the time constant of the “deterministic” period (τ). 35 Figure S1. Diagram illustrating the steps for the computation of the phase through the Hilbert transform. A) Block diagram illustrating the steps for the computation of the phase through the Hilbert transform (green box, H). The analytic signal z(t) is obtained as the real part of the signal x(t) plus the imaginary part obtained through the H transform. The phase ϕ(t) is then calculated by the arctangent function as the angle in the Euclidean plane, given in radiant, between the real and the imaginary part of z(t) (orange box). B) Three-dimensional representation of the analytic signal over time (colored trace), and its real, imaginary, and complex projections (gray traces) of an illustrative signal. Figure S1. Diagram illustrating the steps for the computation of the phase Figure S1. Diagram illustrating the steps for the computation of the phase through the Hilbert transform. A) Block diagram illustrating the steps for the computation of the phase through the Hilbert transform (green box, H). The analytic signal z(t) is obtained as the real part of the signal x(t) plus the imaginary part obtained through the H transform. The phase ϕ(t) is then calculated by the arctangent function as the angle in the Euclidean plane, given in radiant, between the real and the imaginary part of z(t) (orange box). B) Three-dimensional representation of the analytic signal over time (colored trace), and its real, imaginary, and complex projections (gray traces) of an illustrative signal. Figure S1. Diagram illustrating the steps for the computation of the phase through the Hilbert transform. A) Block diagram illustrating the steps for the computation of the phase through the Hilbert transform (green box, H). The analytic signal z(t) is obtained as the real part of the signal x(t) plus the imaginary part obtained through the H transform. The phase ϕ(t) is then calculated by the arctangent function as the angle in the Euclidean plane, given in radiant, between the real and the imaginary part of z(t) (orange box). B) Three-dimensional representation of the analytic signal over time (colored trace), and its real, imaginary, and complex projections (gray traces) of an illustrative signal. 36 Figure S2. Neurophysiol. 104, 1314–1324 (2010). Tau and Down durations from different slices and control conditions. The activity recorded from different cortical slices in vitro may present some dynamical differences due to the conditions of the animal, the cut of the slice, the positioning of the electrodes and more. To avoid biased in the results we show here that, despite some differences, the average tau and Down durations estimated from different cortical slices under control conditions is consistent across experiments (Control 0: N=6, Control DC: N=6, Control BMI: N=6 slices). Figure S2. Tau and Down durations from different slices and control conditions. g The activity recorded from different cortical slices in vitro may present some dynamical differences due to the conditions of the animal, the cut of the slice, the positioning of the electrodes and more. To avoid biased in the results we show here that, despite some differences, the average tau and Down durations estimated from different cortical slices under control conditions is consistent across experiments (Control 0: N=6, Control DC: N=6, Control BMI: N=6 slices). 37 Figure S3. Whole network vs local network dynamics estimation. Scatterplots showing the linear correlation between the τ estimated at whole network level and the one estimated in a single triod in vitro (i.e. three electrodes placed close to each either in the 16-channels MEA used for in vitro recording, see Materials and Methods), in three different slices (Aa: Pearson correlation=0.64, p=0.04; Ba: Pearson correlation=0.59, p=0.002; Ca: Pearson correlation=0.56, p=0.001). Kernel Density Estimation (KDE) of in vitro Down durations vs τ estimated at whole network level in the same three slices (Ab, Bb, Cb). KDE of in vitro Down durations vs τ estimated using data from one triode (see scheme above) in the same three slices (Ac, Bc, Cc). KDE of in vitro Down durations vs τ estimated using data from one recording channel (see scheme above) in the same three slices (Ad, Bd, Cd). Figure S3. Whole network vs local network dynamics estimation. Scatterplots g y p showing the linear correlation between the τ estimated at whole network level and the one estimated in a single triod in vitro (i.e. three electrodes placed close to each either in the 16-channels MEA used for in vitro recording, see Materials and Methods), in three different slices (Aa: Pearson correlation=0.64, p=0.04; Ba: Pearson correlation=0.59, p=0.002; Ca: Pearson correlation=0.56, p=0.001). Neurophysiol. 104, 1314–1324 (2010). Kernel Density Estimation (KDE) of in vitro Down durations vs τ estimated at whole network level in the same three slices (Ab, Bb, Cb). KDE of in vitro Down durations vs τ estimated using data from one triode (see scheme above) in the same three slices (Ac, Bc, Cc). KDE of in vitro Down durations vs τ estimated using data from one recording channel (see scheme above) in the same three slices (Ad, Bd, Cd). 38
https://openalex.org/W2977641185	https://europepmc.org/articles/pmc6835421?pdf=render	English	null	Progress and Perspectives on Ceramic Membranes for Solvent Recovery	Membranes	2,019	cc-by	20,062	Received: 6 May 2019; Accepted: 23 September 2019; Published: 4 October 2019 Abstract: With the increase in demand for commodities in the world, it is advisable to conserve resources. In the case of liquid wastes generated from pharmaceutical and petroleum industries, an unconventional solution is provided for the regeneration of solvents. However, this solvent recovery can be carried out using various eﬃcient methods. Recently, Mixed Matrix Membranes (MMM) obtained by the addition of nanoparticles into a polymer matrix as reinforcements, or using a material with a well-deﬁned inorganic network as a membrane like zeolite, silica based, Zeolite imidazolate frameworks (ZIFs) and Metal organic frameworks (MOFs), were explored for a solvent recovery process. These membranes possess characteristics such as high selectivity, ﬂux and stability at various environmental conditions for the solvent recovery process. In this review, we have covered the polymer, nanocomposites, and ceramic membranes for solvent recovery through the pervaporation and organic solvent nanoﬁltration processes. The key challenges faced by the materials such as MOFs, zeolite, silica, zeolite and ZIFs when they are fabricated (through in situ synthesis or secondary growth process) as membranes and separation of solvents to explore for the solvent recovery process are reviewed. Keywords: solvent dehydration; solvent recovery; pervaporation Membranes 2019, 9, 128; doi:10.3390/membranes9100128 www.mdpi.com/journal/membranes membranes membranes membranes membranes membranes Progress and Perspectives on Ceramic Membranes Solvent Recovery Senthilnathan Ruthusree, Subramanian Sundarrajan * and Seeram Ramakrishna * Center for Nanoﬁbers and Nanotechnology Lab, Mechanical Engineering, National University of Singapore, Blk E3 05-12, 2 Engineering Drive 3, Singapore 117581, Singapore; ruthusree6@gmail.com * Correspondence: sundar@nus.edu.sg (S.S.); seeram@nus.edu.sg (S.R.) Received: 6 May 2019; Accepted: 23 September 2019; Published: 4 October 2019 1. Introduction Organic materials contribute the polymeric membranes which are both natural and synthetic. The inorganic ones consist of artiﬁcial ceramic, metallic and zeolite membranes [11]. Ceramic membranes consist of materials like alumina and titania; they also contain compounds like carbides and nitrides. Due to their inertness, they are used for highly acidic or basic environments [11]. They fail due to the highly sensitive temperature gradient, which leads to the cracking of membrane. Zeolite membranes are synthesized hydrothermally on to a porous ceramic or metal support in the presence of heat [12]. They also require a thicker layer to protect them from the formation of cracks and pinholes. Inorganic membranes possess few advantages like high thermal and chemical stabilities, inertness to microbiological degradation and reduction in cleaning requirements [13]. The membrane is fabricated on a porous support to improve the permeability. The establishment of a selective layer on a porous support enhances the permeability and selectivity of the membrane. Despite its advantages, they face complications arising because of the fall in the thermal compatibility between the porous support and the selective layer [14]. Coeﬃcient of thermal expansion in metal oxides and membranes play a major role in the material selection criteria [10]. Although the process of solvent recovery is very advantageous in preserving and conserving the waste solvent, it has obstacles to overcome in order to be explored in real application. The major drawback impeding this process is the existence of azeotropic compositions in the liquid discharge. Azeotrope is a mixture having the same composition in both liquid and vapor phase. Hence, the separation of the mixture via fractional distillation was a failure. Alternative solutions for resolving the hurdle are azeotropic distillation, molecular sieve and membranes for azeotropic mixture separation [13]. In the case of azeotropic distillation, there is an addition of a third component to the mixture to generate a contemporary lower body azeotrope, which is heterogeneous and forms a separate phase for extractive distillation. Molecular sieve absorbents have pores of uniform size which are identical to the dimensions of small molecules; they also restrict the entry of large molecules and, with heat treatment, the sieves can be regenerated [15]. Although reviews have already been published in the concept of solvent recovery, they are focused on the general aspects [4,16–19]. The study conducted by Yao et al. 1. Introduction Solvents ﬁnd their applications in an ample variety of pharmaceutical and other industrial products. The liquid wastes generated from the manufacturers can be hazardous, making their disposal a costly aﬀair [1–5]. Storage of the waste occupies plethora of workspace and, in addition results in occupational hazard. When the waste generated is on a small scale, disposal can be easy; however, an alternative solution of recycling the liquid discharge can be considered. In order to fulﬁll the demands of living things, conserving and recycling of sources would be an easier, greener- and more economical approach. Recycling of solvent eﬄuent waste can help to the recover the valuable solvents, termed as solvent recovery. Solvent recovery can considerably not only reduce the waste generation, but also the transport cost, especially when the solvents are expensive and large volumes are involved. The recycling of solvent waste can dramatically bring down the cost of solvents by 80%, with their ability to recycle up to 95% of waste and using them for 10 cycles. Solvent recycling is typically used in manufacturing units generating wastes such as pharmaceutical industry, paint lacquer, metal ﬁnishing and automotive coatings. This recycling concept has been implemented in industry for many decades and is a routine practice when it comes to the pharmaceutical industry; they serve as an economically viable solution for the regeneration of a particular solvent waste [6,7]. Recovery of solvents from the eﬄuent can be carried out in various processes or techniques, subjected to size of the batch and also the automation [8]. This technique has limitations such as high initial cost to setup the process, technological barrier and process complexity. The traditional method Membranes 2019, 9, 128; doi:10.3390/membranes9100128 www.mdpi.com/journal/membranes 2 of 24 Membranes 2019, 9, 128 used for recycling is fractional distillation, but, with the advances in techniques, liquid–liquid extraction, absorbing systems, ﬁlm evaporation, crystallization and separation process using membranes can be used for the particular application [9]. The liquid waste that can be treated can be: (1) non-azeotropic solution, (2) aqueous non azeotropic solution, (3) heterogeneous azeotropic solution, or (4) aqueous homogeneous azeotropic solution. g p Recently, membrane technology has found their widespread adoption in the solvent recovery process due to their improved eﬃciency and low energy cost [10]. The membranes used in this process for solvent recovery are generally made of either organic or inorganic materials. 2.1. Zeolite-Based Membranes 2.1. Zeolite-Based Membranes Zeolites are artiﬁcially or naturally occurring materials with a uniform and well-deﬁned network of pores. They are crystalline in nature and are composed of materials like silicon, aluminum and oxygen, as well as other cations required for the framework. They have pore sizes varying from 0.3 to 1.3 nm, depending on the type of framework. In case of the synthetic zeolite, the synthesis procedure determines the ratio of materials used (Si/Al), and it simultaneously aﬀects the material properties such as polarity and adsorption. Zeolite membranes are inorganic in nature and have inter grown zeolite crystals on the porous membranes. They have incredible properties like high thermal and chemical stabilities to stand over polymeric membranes, and also possess high ﬂux and selectivity through the well-deﬁned pore size (Figure 1). During a pervaporation process, the zeolitic and non-zeolitic pores of the Linde type A Sodium (zeolite NaA) membranes strongly adsorb the water vapor and undergo condensation. As a result, the water permeates with high permeance though the capillary condensation mechanism and hinders other molecules permeation by obstructing their entry into the pores. Zeolite membranes have found their application on a pharmaceutical industrial scale for solvent dehydration. Another factor determining the productivity of the membranes are thickness and defects found in them. The permeability of solvents can be diminished with the high ﬁlm thickness and selectivity due to defects. Hedlund et al. [20] reported that, in order to obtain high ﬂux, the thickness of membrane should be 1 µm and ideally supported by a substrate. Zeolite ﬁlms on substrate can be fabricated using in situ synthesis and secondary growth with hydrothermal treatment. Many recent publications show that the secondary growth method favors high reproducibility and good control over the membrane structure [18,21]. Zeolites are artificially or naturally occurring materials with a uniform and well-defined network of pores. They are crystalline in nature and are composed of materials like silicon, aluminum and oxygen, as well as other cations required for the framework. They have pore sizes varying from 0.3 to 1.3 nm, depending on the type of framework. In case of the synthetic zeolite, the synthesis procedure determines the ratio of materials used (Si/Al), and it simultaneously affects the material properties such as polarity and adsorption. Zeolite membranes are inorganic in nature and have inter grown zeolite crystals on the porous membranes. 2.1. Zeolite-Based Membranes 2.1. Zeolite-Based Membranes They have incredible properties like high thermal and chemical stabilities to stand over polymeric membranes, and also possess high flux and selectivity through the well-defined pore size (Figure 1). During a pervaporation process, the zeolitic and non-zeolitic pores of the Linde type A Sodium (zeolite NaA) membranes strongly adsorb the water vapor and undergo condensation. As a result, the water permeates with high permeance though the capillary condensation mechanism and hinders other molecules permeation by obstructing their entry into the pores. Zeolite membranes have found their application on a pharmaceutical industrial scale for solvent dehydration. Another factor determining the productivity of the membranes are thickness and defects found in them. The permeability of solvents can be diminished with the high film thickness and selectivity due to defects. Hedlund et al. [20] reported that, in order to obtain high flux, the thickness of membrane should be 1 μm and ideally supported by a substrate. Zeolite films on substrate can be fabricated using in situ synthesis and secondary growth with hydrothermal treatment. Many recent publications show that the secondary growth method favors high reproducibility and good control over the membrane structure [18,21]. Figure 1 Separation mechanism in zeolite membrane Figure 1. Separation mechanism in zeolite membrane. Figure 1 Separation mechanism in zeolite membrane Figure 1. Separation mechanism in zeolite membrane. LTA (Linde type A) zeolite membranes are the first membranes introduced commercially in the market for commercial use. They have a three-dimensional porous structure with pores running perpendicular to all the planes. Zeolite A is synthesized using a sol-gel technique by the reaction of alumina and silica in an alkaline medium. The super cage structure possessed by the zeolite makes the membrane enhance both the selectivity and flux. The presence of alumina in the membrane i th h d hili it f th t LTA (Linde type A) zeolite membranes are the ﬁrst membranes introduced commercially in the market for commercial use. They have a three-dimensional porous structure with pores running perpendicular to all the planes. Zeolite A is synthesized using a sol-gel technique by the reaction of alumina and silica in an alkaline medium. The super cage structure possessed by the zeolite makes the membrane enhance both the selectivity and ﬂux. The presence of alumina in the membrane improves the hydrophilicity of the system. improves the hydrophilicity of the system. 1. Introduction [16] has highlighted the synthesis of Zeolitic imidazolate framework (ZIF) membranes, thin ﬁlm formation and their application as pervaporation membranes for dehydration (till the year 2014). The review by Betard et al. [17] has stated some views on the fundamental concepts and applications of MOF thin ﬁlms, such as sensing and gas separation till 2012. The puriﬁcation and recovery of solvent waste in the manufacture of active pharmaceutical ingredients (API’s) and three case studies are presented by Cavanagh et al. [4]. To date, there has not been any solid comprehensive review that reports the progress in the aspects of solvent recovery by membranes. In fact, the most recent developments have been published over the past few years. Lately, the synthesis of continuous ZIF-68 membranes on alumina support by reactive seeding was developed by Kasik et al. [18]. More recently, ZIFs with higher pore opening materials (largest pore opening of 22.5 Å and the largest cage size of 45.8 Å) were synthesized and tested for the removal of organic mixtures such as octane and p-xylene from humid air [19]. Hence, in this review, mixed matrix membrane, Zeolite based membranes, Mordenite Framework Inverted (MFI) Type Zeolite, metal organic frameworks (MOF), silica-based membranes and Zeolite imidazolate framework (ZIF) for the separation of organic solvents from their corresponding solvent mixture (or water) is highlighted. 3 of 24 Membranes 2019, 9, 128 2.1. Zeolite-Based Membranes 2.1. Zeolite-Based Membranes Therefore, the size of cr reduced to enhance the aﬃnity of water which leads to stronger inte and permeation by approximately 5 times (8 to 39.9 µmole·m−2·s−1· The pervaporation permeation of diﬀerent organic/water liquid mixtur tabulated in Table 1 [24]. Table 1. Pervaporation (PV) performance of the zeolite NaA membranes systems; Reproduced with permission from [24]. Separation System Temp (K) XW (wt%) Q (kg/(m Ethanol 348 10 2.1 348 5 1.1 348 0.5 0.01 Methanol 323 10 0.5 323 5 0.2 n-propanol 348 10 1.9 i-propanol 348 10 1.7 Acetone 323 10 0.9 323 5 0.8 Dioxane 333 10 1.8 DMF 333 10 0.9 Temp—Temperature; Xw—Weight percentage (wt%); Q—Total Permeation The membrane pores are smaller than the size of the small organic mixtures. The diﬀusion rates of water, 2-propanol, methanol, ethanol 5 zeolite membrane by using isotopic- transient pervaporation were In the methanol/ethanol mixtures, the presence of methanol increas ethanol. In the feed of methanol/ethanol mixtures containing 95 wt% t d f th l d th l 3 8 d 0 2 l/ 2 h ti content) and tested separation performance by pervaporation experiment The synthesized membranes exhibited high ﬂux rates at diﬀerent temperatures (water/isopropanol (up to 16 kg/(m2·h), 120 ◦C), water/ethanol (4.5 kg/(m2·h), 105 ◦C) and water/dioxane (7.8 kg/(m2·h), 105 ◦C)) indicating that temperature plays a vital role in governing the permeate ﬂux. The separation factor of more than 1000 was observed, indicating that high selectivity of the separation can be achieved. However, the membranes showed enhancement in solvent ﬂux, but the usage of tubular zeolite membranes seems to be costly [11]. To overcome this issue, Zhang et al. [22] introduced thin, porous metal sheets to reinforce the zeolite membrane. The fabrication of these high ﬂux and selective zeolite NaA membrane is formed from the porous metal sheets along with the two-time dip coating–wiping seed deposition procedure. The substrate used has a smooth and uniform porous structure over the surface. Apart from this, the porous metal sheets provide beneﬁcial properties such as high permeability, chemical stability, mechanical strength, membrane packing density and low cost. With increase in separation temperature during the pervaporation process in the setup, the selectivity and permeability of solvents also increase gradually. With an elevation in the separation temperature from 75 ◦C to 135 ◦C, there was a uniform increase in the water/ethanol permeation ﬂux and selectivity. The membrane pores are smaller than the size of the small organic molecules for the separation of mixtures. The diﬀusion rates of water, 2-propanol, methanol, ethanol and acetone through a Ge-ZSM 5 zeolite membrane by using isotopic- transient pervaporation were measured by Bowen et al. [25]. In the methanol/ethanol mixtures, the presence of methanol increased the rate of the diﬀusion of ethanol. In the feed of methanol/ethanol mixtures containing 95 wt% of ethanol at 313 K, the ﬂuxes reported for ethanol and methanol are 3.8 and 0.2 mol/m2·h, respectively. 2.1. Zeolite-Based Membranes 2.1. Zeolite-Based Membranes Zeolite membranes were commercially first developed by the Mitsui Engineering and Shipbuilding Co. (Tokyo, Japan) [11]. In association with Yamaguchi University, for the purpose of organic solvent degradation in an expansive scale, they fabricated the zeolite membrane on the surface of ceramic alumina tube of 1 2 cm OD and 80 cm length for ethanol and solvent dehydration Zeolite membranes were commercially ﬁrst developed by the Mitsui Engineering and Shipbuilding Co. (Tokyo, Japan) [11]. In association with Yamaguchi University, for the purpose of organic solvent degradation in an expansive scale, they fabricated the zeolite membrane on the surface of ceramic alumina tube of 1.2 cm OD and 80 cm length for ethanol and solvent dehydration (10 wt% of water 4 of 24 Membranes 2019, 9, 128 content) and tested separation performance by pervaporation experime exhibited high ﬂux rates at diﬀerent temperatures (water/isopropan water/ethanol (4.5 kg/(m2·h), 105 ◦C) and water/dioxane (7.8 kg/(m temperature plays a vital role in governing the permeate ﬂux. The 1000 was observed, indicating that high selectivity of the separation membranes showed enhancement in solvent ﬂux, but the usage of tub to be costly [11]. To overcome this issue, Zhang et al. [22] introduce reinforce the zeolite membrane. The fabrication of these high ﬂux and s is formed from the porous metal sheets along with the two-time dip c procedure. The substrate used has a smooth and uniform porous structu this, the porous metal sheets provide beneﬁcial properties such as high mechanical strength, membrane packing density and low cost. With inc during the pervaporation process in the setup, the selectivity and perm gradually. With an elevation in the separation temperature from 75 ◦C increase in the water/ethanol permeation ﬂux and selectivity. The wate between 10,000 and 70,000 and displayed water permeation ﬂux of abo with a feed of 10% (w/w) water in ethanol. The selectivity of water/eth in water content. However, these membranes fail to possess enough potential industrial application. To counter this problem, Mastropietro for simple synthesis of FAU membranes composed by hierarchically ass these membranes demonstrated high water vapor ﬂuxes as well as h vapor, which are mostly due to the higher rate of diﬀusion for water w probe within the hydrophilic zeolite network. Membranes 2019, 9, 128 Membranes 2019, 9, 128 The separation of permeating molecules through zeolite membranes takes place because of the diﬀerence in chemical aﬃnities, along with the shape and size with respect to the pores. MFI-type zeolite is a high silica zeolite synthesized using hydroxide of tetra propylammonium as a template. This avenue of research played a remarkable role in the petrochemical industry. For the separation of liquid mixture through pervaporation process, a liquid feed is used and the permeate is a vapor. It is also reported that, with an increase in partial pressure of feeds, the separation factor decreases. In the pervaporation experiments conducted by Algieri et al. [26], thin MFI zeolite membranes were synthesized by in situ nucleation and secondary growth at 70 ◦C. They fed 9.4 wt% of ethanol–water mixtures and high ﬂuxes (2.1 kg/m2·h), and separation factor as low as 1.3 were obtained. They also reported ﬂuxes and separation factor for NaA type zeolite membrane varying from 0.23 to 5.60 kg/m2·h and 3600 to 10,000, respectively. Kanezashi et al. [27] reported the usage of MFI type zeolite membrane for the pervaporation of p- and o-xylene binary mixture. At 25 ◦C and 1.1 kPa partial pressure, the obtained p-xylene ﬂux and p/o-xylene selectivity were 7.6 × 10−4 and 22 mol/m2·s, respectively. Wang et al. [28] reported the utilization of MFI Zeolite membranes for the separation of CO2/Xe gas mixtures with a separation factor of 5.6, and separating CO2 molecules of 3.3 Å from Xe molecules of 4.1 Å. The most important industrially explored zeolite structures are given in Figure 2 [29]. Membranes 2019, 9, x 5 of 24 This avenue of research played a remarkable role in the petrochemical industry. For the separation of liquid mixture through pervaporation process, a liquid feed is used and the permeate is a vapor. It is also reported that, with an increase in partial pressure of feeds, the separation factor decreases. In the pervaporation experiments conducted by Algieri et al. [26], thin MFI zeolite membranes were synthesized by in situ nucleation and secondary growth at 70 °C. They fed 9.4 wt% of ethanol–water mixtures and high fluxes (2.1 kg/m2·h), and separation factor as low as 1.3 were obtained. They also reported fluxes and separation factor for NaA type zeolite membrane varying from 0.23 to 5.60 kg/m2·h and 3600 to 10,000, respectively. Kanezashi et al. Membranes 2019, 9, 128 This study showed that methanol selectivity can be achieved h h FAU li b i i i h l fl f b 2 0 k 2 h 1 Kumakiri et al. [30] studied the FAU zeolite membranes to selectively separate the methanol solvent from various feed mixtures such as methanol–MMA–butanol–BMA. When a mixture of 20.2/29.7/9.2/40.9 wt% of butanol–MMA–methanol–BMA, respectively, was fed at 60 ◦C through a FAU zeolite membrane, the permeate had a composition of 1.60/0.17/98.1/0.17 wt% of butanol–MMA–methanol–BMA, respectively. This study showed that methanol selectivity can be achieved through FAU zeolite membrane in a quaternary mixture with total ﬂux of about 2.0 kg·m−2·h−1. Kumakiri et al. [30] studied the FAU zeolite membranes to selectively separate the methanol solvent from various feed mixtures such as methanol–MMA–butanol–BMA. When a mixture of 20.2/29.7/9.2/40.9 wt% of butanol–MMA–methanol–BMA, respectively, was fed at 60 °C through a FAU zeolite membrane, the permeate had a composition of 1.60/0.17/98.1/0.17 wt% of butanol– MMA–methanol–BMA, respectively. This study showed that methanol selectivity can be achieved through FAU zeolite membrane in a quaternary mixture with total flux of about 2.0 kg·m−2·h−1. Dip coating, rubbing, and reactive seeding are some of the methods that are applied effectively for seeding the outer surface of the support material [31–33]. In the zeolite membrane synthesis, rubbing alone or a rubbing-dip coating technique were applied for seeding the zeolite on support materials such as α-alumina and secondary growth followed by pervaporation study by various authors. Applying the rubbing method, for the ethanol/water (90%) as feed, the obtained separation factors and fluxes were 3603, >10,000, >10,000 and 3.8, 3.17, 3.6 kg·m−2·h−1, respectively, by Pina, Ma and Wang et al. [31–33]. Similarly, when the rubbing-dip coating method was applied, for the ethylene glycol/water system as feed the obtained separation factors and fluxes at 80 °C were 10 996 Kumakiri et al. [30] studied the FAU zeolite membranes to selectively separate the methanol solvent from various feed mixtures such as methanol–MMA–butanol–BMA. When a mixture of 20.2/29.7/9.2/40.9 wt% of butanol–MMA–methanol–BMA, respectively, was fed at 60 ◦C through a FAU zeolite membrane, the permeate had a composition of 1.60/0.17/98.1/0.17 wt% of butanol–MMA–methanol–BMA, respectively. This study showed that methanol selectivity can be achieved through FAU zeolite membrane in a quaternary mixture with total ﬂux of about 2.0 kg·m−2·h−1. Membranes 2019, 9, 128 [27] reported the usage of MFI type zeolite membrane for the pervaporation of p- and o-xylene binary mixture. At 25 °C and 1.1 kPa partial pressure, the obtained p-xylene flux and p/o-xylene selectivity were 7.6 × 10−4 and 22 mol/m2·s, respectively. Wang et al. [28] reported the utilization of MFI Zeolite membranes for the separation of CO2/Xe gas mixtures with a separation factor of 5.6, and separating CO2 molecules of 3.3 Å from Xe molecules of 4.1 Å. The most important industrially explored zeolite structures are given in Figure 2 [29]. Figure 2. Industrially most important zeolite structures: (A) LTA; (B) FAU; (C) ZSM-5 (Zeolite Socony Mobil-5); (D) MOR (Mordenite Framework); (E) FER (Ferrierite); and (F) BEA (Beta Polymorph). The lines represent the O atoms and the corners Si or Al atoms, and the exchangeable cation sites in LTA and FAU are indicated; Reproduced with permission from [29]. Figure 2. Industrially most important zeolite structures: (A) LTA; (B) FAU; (C) ZSM-5 (Zeolite Socony Mobil-5); (D) MOR (Mordenite Framework); (E) FER (Ferrierite); and (F) BEA (Beta Polymorph). The lines represent the O atoms and the corners Si or Al atoms, and the exchangeable cation sites in LTA and FAU are indicated; Reproduced with permission from [29]. Figure 2. Industrially most important zeolite structures: (A) LTA; (B) FAU; (C) ZSM-5 (Zeolite Socony Mobil-5); (D) MOR (Mordenite Framework); (E) FER (Ferrierite); and (F) BEA (Beta Polymorph). The lines represent the O atoms and the corners Si or Al atoms, and the exchangeable cation sites in LTA and FAU are indicated; Reproduced with permission from [29] Figure 2. Industrially most important zeolite structures: (A) LTA; (B) FAU; (C) ZSM-5 (Zeolite Socony Mobil-5); (D) MOR (Mordenite Framework); (E) FER (Ferrierite); and (F) BEA (Beta Polymorph). The lines represent the O atoms and the corners Si or Al atoms, and the exchangeable cation sites in LTA and FAU are indicated; Reproduced with permission from [29]. Kumakiri et al. [30] studied the FAU zeolite membranes to selectively separate the methanol solvent from various feed mixtures such as methanol–MMA–butanol–BMA. When a mixture of 20.2/29.7/9.2/40.9 wt% of butanol–MMA–methanol–BMA, respectively, was fed at 60 °C through a FAU zeolite membrane, the permeate had a composition of 1.60/0.17/98.1/0.17 wt% of butanol– MMA–methanol–BMA, respectively. 2.1. Zeolite-Based Membranes 2.1. Zeolite-Based Membranes The water/ethanol selectivity ﬂuctuated between 10,000 and 70,000 and displayed water permeation ﬂux of above 4kg/(m2·h) starting at 75 ◦C with a feed of 10% (w/w) water in ethanol. The selectivity of water/ethanol improved with an increase in water content. However, these membranes fail to possess enough selectivity with high ﬂuxes for potential industrial application. To counter this problem, Mastropietro et al. [23] modiﬁed a procedure for simple synthesis of FAU membranes composed by hierarchically assembled Nano-zeolites. Initially, these membranes demonstrated high water vapor ﬂuxes as well as high selectivity values for water vapor, which are mostly due to the higher rate of diﬀusion for water with respect to the N2 molecular probe within the hydrophilic zeolite network. Therefore, the size of crystallites of the membrane was reduced to enhance the aﬃnity of water which leads to stronger interactions with water molecules and permeation by approximately 5 times (8 to 39.9 µmole·m−2·s−1·Pa−1) times at the steady state. The pervaporation permeation of diﬀerent organic/water liquid mixtures studied by Okamoto et al. is tabulated in Table 1 [24]. Table 1. Pervaporation (PV) performance of the zeolite NaA membranes toward water/ organic liquid systems; Reproduced with permission from [24]. Table 1. Pervaporation (PV) performance of the zeolite NaA membranes toward water/ organic liquid systems; Reproduced with permission from [24]. Separation System Temp (K) XW (wt%) Q (kg/(m2·h)) α [-] Ethanol 348 10 2.15 10,000 348 5 1.10 16,000 348 0.5 0.012 5100 Methanol 323 10 0.57 2100 323 5 0.23 2500 n-propanol 348 10 1.91 18,000 i-propanol 348 10 1.76 10,000 Acetone 323 10 0.91 5600 323 5 0.83 6800 Dioxane 333 10 1.87 >9000 DMF 333 10 0.95 >9000 Temp—Temperature; Xw—Weight percentage (wt%); Q—Total Permeation Flux; α—Separation factor. The membrane pores are smaller than the size of the small organic molecules for the separation of mixtures. The diﬀusion rates of water, 2-propanol, methanol, ethanol and acetone through a Ge-ZSM 5 zeolite membrane by using isotopic- transient pervaporation were measured by Bowen et al. [25]. In the methanol/ethanol mixtures, the presence of methanol increased the rate of the diﬀusion of ethanol. In the feed of methanol/ethanol mixtures containing 95 wt% of ethanol at 313 K, the ﬂuxes reported for ethanol and methanol are 3.8 and 0.2 mol/m2·h, respectively. 5 of 24 Membranes 2019, 9, 128 [34], and for ethanol/water (90%) as feed at 70 ◦C, the obtained separation factors and ﬂuxes were >15,000, 3.26 kg·m−2·h−1, respectively, by Liu et al. [35]. An ensemble synthesis strategy was applied on hollow ﬁber supported T-type zeolite membrane modules by the secondary growth method by Ji et al. [36]. Optimization of seed particles size, seed concentration, coating time and crystallization time were studied. The high-quality membrane modules with membrane areas of 0.03 m2 showed an average ﬂux of 2.25 kg·m−2·h−1 and separation factor of 1348 for the dehydration of 90 wt% ethanol/water solutions at 348 K, whereas a pilot-scale apparatus of two 0.54 m2 membrane modules connected serially showed pervaporation dehydration of 90 wt% isopropanol/water mixture to 99.3 wt%. p p Two diﬀerent hydrophilic topologies of zeolites such as Faujasite (FAU) and mordenite (MOR) were investigated as membrane layers on tubular mullite and disk-shaped α-alumina supports for PV dehydration of ethanol by Asghari et al. [37]. Various synthesis parameters such as ceramic support, repetition of coating, seeding method, crystallization time (14 and 18 h), temperature (160, 170 and 180 ◦C), and Si/Al ratio of the precursor gel formulation (12 and 16) on the membranes structures and their PV performances were studied. Permeation ﬂux (43.43%), separation factor (30.39%) and PSI (62.35%) decrease with increasing ethanol concentration in feed (50–90 wt%) was observed and the MOR layer on α-alumina (MOR_D-II) was reported as the best membrane for ethanol/water PV system. NaA zeolite pervaporation membranes was synthesized on the alumina hollow ﬁber inner-surface in a continuous ﬂow system by Cao et al. [38]. The optimal PV performance of up to 19.7 kg/m2·h for the permeation ﬂux and more than 80,000 for the separation factor at the ﬂow rate of 148 mL/min with 90 wt% ethanol/water solutions at 348 K was reached and yielded an unprecedented performance. High-quality hollow ﬁber supported Decadodecasil 3R zeolite membranes were prepared on four-channel ceramic hollow ﬁbers and successfully tested in pervaporation dehydration of acetic acid (AcOH) for the ﬁrst time by Zhang et al. [39]. The membrane showed a water permeation ﬂux of 0.58 kg·m−2·h−1 and a separation factor of 800 for the dehydration of 70 wt% water/AcOH mixture at 368 K and the stable permeation ﬂux and separation factor was observed even in the presence of inorganic acid, thereby showing outstanding acid resistance. Membranes 2019, 9, 128 Dip coating, rubbing, and reactive seeding are some of the methods that are applied eﬀectively for seeding the outer surface of the support material [31–33]. In the zeolite membrane synthesis, rubbing alone or a rubbing-dip coating technique were applied for seeding the zeolite on support materials such as α-alumina and secondary growth followed by pervaporation study by various authors. Applying the rubbing method, for the ethanol/water (90%) as feed, the obtained separation factors and ﬂuxes were 3603, >10,000, >10,000 and 3.8, 3.17, 3.6 kg·m−2·h−1, respectively, by Pina, Ma and Wang et al. [31–33]. Similarly, when the rubbing-dip coating method was applied, for the ethylene glycol/water system as feed, the obtained separation factors and ﬂuxes at 80 ◦C were 10,996, through FAU zeolite membrane in a quaternary mixture with total flux of about 2.0 kg·m−2·h−1. Dip coating, rubbing, and reactive seeding are some of the methods that are applied effectively for seeding the outer surface of the support material [31–33]. In the zeolite membrane synthesis, rubbing alone or a rubbing-dip coating technique were applied for seeding the zeolite on support materials such as α-alumina and secondary growth followed by pervaporation study by various authors. Applying the rubbing method, for the ethanol/water (90%) as feed, the obtained separation factors and fluxes were 3603, >10,000, >10,000 and 3.8, 3.17, 3.6 kg·m−2·h−1, respectively, by Pina, Ma and Wang et al. [31–33]. Similarly, when the rubbing-dip coating method was applied, for the ethylene glycol/water system as feed the obtained separation factors and fluxes at 80 °C were 10 996 Dip coating, rubbing, and reactive seeding are some of the methods that are applied eﬀectively for seeding the outer surface of the support material [31–33]. In the zeolite membrane synthesis, rubbing alone or a rubbing-dip coating technique were applied for seeding the zeolite on support materials such as α-alumina and secondary growth followed by pervaporation study by various authors. Applying the rubbing method, for the ethanol/water (90%) as feed, the obtained separation factors and ﬂuxes were 3603, >10,000, >10,000 and 3.8, 3.17, 3.6 kg·m−2·h−1, respectively, by Pina, Ma and Wang et al. [31–33]. Similarly, when the rubbing-dip coating method was applied, for the ethylene glycol/water system as feed, the obtained separation factors and ﬂuxes at 80 ◦C were 10,996, 6 of 24 Membranes 2019, 9, 128 7.16 kg·m−2·h−1, respectively by Jafari et al. 2.2. Silica-Based Membranes Silica has the ability to link together with diﬀerent amorphous or crystalline solids, to give rise to porous structures. The pore sizes of the material can be ﬁne-tuned by using surfactant micelles (cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS)) to separate molecules based on their pore size with the property of size speciﬁcity through the pervaporation process. Elements like zirconia, alumina and titania are chosen as secondary elements for inclusion in the silica membrane to enhance the hydrothermal stability of membrane. The solvent used determines the rate of rejection. Silica membranes are generally casted using both chemical- and physical routes, where the sol-gel technique contributes to a chemical route and chemical vapour deposition to a physical one. Membranes are required to be used in non-aqueous system for the purpose of regenerating organic solvents. Tsuru et al. [44] made use of porous silica-zirconia membrane with nanopores in the range of 1–4 nm for the application of separating solvents like ethanol and methanol from non-aqueous solutions such as polyethylene glycol and ethylene glycol. Silica membranes were synthesized on α-alumina, γ-alumina, alumina, support by various researchers [45–48]. Silica MEL membrane was synthesized on a porous α-alumina hollow ﬁber support by a secondary growth approach. Kosinov et al. [45] have reported that silicalite-2 membrane showed higher ﬂuxes without compromising selectivity for ethanol/water separation by pervaporation. However, only diﬀerent selectivity was obtained for n-/i-butane mixture separation, in which MEL structure showed favorable diﬀusion to branched alkane compared to the MFI one due to the diﬀerent pore topologies. It further showed that hydrophobic silicalite-2 membranes have the potential for the removal of organics from the aqueous solutions. Similarly, Boutikos et al. [47] applied silica membranes on γ-alumina for the separation of n-butanol and water mixture with a ﬂux of 196 mol/m3.hr and separation factor of 150. Cobalt-doped silica was explored on α-alumina support for the separation of ethanol/water mixture with ﬂux of 60 mol/m3·h and separation factor of 2530 by Wang et al. [49]. p y g Hydrophobicity was introduced onto alumina and titania micro and meso porous ceramic membranes by grafting of C6F13C2H4Si(OEt)3 (C6) molecules by Kujawa et al. [50] and tested for the removal of hazardous volatile organic solvents (methyl tert-butyl-ether (MTBE), ethyl acetate (EtAc) and butanol (BuOH)) from binary aqueous solutions by the pervaporation process. Membranes 2019, 9, 128 Although various developments in the zeolite synthesis were achieved by giving importance to techno-economic criteria with an aim of industrial scaling, the environmental impact of these processes has not been studied. Normally, aggressive solvents were used, which has resulted in the environmental degradation. A life cycle assessment (LCA) tool has been most widely used to evaluate the environmental impacts of the manufacturing processes, which has been used in gas separation, water treatment, and alcohol puriﬁcation by pervaporation. Navajas et al. [40] applied the LCA for the ﬁrst time to zeolite membrane synthesis and quantiﬁed the eﬀect of: (i) seed layers that allow membranes of submicron thickness; (ii) gel-less secondary treatments that avoid the use of large amounts of expensive structure directing agents; and (iii) use of low-cost polymer supports instead of conventional ceramic supports using GaBi 8.7 Pro software. They found that most of the impacts were given by the support layer and progresses in the synthesis of hollow ﬁbers (thinner ﬁbers) and use of less-aggressive solvents could signiﬁcantly reduce the environmental impacts related with overall membrane synthesis. y The cost of the zeolite membranes are about ten times more expensive than polymeric membranes [41]. Although the inorganic membranes are costlier, their longer lifetime, high water concentration operating range and high thermal stability are added advantages when compared to the polymeric counterparts. In the early stages of zeolite membranes development, in addition to cost, the membranes developed were of low water flux due to thicker zeolite layers required and low water selectivity due to grain boundary/intercrystalline defects. However, these disadvantages were overcome by the deposition of a thin and uniform layer of zeolite seed crystals onto the porous support followed by secondary crystal growth treatment, which resulted in a thinner defect-free zeolite layer [42,43]. 7 of 24 7 of 24 Membranes 2019, 9, 128 2.2. Silica-Based Membranes In recent years, the development of the high-silica zeolite beta (HSZB) membrane has received much attention in the literature due to its high hydrophobicity and excellent mechanical, thermal, and chemical stabilities [56]. Membranes 2019, 9, x 8 of 24 Li et al. [57] have fabricated HSZB membranes with controllable orientation (higher H2O/SiO2 ratio) on randomly oriented seed layers via a secondary growth method. Preferential (h0l)-oriented HSZB membrane was achieved when the H2O/SiO2 ratio was 4, whereas the c-oriented was obtained when the H2O/SiO2 ratio of 7 (Figure 3). The (h0l)-oriented HSZB membrane exhibited high ﬂuxes up to 1.45 and 1.05 kg·m−2·h−1 with respect to 1 and 5 wt% n-butanol/water mixtures, and the corresponding separation factors were 36.5 and 32.5, respectively. This study indicates that (h00l)-oriented HSZB membranes can be prepared at higher H2O/SiO2 ratio and would be more promising for n-butanol recovery from dilute aqueous solution due to high ﬂuxes with relatively high separation factors because of high hydrophobicity. Li et al. [57] have fabricated HSZB membranes with controllable orientation (higher H2O/SiO2 ratio) on randomly oriented seed layers via a secondary growth method. Preferential (h0l)-oriented HSZB membrane was achieved when the H2O/SiO2 ratio was 4, whereas the c-oriented was obtained when the H2O/SiO2 ratio of 7(Figure 3). The (h0l)-oriented HSZB membrane exhibited high fluxes up to 1.45 and 1.05 kg·m−2·h−1 with respect to 1 and 5 wt% n-butanol/water mixtures, and the corresponding separation factors were 36.5 and 32.5, respectively. This study indicates that (h00l)- oriented HSZB membranes can be prepared at higher H2O/SiO2 ratio and would be more promising for n-butanol recovery from dilute aqueous solution due to high fluxes with relatively high separation factors because of high hydrophobicity. Figure 3. The influence of H2O/SiO2 ratio on the formation process of HSZB membranes with preferred orientation during an evolutionary growth process. Reproduced with Permission from [57]. Figure 3. The inﬂuence of H2O/SiO2 ratio on the formation process of HSZB membranes with preferred orientation during an evolutionary growth process. Reproduced with Permission from [57]. Figure 3. The influence of H2O/SiO2 ratio on the formation process of HSZB membranes with preferred orientation during an evolutionary growth process. Reproduced with Permission from [57]. Figure 3. The inﬂuence of H2O/SiO2 ratio on the formation process of HSZB membranes with preferred orientation during an evolutionary growth process. Reproduced with Permission from [57]. Taleb et al. 2.2. Silica-Based Membranes They reported that the highest eﬃciency was achieved using a Titania membrane, which was characterized by the highest value of the pervaporation separation index (PSI) and the highest value of permeate ﬂux of organic compounds in water–EtAc (JEtAc = 1.1 kg·m−2·h−1; PSIEtAc = 140 kg·m−2·h−1) and water–MTBE (JMTBE = 1.0 kg·m−2·h−1; PSIMTBE = 194 kg·m−2·h−1) systems. Tres et al. [51] have studied the potential applicability of ceramic membrane technology in vegetable oil processing and biodiesel industries in the solvent recovery step, in which separations of mixtures of reﬁned soybean oil/n-hexane, crude soybean oil/n-hexane (industrial miscella) and reﬁned soybean oil/pressurized n-butane were studied. When the commercial ceramic membranes with molecular weight cut-oﬀs between 5 and 10 kDa were investigated, oil rejections up to 100%, total permeate ﬂuxes (oil + solvent) up to 42.97 kg·m−2·h−1 with oil permeate ﬂuxes up to 1.4 kg·m−2·h−1 were observed. p p g Amelio et al. [52] applied a hybrid process of distillation/pervaporation with the ceramic membrane, hybrid silica (HybSi) and showed that it can give both economic beneﬁt and low environmental impact (with life cycle assessment) in solvent recovery (acetone from water) when compared to conventional waste solvent incineration. Furthermore, this hybrid process will bring an economic beneﬁt of the replacement cost of fresh solvent (about 850 U$S/ton), which is considered as a credit value. Nagasawa [53] applied the same HybSi membranes for the water/ isopropyl alcohol (IPA) separation, in which their water ﬂuxes were like that of NaA zeolite membranes, but their separation factors are lower than NaA zeolite membranes. Clay-alumina-based tubular MF membranes were explored as a viable option for solvent separation and possess advantages such as high micronutrient content (1.56% oryzanol) and negligible oil loss (2.6%) by Roy et al. [54]. When the membranes were operated for 10 h with a 0.7 bar trans-membrane pressure, permeate ﬂuxes of 15 and 8 L/m2·h were achieved for the degumming-dewaxing and deacidiﬁcation operations. Maitlo et al. [55] introduced 8 of 24 Membranes 2019, 9, 128 hydrophobic-oleophilic nature into silica membranes and the membrane showed 100% eﬃciency for toluene solvent from water within 50 min. All the other membranes tested also showed good eﬃciency for solvents and no permeability for water, which can be from potential candidates for oil–water and organic solvent–water separation. 2.2. Silica-Based Membranes [58] has used natural Moroccan clay for the development of a ceramic support for the purpose of microfiltration. This clay mostly consists of Al2O3 and SiO2, and exhibits properties like high mechanical resistance, high chemical and thermal stability. The macroporous tubular support uses an intermediate layer of ZrO2 material to serve the purpose of microfiltration of methylene blue. The supports are fabricated by extrusion process along with heat treatment and then they are coated with dispersed ZrO2 and dried. The support displays a permeability of 1926 L/h·m2·bar for pure distilled water and a maximum rejection rate of 3.8% for the treatment of methylene blue in the microfiltration process. Taleb et al. [58] has used natural Moroccan clay for the development of a ceramic support for the purpose of microﬁltration. This clay mostly consists of Al2O3 and SiO2, and exhibits properties like high mechanical resistance, high chemical and thermal stability. The macroporous tubular support uses an intermediate layer of ZrO2 material to serve the purpose of microﬁltration of methylene blue. The supports are fabricated by extrusion process along with heat treatment and then they are coated with dispersed ZrO2 and dried. The support displays a permeability of 1926 L/h·m2·bar for pure distilled water and a maximum rejection rate of 3.8% for the treatment of methylene blue in the microﬁltration process. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane In this case, the reinforcement present in the membrane was 2 wt% and heat was provided to the membrane via light irradiation. Stronger heat treatment for the composite can be given via laser radiation when the gold nanoparticles are well dispersed in the polymer matrix during in situ synthesis with a mean particle size of 5 nm and larger particles with a maximum size of 20 nm. At high pressures of 3.5, 4.5 and 10 bar, the ethanol ﬂux displayed an average of 0.25 L·m−2·h−1, 0.4 L·m−2·h−1 and 1 L·m−2·h−1, respectively. The authors concluded that the membrane ﬂux is improved by photothermal heating, without a major reduction in the rejection. The gradual increase in intensity of radiation determines the increase in permeance. Functionalization of carbon nanotubes has found its application in various ﬁelds of research and is capable of enhancing the separation process, due to their unique properties such as high ﬂexibility, low density and existence of substantial nanochannels. The design of amine functionalized multi walled carbon nanotubes (NH2-MWCNT) with the matrix of P84 polyimide by Farahani et al. [60] has led to the formation of cross-linked mixed matrix membrane with enhanced ﬂux for organic solvent nanoﬁltration (OSN) purposes. The hydrophilic carbon nanotubes not only enhance the liquid sorption and transportation in the membrane, but also increase the porosity and pore size, which in turn elevates the solvent ﬂuxes. Although the higher loading of ﬁllers could decline the rejection, they could also lead to agglomeration. The same group also fabricated MMM with the functionalization of MWCNT consisting of carboxyl group in P84 polyimide matrix for OSN applications. The properties such as transfer, sorption and porosity were enhanced with the hydrophilic functional groups on the MWCNT ﬁllers. With the maximum embedding of 0.075 wt% of the filler, the permeance of solvents such as water, ethanol and isopropanol across the membranes increases, as a higher concentration of carbon nanotubes can lead to agglomeration and reduce separation performance. A similar crosslink of MWCNT-COOH has been introduced for the purpose of organic solvent nanofiltration by Farahani et al. [61]. He reported the permeance of solvents like pure water, isopropanol and ethanol, and rejection of rose Bengal in ethanol and isopropanol solutions. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane The customization of organic-inorganic polymer hybrids, which involves the combination of properties of both the materials, gives rise to a Mixed Matrix membrane (MMM) or nanocomposite membrane. These membranes consist of two phases: one being the continuous phase acts as the support medium and the other is a filler in a stationary phase. The supporting medium is also known as the matrix. It is usually made of polymers, while the reinforcements provided are in nanoscale dimensions. The addition of nanoparticles to the membranes tends to modify their structure as well The customization of organic-inorganic polymer hybrids, which involves the combination of properties of both the materials, gives rise to a Mixed Matrix membrane (MMM) or nanocomposite membrane. These membranes consist of two phases: one being the continuous phase acts as the support medium and the other is a ﬁller in a stationary phase. The supporting medium is also known as the matrix. It is usually made of polymers, while the reinforcements provided are in nanoscale dimensions. The addition of nanoparticles to the membranes tends to modify their structure as well as Membranes 2019, 9, 128 9 of 24 enhance their properties. Incorporation of nanoparticles in the membrane for modiﬁcation purpose can be done by the following methods: (1) addition of nanoparticles into the polymeric solution before casting, (2) deposition of nanoparticles on the surface of the membrane, and (3) nanoparticles are used to ﬁll the pores on the polymeric membrane. Livingston et al. [19] prepared a nanocomposite with TiO2 nanoparticles incorporated in the crosslinked Polyimide membrane matrix and reported that the addition of reinforcement before the casting process resulted in suppression of macro sized pores on the membrane morphology. The TiO2 incorporation also enhances the hydrophilicity and mechanical properties of the system, with the water contact angle decreasing from 81◦to 54◦and steady ethanol ﬂux settling at 128 L·m−2·h−1 and 105 L·m−2·h−1 for DMF. In the case of Organic Solvent Nanoﬁltration (OSN) membranes, the rejection and ﬂux rates might not be satisfactory due to the pores in nanoscale, but the embedding of gold nanoparticles in cellulose acetate membranes with the absence of defects on the active layer was approached by Vanherck et al. [59], with a 15% increment in the water ﬂux and 400% for pure solvents like ethanol and isopropanol. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane The permeance of the solvents depend on the MWCNT-COOH loadings, where the permeance increases with an increase in the content of fillers in the membrane from 0 to 0.05 wt% and the permeance decreases with further increase in the filler loading. However, the cross-linked MMM with loading of 0.05 wt% of MWCNT-COOH results in 99% rejection of rose Bengal in isopropanol and 85% in ethanol with a permeance of 9.6 L/m2·h bar at 5 bar. Defect free layers of poly(dimethyl siloxane) (PDMS) were incorporated with ZIF-8 nanoparticles for the application of permselective pervaporation. Mao et al. [15] stated that the uniform dispersion of ZIF-8 nanoparticles enhanced the hydrophobicity, thermal stability and aﬃnity towards ethanol, which results in selectivity of larger content. The MMM was synthesized by in situ fabrication, where the synthesis time played a vital role in regulating the contact angle of the hydrophobic surface of the membrane. With increase in time from 3–10 min, the contact angle varied from 124◦to 138◦, but, with variation of time from 10–30 min, there was a decline in the contact angle to 127◦. The increase in feed ﬂow rate results in a comparative rise in permeation and separation ﬂux of the membrane. 10 of 24 10 of 24 Membranes 2019, 9, 128 Concurrently, the permeability of water and ethanol was enhanced with feed ﬂow rate; at 40 ◦C, the MMM separating 5 wt% ethanol aqueous solution displayed a ﬂow rate of 90 Lh−1, along with a separation factor of 12.1 due to the defect free active layer and also achieved a high permeation ﬂux of 1778 g·m−2·h−1. As a result, durable membranes with the ability to perform under diﬀerent conditions were fabricated. Concurrently, the permeability of water and ethanol was enhanced with feed ﬂow rate; at 40 ◦C, the MMM separating 5 wt% ethanol aqueous solution displayed a ﬂow rate of 90 Lh−1, along with a separation factor of 12.1 due to the defect free active layer and also achieved a high permeation ﬂux of 1778 g·m−2·h−1. As a result, durable membranes with the ability to perform under diﬀerent conditions were fabricated. Metal Organic Frameworks (MOFs) are in aﬃnity with the polymeric chains rather than the inorganic ones due to the presence of organic linkers. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane The embedding of hydrophobic and hydrophilic MOF nanoparticles (MIL-53-Al, MIL-101-Cr, ZIF-8, and NH2-MIL-53(Al), Table 2) into polyamide layer in order to form thin ﬁlm nanocomposite membranes were reported by Sorribas et al. [62]. Around 0.2 wt% of synthesized MOFs was dispersed in the organic phase to produce thin ﬁlm nanocomposite membranes (TFN-MOF membrane). ZIF-8 nanoparticles being a hydrophobic MOF displayed an increase in contact angle, while MIL-53 Al, NH2-MIL-53(Al) and MIL-101-Cr displayed small contact angle values due to the hydrophilic nature. The changes in hydrophilicity can lead to changes in the chemical structure of the thin ﬁlm, which can either hydrate or release heat when in contact with organic solvents. The observed permeance results indicate that all the membranes exhibited a rejection greater than 90%, with the permeance varying from 1.5 to 3.9 L·m−2·h−1·bar−1 (Figure 4, Table 3). The order of increase in permeance of methanol/PS (Polystyrene) in the TFN is NH2-MIL-53 (Al) < MIL-53(Al) < ZIF-8 < MIL-101(Cr). Membranes 2019, 9, x 10 of 24 al. [62]. Around 0.2 wt% of synthesized MOFs was dispersed in the organic phase to produce thin film nanocomposite membranes (TFN-MOF membrane). ZIF-8 nanoparticles being a hydrophobic MOF displayed an increase in contact angle, while MIL-53 Al, NH2-MIL-53(Al) and MIL-101-Cr displayed small contact angle values due to the hydrophilic nature. The changes in hydrophilicity can lead to changes in the chemical structure of the thin film, which can either hydrate or release heat when in contact with organic solvents. The observed permeance results indicate that all the membranes exhibited a rejection greater than 90%, with the permeance varying from 1.5 to 3.9 L·m−2·h−1·bar−1 (Figure 4, Table 3) . The order of increase in permeance of methanol/PS (Polystyrene) in the TFN is NH2-MIL-53 (Al) < MIL-53(Al) < ZIF-8 < MIL-101(Cr). Table 2. Contact angle of thin film composite (TFC) and thin film nanocomposite MOF (TFN-MOF) membranes with 0 2% (w/v) concentration in the organic phase before Interfacial polymerization (IP) Table 2. Contact angle of thin ﬁlm composite (TFC) and thin ﬁlm nanocomposite MOF (TFN-MOF) membranes with 0.2% (w/v) concentration in the organic phase before Interfacial polymerization (IP) reaction. For TFN-MIL-101, concentration was changed from 0.05 to 0.4% (w/v); Reproduced with permission from [62]. reaction. For TFN-MIL-101, concentration was changed from 0.05 to 0.4% (w/v); Reproduced with permission from [62]. Membrane Contact Angle (°) TFC ( without MOF) - - ~73 - Table 2. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane Contact angle of thin ﬁlm composite (TFC) and thin ﬁlm nanocomposite MOF (TFN-MOF) membranes with 0.2% (w/v) concentration in the organic phase before Interfacial polymerization (IP) reaction. For TFN-MIL-101, concentration was changed from 0.05 to 0.4% (w/v); Reproduced with permission from [62]. reaction. For TFN-MIL-101, concentration was changed from 0.05 to 0.4% (w/v); Reproduced with permission from [62]. Membrane Contact Angle (°) TFC ( without MOF) - - ~73 - Membrane Contact Angle (◦) TFC (without MOF) - - ~73 - TFN-NH2-MIL-53 (Al) - - ~49 - TFN-MIL-53 (Al) - - ~54 - TFN-ZIF-8 - - ~75 - TFN-MIL-101 (Cr) ~53 (0.05) ~52 (0.1) ~50 (0.2) ~43 (0.4) The values in parentheses are subjected to MOF loading in %. ( ) TFN-NH2-MIL-53 (Al) - - ~49 - TFN-MIL-53 (Al) - - ~54 - TFN-ZIF-8 - - ~75 - TFN-MIL-101 (Cr) ~53 (0.05) ~52 (0.1) ~50 (0.2) ~43 (0.4) # The values in parentheses are subjected to MOF loading in %. Table 3. Organic Solvent nanofiltration (OSN) results of TFN membranes for MeOH at 30 °C and Table 3. Organic Solvent nanoﬁltration (OSN) results of TFN membranes for MeOH at 30 ◦C and 30 bar. (The values given are an average of 3–4 diﬀerent membranes); Reproduced with permission from [63]. bar. (The values given are an average of 3-4 different membranes); Reproduced with permission from [63]. Table 3. Organic Solvent nanoﬁltration (OSN) results of TFN membranes for MeOH at 30 ◦C and 30 bar. (The values given are an average of 3–4 diﬀerent membranes); Reproduced with permission from [63]. bar. (The values given are an average of 3-4 different membranes); Reproduced with permission from [63]. Table 3. Organic Solvent nanoﬁltration (OSN) results of TFN membranes for MeOH at 30 ◦C and 30 bar. (The values given are an average of 3–4 diﬀerent membranes); Reproduced with permission from [63]. Membrane Permeance Methanol (L·m−2·h−1·bar−1) Permeance Methanol/PS (Polystyrene) (L·m−2·h−1·bar−1) TFC (without MOF) 1.8 1.5 TFN-NH2-MIL-53 (Al) 2.3 1.8 TFN-MIL-53 (Al) 2.3 1.9 TFN-ZIF-8 2.5 2.1 TFN-MIL-101 (Cr) 4.2 3.9 bar. (The values given are an average of 3 4 different membranes); Reproduced with permission from [63]. Membrane Permeance Methanol (L·m−2·h−1·bar−1) Permeance Methanol/ PS (Polystyrene) (L·m−2·h−1·bar−1) TFC ( without MOF) 1.8 1.5 TFN-NH2-MIL-53 (Al) 2.3 1.8 TFN-MIL-53 (Al) 2.3 1.9 TFN-ZIF-8 2.5 2.1 TFN-MIL-101 (Cr) 4.2 3.9 Figure 4. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane (a) Building blocks of ZIF-8 with ZnN4; (b) pore system in NH2-MIL-53 with AlO6; (c) building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63] Figure 4. (a) Building blocks of ZIF-8 with ZnN4; (b) pore system in NH2-MIL-53 with AlO6; (c) building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63]. Membrane Permeance Methanol (L·m−2·h−1·bar−1) Permeance Methanol/PS (Polystyrene) (L·m−2·h−1·bar−1) TFC (without MOF) 1.8 1.5 TFN-NH2-MIL-53 (Al) 2.3 1.8 TFN-MIL-53 (Al) 2.3 1.9 TFN-ZIF-8 2.5 2.1 TFN-MIL-101 (Cr) 4.2 3.9 Membrane Permeance Methanol (L·m−2·h−1·bar−1) Permeance Methanol/ PS (Polystyrene) (L·m−2·h−1·bar−1) TFC ( without MOF) 1.8 1.5 TFN-NH2-MIL-53 (Al) 2.3 1.8 TFN-MIL-53 (Al) 2.3 1.9 TFN-ZIF-8 2.5 2.1 TFN-MIL-101 (Cr) 4.2 3.9 Figure 4. (a) Building blocks of ZIF-8 with ZnN4; (b) pore system in NH2-MIL-53 with AlO6; (c) building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63]. Figure 4. (a) Building blocks of ZIF-8 with ZnN4; (b) pore system in NH2-MIL-53 with AlO6; (c) building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63]. P ti b d f th ti f li id i t h ﬁlt ti f il t d Membrane Permeance Methanol (L·m−2·h−1·bar−1) Permeance Methanol/PS (Polystyrene) (L·m−2·h−1·bar−1) TFC (without MOF) 1.8 1.5 TFN-NH2-MIL-53 (Al) 2.3 1.8 TFN-MIL-53 (Al) 2.3 1.9 TFN-ZIF-8 2.5 2.1 TFN-MIL-101 (Cr) 4.2 3.9 Membrane Permeance Methanol (L·m−2·h−1·bar−1) Permeance Methanol/ PS (Polystyrene) (L·m−2·h−1·bar−1) TFC ( without MOF) 1.8 1.5 TFN-NH2-MIL-53 (Al) 2.3 1.8 TFN-MIL-53 (Al) 2.3 1.9 TFN-ZIF-8 2.5 2.1 TFN-MIL-101 (Cr) 4.2 3.9 Figure 4. (a) Building blocks of ZIF-8 with ZnN4; (b) pore system in NH2-MIL-53 with AlO6; (c) Figure 4. (a) Building blocks of ZIF-8 with ZnN4; (b) pore system in NH2-MIL-53 with AlO6; (c) building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63]. building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63]. Pervaporation can be used for the separation of liquid mixtures when filtration fails to do s So iba et al [62] di u ed that i the e a atio of etha ol/ ate i tu e HKUST 1 hyd o hil Pervaporation can be used for the separation of liquid mixtures when ﬁltration fails to do so Sorribas et al. [62] discussed that, in the separation of ethanol/water mixture, HKUST-1 hydrophilic building blocks of MIL-101 with mesoporous cages; Reproduced with permission from [63]. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane Pervaporation can be used for the separation of liquid mixtures when filtration fails to do so Sorribas et al [62] discussed that in the separation of ethanol/ ater mixture HKUST 1 hydrophil Pervaporation can be used for the separation of liquid mixtures when ﬁltration fails to do so Sorribas et al. [62] discussed that, in the separation of ethanol/water mixture, HKUST-1 hydrophilic 11 of 24 Membranes 2019, 9, 128 MOF is used in 40 wt% mixed membrane matrices. The incorporation of MOF in the membrane matrix increased the ﬂux by two times and left the selectivity and permeability of water unaltered in the membrane. Su et al. [64] presented a high separation performance of alcohol/water distillation when ZIF-8 nanoparticles were incorporated into PDMS hollow ﬁber membrane modules. The ZIF-8 nanoparticles enhanced the permeability of both isopropanol and water molecules through the membrane due to high surface area and porosity; this eventually increased the mass transfer eﬃciency between the phases from 0.95 cm/s to 1.65 cm/s. This membrane displayed higher IPA concentration of 62.2% (mol/mol) with height mass transfer unit (HTU) value of 4.9 cm, when compared with the pure PDMS membrane module. The addition of Mesoporous silica sphere (MSS) with ZIF-71 into PDMS matrix was studied by Naik et al. [65]. The incorporation of composite spheres in PDMS showed relatively improved ﬂux and separation factors for water and ethanol mixtures under pervaporation, in comparison with the pure and MSS ﬁlled PDMS membranes. For the separation of 6% aqueous ethanol at 40 ºC for various ﬁller loading (0, 10, 15 and 20 wt%), the normalized ﬂux and separation factor were enhanced by addition of the ﬁller. The reported ﬂuxes and separation factor for the loadings 0, 10, 15 and 20 wt% of the ﬁller are 0.32, 0.91, 0.95 and 1 kg/m2·h, and 8, 8.2, 11 and 13 ß, respectively. Similarly, for the addition of ZIF-8 along with MSS in the PDMS matrix, the ﬂux and separation factor displayed values of 0.63 kg/m2·h and 14 ß, respectively, for the loading of 20 wt% of the ﬁller. Fan et al. [66] studied the fabrication of ZIF-8-PDMS membrane for the recovery of n-butanol from aqueous solution. The ZIF-8 nanoparticles were added to the membrane surface through the process of simultaneous spray self-assembly. 2.4. Metal Organic Frameworks Metal organic frameworks (MOF) are hybrid crystalline materials having metal ions linked with a network of organic ligands. They have a porous structure due to the presence of potential voids in the organic network and have large surface area with new topologies. These pores serve the purpose of storing molecules and the structure might experience instability during the elimination of molecules. These functional materials have found various applications such as ion exchange, adsorption and catalysis. The organic solvent nanoﬁltration (OSN) membranes face a major challenge in establishing materials that are stable and suitable for producing high performance over a long span of time, as the early MOFs displayed poor chemical stability with atmospheric moisture along with easy deterioration of framework. On overcoming their disadvantages, MOFs presented a structural diversity greater than that of zeolite and exhibited surface areas of 7000 m2·g−1. The membrane performance can be varied based on the post treatments conditions as well as the MOF loadings. MOFs have pore size ranging from 0.3 nm to 10 nm depending on composition and positioning MOF membranes in the nanoﬁltration establishment. The membranes involved in the organic solvent nanoﬁltration are evaluated based on the permeance and rejection of chemical compounds. The selective permeability in MOF membranes can be achieved by molecular sieving, and the presence of high porosity within them favors the permeates’ high ﬂux with a high rejection of contaminants. The integration of selective pores and molecular sieving concept in the membranes incorporates both the desirable properties required for the membranes like, high selectivity and ﬂux. In the case of ZIF-78, Kasik et al. reported [67] the separation of cyclohexanone–cyclohexanol mixture through pervaporation, with 50:50 ratios experiencing a permselectivity of 1:2 and a total ﬂux of around 8.7 × 10−2 kg·m−2·h−1 at room temperature. 2.3. Mixed Matrix Membrane 2.3. Mixed Matrix Membrane At extremely high loading of 40 wt% of ZIF-8 in PDMS, high total ﬂux of 4846.2 kg/m2·h and separation factor of 81.6 were obtained at a feed temperature of 80 ◦C. The ﬂux and separation factor values attained for pure PDMS are 1000 kg/m2·h and 38 and tend to increase with addition of ZIF-8 up to 40 wt%. 2.4.2. MIL-53 Materials Institute Lavoisier is a metal organic framework based on aluminium that is linked with carboxylate groups into a three-dimensional network. These ﬂexible MOFs are capable of altering their shape and size with heat treatment and adsorption of CO2. Van der Bruggen et al. [68] conducted a study on the incorporation of MIL-53 (Al) into a polymer matrix to perform the ﬁltration of organic solvents with pore size distribution between 0.4 nm to 1.1 nm, and they obtained results with increased permeance of solvents like ethanol. MIL-53 was embedded into the polyamide matrix with diﬀerent concentrations of the ﬁller (0.3, 0.5, 1.0 and 1.5 wt%). The membranes displayed ﬂuctuations on exposure to the organic solvents. In the case of the membrane with 0.5 wt% concentration of the ﬁller, the membrane observed stability was 10 days, and also showed the increase in ethanol permeance from 0.2 L·m−2·h−1·bar−1 to 0.7 L·m−2·h−1·bar−1 (up to 289%) and a slight reduction in the brilliant blue rejection from 97% to 94% (3% less). Zhu et al. [68] reported the application of MIL-53 for the dehydration of water–ethyl acetate mixtures through pervaporation at a temperature of 60 ◦C, where the high water selectivity led to the permeate containing 99% water and the 7% feed. Furthermore, the membrane with the ﬁllers exhibited high stability of 200 h of operation. The ﬂux of the membrane rises with increase in the MOF content (0–0.5 wt%), and the increase in ﬂux is a result of the hydrophilicity of MIL-53 (Al) (Table 4). Increased inclusion of MIL-53(Al) particles has resulted in aggregation of them and, in turn, decreased the ﬂux of the membrane. Decrease in the contact angle was observed for the membranes due to hydrophilicity of the added MIL-53, which has increased the aﬃnity of the matrix to water molecules. Table 4. Performance of membranes with diﬀerent MIL-53 (Al) contents; Reproduced with permission from Ref [68]. MOF Content (wt%) Contact Angle (◦C) 0 86 ± 2 0.3 79 ± 2 0.5 76 ± 1 1.0 73 ± 1 1.5 71 ± 2 Table 4. Performance of membranes with diﬀerent MIL-53 (Al) contents; Reproduced with permission from Ref [68] Li et al. [69] applied the micropatterning concept for the ﬁrst time to MOF membrane with enhanced molecular sieving property, which can used in compact devices. 2.4.1. MIL-101 (Cr) MIL (Materials Institute Lavoisier) are MOFs with chromium as the metal linking to organic linkers. Sorribas et al. [63] loaded diﬀerent concentrations of MIL-101-Cr in thin ﬁlm nanocomposite, 12 of 24 Membranes 2019, 9, 128 and noticed that the permeance and rejection varied generally. The concentration was varied between 0.05–0.4 wt%, and the permeance of tetrahydrofuran and methanol increased gradually up to 0.2 wt% loading. Nonetheless, there was a slight increase in permeance from 0.2 to 0.4 wt% as a result of aggregation of particles at high concentration, and not many changes were observed for the rejection on the basis of MOF loading. The nanoparticles embedded a TFN membrane with 0.2 wt%. MOF displayed an increase of 160% permeance compared to the thin ﬁlm composite in the case of methanol/PS with 3.9 L·m−2·h−1·bar−1 (from 1.5 L·m−2·h−1·bar−1), whereas the tetrahydrofuran (THF)/PS permeance increased to 488% with permeance up to 10.0 L·m−2·h−1·bar−1 (1.7 L m−2·h−1·bar−1), which is due to low viscosity of THF. For the loading of 0.4 wt% of MIL-101 (Cr), the permeance was reported as 11 L·m−2·h−1·bar−1 with a rejection of THF greater than 90%. This TFNC achieved high performance in organic solvent nanoﬁltration with increasing permeance and preserving the high rejection. 2.4.2. MIL-53 Ultrathin polycrystalline zirconium-MOF UiO-66 membranes with a thickness down to 250 nm on a patterned porous yttria-stabilized zirconia (YSZ) ceramic substrates were grown by a bottom-up procedure. The patterned UiO-66 membranes exhibited good molecular separation property (separation factor of over 1000) and 100% improvement in the apparent permeation ﬂux of 2.96 kg·m−2·h−1 (patterned UiO-66 membranes were considerably higher than the unpatterned membrane, Figure 5) in butanol dehydration through 13 of 24 Membranes 2019, 9, 128 the pervaporation process and showed that growing high-quality MOF thin ﬁlms (defect-free) on complex surfaces can be achieved in MOF membrane formation. Membranes 2019, 9, x 13 of 24 Figure 5. Separation performance of UiO-66 membranes with different patterns; Reproduced with permission from [69]. Figure 5. Separation performance of UiO-66 membranes with diﬀerent patterns; Reproduced with permission from [69]. Figure 5. Separation performance of UiO-66 membranes with different patterns; Reproduced with permission from [69]. Figure 5. Separation performance of UiO-66 membranes with diﬀerent patterns; Reproduced with permission from [69]. In MOF, organic linkers are attached with metal ions that are sufficiently strong to make the MOF structure adequately robust and uniform distribution of metal active sites. Zirehpour et al [70] immobilized MOF nanocrystals into the active layer of the forward osmosis (FO) membranes via dispersion in the organic solution followed by interfacial polymerization. The immobilization improved biofouling resistance in the membranes, with 8% decline in flux after 24 h of operation in biofouling experiments, whereas control membranes had a greater decline of ~21%. A similar approach can be applied in MOF membranes in the solvent recovery applications, as membrane antifouling behaviour will improve membrane performance, which will decrease operational costs, and will also increase the membrane life In MOF, organic linkers are attached with metal ions that are suﬃciently strong to make the MOF structure adequately robust and uniform distribution of metal active sites. Zirehpour et al. [70] immobilized MOF nanocrystals into the active layer of the forward osmosis (FO) membranes via dispersion in the organic solution followed by interfacial polymerization. The immobilization improved biofouling resistance in the membranes, with 8% decline in ﬂux after 24 h of operation in biofouling experiments, whereas control membranes had a greater decline of ~21%. A similar approach can be applied in MOF membranes in the solvent recovery applications, as membrane antifouling behaviour will improve membrane performance, which will decrease operational costs, and will also increase the membrane life. 2.4.2. MIL-53 and will also increase the membrane life. Although MOFs are synthesized in the form of large crystals or micro-crystalline powders, they have few drawbacks such as inferior mechanical properties (fragile, brittle) and low processability, thereby hindering their utilization of defect-free polycrystalline membranes for industrial scale applications. Satheeshkumar et al. [71] have overcome these issues, by the fabrication of a free- standing mixed-matrix membrane (MMM) containing covalently incorporated metal–organic framework (MOF) particles (up to 60 wt%) consisting of vinyl functionality by applying the thiol-ene photopolymerization technique. The free standing MMM was readily produced by irradiation of a polymerization mixture containing UiO-66-CH=CH2 (synthesised from 2-vinyl-1,4-dicarboxylic acid with ZrCl4) poly(ethylene glycol) divinyl ether (PEO-250), pentaerythritol tetra(3- mercaptopropionate) (PETM), 2,20-(ethylenedioxy)diethanethiol (EDDT), and 2,2-dimethoxy-2- phenylacetophenone (DMPA) as a photoradical initiator Although MOFs are synthesized in the form of large crystals or micro-crystalline powders, they have few drawbacks such as inferior mechanical properties (fragile, brittle) and low processability, thereby hindering their utilization of defect-free polycrystalline membranes for industrial scale applications. Satheeshkumar et al. [71] have overcome these issues, by the fabrication of a free-standing mixed-matrix membrane (MMM) containing covalently incorporated metal–organic framework (MOF) particles (up to 60 wt%) consisting of vinyl functionality by applying the thiol-ene photopolymerization technique. The free standing MMM was readily produced by irradiation of a polymerization mixture containing UiO-66-CH=CH2 (synthesised from 2-vinyl-1,4-dicarboxylic acid with ZrCl4) poly(ethylene glycol) divinyl ether (PEO-250), pentaerythritol tetra(3-mercaptopropionate) (PETM), 2,20-(ethylenedioxy)diethanethiol (EDDT), and 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photoradical initiator. phenylacetophenone (DMPA) as a photoradical initiator. Although studies on MOF films on various copper supports such as plates, disks, meshes, beads and copper coated silicon wafers, using gold and glass carbon electrodes, were reported, electrochemical deposition of MOFs directly on porous hollow fibers (HFs) have not been reported. Demirel et al. [72] reported on the thin metal organic framework (Cu-BTC, (Cu3(BTC)2,BTC =benzene- 1,3,5-tricarboxylate) films on Cu-HFs, to study the effect of the presence of a supporting electrolyte and the magnitude of the applied electrical potential on the formation and the morphology of the Although studies on MOF ﬁlms on various copper supports such as plates, disks, meshes, beads and copper coated silicon wafers, using gold and glass carbon electrodes, were reported, electrochemical deposition of MOFs directly on porous hollow ﬁbers (HFs) have not been reported. Demirel et al. 2.4.3. ZIF (1) ZIF-8 Zeolite imidazolate framework (ZIF) is a metal organic framework consisting of four imidazolate rings linked to zinc ions. The metal ions are covalently bridged with imidazolate in a similar pattern to silicon or aluminum with oxygen in zeolites. ZIF-8 has a porous structure with pores in micro to nanometer range and topology that is indistinguishable to zeolite. ZIFs exhibit properties like high surface area, robust porosity, thermal and chemical stability, and high speciﬁcity. Studies have showed their potential in separation of hydrocarbon mixtures like ethane/propane, ethylene/propylene and alcohols. They tend to oﬀer high separation factor at a low energy consumption. The hydrophobic nature makes it diﬃcult for the extraction of pollutants in an aqueous phase due to the poor contact between phases. Therefore, Maya et al. [73] engineered dispersions of ZIF-8 crystals in binary solvent mixtures for the extraction of solvents, identical to the dispersive liquid-phase microextraction technique. However, ZIF-8, with hydrophobic pore surface and hydrophilic crystal surface, along with aperture size of ~3.4 Å seems to be unfavorable for the transfer of organic molecules. Xu et al. [74] incorporated β-CD@ZIF-8 into various polymer supports, and tested these membranes for their dye/solvent separation performances. The presence of ZIF in the selective layer enhances the permeance of solvent. In the separation of rose Bengal and the solvent using PA membranes with β-CD@ZIF-8 nanoparticles embedded in them, they attained rejection values of 96.2 ± 1.6% of RB/MeOH, and 95.0 ± 1.1% of RB/THF. Similarly, for PPA-05matrix, -β-CD@ZIF-8 rejection of 96.6 ± 1.8% of RB/MeOH and 94.5 ± 0.5% of RB/THF were observed. With the addition of 25 wt% β-CD@ZIF-8 in PA, the water contact angle was minimized, which reduced the resistance to mass transfer of solvents and also resulted in the highest methanol ﬂux in MF (mass ﬂux) scale. ZIF-68 membranes with large pores were applied for the molecular sieving of large sized liquid molecules. Kasik et al. [18] reported the pervaporation of p-xylene using ZIF-68, synthesized by reactive seed method, which produced ﬂux of 492 × 10−5 mol/m2·s, 5.4 times greater than MOF-5 membranes. However, pervaporation ﬂux of the larger size molecules using ZIF-68 membranes was lower than MOF-5. (2) ZIF-90 MOFs with linkers of transition metals and imidazolate have porous structure, making them desirable candidates for molecular sieve membranes. 2.4.2. MIL-53 [72] reported on the thin metal organic framework (Cu-BTC, (Cu3(BTC)2,BTC =benzene-1,3,5-tricarboxylate) ﬁlms on Cu-HFs, to study the eﬀect of the presence of a supporting electrolyte and the magnitude of the applied electrical potential on the formation and the morphology of the ﬁlms. In the presence of a 14 of 24 Membranes 2019, 9, 128 supporting electrolyte, formation of less homogeneous ﬁlms and the growth of MOF crystals were observed in the liquid, whereas, in the absence of a supporting electrolyte, and at low potential, more uniform ﬁlms with smaller particles were obtained. ( (3) ZIF-71 Yin et al. [13] reported the recovery of solvents like ethanol and 1-butanol using ZIF-71/PDMS nanocomposite membranes. Being a hydrophobic polymer with the ability to perform the pervaporation process, polydimethylsiloxane (PDMS) was embedded with different quantities of ZIF-71 fillers. The content of fillers was optimized for 40 wt% load of ZIF-71 in PDMS attaining the maximum selectivity of 0.81 ± 0.04 (separation factor of 12.5 ± 0.3 for 2 wt% ethanol feed) and maximum 1-butanol/water selectivity of 5.64 ± 0.15 (separation factor of 69.9 ± 1.8 for 2 wt% 1-butanol feed). The composites displayed a decrease in fracture strain from 381.4 to 35.6 J/cm2 when formed through a condensation cure rather than an addition cure. Membranes with 40 wt% ZIF-71exhibited maximum permeability and selectivity, for ethanol/water mixture, with ethanol permeability of 24,809 ± 4374 Barrer, water permeability of 30,661 ± 4015 Barrer and selectivity of 0.81 ± 0.04. For 1- butanol/water separation, 40 wt% ZIF-71 loading MMMs had the highest 1-butanol permeability of 123,045 ± 17,118 Barrer, water permeability of 21,758 ± 2497 Barrer and selectivity of 5.64 ± 0.15. In addition to the above ZIFs, several other ZIF materials with increased pore sizes were synthesized [7] and tested for various applications (Figure 7). Some of the notable ZIF materials pore sizes are reported below in Table 5. It is to be noted here that ZIF membranes of smaller pore ZIF are synthesized extensively and studied for the separation of smaller molecules such as H2 purification Yin et al. [13] reported the recovery of solvents like ethanol and 1-butanol using ZIF-71/PDMS nanocomposite membranes. Being a hydrophobic polymer with the ability to perform the pervaporation process, polydimethylsiloxane (PDMS) was embedded with diﬀerent quantities of ZIF-71 ﬁllers. The content of ﬁllers was optimized for 40 wt% load of ZIF-71 in PDMS attaining the maximum selectivity of 0.81 ± 0.04 (separation factor of 12.5 ± 0.3 for 2 wt% ethanol feed) and maximum 1-butanol/water selectivity of 5.64 ± 0.15 (separation factor of 69.9 ± 1.8 for 2 wt% 1-butanol feed). The composites displayed a decrease in fracture strain from 381.4 to 35.6 J/cm2 when formed through a condensation cure rather than an addition cure. Membranes with 40 wt% ZIF-71exhibited maximum permeability and selectivity, for ethanol/water mixture, with ethanol permeability of 24,809 ± 4374 Barrer, water permeability of 30,661 ± 4015 Barrer and selectivity of 0.81 ± 0.04. ( (3) ZIF-71 For 1-butanol/water separation, 40 wt% ZIF-71 loading MMMs had the highest 1-butanol permeability of 123,045 ± 17,118 Barrer, water permeability of 21,758 ± 2497 Barrer and selectivity of 5.64 ± 0.15. synthesized extensively and studied for the separation of smaller molecules, such as H2 purification and CO2 sequestration. On the other hand, few research studies were focused on the synthesis of larger pore ZIFs and investigated for the separation of large size gas molecules and liquid separations (by adsorption mechanism). Table 5. Preparation and pore sizes of different ZIFs reported in the literature [7]. Material Molecular Name Pore Size (Å) References ZIF-7 Zn (benzimidazole)2 3.0 [16,76,77] p y y In addition to the above ZIFs, several other ZIF materials with increased pore sizes were synthesized [7] and tested for various applications (Figure 7). Some of the notable ZIF materials pore sizes are reported below in Table 5. It is to be noted here that ZIF membranes of smaller pore ZIF are synthesized extensively and studied for the separation of smaller molecules, such as H2 puriﬁcation and CO2 sequestration. On the other hand, few research studies were focused on the synthesis of larger pore ZIFs and investigated for the separation of large size gas molecules and liquid separations (by adsorption mechanism). ZIF 8 Zn(2 methylimidazole)2 3.4 [16,76,77] ZIF-90 Zn(imidazolate-2- b ld h d ) 3.5 [16,76,77] Table 5. Preparation and pore sizes of diﬀerent ZIFs reported in the literature [7]. ZIF 8 Zn(2 methylimidazole)2 3.4 [16,76,77] ZIF-90 Zn(imidazolate-2- carboxaldehyde) 3.5 [16,76,77] ZIF-71 Zn(4,5-dichloroimidazole)2 4.2 [16,76,77] ZIF-69 Zn(5chlorobenzimidazole)(2- nitroimidazole) 4.4 [16,76,77] ZIF 68 Zn(benzimidazole)(2- nitroimidazole) 7.5 [18] ZIF 22 Zn(5-azabenzimidazolate)2 0.44 [77] ZIF 78 Zn(5-nitrobenzimidazole)(2- nitroimidazole) 0.38 [78] Table 5. Preparation and pore sizes of diﬀerent ZIFs reported in the literature [7]. Material Molecular Name Pore Size (Å) References ZIF-7 Zn (benzimidazole)2 3.0 [16,76,77] ZIF-8 Zn(2-methylimidazole)2 3.4 [16,76,77] ZIF-90 Zn(imidazolate-2-carboxaldehyde) 3.5 [16,76,77] ZIF-71 Zn(4,5-dichloroimidazole)2 4.2 [16,76,77] ZIF-69 Zn(5chlorobenzimidazole)(2-nitroimidazole) 4.4 [16,76,77] ZIF 68 Zn(benzimidazole)(2-nitroimidazole) 7.5 [18] ZIF 22 Zn(5-azabenzimidazolate)2 0.44 [77] ZIF 78 Zn(5-nitrobenzimidazole)(2-nitroimidazole) 0.38 [78] ZIF 95 Zn(5-chlorobenzimidazole)2 0.37 [79] ZIF 360 Zn(bIM)1.00 (nIM)0.70(IM)0.30 4.8 [7] ZIF 365 Zn(cbIM)0.95 (nIM)0.60(IM)0.45 5.0 [7] ZIF-410 Zn(cbIM)1.0 (aIM)0.90 5.0 [7] ZIF486 Zn(nbIM)0.20 (mIM)0.65(IM)1.15 6.0 [7] ZIF412 Zn(bIM)1.13 (nIM)0.62(IM)0.25 8.2 [7] ZIF413 Zn(mIM)1.03 (nIM)0.64(IM)0.33 6.8 [7] ZIF414 Zn(nbIM)0.921 (mIM)0.62(IM)0.47 4.6 [7] ZIF725 Zn(bbIM)1.35 (nIM)0.40(IM)0.25 22.5 [7] List of abbreviations used in the above table. 2.4.3. ZIF (1) ZIF-8 The potential to adjust the pore size and surface properties of the ZIF has found its application in the recovery of bio-alcohols by using them as adsorbents. Liu et al. [75] reported the amine aggregate functionalisation on ZIF-90 by covalently connecting with free aldehyde functional groups present in the structure through host–guest interaction. Liu et al. reported the combination of a superhydrophobic ZIF-90 for the purpose of bioalcohol recuperation through the post-functionalization of ZIF-90 with penta-ﬂuorobenzyl amine by means of an amine group. It is generally presumed that the ﬂuorinated ZIF-90 will display a high hydrophobicity and, in this way, it is a promising candidate for bioalcohol recuperation. The reaction between pentaﬂurobenzylamine and ZIF-90 takes place on the external surface of ZIF-90 through the condensation of amine groups. The surface of the ﬂuorinated ZIF-90 showed a water contact angle of about 152.41; while the contact angle of water on the surface of the as-arranged ZIF-90 is 93.91◦, it indicates that the hydrophobicity of the ZIF-90 can be increased through post-functionalization with pentaﬂuorobenzyl amine. About 98% ethanol was removed from the mixture of ethanol/water from the superhydrophobic ZIF-90 (Figure 6). In contrast, only 7% ethanol can be removed while applying as-prepared ZIF-90 as the adsorbent. Besides ethanol, the superhydrophobic ZIF-90 displays high adsorptive separation performances for the other bio-alcohols such as methanol, iso-propanol and butanol, along with its mixtures. 15 of 24 Membranes 2019, 9, 128 Membranes 201 Figure 6. Adsorptive separation performance from of the superhydrophobic ZIF-90 for the removal of alcohols from alcohol/water mixtures; Reproduced with permission from [75]. Figure 6. Adsorptive separation performance from of the superhydrophobic ZIF-90 for the removal of alcohols from alcohol/water mixtures; Reproduced with permission from [75]. Figure 6. Adsorptive separation performance from of the superhydrophobic ZIF-90 for the removal of alcohols from alcohol/water mixtures; Reproduced with permission from [75]. Figure 6. Adsorptive separation performance from of the superhydrophobic ZIF-90 for the removal of alcohols from alcohol/water mixtures; Reproduced with permission from [75]. List of abbreviations used in the above table. bIM- benzimidazole; nIM -nitroimidazole; IM- Imidazole; cbIM- 5-chlorobenzimidazole; aIM- Imidazole-2-carboxaldehyde; nbIM-6-nitrobenzimidazole; mIM -2-methylimidazole; bbIM - 6-bromobenzimidazole. ( (3) ZIF-71 (c) Space-filling view for the largest cage i ucb ZIFs (illustrated by ZIF-412) is shown: zinc, blue; N, light blue; C, gray; O, red. The 12 MR openin (ucb ZIF-412, 48 atoms) is highlighted in yellow. Reproduced with permission from [7]. Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest cages are presented with adjacent small cages, and characteristic cages are shown with ball-and-stick structures for linkers (N, blue; C, dark; O, red; Cl, green; Br, orange, H, omitted for clarity) and blue tetrahedra for ZnN4 units. (a) KFI, ZIF-360; AFX, ZIF-386; ykh, ZIF-516; gcc, ZIF-615; bam, ZIF-725; ucb, ZIF-412. Largest openings for each cage are highlighted. (b) Space-ﬁlling views for the channel in bam ZIF (ZIF-725) are shown (zinc, blue; N, light blue; C, gray; O, red; Br, orange). The 24-MR aperture (bam ZIF-725, 96 atoms) is highlighted in yellow. (c) Space-ﬁlling view for the largest cage in ucb ZIFs (illustrated by ZIF-412) is shown: zinc, blue; N, light blue; C, gray; O, red. The 12 MR opening (ucb ZIF-412, 48 atoms) is highlighted in yellow. Reproduced with permission from [7]. ( (3) ZIF-71 The largest cage are presented with adjacent small cages, and characteristic cages are shown with ball-and-stic structures for linkers (N, blue; C, dark; O, red; Cl, green; Br, orange, H, omitted for clarity) and blu tetrahedra for ZnN4 units. (a) KFI, ZIF-360; AFX, ZIF-386; ykh, ZIF-516; gcc, ZIF-615; bam, ZIF-725 ucb, ZIF-412. Largest openings for each cage are highlighted. (b) Space-filling views for the channe in bam ZIF (ZIF-725) are shown (zinc, blue; N, light blue; C, gray; O, red; Br, orange). The 24-MR aperture (bam ZIF-725, 96 atoms) is highlighted in yellow. (c) Space-filling view for the largest cage in ucb ZIFs (illustrated by ZIF-412) is shown: zinc, blue; N, light blue; C, gray; O, red. The 12 MR openin (ucb ZIF-412, 48 atoms) is highlighted in yellow. Reproduced with permission from [7]. Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest cages are presented with adjacent small cages, and characteristic cages are shown with ball-and-stick structures for linkers (N, blue; C, dark; O, red; Cl, green; Br, orange, H, omitted for clarity) and blue tetrahedra for ZnN4 units. (a) KFI, ZIF-360; AFX, ZIF-386; ykh, ZIF-516; gcc, ZIF-615; bam, ZIF-725; ucb, ZIF-412. Largest openings for each cage are highlighted. (b) Space-ﬁlling views for the channel in bam ZIF (ZIF-725) are shown (zinc, blue; N, light blue; C, gray; O, red; Br, orange). The 24-MR aperture (bam ZIF-725, 96 atoms) is highlighted in yellow. (c) Space-ﬁlling view for the largest cage in ucb ZIFs (illustrated by ZIF-412) is shown: zinc, blue; N, light blue; C, gray; O, red. The 12 MR opening (ucb ZIF-412, 48 atoms) is highlighted in yellow. Reproduced with permission from [7]. Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest cage are presented with adjacent small cages, and characteristic cages are shown with ball-and-stic structures for linkers (N, blue; C, dark; O, red; Cl, green; Br, orange, H, omitted for clarity) and blu tetrahedra for ZnN4 units. (a) KFI, ZIF-360; AFX, ZIF-386; ykh, ZIF-516; gcc, ZIF-615; bam, ZIF-72 ucb, ZIF-412. Largest openings for each cage are highlighted. (b) Space-filling views for the channe in bam ZIF (ZIF-725) are shown (zinc, blue; N, light blue; C, gray; O, red; Br, orange). The 24-M aperture (bam ZIF-725, 96 atoms) is highlighted in yellow. ( (3) ZIF-71 bIM- benzimidazole; nIM -nitroimidazole; IM- Imidazole; cbIM 5-chlorobenzimidazole; aIM- Imidazole-2-carboxaldehyde; nbIM-6-nitrobenzimidazole; mIM -2-methylimidazole bbIM - 6-bromobenzimidazole. 16 of 24 nbIM-6 Membranes 2019, 9, 128 Imidazole; cb it b i id nitrobenzimidazole; mIM -2-methylimidazole; bbIM - 6-bromobenzimidazole;. Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest cage are presented with adjacent small cages, and characteristic cages are shown with ball-and-stic structures for linkers (N, blue; C, dark; O, red; Cl, green; Br, orange, H, omitted for clarity) and blu tetrahedra for ZnN4 units. (a) KFI, ZIF-360; AFX, ZIF-386; ykh, ZIF-516; gcc, ZIF-615; bam, ZIF-725 ucb, ZIF-412. Largest openings for each cage are highlighted. (b) Space-filling views for the channe in bam ZIF (ZIF-725) are shown (zinc, blue; N, light blue; C, gray; O, red; Br, orange). The 24-M aperture (bam ZIF-725, 96 atoms) is highlighted in yellow. (c) Space-filling view for the largest cage i ucb ZIFs (illustrated by ZIF-412) is shown: zinc, blue; N, light blue; C, gray; O, red. The 12 MR openin (ucb ZIF-412, 48 atoms) is highlighted in yellow. Reproduced with permission from [7]. Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest cages are presented with adjacent small cages, and characteristic cages are shown with ball-and-stick structures for linkers (N, blue; C, dark; O, red; Cl, green; Br, orange, H, omitted for clarity) and blue tetrahedra for ZnN4 units. (a) KFI, ZIF-360; AFX, ZIF-386; ykh, ZIF-516; gcc, ZIF-615; bam, ZIF-725; ucb, ZIF-412. Largest openings for each cage are highlighted. (b) Space-ﬁlling views for the channel in bam ZIF (ZIF-725) are shown (zinc, blue; N, light blue; C, gray; O, red; Br, orange). The 24-MR aperture (bam ZIF-725, 96 atoms) is highlighted in yellow. (c) Space-ﬁlling view for the largest cage in ucb ZIFs (illustrated by ZIF-412) is shown: zinc, blue; N, light blue; C, gray; O, red. The 12 MR opening (ucb ZIF-412, 48 atoms) is highlighted in yellow. Reproduced with permission from [7]. Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest cag Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. The largest Figure 7. Crystal structures of the new ZIFs. Topologies are shown in natural tilings. Challenges in the Formation of ZIF Membranes Challenges in the Formation of ZIF Membranes In the recovery of solvent mixtures, the excellent properties of ZIF such as pore size, chemical stability, thermal stability and structural stability have motivated the researchers to explore their industrial application. During the usage for these membranes for industrial applications, they show poor coverage of ZIF on the support membranes due to the growth of large sized ZIF crystals [19]. To overcome this obstacle, researchers have concluded that ZnO coating is ideal for the modiﬁcation of membrane to coat ZIFs [80]. In the synthesis of high quality ZIF-71 crystals using zinc acetate and 4,5-dichloroimidazole (dcIm) as precursors in methanol, the organic linkers were consumed instantly upon their introduction to the zinc ions and produce the crystals of ZIF-71 in the solvent medium rather than on the porous support. The secondary growth for the preparation of ZIF-71 membrane in methanol was observed to be unfavorable by Dong et al. [80] with a contrast of relatively slow and controllable reaction rates 17 of 24 17 of 24 Membranes 2019, 9, 128 of the precursors in DMF solvent, due to the diﬀerence in deprotonation rates of dcIm in the two solvents (Table 6). The high aﬃnity of organic molecules to the membrane surface and channel are the vital roles in the segregation of organic solvents with membrane pore size larger than the molecular size of solvents. ZIF-71 consists of organophilic surfaces on both the sides and can act as desirable membrane material for the separation of organic molecules via pervaporation. Separation studies show that the ZIF-71 membrane displays lower adsorption performance than that of the ZIF-71 crystals due to relatively slow diﬀusion of solvents. In the case of ethanol, slow diﬀusion is observed due to the kinetic diameter of 4.53 Å, which is nearly the size of ZIF-71 (4.8 Å). Organic-organic mixtures can be separated through a hydrophobic membrane, where the weak-polar organic molecules show greater aﬃnity towards the membrane than strong polar molecules. In addition, dimethyl carbonate (DMC) molecules with a size larger than ZIF-71 can diﬀuse through membranes similar to ZIF crystals via ﬂexible frameworks or gate opening eﬀect and showed total ﬂux and separation factor of 271 g·m−1·h−1 and 5.34, respectively, for 5 wt% dimethyl carbonate–methanol separation. Challenges in the Formation of ZIF Membranes [18] reported that secondary growth of ZIF-68 with continuous layer can be synthesized on the alumina support modiﬁed by ZnO via an in situ method (Figure 8) when compared to the ZnO via dip coating (Figure 9). Separation studies show that the ZIF-71 membrane displays lower adsorption performance than that of the ZIF-71 crystals due to relatively slow diffusion of solvents. In the case of ethanol, slow diffusion is observed due to the kinetic diameter of 4.53 Å, which is nearly the size of ZIF-71 (4.8 Å). Organic- organic mixtures can be separated through a hydrophobic membrane, where the weak-polar organic Kasik et al. [18] reported that secondary growth of ZIF-68 with continuous layer can be synthesized on the alumina support modiﬁed by ZnO via an in situ method (Figure 8) when compared to the ZnO via dip coating (Figure 9). Separation studies show that the ZIF-71 membrane displays lower adsorption performance than that of the ZIF-71 crystals due to relatively slow diffusion of solvents. In the case of ethanol, slow diffusion is observed due to the kinetic diameter of 4.53 Å, which is nearly the size of ZIF-71 (4.8 Å). Organic- organic mixtures can be separated through a hydrophobic membrane, where the weak-polar organic Li et al. [81] have overcome the inferior processability of the MOFs (such as ZIF) and scalability, which hinders their application in various domains, by gel coating for precursor dispersion followed by thermal-treatment for crystallization. This is similar to the processing of polymeric membranes that can be applied with controllable thickness on various substrates. The asynchronous crystallization between the bottom and the top of gels that found was critical for the formation of defect-free MOF (such as ZIF-8) membranes. Although prepared ZIF-8 membranes with a ligand/zinc ratio of 4 showed impressive performance in gas separation, they have not been utilized for solvent separation, which can be explored in the future. g p g y p , p g molecules show greater affinity towards the membrane than strong polar molecules. In addition, dimethyl carbonate (DMC) molecules with a size larger than ZIF-71 can diffuse through membranes similar to ZIF crystals via flexible frameworks or gate opening effect and showed total flux and separation factor of 271 g·m−1·h−1 and 5.34, respectively, for 5 wt% dimethyl carbonate–methanol separation. Kasik et al. Challenges in the Formation of ZIF Membranes Membranes 2019, 9, x 17 of 24 Challenges in the Formation of ZIF Membranes In the recovery of solvent mixtures, the excellent properties of ZIF such as pore size, chemical stability, thermal stability and structural stability have motivated the researchers to explore their industrial application. During the usage for these membranes for industrial applications, they show poor coverage of ZIF on the support membranes due to the growth of large sized ZIF crystals [19]. To overcome this obstacle, researchers have concluded that ZnO coating is ideal for the modification of membrane to coat ZIFs [80]. In the synthesis of high quality ZIF-71 crystals using zinc acetate and 4,5-dichloroimidazole Table 6. Pervaporation performance of the ZIF-71 membrane in the separation of 5 wt% ethanol-water, methanol-water and DMC-methanol at 25 ◦C; Reproduced with permission from [80]. System Total Flux (g·m−2·h−1) Separation Factor Alcohol or DMC Permeance (g·m−2·h−1·kPa−1) Selectivity EtOH-water 322.18 6.09 117.43 1.50 MeOH-water 394.64 21.38 260.22 4.32 DMC-MeOH 271.21 5.34 102.89 8.08 (dcIm) as precursors in methanol, the organic linkers were consumed instantly upon their introduction to the zinc ions and produce the crystals of ZIF-71 in the solvent medium rather than on the porous support. The secondary growth for the preparation of ZIF-71 membrane in methanol was observed to be unfavorable by Dong et al. [80] with a contrast of relatively slow and controllable reaction rates of the precursors in DMF solvent, due to the difference in deprotonation rates of dcIm in the two solvents (Table 6). The high affinity of organic molecules to the membrane surface and channel are the vital roles in the segregation of organic solvents with membrane pore size larger than the molecular size of solvents. ZIF-71 consists of organophilic surfaces on both the sides and can act as desirable membrane material for the separation of organic molecules via pervaporation. Table 6. Pervaporation performance of the ZIF-71 membrane in the separation of 5 wt% ethanol-water, methanol-water and DMC-methanol at 25 ◦C; Reproduced with permission from [80]. Im) as precursors in methanol, the organic linkers were consumed instantly upon the oduction to the zinc ions and produce the crystals of ZIF-71 in the solvent medium rather than o porous support The secondary growth for the preparation of ZIF 71 membrane in methanol wa Kasik et al. Challenges in the Formation of ZIF Membranes [18] reported that secondary growth of ZIF-68 with continuous layer can be synthesized on the alumina support modified by ZnO via an in situ method (Figure 8) when compared to the ZnO via dip coating (Figure 9). Figure 8. ZIF-68 membrane synthesis step following in situ growth of ZnO on Al2O3 SEM micrographs showing ZIF-68 membrane layer formed following secondary growth at (a) 1500× and (b) a cross- sectional picture at 660×; Reproduced with permission from [18]. Figure 8. ZIF-68 membrane synthesis step following in situ growth of ZnO on Al2O3 SEM micrographs showing ZIF-68 membrane layer formed following secondary growth at (a) 1500× and (b) a cross-sectional picture at 660×; Reproduced with permission from [18]. Figure 8. ZIF-68 membrane synthesis step following in situ growth of ZnO on Al2O3 SEM micrographs showing ZIF-68 membrane layer formed following secondary growth at (a) 1500× and (b) a cross- sectional picture at 660×; Reproduced with permission from [18]. Figure 8. ZIF-68 membrane synthesis step following in situ growth of ZnO on Al2O3 SEM micrographs showing ZIF-68 membrane layer formed following secondary growth at (a) 1500× and (b) a cross-sectional picture at 660×; Reproduced with permission from [18]. Membranes 2019, 9, 128 18 of 24 Figure 9. ZIF-68 membrane synthesis step following dip-coating of Al2O3 support in ZnO. SEM micrographs of ZIF-68 membrane layer on Al2O3 support following dip coating of ZnO, reactive seeding, and secondary growth at (a) 2000× and (b) a cross-sectional view at 10,000×; Reproduced with permission from [18]. Figure 9. ZIF-68 membrane synthesis step following dip-coating of Al2O3 support in ZnO. SEM micrographs of ZIF-68 membrane layer on Al2O3 support following dip coating of ZnO, reactive seeding, and secondary growth at (a) 2000× and (b) a cross-sectional view at 10,000×; Reproduced with permission from [18]. Figure 9. ZIF-68 membrane synthesis step following dip-coating of Al2O3 support in ZnO. SEM micrographs of ZIF-68 membrane layer on Al2O3 support following dip coating of ZnO, reactive seeding, and secondary growth at (a) 2000× and (b) a cross-sectional view at 10,000×; Reproduced with permission from [18]. Figure 9. ZIF-68 membrane synthesis step following dip-coating of Al2O3 support in ZnO. SEM micrographs of ZIF-68 membrane layer on Al2O3 support following dip coating of ZnO, reactive seeding, and secondary growth at (a) 2000× and (b) a cross-sectional view at 10,000×; Reproduced with permission from [18]. Table 6. MeOH-water 394.64 21.38 DMC-MeOH 271 21 5 34 2.4.4. Metal Azolate Framework (MAF-6) Li et al. [81] have overcome the inferior processability of the MOFs (such as ZIF) and scalability, which hinders their application in various domains, by gel coating for precursor dispersion followed by thermal-treatment for crystallization. This is similar to the processing of polymeric membranes that can be applied with controllable thickness on various substrates. The asynchronous crystallization between the bottom and the top of gels that found was critical for the formation of defect-free MOF (such as ZIF-8) membranes. Although prepared ZIF-8 membranes with a ligand/zinc ratio of 4 showed impressive performance in gas separation, they have not been utilized for solvent separation, which can be explored in the future. Kong et al [82] applied the rubbing method to coat ZnO particles onto the porous α-Al2O3 MAF-6 a hydrophobic MOF with larger pore and aperture size (~7.6 Å) was introduced by Li et al. [14] The nanoporous zeolitic metal azolate framework, RHO-[Zn(eim)2], has the ability to absorb organic solvents like ethanol instantly but is resistant to water. MAF-6 nanoparticles are synthesized at room temperature rather than using high temperature and pressure like most MOFs. The synthesis procedure is highly beneﬁcial because of the high eﬃciency and low energy consumption. Embedding of MAF-6 nanoparticles in PDMS matrix to produce MMM for the ethanol/water pervaporation separation has resulted in high performance with a separation ﬂux of 1200 g/m2·h and separation factor of 14.9, which happens to be 1.5 and 2.3 times of PDMS pristine membrane, respectively. This MAF-6 showed a long-term stability of 120 h, proving their durability. Challenges in the Formation of ZIF Membranes Pervaporation performance of the ZIF-71 membrane in the separation of 5 wt% ethanol- water, methanol-water and DMC-methanol at 25 °C; Reproduced with permission from [80]. System Total Flux (g·m−2·h−1) Separation Factor Alcohol or DMC Permeance (g·m−2·h−1·kPa−1) Selectivity EtOH-water 322.18 6.09 117.43 1.50 Kong et al. [82] applied the rubbing method to coat ZnO particles onto the porous α-Al2O3 support, which could serve three major functions such as modiﬁcation of support, induction (as active seed for homogeneous nucleation) and anchoring of ZIF during membrane synthesis. The formed membranes were continuous, uniform and showed good molecular sieve performance of various gases and the method is simple, convenient and scalable. support, which could serve three major functi active seed for homogeneous nucleation) and 2.5. Grignard Functionalized Ceramic Membranes active seed for homogeneous nucleation) and anchoring of ZIF during membrane synthesis. The formed membranes were continuous, uniform and showed good molecular sieve performance of various gases and the method is simple, convenient and scalable. 2.4.4. Metal Azolate Framework (MAF-6) MAF-6 a hydrophobic MOF with larger pore and aperture size (~7.6 Å) was introduced by Li et al. [14] The nanoporous zeolitic metal azolate framework, RHO-[Zn(eim)2], has the ability to absorb organic solvents like ethanol instantly but is resistant to water. MAF-6 nanoparticles are synthesized at room temperature rather than using high temperature and pressure like most MOFs. The synthesis procedure is highly beneficial because of the high efficiency and low energy consumption. Embedding of MAF-6 nanoparticles in PDMS matrix to produce MMM for the ethanol/water pervaporation separation has resulted in high performance with a separation flux of 1200 g/m2·h and Grignard reaction is an organometallic chemical reaction, where alkyl halides are added to carbonyl groups in ketone or aldehyde. Hosseinabadi et al. [83] discussed the use of membranes modiﬁed with alkyl groups in them as a prospective membrane for organic solvent nanoﬁltration. The change in aﬃnity of solvents and solutes for the modiﬁed membranes varies the retention of them. The membranes were fabricated using TiO2 nanoparticles, which were then functionalized by grafting alkyl groups, using an organometallic Grignard reagent. A solvent with less aﬃnity towards the membrane was chosen to obtain relatively low retentions. For example, toluene is the solvent with lower solvent-membrane aﬃnity in the mixture of toluene/polystyrene. When the mixture was passed down through the Grignard modiﬁed membrane, there is an increase in the hydrophobicity of the membrane which also happened to raise the retention of polystyrene in toluene, whereas the retention was increased to 55% from 30% for polystyrene with 580 Da in toluene. Figure 10. Schematic illustrati 4. Merits of Solvent Recovery 4. Merits of Solvent (I) Economic Beneﬁt separation factor respectively. Thi 3. Pervaporation 2.5. Grignard Functionalized Ceramic Membranes The transportation of components in a pervaporation process across the membrane reported by solution desorption model consists of a series of processes (Figure 10) [9]: 1. Diﬀusion of component to the membrane surface through the liquid boundary layer, 2. Sorption into the membrane, 19 of 24 mponent rane 3 Membranes 2019, 9, 128 solution desorption h b 3. Transportation though the membrane, and 4. Diﬀusion through vapor phase boundary layer into the bulk of the permeance. The transportation of gas, vapor, or liquid through a membrane is given by permeability, which is the product of diﬀusivity and solubility. The eﬃcacy of a pervaporation is majorly determined by the separation factor and the permeate ﬂux. The permeability is dependent on the mass divided by both membrane area and time. The product of diﬀusion coeﬃcient and sorption coeﬃcient equals the ﬂux of dilute solutions. The membrane performance during pervaporation also depends on the following parameters: membrane thickness, temperature and feed concentration. Transportation though the membrane, and 4. Diffusion through vapor phase boundary layer into the bulk of the permeance. The transportation of gas, vapor, or liquid through a membrane is given by permeability, which is the product of diffusivity and solubility. The efficacy of a pervaporation is majorly determined by the separation factor and the permeate flux. The permeability is dependent on the mass divided by both membrane area and time. The product of diffusion coefficient and sorption coefficient equals the flux of dilute solutions. The membrane performance during pervaporation also depends on the following parameters: membrane thickness, temperature and feed concentration. Figure 10. Schematic illustrations for pervaporation method; Reproduced with permission from [18]. Figure 10. Schematic illustrations for pervaporation method; Reproduced with permission from [18]. Fi 10 S h ti ill t ti f ti th d R d d ith i i f [18] Figure 10. Schematic illustrations for pervaporation method; Reproduced with permission from [18]. 4. Merits of Solvent R (I) Economic Beneﬁt (I) Economic Benefit The process of solvent recycling and recovery is schemed to favor the industrial facility with reduction in liquid waste disposal expenses and recycling of solvent wastes to generate solvents. This helps in bringing down the purchases of expensive solvents [1,8]. (II) Environmental Beneﬁt In addition to the reduction in investment for solvents and waste through solvent recycling, it also inﬂuences the environment. The elimination of hazardous wastes through recycling and recovery reduces the quantity of waste disposed. The reduction in cost of disposal and usage of minimum storage space reduces the impact of the waste on the environment [8]. According to the Hazardous Waste regulations enforced in the United Kingdom in 2005, any organization producing hazardous waste of more than 500 kg is considered an oﬀence. The aim of these regulations is to prevent pollution and protect the environment and human health [8]. Membranes 2019, 9, 128 20 of 24 20 of 24 5. Conclusions In the ﬁeld of solvent recovery, the desire to preserve the solvents from the industry after use has led to the development of various materials chemistry and separation processes. Some of the materials explored are inorganic membranes for the process of solvent recovery and recycling in the industrial scale. On a commercial scale, the membranes seem to be dominated by porous ceramic membranes with shapes like disks, tubes or multi-channel monoliths. These membranes are feasible and commercially sold by Mitsui Engineering and Shipbuilding Company, City, Japan and a few other companies around the world. These inorganic membranes consist of layers of various materials such as alumina, zirconia, stainless steel and other ceramics. The polymers modiﬁed with the embedding of nanomaterials have high permeability and selectivity of solvents. By varying the amount of nanomaterials in the matrix, the nanocomposites properties such as the permeability, selectivity and rejection of solvents are varied. Generally, ﬁlms made up of zeolites, ZIFs on substrate can be fabricated using in situ synthesis and secondary growth of a reactive seed with hydrothermal treatment. It has been observed that the secondary growth method favors high reproducibility and good control over the membrane structure [18,45]. Recently, the patterned MOF membranes were developed with good molecular separation property and improvement in the apparent permeation ﬂux, which can be applied in compact devices application. Only very few reports were available on the recovery of solvent mixtures using ZIF materials, which is due to considerable challenges connected with suitable ZIF materials screening and well-connected ZIF membranes formation on support material. This particular area can be exploited further in future. is due to considerable challenges connected with suitable ZIF materials screening and well-connected ZIF membranes formation on support material. This particular area can be exploited further in future. This paper demonstrates the performances of various Mixed Metal Membranes, MOFs and ceramic membranes in the solvent recovery process with respect to permeability, selectivity and rejection ﬂux. This paper demonstrates the performances of various Mixed Metal Membranes, MOFs and ceramic membranes in the solvent recovery process with respect to permeability, selectivity and rejection ﬂux. Funding: This research was funded by the GSK Singapore Partnership 4th Green & Sustainable Manufacturing Award 2017, funded by the GSK-EDB Trust Fund (WBS: 265-000-608-504). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4361903619	https://figshare.com/articles/journal_contribution/Supplementary_Methods_from_Monoclonal_Antibody_against_Cell_Surface_GRP78_as_a_Novel_Agent_in_Suppressing_PI3K_AKT_Signaling_Tumor_Growth_and_Metastasis/22446794/1/files/39897836.pdf	English	null	Supplementary Methods from Monoclonal Antibody against Cell Surface GRP78 as a Novel Agent in Suppressing PI3K/AKT Signaling, Tumor Growth, and Metastasis	null	2,023	cc-by	3,023	Antibodies Antibodies against clathrin, p85 (PI3K regulatory unit, rabbit monoclonal), Akt, phosphorylated Akt (Ser473), S6, phosphorylated S6 (Ser240/244), ERK1/2 (Thr202/Tyr204), phosphorylated ERK1/2, phosphorylated mTOR (Ser2448), Src, phosphorylated Src (Tyr416), were from Cell Signaling (Danvers, MA). Beta-actin and FLAG antibodies were from Sigma (St Louis, MO) and GAPDH and p85 (mouse monoclonal) antibody was from Millipore (Temecula, CA). Rabbit polyclonal HSP70 and GRP78 antibodies were from Santa Cruz (Santa Cruz, CA). CD31 antibody was from BD Biosciences (San Jose, CA). Ki67 antibody was from Abcam (Cambridge, MA). EphB4 antibody was from VasGene Therapeutics Inc. (Los Angeles, CA). Horse radish peroxidase (HRP) and IRDye conjugated secondary antibodies were from Rockland (Gilbertsville, PA). Generation of monoclonal antibodies The human Grp78 cDNA clone was generated as described (1). Human GRP78 (amino acids 1- 650; without KDEL) with hexahistidine tag on C-terminus (designated as GRP78-His) was constructed, transiently expressed in 293T cells and purified through nickel-NTA column (Biorad, Hercules, CA). Female Swiss Webster mice were immunized three times (every second week) intraperitoneally (i.p.) with 50 µg of GRP78-His per mouse. Antigen was injected as 1:1 mixture with Complete Freund’s Adjuvant (Sigma, St. Louis, MO) in the first immunization, and with incomplete Freund’s Adjuvant (Sigma, St. Louis, MO) in the second and third doses. Mice were given a final boost with 20 µg of GRP78-His through tail-vein injection, and splenocytes were harvested 4 days later for fusion with myeloma cell line NS0 from ATCC (Allendale, NJ). Hybridoma supernatants were screened for antibodies that immunoprecipitate GRP78 (without KDEL) fused to alkaline phosphatase (AP). Selected monoclonal antibodies (MAb) were produced in BD CELLine cultivation system (BD biosciences, Bedford, MA) following manufacturer’s protocol. MAbs were purified with protein G affinity chromatography. Estimated purity of MAbs was higher than 95% based on HPLC analysis and SDS-PAGE Coomassie staining. Western blotting For Western blot, typically 20 µg of whole-cell lysates were run on 4-20% Tris-glycine gradient gel (Biorad, Hercules, CA) and transferred onto nitrocellulose membrane (BioRad, Hercules, CA). The membrane was blocked with 5% non-fat dry milk in TBS and 0.05% Tween-20 (TBST) for 40 min, and then incubated with 1 µg/ml primary antibody at 4°C overnight. Membrane was washed three times for 10 min each and incubated with secondary HRP-labeled or IRDye labeled secondary antibody for 40 min. After three times wash with TBST, HRP signal was detected using Femto Maximum Sensitivity chemiluminescent substrate from Thermo Scientific, and IRDye signal was detected by Odyssey (LICOR, Lincoln, NE). Surface protein biotinylation and immunoprecipitation 293T cells growing on 10 cm dish were transfected with Flag tagged GRP78 (without KDEL) alone or co-transfected with a human EphB2 full length expression vector. 3 days after transfection, cells were rinsed with ice cold PBS twice and incubated with 0.5 mg/mL EZ-link Sulfo-NHS-LC-Biotin (Thermo scientific) with gentle shake at 4 °C for 30 min. Tris-HCl (pH 7.5) was added to a final concentration of 100 nM to stop the biotinylation reaction. The cells were rinsed with ice cold PBS twice and then lysed with lysis buffer (PBS, 2mM NaF, 1mM Na3VO4, 1 x protease inhibitor cocktail, and 1% Triton X-100). The biotinylated surface proteins were purified with monomeric avidin-agarose beads (Thermo scientific, Rockford, IL) following manufacturer’s instructions. The eluted biotinylated proteins were further incubated with beads conjugated with anti-Flag antibody M2 (Sigma, St. Louis, MO) at 4°C overnight to pull down Flag-tagged GRP78. Beads were then washed two times with lysis buffer and the bound proteins were eluted with Laemmli SDS buffer (BioRad, Hercules, CA) supplemented with 50 mM dithiothreitol, followed by incubation for 5min at 95°C. The negative control for IP was beads 37 conjugated with normal mouse IgG from Rockland. To examine endogenous interaction of p85 and surface GRP78, before biotin labeling, 293T cells were treated with 300nM thapsigargin for 16 hours instead. The immunoprecipitation was performed with protein G beads (Genescript, Piscataway, NJ) and p85, GRP78, and beta-actin antibodies (all mouse IgG1, 5 ug per reaction). To study the surface GRP78 ratio in different cancer cell lines, cells were first starved in glucose depleted medium for 3 days and then biotinylated as described above. The biotinylated surface proteins were enriched with streptavidin sepharose (Thermo Scientific). Cell viability assay Cancer cells were seeded in 24-well plates at a density of 2x104 cells/well in a total volume of 500 µL. One day later, the medium was changed to growth medium without glucose. Triplicate wells were treated with 50 µg/mL MAb159 or control mouse IgG (Rockland, Gilbertsville, PA). 5 days after treatment, cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) as described previously (2). Cells were also processed for apoptosis analysis with TdT-mediated dUTP nick-end labeling (TUNEL) assay kit (Promega, Madison, WI), Caspase-8/9 colorimetric assay kit (R&D systems, Minneapolis, MN), M30 CytoDEATH kit (Roche , Mannheim, Germany) following manufacturers’ instructions. For colonogenic assay, HT29 cells were starved and treated with MAb159 for 5 days as described above, and then plated on 6-well plates in triplicate at 200 cells/well in complete growth medium. The 6-well plates were pre-coated with gelatin and fibronectin (Sigma) to facilitate cell attachment. Medium was changed 4 days later. 8 days later, the cells were fixed with acetic acid/methanol 1:7 (v/v) for 20 min and stained with 0.5% Crystal Violet for 2 hours. The plates were rinsed with water and then air dried. The colonies were counted with naked eye. Murine xenograft tumor models HT29, A549, colo205, and H249 cells were propagated, collected after trypsin digestion, and resuspended in serum-free medium. Cells (2 x 106) were injected subcutaneously bilaterally in the flanks of 8-week-old Balb/C nu/nu mice. Tumor growth was measured 3 times a week and volume was estimated as 0.52 x a x b2, where a and b are the largest and smallest lengths of the tumor. Once tumors were about 100mm3 (day 0), animals were distributed into treatment and control groups (n = 10 tumors per group) such that the mean tumor volume of each group was comparable and the standard error between groups was minimal. Each group was treated by i.p. injection of antibody two times a week at a dose of 10 mg/kg. At the end of the experiment, mice were sacrificed for tissue analysis. All procedures were approved by Institutional Animal Care and Use Committee and performed in accordance with the Animal Welfare Act regulations. Antibody endocytosis MAb159 was biotinylated with EZ-link biotin hydrazide from Thermo Scientific (Rockford, IL) following manufacturer’s procedure. MCF7 cells were treated with 50 µg/mL biotinylated MAb159 for 1 hour at 37°C or at 4°C. The cells were then fixed with 4% paraformaldehyde for 20 min and washed with PBS for 3 times. Cells were permeabilized with 0.1% Triton X-100 and washed with PBS for 3 times. Subsequently cells were stained with streptavidin-FITC (Invitrogen) for 30 min at room temperature. Nuclei were counterstained with 6-diamidino-2- phenylindole dihydrochloride hydrate (DAPI). Images were taken with a 100x objective on a Zeiss LSM 510 confocal microscope. To study the colocalization of internalized MAb159 with clathrin coated endosomes, cells were costained with MAb159 and clathrin antibody after fixation and permeabilization. Images were obtained with a Nikon Eclipse 80i fluorescence microscope and Meta Morph imaging series system. To examine the effect of MAb159 treatment on cell surface GRP78, MCF7 cells were starved for overnight and then treated with normal mouse IgG or MAb159 (50 µg/mL) for 48 hours. For immunostaining, cells were incubated with the following reagents stepwise on ice: goat serum for 30 min, a polyclonal anti-GRP78 antibody (clone H129, Santa Cruz Biotechnology) for one hour, and an AlexaFluor conjugated anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, CA) for 30 min. Cells were washed with cold PBS between steps. Images were obtained with a Nikon Eclipse 80i fluorescence microscope and Meta Morph imaging series system. To study the effect of chlorpromazine (the specific inhibitor for clathrin mediated endocytosis) on MAb159/GRP78 endocytosis, cells were treated with 2 or 5 μg/mL chlorpromazine along with 50 μg/mL MAb159 for 1 hour at 37°C, followed by staining of MAb159 without fixation and permeabilization as described above. EphB4, a receptor tyrosine kinase located on plasma membrane, was also stained to mark the cell surface. Images were taken with a 100x objective on a Zeiss LSM 510 confocal microscope. 36 PTEN-null prostate cancer model This prostate specific PTEN knock out model was described previously (4). 2-month-old Cre- PTEN-/-Luc mice were randomized into two groups (n=4) and administered intraperitoneally with either 20 mg/kg normal mouse IgG or MAb159, twice a week. Prostate tumors were monitored by luminescence imaging (xenogen) immediately before treatment and 7 weeks after treatment. PTEN-null leukemia model The Pten (floxed/floxed); Mx-1 cre leukemic model and the protocols for flow cytometry for analysis of leukemic blasts and peripheral blood counts have been described (5). Intraperitoneal administration of polyinosinic-polycytidylic acid (pIpC, 20 µg/gW) was given to 6-8 week old Ptenf/f;Mx1-Cre mice every other day for a total of 7 doses to induce Cre expression. 10 mg/kg of normal mouse IgG (n=5) or MAb159 (n=8) was co-administered with pIpC for these 7 doses. Three days after the last pIpC injection, MAb159 or IgG were administrated for the last time and the mice were analyzed after another three days. Orthotopic 4T1 breast cancer model A total of 6.3 X 105 4T1 cells in 10 µL was injected into the #4 mammary fat pad using Hamilton microsyringe and custom 29 gauge removable needles (Hamilton Catalog # 7653-01 and 7803-06 RN/ 29GA/0.5”/12 degrees). Mouse nipples served as positional cues for fat pad location. Antibody MAb159 or control normal mouse IgG was administered at 10 mg/kg twice weekly by intra-peritoneal injection and mouse body weight and body condition were monitored to assess metastatic burden and euthanasia end points. The first does was given 30 minutes after 4T1 cell injection. Metastasis in contralateral # 9 fat pad, lung, and liver was determined upon terminal necropsy on days 7, 13 and 14 after orthotopic injections. Upon surgical incision, the dermis was separated from the body wall and pinned back to reveal the mammary fat pads. Primary tumor and metastatic mammary fat pads and other organs with visible metastasis were fixed in 10% neutral buffered formalin for 24 hours followed by reconstitution in 30% sucrose at 4°C. These organs were embedded in paraffin and 7.5 micron sections were obtained using Leica RM2235 ultra-microtome. To determine maximum tumor size in mouse fat pads and to evaluate metastasis at different depths of organ tissue, step-cuts were generated through the entire specimen. 7.5 µm sections 37 were generated at intervals of 150 µm into the specimen and 3-4 slides at multiple depths were analyzed by hematoxylin-eosin staining. Slides were deparrafinized by sequential incubation in in xylene (3 min), xylene (3 min), 100% ethanol (1 min), 90% ethanol (1 min), 80% ethanol (1 min) and 75% ethanol (1 min), followed by de-ionized water rinses. Slides were stained for 4 min in CAT Hematoxylin (Biocare Medical) to allow penetration in fatty tissue such as mammary fat pads and de-stained for 1 min using 0.3% acid alcohol. Eosin Y (Fisher Scientific) staining was performed for 3 hours to allow stain penetration into fatty tissue. H&E stained slides were evaluated by microscopy and 4X pictures were taken at the depth where the tumor’s diameter was maximum to allow for an accurate comparison. Similarly for evaluation of lung and liver, metastatic burden at multiple depths was evaluated and representative 4X pictures were depicted. B16 lung metastasis model The B16 lung metastasis model was described previously (3). Briefly, B16-Fluc-A1 melanoma cells (5x105) were injected through the lateral tail vein. Lung metastasis was monitored by luminescence imaging (Xenogen) every week starting from week 3. The treatment with MAb159 (10 mg/kg, twice a week) started right after the injection of melanoma cells. Following sacrifice at week 4, the lungs were removed to visualize the tumors. Normal mouse IgG (Rockland) was used as control. There were 4 mice in each group. Immunofluorescence and immunohistochemistry For immunofluorescence, fresh frozen tissue embedded in OCT was sectioned at 5 µm and fixed in phosphate-buffered 4% paraformaldehyde and washed in PBS. Sections were then blocked with goat serum and incubated with primary antibody overnight at 4°C. After washing in PBS, antibody binding was localized with AlexaFluor conjugated appropriate secondary antibodies (Invitrogen, Carlsbad, CA). Nuclei were counterstained with DAPI. Images were obtained with a Nikon Eclipse 80i fluorescence microscope and Meta Morph imaging series system. For immunohistochemistry, the frozen sections were fixed with 3% formaldehyde for 15 minutes at room temperature, following by two PBS washes. The sections were treated with 3% 38 H2O2 for 10 min, blocked with goat serum for 1 hour, and incubated with primary antibody for overnight at 4°C. The sections were then washed with PBS and processed with ABC kit (Vector labs, Burlingame, CA). The images were obtained with an Olympus BX51 microscope and Image-pro plus 6.0 system. H2O2 for 10 min, blocked with goat serum for 1 hour, and incubated with primary antibody for overnight at 4°C. The sections were then washed with PBS and processed with ABC kit (Vector labs, Burlingame, CA). The images were obtained with an Olympus BX51 microscope and Image-pro plus 6.0 system. Four representative pictures were taken for each sample and quantification was performed with Image J (NIH). P value was determined by an unpaired 2-tail student T-test. In vivo and ex vivo near-infrared fluorescence imaging When the established xenograft tumors (H249) reached about 400 mm3 in volume, the mice (two per group) were put on Teklad Global 18% Protein Rodent Diet for 3 days and then injected intravenously (i.v.) with 3 mg/kg Cy5.5 labeled antibody (labeled through primary amine). In vivo fluorescence imaging was performed using the Xenogen Lumina XR Imaging System and analyzed using the IVIS Living Imaging 3.0 software (Caliper Life Sciences, Alameda, CA, USA). A Cy5.5 filter set was used for acquiring the fluorescence of Cy5.5-conjugated antibody. Identical illumination settings (lamp voltage, filters, f/stop, field of views, binning) were used for acquiring all images. Fluorescence emission images were normalized and reported as photons per second per centimeter squared per steradian (p/s/cm2/sr). All near-infrared fluorescence images were acquired using 1 s exposure time (f/stop = 4). 28 hours after injection, the mice were perfused with PBS, followed by formalin as described previously (6). The tumors and organs were then dissected and subjected to ex vivo fluorescence imaging with the same settings as the in vivo fluorescence imaging. The control IgG used in this study is human IgG purified from normal serum (Rockland). Pharmacokinetics A single 10 mg/kg dose of humanized MAb159 was administered i.v. into three C57BL/7 mice and serum samples were collected at 30 min, 4 hours, 1 day, 2 days and 5 days after injection. The serum levels of the antibody were measured with a capture ELISA assay. Briefly, protein A (1 µg/well) was coated on ELISA plate (Thermo Scientific) in PBS overnight at 4°C. Wells were then blocked with 0.5% BSA in PBS, followed by application of serum and 100 ng AP fused GRP78. The bound antibody-antigen complexes were detected with optical reading at 405nm following application of AP substrate p-Nitrophenyl Phosphate (PNPP). The pharmacokinetics parameters were calculated using a non-compartmental analysis, where Cmax, Tmax, serum half- life and AUC were determined using the trapezoidal rule. Toxicology study Toxicology study was conducted with C57BL/7 mice, which were injected i.v. twice a week for 5 weeks with PBS, 10 mg/kg humanized MAb159, or 10 mg/kg of an isotype control antibody (n=3). The blood was harvested for Chemistry Panel and Complete Blood Count analysis. The major organs (heart, lung, liver, kidney, pancreas, and thymus) were harvested for tissue analysis. At the end of the experiment, animals were anesthetized using isofluorane and euthanized by exsanguination per protocol approved by the University of Southern California IACUC. During necropsy, all animals were subjected to a full gross examination and observations were noted. Blood for hematology and serum chemistry analyses was collected by cardiac puncture from the vena cava. Blood was collected via a single draw and then 0.2-0.5 mL 39 blood was aliquoted into an EDTA tube for hematology and 0.2-0.5 mL blood was placed into a serum separator tube for serum chemistry. Hematology tests included white blood cell count, red blood cell count, platelet count, leukocyte count (basophils, eosinophils, lymphocytes, neutrophils and monocytes), hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), polychromasia, and blood parasites. Serum chemistry parameters analyzed included albumin, globulin, aspartate transaminase, alanine transaminase, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine, phosphorus, calcium, sodium, chloride, cholesterol, and creatinine phosphokinase. Animal spleen, testis, thymus, mesenteric lymph node, pancreas, heart, liver, esophagus, lung, kidney, and bone marrow smears from the femur were collected for histopathology examination. They were fixed immediately using 10% neutral buffered formalin, and were subjected to hematoxylin-eosin staining and microscopic examination. Hematology, serum chemistry and histopathology experiments were performed commercially at Antech Diagnostics (Irvine, CA). All results were compiled, analyzed, and statistical analysis was performed using student’s T Test and one-way analysis of variance (ANOVA). REFERENCES 1. Ni M, Zhou H, Wey S, Baumeister P, Lee AS. Regulation of PERK signaling and leukemic cell survival by a novel cytosolic isoform of the UPR regulator GRP78/BiP. PLoS One 2009;4(8):e6868. 2. Kumar SR, Singh J, Xia G, Krasnoperov V, Hassanieh L, Ley EJ, et al. Receptor tyrosine kinase EphB4 is a survival factor in breast cancer. Am J Pathol 2006;169(1):279-93. 3. Dong D, Stapleton C, Luo B, Xiong S, Ye W, Zhang Y, et al. A critical role for GRP78/BiP in the tumor microenvironment for neovascularization during tumor growth and metastasis. Cancer Res 2011;71(8):2848-57. 4. Liao CP, Zhong C, Saribekyan G, Bading J, Park R, Conti PS, et al. Mouse models of prostate adenocarcinoma with the capacity to monitor spontaneous carcinogenesis by bioluminescence or fluorescence. Cancer Res 2007;67(15):7525-33. 5. Wey S, Luo B, Tseng CC, Ni M, Zhou H, Fu Y, et al. Inducible knockout of GRP78/BiP in the hematopoietic system suppresses pten-null leukemogenesis and AKT oncogenic signaling. Blood 2012;119(3):817-25. 6. 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https://openalex.org/W4382491818	https://zenodo.org/records/8096034/files/Linguistic_Vanguard_camera_ready-prePrint.pdf	English	null	Why is this language complex? Cherry-pick the optimal set of features in multilingual treebanks	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	8,493	Linguistics Vanguard 2022 Corresponding author: Dominique Brunato, Institute for Computational Linguistics "A. Zampolli" (ILC-CNR) - ItaliaNLP Lab, Pisa (Italy) Corresponding author: Giulia Venturi, Institute for Computational Linguistics "A. Zam- polli" (ILC-CNR) - ItaliaNLP Lab, Pisa (Italy) Dominique Brunato* and Giulia Venturi* Why is this language complex? Cherry-pick the optimal set of features in multilingual treebanks Dominique Brunato* and Giulia Venturi* Abstract: This paper investigates linguistic complexity across natural languages from a corpus-based perspective and relies on the assumptions of linguistic profiling as a methodological framework. We focus in particular on the domain of syntactic complexity and analyze the distribution of a set of features taken as proxies of complexity phenomena at sentence level, which were extracted from 63 treebanks annotated according to the Universal Dependencies formalism. This dataset guar- antees that the features considered are modeling the same linguistic phenomena in different treebanks, allowing reliable comparison among languages. We show that our approach is able to identify tendencies of structural proximity between languages not necessarily in line with typologically-supported classification, thus shedding light on new corpus-based findings. Keywords: Linguistic Complexity; Linguistic Profiling; Universal Dependencies; Syntactic Domain Brunato and Venturi 2 were theoretically possible, it would be beyond the capacities of the mortal linguist to exhaustively count all grammatical details of the languages studied, especially in a large-scale cross-linguistic study”. Accordingly, if studying the global complexity of a language is perceived as a very ambitious and probably hopeless endeavor, the dominant and more feasible approach addresses local complexity, i.e. the complexity in the different sub–domains of the language (Kortmann & Szmrecsanyi 2012). were theoretically possible, it would be beyond the capacities of the mortal linguist to exhaustively count all grammatical details of the languages studied, especially in a large-scale cross-linguistic study”. Accordingly, if studying the global complexity of a language is perceived as a very ambitious and probably hopeless endeavor, the dominant and more feasible approach addresses local complexity, i.e. the complexity in the different sub–domains of the language (Kortmann & Szmrecsanyi 2012). In this scenario, the growing availability of linguistically annotated corpora for many languages has promoted the exploitation of data-driven approaches focused on detecting and measuring a large variety of complexity phenomena across corpora representative of different languages and language varieties, with a particular emphasis on syntactic-related peculiarities showing to be consistent across many languages. It is the case, for example, of dependency length – the distance between syntactically related words in a sentence –, which is considered as a reliable measure of sentence complexity according to both experimental and theoretical language research, see e.g. Gibson (1998, 2000), Futrell, Mahowald & Gibson (2015) and Liu (2017). With this respect, the benefits of acquiring information about linguistic com- plexity from multilingual treebanks have been recently promoted by the Universal Dependencies (UD) project,1 an international initiative with over 300 contributors producing nearly 200 treebanks in over 100 languages. The project allowed the definition of a framework for cross-linguistically consistent treebank annotation aiming to capture similarities as well as idiosyncrasies among typologically different languages (Nivre 2015). The first related initiative took place in 2018 as a satellite event of the “Evolution of Language International Conferences” (EVOLANG), named “Workshop on Measuring Language Complexity (MLC)”.2 By relying on the morpho-syntactic and syntactic formalism of the UD treebanks, seven teams of researchers designed 34 different measures of linguistic complexity for 37 language varieties belonging to seven families (Berdicevskis et al. 2018). 1 Introduction Linguistic complexity, along with its detection, evaluation and processing, is a topic that has long attracted researchers embracing different perspectives ranging from typological linguistics (Miestamo, Sinnemaki & Karlsson 2008), first and second language acquisition (Kortmann & Szmrecsanyi 2012), computational linguistics and related fields (Brunato et al. 2016). Despite the debated and multidimensional nature of the notion, a quite established theoretical distinction identifies an “absolute complexity”, that refers to the formal properties of linguistic systems, and a “relative complexity”, that defines complexity in relation to the language user (e.g. speaker, listener or learner) thus considering complexity in terms of processing difficulty (Miestamo 2008). The absolute viewpoint encounters itself a main methodological obstacle that, in Miestamo’s words, can be summarized as follows: “even if this 1 https://universaldependencies.org/ 2 http://www.christianbentz.de/MLC_index.html 3 http://christianbentz.de/MLC2019_index.html Brunato and Venturi 2 3 extend the preliminary findings presented in that context. As described in the following section, the core of this approach is the extraction from multilingual treebanks of a same set of features modeling phenomena of sentence complexity in different sub-domains of language, with a main focus on the syntactic one. From this perspective, our study lies in the framework of linguistic research aiming at acquiring quantitative evidence about linguistic complexity from large-scale data representative of real language usage, and in particularly dependency annotated corpora. The rich variety of features here considered aims to empirically prove that the notion of syntactic complexity is not monolithic. As previously observed, there is a wide consensus in considering it as a multifaceted notion covering several aspects also within the same domain. Thus, with our perspective we would like to underline the need for ‘cherry-picking’ which feature is more reliable to model a specific aspect of complexity. The approach has been tested on 63 UD treebanks, presented in Section 2.1, while the linguistic features are illustrated in Section 2.2. The choice of considering multi-lingual treebanks possibly containing different textual genres is also motivated by our intention of showing that treebanks may be only partially representative of a given language and that, as a consequence, any quantitative evidence about the complexity of a language cannot be generalized to the whole system but instead should be related to the text typologies of its representative corpus. In Section 3.1 our set of features is first analyzed separately, that is considering each feature as a distinct complexity metric. In Section 3.2, we inspect the results of a cluster analysis based on the combination of all features showing that our approach is able to identify tendencies of structural proximity between languages not necessarily in line with typologically-supported classifications. Why is this language complex? 3 Why is this language complex? 3 Why is this language complex? 3 Brunato and Venturi The 2019 “Interactive Workshop on Measuring Language Complexity (IWMLC)”3 allowed a continuation of the debate about cross-linguistic complexity research prompted by the use of UD treebanks as source corpora. Our contribution: The present contribution stems from our participation in the 2019 IWMLC workshop, where we originally presented our approach based on linguistic profiling to measure and compare languages according to their absolute complexity. In this paper we illustrate the fundamentals of this approach and 4 http://www.italianlp.it/demo/profiling-UD/ 5 http://wals.info 4 linguistic complexity. Accordingly, similar sets of features have been used to assess the readability level of texts (Collins-Thompson 2014), to predict human judgments on sentence complexity (Brunato et al. 2018) or to study diachronic variation in syntactic complexity (Lei & Wen 2020). linguistic complexity. Accordingly, similar sets of features have been used to assess the readability level of texts (Collins-Thompson 2014), to predict human judgments on sentence complexity (Brunato et al. 2018) or to study diachronic variation in syntactic complexity (Lei & Wen 2020). In this study we relied on the linguistic profiling methodology described in Brunato et al. (2020) and implemented in Profiling-UD,4 the first web-based tool conceived to linguistically profile multilingual texts by relying on the UD formalism. This tool computes a very large set of linguistic features either extracted from a document or a single sentence. The application of profiling at sentence-level allows focusing on specific instances of phenomena which might be flattened when computed at document level. This is precisely the case of the corpora we are analyzing, since UD treebanks are not homogeneous with respect to textual genres (Plank 2016) and thus linguistic features are unevenly distributed across each corpus. Moreover, for our investigation, we selected only the features particularly relevant for operationalizing sentence complexity in the syntactic sub-domain and we computed their value for each sentence of the considered UD treebanks. The final value for each language corresponds to the average value that the feature has in all sentences of the reference treebank(s) for that language. Finally, following the outcome of the literature on sentence complexity from different perspectives (cognitive, corpus-based, computational), we assumed that the higher this value, the more complex the language usage observed in the treebank with respect to each feature. 2 Linguistic profiling of multilingual treebanks The approach presented here has been inspired by research on “linguistic profiling” which is grounded on two main ingredients: i) large-scale (automatically or manually) annotated corpora representative of a given language variety and ii) counts of linguistic features, extracted from different levels of annotation, which all together model properties related to the form of a text (van Halteren 2004). Although it was originally developed for authorship recognition or verification purposes, this methodology proved to be effective in multiple scenarios, for example to study variations related to genre and register (Argamon et al. 2003) or to the social dimension of language (Nguyen et al. 2016), or also to model stylometric characteristics (Daelemans 2013). It is worth mentioning here that many of the linguistic features used for profiling purposes include fine-grained predictors of 2.1 Universal Dependencies Treebanks As aforementioned, our investigation was based on a subset of UD treebanks released in version 2.3. The UD project is aimed not only at promoting the development and comparative evaluation of multilingual Natural Language Processing systems but also at enabling comparative linguistic studies (Nivre 2015). In fact, corpora annotated with the same inventory of morpho-syntactic categories and dependency relations are paving the way toward methods able to track and quantify linguistic variation across languages avoiding possible interference due to multiple annotation schemata. Table 1 reports the languages considered, together with the corresponding language family and genus according to the World Atlas of Language Structures (WALS) (Dryer & Haspelmath 2013),5 the most commonly-used and broadest Why is this language complex? 5 Language Family Genus TB Tokens Arabic (ARA) Afroasiatic Semitic 1 282k Hebrew (HEB) Afroasiatic Semitic 1 161k Turkish (TUR) Altaic Turkic 1 57k Uyghur (UIG) Altaic Turkic 1 40k Vietnamese (VIE) Austroasiatic Viet-Muong 1 43k Indonesian (IND) Austronesian Malayo-Sumbawan 1 121k Basque (BAQ) Basque Basque 1 121k Latvian (LAV) Indo-European Baltic 1 152k Afrikaans (AFR) Indo-European Germanic 1 49k Danish (DAN) Indo-European Germanic 1 100k German (GER) Indo-European Germanic 1 292k English (ENG) Indo-European Germanic 4 465k Dutch (DUT) Indo-European Germanic 2 326k Norwegian (NOR) Indo-European Germanic 2 301k Swedish (SWE) Indo-European Germanic 2 175k Greek (GRE) Indo-European Greek 1 63k Hindi (HIN) Indo-European Hindi 1 351k Urdu (URD) Indo-European Indic 1 138k Persian (PER) Indo-European Iranian 1 152k Catalan (CAT) Indo-European Romance 1 531k French (FRE) Indo-European Romance 2 470k Italian (ITA) Indo-European Romance 3 477k Portuguese (POR) Indo-European Romance 1 227k Romanian (RUM) Indo-European Romance 2 413k Spanish (SPA) Indo-European Romance 2 980k Bulgarian (BUL) Indo-European Slavic 1 156k Czech (CZE) Indo-European Slavic 3 2167k Croatian (HRV) Indo-European Slavic 1 197k Polish (POL) Indo-European Slavic 2 213k Russian (RUS) Indo-European Slavic 2 99k Slovak (SLO) Indo-European Slavic 1 106k Slovenian (SLV) Indo-European Slavic 1 140k Serbian (SRP) Indo-European Slavic 1 86k Ukrainian (UKR) Indo-European Slavic 1 116k Japanese (JPN) Japanese Japanese 1 184k Korean (KOR) Korean Korean 2 430k Chinese (CHI) Sino-Tibetan – 1 123k Estonian (EST) Uralic Finnic 1 434k Finnish (FIN) Uralic Finnic 2 361k Hungarian (HUN) Uralic Ugric 1 42k Ancient languages Gothic (GOT) Indo-European Germanic 1 55k Ancient Greek (GRC) Indo-European Greek 2 416k Old Church Slavonic (CHU) Indo-European Slavic 1 57k Latin (LAT) Indo-European – 2 552k Tab. Brunato and Venturi 6 6 database of structural (phonological, grammatical, lexical) properties of languages. The rationale behind the choice of these languages lies in our participation in the shared task organized in conjunction with the 2019 “Interactive Workshop on Measuring Language Complexity (IWMLC)”, where these languages were considered as a reasonable test-bed to compare different measures of linguistic complexity. For each language we also specify the number of available treebanks and their size in number of tokens. As it can be seen, the majority of languages (31 out of 44, i.e. 70%) belongs to the Indo-European family, which is internally distinguished into eight genera with three major groups, i.e. Slavic, Germanic and Romance. Concerning the number of treebanks per language, 66% of the languages (29 languages) is represented by one treebank and 29% (13 languages) by two. As it will be discussed in the following sections, it is not always the case that different treebanks of the same language have similar linguistic features. This has a well- known impact on cross-linguistic studies grounded on corpora which may be biased by corpora variations (Chen & Gerdes 2017) mostly due to the multiple genres and domains contained in the different treebanks available for each language (Plank 2016). database of structural (phonological, grammatical, lexical) properties of languages. The rationale behind the choice of these languages lies in our participation in the shared task organized in conjunction with the 2019 “Interactive Workshop on Measuring Language Complexity (IWMLC)”, where these languages were considered as a reasonable test-bed to compare different measures of linguistic complexity. For each language we also specify the number of available treebanks and their size in number of tokens. As it can be seen, the majority of languages (31 out of 44, i.e. 70%) belongs to the Indo-European family, which is internally distinguished into eight genera with three major groups, i.e. Slavic, Germanic and Romance. Concerning the number of treebanks per language, 66% of the languages (29 languages) is represented by one treebank and 29% (13 languages) by two. As it will be discussed in the following sections, it is not always the case that different treebanks of the same language have similar linguistic features. 2.2 Linguistic Features The set of features here considered is a subset of the ones described by Brunato et al. (2020) and has been chosen to be representative of different macro-areas of language complexity phenomena. In what follows, we will describe how they were computed using the following sample sentence taken from the English treebank (EWT). (Figure 1 shows the tree graphical representation): (1) You wonder if he was manipulating the market with his bombing targets. This has a well- known impact on cross-linguistic studies grounded on corpora which may be biased by corpora variations (Chen & Gerdes 2017) mostly due to the multiple genres and domains contained in the different treebanks available for each language (Plank 2016). Basic text properties – Sentence length (sent_length): it is calculated as the average number of words per sentence. Sentence length is typically used as an approximation of syntactic complexity, for example in traditional formulas developed for the automatic assessment of text readability (Kincaid et al. 1975). (1) is 13 tokens long. – Word length (word_length): it is calculated as the average number of characters per word (excluded punctuation). It is a basic indicator of word complexity and, similarly to sentence length, it is used by traditional readability formulas as an approximation of lexical complexity. (1) contains words that are 4.83 characters long on average. 2.1 Universal Dependencies Treebanks 1: Overview of languages (with their ISO-639-2 code), corresponding WALS language family and genus, number of treebanks per language (TB) and treebank size in k of tokens. Tab. 1: Overview of languages (with their ISO-639-2 code), corresponding WALS language family and genus, number of treebanks per language (TB) and treebank size in k of tokens. 6 The syntactic head is always marked in italic. Parse-tree structure Why is this language complex? 7 Fig. 1: Linguistic annotation of the example sentence. Fig. 1: Linguistic annotation of the example sentence. – Average length of clauses (clause_length): it is measured as the number of tokens per clause, which is calculated as the ratio between the number of tokens in a sentence and the number of either verbal or copular heads. Syntactic metrics relying on clause length, such as T-Unit (Hunt 1966), are widely used in studies on human production and comprehension of complex sentences, as well as in first and second language acquisition to assess the development of syntactic competence. In the sample sentence the average clause length is 6.5 tokens, since there are two verbal heads (‘wonder’ and ‘manipulating’). – Average length of dependency links (dep_links_len): this is calculated as the average number of words occurring between the syntactic head and the dependent. As mentioned in Section 1, longer dependencies represent a source of greater processing difficulties for both humans, see (Gibson 1998, 2000), and Demberg & Keller (2008), and statistical parsers, see McDonald & Nivre (2007), Rimell, Clark & Steedman (2009), Nivre et al. (2010), and Gulordava & Merlo (2015). This measure is also considered as a universal property by typological studies, which demonstrate that dependency length is actually minimized in real utterances across many languages and language families, even if with some differences due to language-specific grammatical constraints, syntactic choices (Temperley & Gildea 2018) or diachronic changes (Gulordava & Merlo 2015). The average value in (1) is 2.36: four links have a distance of one from their syntactic head (‘You’ ‘wonder’; ‘was’ ‘manipulating’; ‘the’ ‘market’; ‘bombing’ ‘targets’),6 three links have a distance of two (‘he’ ‘manipulating’; ‘market’ ‘manipulating’; ‘his’ ‘targets’), two have a distance of three (‘if’ ‘manipulating’; – Average length of clauses (clause_length): it is measured as the number of tokens per clause, which is calculated as the ratio between the number of tokens in a sentence and the number of either verbal or copular heads. Syntactic metrics relying on clause length, such as T-Unit (Hunt 1966), are widely used in studies on human production and comprehension of complex sentences, as well as in first and second language acquisition to assess the development of syntactic competence. In the sample sentence the average clause length is 6.5 tokens, since there are two verbal heads (‘wonder’ and ‘manipulating’). Subordination – Percentage distribution of subordinate clauses (subord_dist): it is calculated as the percentage distribution of main vs subordinate clauses, where the latter are identified on the basis of the UD guidelines that distinguish four types.7 We included this and the following feature as the use of subordination is a broadly studied marker of structural complexity, for example for text simplification purposes (Bott & Saggion 2014). (1) is articulated into a main (‘wonder’) and a subordinate clause (‘manipulating’), headed by the verbal root ‘wonder’ and marked as an adverbial clause modifier (advcl). Thus, the percentage distribution of this features is 50%. – Percentage distribution of subordinate clauses (subord_dist): it is calculated as the percentage distribution of main vs subordinate clauses, where the latter are identified on the basis of the UD guidelines that distinguish four types.7 We included this and the following feature as the use of subordination is a broadly studied marker of structural complexity, for example for text simplification purposes (Bott & Saggion 2014). (1) is articulated into a main (‘wonder’) and a subordinate clause (‘manipulating’), headed by the verbal root ‘wonder’ and marked as an adverbial clause modifier (advcl). Thus, the percentage distribution of this features is 50%. – Average depth of ‘chains’ of embedded subordinate clauses (subord_chain_len): once the sub-tree of the subordinate clause is identified, a subordinate ‘chain’ is calculated as the number of subordinate clauses recursively embedded in the top subordinate clause. In the sample sentence, the value of this feature is equal to one, since it contains only one single subordinate clause. – Average depth of ‘chains’ of embedded subordinate clauses (subord_chain_len): once the sub-tree of the subordinate clause is identified, a subordinate ‘chain’ is calculated as the number of subordinate clauses recursively embedded in the top subordinate clause. In the sample sentence, the value of this feature is equal to one, since it contains only one single subordinate clause. 8 ‘with’ ‘targets’), one has a distance of four (‘manipulating’ ‘wonder’) and the longest one, i.e. a distance of six, links ‘targets’ to manipulating. ‘with’ ‘targets’), one has a distance of four (‘manipulating’ ‘wonder’) and the longest one, i.e. a distance of six, links ‘targets’ to manipulating. – Depth of the whole parse tree (tree_depth): it corresponds to the longest path from the root of the dependency tree to some leaf nodes. The measure originates from studies on “relative complexity” showing that deeper syntactic trees hamper human sentence processing (Frazier 1985). In (1), this feature is equal to 3, corresponding to the three intermediate dependency links that are crossed in the path going from the root of the sentence (‘wonder’) to each of the more distant leaf nodes, represented by the words ‘the’, ‘with’, ‘with’, ‘his’ and ‘bombing’. – Depth of the whole parse tree (tree_depth): it corresponds to the longest path from the root of the dependency tree to some leaf nodes. The measure originates from studies on “relative complexity” showing that deeper syntactic trees hamper human sentence processing (Frazier 1985). In (1), this feature is equal to 3, corresponding to the three intermediate dependency links that are crossed in the path going from the root of the sentence (‘wonder’) to each of the more distant leaf nodes, represented by the words ‘the’, ‘with’, ‘with’, ‘his’ and ‘bombing’. Parse-tree structure – Average length of clauses (clause_length): it is measured as the number of tokens per clause, which is calculated as the ratio between the number of tokens in a sentence and the number of either verbal or copular heads. Syntactic metrics relying on clause length, such as T-Unit (Hunt 1966), are widely used in studies on human production and comprehension of complex sentences, as well as in first and second language acquisition to assess the development of syntactic competence. In the sample sentence the average clause length is 6.5 tokens, since there are two verbal heads (‘wonder’ and ‘manipulating’). Average length of dependency links (dep_links_len): this is calculated as the average number of words occurring between the syntactic head and the dependent. As mentioned in Section 1, longer dependencies represent a source of greater processing difficulties for both humans, see (Gibson 1998, 2000), and Demberg & Keller (2008), and statistical parsers, see McDonald & Nivre (2007), Rimell, Clark & Steedman (2009), Nivre et al. (2010), and Gulordava & Merlo (2015). This measure is also considered as a universal property by typological studies, which demonstrate that dependency length is actually minimized in real utterances across many languages and language families, even if with some differences due to language-specific grammatical constraints, syntactic choices (Temperley & Gildea 2018) or diachronic changes (Gulordava & Merlo 2015). The average value in (1) is 2.36: four links have a distance of one from their syntactic head (‘You’ ‘wonder’; ‘was’ ‘manipulating’; ‘the’ ‘market’; ‘bombing’ ‘targets’),6 three links have a distance of two (‘he’ ‘manipulating’; ‘market’ ‘manipulating’; ‘his’ ‘targets’), two have a distance of three (‘if’ ‘manipulating’; Brunato and Venturi Verbal predicate structure – Average number of dependency links of a verbal head (verb_arity): this corre- sponds to the average number of instantiated dependency links (both arguments and modifiers) sharing the same verbal head, excluding auxiliaries bearing the syntactic role of copula according to the UD scheme. This feature reflects the richness of verbal predicates, i.e the higher the score the richer the verbal predicate.8 Note that this measure might be highly sensitive to language: pro-drop languages, which do not obligatorily require an explicit subject, can – Average number of dependency links of a verbal head (verb_arity): this corre- sponds to the average number of instantiated dependency links (both arguments and modifiers) sharing the same verbal head, excluding auxiliaries bearing the syntactic role of copula according to the UD scheme. This feature reflects the richness of verbal predicates, i.e the higher the score the richer the verbal predicate.8 Note that this measure might be highly sensitive to language: pro-drop languages, which do not obligatorily require an explicit subject, can 7 https://universaldependencies.org/u/overview/complex-syntax.html#subordination 8 This measure could be refined if corpora had a further level of annotation making explicit the verb argument structure (allowing one to distinguish arguments from adjuncts) or an external sub-categorization lexicon serving as a reference resource. Why is this language complex? 9 9 have fewer dependents since null subjects are not explicitly marked in the UD annotation scheme. In (1) the average arity score is 3, since the main verb ‘wonder’ has two dependents (‘You’ and ‘manipulating’) and the first embedded verb ‘manipulating’ has four (‘if’, ‘he’, ‘was’ and ‘market’). 3 Comparison of multilingual treebanks Based on the analysis of the selected features, our approach to the study of linguistic complexity allowed attaining several outcomes which can be categorized in two main groups. The first one, described in Section 3.1, is meant to investigate how the considered features are able to intercept different aspects of sentence complexity, whether and to what extent their values are stable within each UD treebank, and how they change across languages and also across multiple treebanks available for a language. Through the second group of results (see Section 3.2), we looked at these features from an holistic perspective and used them to cluster all the treebanks considered. 3.1 A feature-based comparison Figure 2 reports the average distribution of each feature extracted from all sentences of a given treebank. The heatmap provides a direct visualization of which treebank has the higher feature value (darker color) and thus presents a more complex usage with respect to that feature. In each cell we also report in parentheses the position that the treebank occupies in the ranking of all treebanks established by the coefficient of variation for each feature. The coefficient of variation represents a standardized measure of the dispersion of data points around the mean and it is particularly useful for comparing series of data calculated on different scales. Being calculated as the ratio between the standard deviation and the mean, we considered it as particularly appropriate for accounting for the nature of our data as well as for the aim of the analysis. On the one hand, it allows normalizing standard deviation, thus preventing the impact of extreme values, and it turned out to be a reliable index to compare values of linguistic features which can have quite different scales and ranges, such as sentence length (absolute number) and subordinate clauses (distribution). On the other hand, it quantifies the degree of variation within the composition of the considered treebank, on the assumption that the more stable a feature is, the more representative it is for a given language (when we have a 10 Brunato and Venturi unique treebank per language) or for a language variety (when we have more than one). unique treebank per language) or for a language variety (when we have more than one). Let us start analyzing the treebanks with respect to our basic features of complexity, i.e. sentence and word length. For the former, a distinct result emerges that Romance languages treebanks tend to have longer sentences with respect to the other languages, although the treebank with the longest sentences (∼37 tokens) belongs to the Semitic genus and it represents the Arabic language. The different degree of affixation in word formation clearly affects the resulting length of words. In this respect, our data confirm the ‘coarse’ distinction into analytical vs synthetic languages as we find Chinese and Finnish in the lowest and highest positions of ranking by word length, respectively (Finnish_TDT: 7.34; Chinese: 1.69). 3.1 A feature-based comparison Like Finnish, other typical examples of agglutinative languages like Turkish, Basque and Hungarian are similarly highly ranked, followed by the majority of languages of the Germanic and Romance group which still have a rich inflectional morphology but often realized with fusional suffixes. However, it is generally agreed that the distinction into different morphological types should be considered as more gradient rather than categorical and that the same language can exhibit patterns of a different nature (Haspelmath 2009). Japanese, for instance, is highly synthetic with a complex system of verb inflection, but also highly analytic in not having noun inflection; and this might explain the lower position in our raking. Interestingly, focusing merely on these raw text features we observe that languages with more than one treebank have different behaviors. Consider for example the case of Italian, for which there are three treebanks, two of them (ISDT and ParTUT) containing miscellaneous textual genres (i.e. legal texts, newspaper articles and Wikipedia pages) and PoSTWITA, a collection of Italian tweets. The limited number of characters for tweets allowed by the Twitter platform necessarily yields shorter sentences in PoSTWITA (18.54) than the other two treebanks (21.06 in ISDT and 26.58 in ParTUT). Such a constraint in terms of length is also reflected by the ranking position established by the coefficient of variation: the first position of PoSTWITA suggests that it is the most stable treebank regarding this feature. Since it is well-known that sentence length is highly related to features extracted from the syntactic level of annotation, we observe that treebanks can be grouped quite similarly when we consider complexity measures accounting for the parse tree structure. Thus, Arabic, the language with the longest sentences, is also the language with the deepest syntactic trees (7.14). As expected, the genus with the highest tree_depth values is the Romance one, with an average depth of 4.59 in the corresponding treebanks, even though Afrikaans, a Germanic language, has the second greatest tree depth (3.1). Also in this case, the Italian Twitter treebank (PoSTWITA) is among the most stable language variety. A slightly different trend can be observed if we focus on the average length of dependency Why is this language complex? 11 Why is this language complex? 11 11 Fig. 2: Distribution of linguistic features for each treebank. Brunato and Venturi 12 links, a feature similarly extracted from the syntactic annotation, but accounting for the linear structure. The greatest average dependency lengths occur in Chinese (3.28), Persian (3.2), Urdu (3.17) and Hindi (3.02) sentences. However, because this feature is highly related to the two aforementioned ones, it is to be expected that the treebanks belonging to the Romance genus are still those with longer links (with an average length of 2.52). Romance treebanks are also the most stable with respect to this feature, as we find six Romance languages (Romanian-RRT, Italian-ParTUT, Catalan, French-GSD, Spanish-GSD and Spanish-AnCora) in the top ten ranked treebanks for coefficient of variation. Treebanks representative of the Romance languages are confirmed to be the most complex ones in terms of sentence structure also when we consider the average clause length. However, the computation of this feature does not allow us to take into account any distinction among the typology of clauses, e.g. subordinate vs coordinate ones. To inspect this aspect we need to examine the values of features explicitly modeling the use of subordination. As it can be seen, this feature does not strictly follow the distribution of the other features. The languages with the most complex use of subordination are Chinese, Korean (GSD), Vietnamese, Latin (ITTB), Spanish (AnCora), Japanese. The Arabic language turns out to be the second most complex one only with respect to the distribution of subordinate clauses, but not when the internal subordinate clause structure is considered (subord_chain_len). Interestingly, Chinese, Korean (GSD), Vietnamese, Latin (ITTB) are also the top-four most stable languages for this feature in terms of coefficient of variation. We conclude this part with some observations about the verbal arity property. As we observed when we explained how it is computed, our intuition is that this measure is highly sensitive to language-specific constraints also related to the obligatory expression of nominal (or pronominal) subject. To verify this hypothesis, we checked in the WALS Online database the feature “Expression of Pronominal Subjects (101A)” and we found that the languages obtaining the highest verbal arity in our analysis, i.e. Urdu (3.22), Hungarian (3.13), Hindi (3.1), Afrikaans (3.01), are not marked for the “Obligatory pronous” value in WALS. This suggests that our feature is able to intercept information not only limited to the nuclear verb structure. Hindi, Urdu and Afrikaans are also among the top-five ranked languages in terms of coefficient of variation. 3.1 A feature-based comparison In each cell is reported the average value of the feature in the corresponding treebank and the number (in parentheses) indicating the ordinal position that the treebank has in the ranking of all treebanks given by the coefficient of variation for each feature. The lower the number, the more stable the feature in a given treebank. Fig. 2: Distribution of linguistic features for each treebank. In each cell is reported the average value of the feature in the corresponding treebank and the number (in parentheses) indicating the ordinal position that the treebank has in the ranking of all treebanks given by the coefficient of variation for each feature. The lower the number, the more stable the feature in a given treebank. Fig. 2: Distribution of linguistic features for each treebank. In each cell is reported the average value of the feature in the corresponding treebank and the number (in parentheses) indicating the ordinal position that the treebank has in the ranking of all treebanks given by the coefficient of variation for each feature. The lower the number, the more stable the feature in a given treebank. Why is this language complex? 13 and specifically we perform a hierarchical clustering using the Ward algorithm on normalized data. We first apply the cluster analysis on all languages of our dataset and then we focus on the most representative language family, i.e. Indo-European. The purpose of a cluster analysis applied to natural languages is to identify coherent groups of languages (i.e. clusters) whose members are more related each other (in some sense) than members in other groups. In many previous works, clustering has been framed in a typological perspective and informed by properties of languages pointing to different aspects of cross-linguistic diversity typically available in descriptive materials. One of the most informative sources used for this purpose is again WALS, which has been used e.g. by Daumé III & Campbell (2007), who proposed a Bayesian approach for automatically uncovering universal implications from sparse data, and by Georgi, Xia & Lewis (2010) to compare phylogenetic groupings to clusters derived from typological features. Other works have studied graph-theoretic properties of dependency trees for language classification. Liu & Li (2010) proposed a method to cluster languages according to parameters derived from complex network analysis. Features derived from labeled dependency parses were also used by Chen & Gerdes (2017) and applied to UD treebanks, which were clustered according to two quantitative measures of syntactic order variation, i.e. dependency direction and head-dependent distance for each order. In line with the authors of this study, we share the assumption that our cluster-based analysis is not expected to find a categorical answer of grouping languages into fixed language groups – as our complexity measures only partially cover the whole spectrum of language variation –, but rather to identify tendencies of structural proximity between treebanks. In this sense, we were inspired by the most recent developments of the Distributional Typology framework for comparative linguistics (see e.g. Bickel (2015) and Gerdes, Kahane & Chen (2021)), which is mainly focused on the use of statistical methods applied to large sets of fine-grained variables in order to identify quantitative trends across languages. Figure 3 shows the hierarchical similarity tree resulting from clustering all treebanks of the dataset. The horizontal axis corresponds to the distance between each cluster using the Ward method. 3.2 A cluster-based comparison In this last section, we try to understand how languages tend to cluster on the basis of our complexity metrics. To this end, we employ cluster analysis techniques 9 The Latin PROIEL treebank contains most of the Vulgate New Testament translations plus selections from Caesar’s Gallic War, Cicero’s Letters to At- As it can be seen, starting from the bottom of the hierarchy, Croatian and Serbian, Ukranian and Czech (PDT), and Latin (PROIEL) and Gothic are the first merged pairs, which are clustered together at a distance lower than 0.2, while Chinese and Japanese are similarly paired together but at a higher distance (about 0.7). As we move up the dendrogram at a distance of about 1.1, we see that treebanks belonging to the Romance genus tend to group into a quite homogeneous cluster, even though with some exceptions represented by treebanks not representative of Romance languages (e.g. Greek, Hebrew, Serbian, etc.). It can also be observed that two Italian treebanks (i.e. ISDT and POSTWITA) and the French Sequoia treebank form a bigger and more heterogeneous cluster, 14 Brunato and Venturi Fig. 3: Hierarchical clustering for all languages. Fig. 3: Hierarchical clustering for all languages. merged with the former only in a subsequent step (at a distance of about 3.0). We also observe an isolated cluster grouping together Japanese, Chinese, Vietnamese and Korean, which would deserve more in-depth investigation. In fact, despite belonging to different families according to traditional comparative literature, their distance is relatively small with respect to the distribution of the features considered. Among the many possible reasons, a role might be played by the specific annotation criteria defined in the UD project. merged with the former only in a subsequent step (at a distance of about 3.0). We also observe an isolated cluster grouping together Japanese, Chinese, Vietnamese and Korean, which would deserve more in-depth investigation. In fact, despite belonging to different families according to traditional comparative literature, their distance is relatively small with respect to the distribution of the features considered. Among the many possible reasons, a role might be played by the specific annotation criteria defined in the UD project. The case of Italian and French, whose different treebanks are clustered far away in the tree, also affects other languages for which more than one treebank is available. Note, for instance, the case of English, whose four treebanks are clustered together only at higher levels. Similarly, the Latin PROIEL treebank9 appears in a small cluster with Gothic and Old Church Slavonic, while the Latin IITB treebank, Why is this language complex? 15 Fig. 4: Hierarchical clustering for Indo-European languages. Fig. 4: Hierarchical clustering for Indo-European languages. ticus, Palladius’ Opus Agriculturae and the first book of Cicero’s De officiis (https://universaldependencies.org/treebanks/la_proiel/index.html). 10 https://universaldependencies.org/treebanks/la_ittb/index.html 16 represented in each treebank. In fact, there are also languages, such as Norwegian and Korean, which have both their treebanks closely grouped together. represented in each treebank. In fact, there are also languages, such as Norwegian and Korean, which have both their treebanks closely grouped together. Similar observations hold from inspection of the dendogram resulting from the hierarchical clustering of the Indo-European (Figure 4). Starting from the bottom, languages sharing the same genus and having similar feature values are grouped together. This is the case, for example, of Croatian and Serbian or Catalan and Spanish both represented by the AnCora treebank. We still observe homogeneous groups of languages when we focus on clusters at 0.5 distance. These clusters join together many Slavic, (i.e. Ukrainian, Czech and Russian), and Germanic, (i.e. Swedish, Slovenian, Norwegian, two of the four English treebanks, Danish), languages. In addition, all the treebanks representative of ancient languages are grouped together: the two Latin and Ancient Greek treebanks as well as the Gothic and Old Church Slavonic ones. In this case, the similarity concerns the diachronic variation of language rather than the WALS genus. Interestingly, this cluster also includes one of the two Romanian treebanks, i.e. the NonStandard one, which also contains documents of Old Romanian and folklore. which is based on data from the Index Thomisticus corpus,10 is clustered together with the two treebanks representative of Ancient Greek. These findings suggest that the observed proximity of the considered treebanks may be due not only to language-specific properties, but also to genre-specific features. However, we are aware that a thorough analysis is required to unravel the relationship between genre and complexity starting from an in-depth survey of the textual genres mostly ticus, Palladius’ Opus Agriculturae and the first book of Cicero’s De officiis (https://universaldependencies.org/treebanks/la_proiel/index.html). 10 https://universaldependencies.org/treebanks/la_ittb/index.html Brunato and Venturi REFERENCES 17 languages contain sentences with the highest use of subordination, and Chinese, Hindi, Urdu and Persian treebanks have the longest dependency links. languages contain sentences with the highest use of subordination, and Chinese, Hindi, Urdu and Persian treebanks have the longest dependency links. The study raises several issues that we believe deserve a thorough analysis. One of these is to establish the effect of textual genre on the assessment of ‘general- purpose’ language complexity features. We often noticed, in fact, that languages represented by more than one treebank behave quite differently with respect to the same features. Having a better understanding of the relationship between genre and complexity is relevant not only for informing research on genre variation but also from an application perspective: for instance, in the field of readability assessment, to enable the collection of textual resources labeled for genre-specific complexity levels, which can be used as training dataset for machine learning systems. In this respect, a related issue worth investigating concerns the correlation between highest values of the considered features and their variation in a treebank. In our study, we found that in many cases treebanks highly complex for a given feature are also those for which the feature is more stable. This is the case for example for the average length of dependency links or of the use of subordination. Conversely, this does not hold for example for sentence or word length, as treebanks with longer sentences show a high variability. Our approach has also some limitations which we would like to tackle in the future, starting from the operationalization of some features. For instance, verbal arity as calculated here gives only an approximation of the valency structure of verbal predicates thus not allowing to discriminate obligatory arguments from redundant adjuncts possibly affecting complexity. Similarly, with respect to the use of subordination, it could be also informative to calculate separately the distribution of subordinate clauses of distinct typologies, as well as their relative position with respect to the main clause. Since these properties are known to be related to the interaction between structural and discourse-pragmatic factors (Diessel 2005), they can be relevant also from a language complexity perspective. Argamon, Shlomo, Moshe Koppel, Jonathan Fine & Anat Rachel Shimoni. 2003. Gender, genre, and writing style in formal written texts. Text 23(3). 321–346. 4 Conclusion In this study we have proposed a cross-language investigation on linguistic com- plexity covering more than 60 languages distinguished into different families and genera. We motivated our analysis within the framework of linguistic profiling, a data-driven methodology favored by the availability of large-scale corpora, which assumes that a given language and language variety can be characterized by count- ing the distribution of a wide set of features representative of phenomena spanning across language domains. We focused here on a rather small subset of features among those that are typically used in linguistic profiling, whose selection has been informed by cognitive, corpus-based and computational linguistics literature on sentence complexity. The availability of multi-lingual treebanks annotated with the same morpho-syntactic and syntactic formalism has guaranteed reliable compar- isons since the selected proxies of sentence complexity were computed in the same way across corpora. We identified tendencies of structural proximity between languages, not always expected in light of typologically-driven classifications. For instance, we observed that languages belonging to the Romance group show a quite homogeneous behav- ior with respect to several features but also that languages belonging to different language families share a number of characteristics. For example, treebanks rep- resentative of the Chinese, Korean, Vietnamese, Japanese, Latin and Spanish Berdicevskis, Aleksandrs, Çağrı Çöltekin, Katharina Ehret, Kilu von Prince, Daniel Ross, Bill Thompson, Chunxiao Yan, Vera Demberg, Gary Lupyan, Taraka Rama & Christian Bentz. 2018. Using Universal Dependencies in cross-linguistic complexity research. 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https://openalex.org/W2081297032	https://revistas.ucr.ac.cr/index.php/rbt/article/download/5965/5675	Spanish; Castilian	null	Arañas (Arachnida: Araneae) asociadas a dos bosques degradados del Chaco húmedo en Corrientes, Argentina	Revista de Biología Tropical	2,007	cc-by	5,724	Recibido 24-v-2006. Corregido 08-Xii-2006. Aceptado 07-v-2007. Abstract: Spiders (Arachnida: Araneae) associated with two degraded forests in the humid Chaco of Corrientes, Argentina. The advancing degradation of the forest in the biogeographic Chaco province (Argentina) produces an important loss of its little known biodiversity. We studied the spider biodiversity in two forests of Corrientes, Argentina’s “Distrito Oriental Húmedo del Chaco”: Laguna Brava and El Perichón. Seasonal samplings of foliage and fallen leaves between 2001 and 2002 produced 2 067 individuals from 33 families and 226 species/ morphospecies). The families Araneidae, Anyphaenidae, Salticidae and Theridiidae were the most abundant in both forests. The “orb weavers” guild had the highest number of specimens (n=382) and “stalkers” the highest richness (S=56). In Brava, highest abundance was in the summer (n=287) and spring (n=273), in Perichón, it was in winter (n=315). The specific richness and the diversity indexes were higher in Brava (S=134, H´=4.23, E=0.86, D=0.023) than in Perichón (S=127, H´=4.08, E=0.84, D=0.029). The similarity value between both forests was MH=0.611. Rev. Biol. Trop. 55 (3-4): 899-909. Epub 2007 December, 28. Key words: Araneae, biodiversity, humid forests, Corrientes, Argentina. Las arañas comprenden un grupo faunís- tico diverso y ampliamente distribuido en todos los ecosistemas terrestres, incluso el dulceacuícola (Turnbull 1973). Su diversidad es particularmente elevada en los bosques tropicales, donde se puede encontrar casi el 80 % de las especies conocidas (Coddington y Levi 1991). Son depredadores generalistas importantes en las redes tróficas por su abun- dancia, biomasa y diversidad de especies. Por sus hábitos depredadores, influyen en la densidad y actividad de la fauna de detritívo- ros y fungívoros, afectando los procesos de descomposición (Wise 2002). comunidades de arañas han mostrado ser fuer- temente influenciadas, y de manera predecible, por el tipo de hábitat y el patrón de uso de la tierra (Weeks y Holtzer 2000). La arquitectura de la vegetación juega un papel importante en la composición de especies encontradas dentro de un hábitat (Scheidler 1990), por lo que una vegetación estructuralmente compleja puede contener una abundancia y diversidad mayor de arañas (Hatley y MacMahon 1980). A pesar de ser consideradas uno de los grupos entomófagos más abundantes en la naturaleza (Nyffeler et al. 1994) y del esfuer- zo de algunos autores (Höfer 1990, Silva 1996, Silva y Coddington 1996), que en los últimos años contribuyeron al conocimiento de las comunidades de arañas en bosques del Neotrópico, el conocimiento que de ella se tiene es aún incipiente. Arañas (Arachnida: Araneae) asociadas a dos bosques degradados del Chaco húmedo en Corrientes, Argentina Gilberto Avalos1, Gonzalo D. Rubio1, María E. Bar1 & Alda González2 , , 1 Cátedra de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional del Nordeste, Avda. Libertad 5470 (3400) Corrientes, Argentina; gilbertoa@exa.unne.edu.ar 2 Centro de Estudios Parasitológicos y de Vectores (CEPAVE)(UNLP), Calle 2 Nº 584 (1900), La Plata, Argentina; asgonzalez@cepave edu ar 1 Cátedra de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional del Nordeste, Avda. Libertad 5470 (3400) Corrientes, Argentina; gilbertoa@exa.unne.edu.ar 5470 (3400) Corrientes, Argentina; gilbertoa@exa.unne.edu.ar 2 Centro de Estudios Parasitológicos y de Vectores (CEPAVE)(UNLP), Calle 2 Nº 584 (1900), La Plata, Argentina; asgonzalez@cepave.edu.ar 2 Centro de Estudios Parasitológicos y de Vectores (CEPAVE)(UNLP), Calle 2 Nº 584 (1900), La Plata, Argentina; asgonzalez@cepave.edu.ar 2 Centro de Estudios Parasitológicos y de Vectores (CEPAVE)(UNLP), Calle 2 Nº 584 (1900), La Plata, Argentina; asgonzalez@cepave.edu.ar Recibido 24-v-2006. Corregido 08-Xii-2006. Aceptado 07-v-2007. Las arañas han ganado una amplia acep- tación en los estudios ecológicos como indi- cadores de calidad ambiental (Clausen 1986, Maelfait et al. 1990, Willett, 2001, Pinkus- Rendón et al. 2006, Tsai et al. 2006), ya que las Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 899 cactáceas y helechos), encontrándose pequeños charcos temporarios. En Argentina el conocimiento taxonómico y sistemático de algunas familias de arañas es amplio. Sin embargo, las investigaciones refe- ridas a aspectos ecológicos de comunidades de arañas asociadas a áreas naturales o alteradas son escasas. Se destacan los estudios efectua- dos en un área natural protegida de la provincia de Formosa (Corronca y Abdala 1994). En los dos bosques el estrato de árboles se caracteriza por la presencia de quebracho colorado (Schinopsis balansae Engl.), urun- day (Astronium balansae Engl.) y ejempla- res de Tabebuia heptaphylla (Vell.) Toledo, Enterolobium contortisiliquum (Vell.) Morong y Patagonula americana L.; en el estrato arbustivo predominan Schaefferia argenti- nensis Speg., Phylira brasiliensis Klotzsch, Trichilia elegans A. Juss. y Celtis iguanaea (Jacq.) Sarg.; y en el estrato herbáceo se encuentran Oplismenus hirtellus (L.) P. Beauv., Pseudananas sagenarius (Arruda) Camargo, Anemia tomentosa (Savigny) Sw. y Adiantopsis chlorophylla (Sw.) Fée. Las actividades humanas han causado severos cambios en la composición y la diversi- dad de la mayoría de los ecosistemas conocidos (Barnes et al. 1998), llevando a la modificación de las poblaciones, la distribución de las espe- cies, la estructura y el funcionamiento de las comunidades, pudiendo llegar aún a la extin- ción (Meffe y Carroll 1994). Por lo menciona- do, este trabajo tiene como objetivos conocer la fauna de arañas en dos bosques degradados del chaco húmedo argentino de la provincia de Corrientes y analizar comparativamente la diversidad, riqueza específica y abundancia según las estaciones climáticas. y Trabajo de campo: los muestreos se efec- tuaron desde mayo 2001 hasta marzo de 2002, de acuerdo al siguiente esquema: otoño (mayo y junio), invierno (agosto y septiembre), prima- vera (noviembre y diciembre) y verano (febre- ro y marzo). Las arañas fueron recolectadas mediante la técnica de golpeteo del follaje en los estratos arbóreo y arbustivo y el tamizado de la hojarasca en el mantillo (Coddington et al. 1991). Recibido 24-v-2006. Corregido 08-Xii-2006. Aceptado 07-v-2007. En cada bosque, se muestreó entre las 9:00-12:00 h, en un área de 90 000 m2 elegida al azar donde se tomaron, también al azar, cuatro muestras con cada una de las técnicas de colecta mencionadas. Cada muestra de vegetación con- sistió en 15 golpes con una vara sobre la vegeta- ción arbustiva y porción baja del estrato arbóreo y el material fue recolectado sobre una sábana blanca de 2.25 m2. Las arañas de hojarasca fue- ron obtenidas por tamizado de una superficie de 0.25 m2 por muestra, las que fueron colocadas en bolsas de polipropileno debidamente rotula- das y fijadas en alcohol al 70 % hasta su traslado al laboratorio. MATERIALES Y MÉTODOS Área de estudio: el área estudiada pertene- ce al Distrito Oriental Húmedo de la Provincia Fitogeográfica Chaqueña (Cabrera y Willink 1980, Morrone 2001); con clima subtropical húmedo con veranos calurosos y lluviosos e inviernos fríos y secos, temperatura y humedad media de 21.3 °C y 1 200 mm, respectivamente (Bruniard 1981). Los bosques se ubican en las localidades de Laguna Brava (27º31’ S, 58º41’ W) y El Perichón (27º24’ S, 58º44’ W), depar- tamento Capital, Corrientes, Argentina. Ambos bosques están degradados por tala de árboles de importancia económica y por pastoreo. El Perichón se encuentra sobre el Río Paraná y presenta una vegetación más cerrada y densa, con un microclima húmedo, fresco y con poca luz. Laguna Brava está ubicada en una loma entre zonas deprimidas ocupadas por bajos inundables con vegetación higrófila. Un mato- rral de abrigo muy denso dificulta la entrada, la luz es muy pobre y el microclima muy húme- do, con abundancia de epifitos (orquídeas, En el laboratorio el material fue separado en familias y se identificó a nivel de especies y/o morfoespecies (msp) mediante el uso de claves y en algunos casos con la colaboración de espe- cialistas del país. Las arañas fueron separadas por estado del desarrollo, sexo y clasificado por gremios (Enders 1976, Dippenaar-Schoeman et al.1989, Marc y Canard 1997, Uetz et al. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 900 1999). El material recolectado se depositó en la colección de la Cátedra de Artrópodos de la Facultad de Ciencias Exactas y Naturales, Universidad Nacional del Nordeste (UNNE) (CARTROUNNE). una abundancia menor a n=80 en los dos bos- ques estudiados. Entre las familias de mayor riqueza específica se destacaron Araneidae (S=26), Salticidae (S=22), Theridiidae (S=12) y Linyphiidae (S=10) en Laguna Brava; y Salticidae (S=23), Araneidae (S=18) y Theridiidae (S=13) en El Perichón. El resto de las familias mostraron una riqueza específica menor a S=10 en los dos bosques estudiados. En Laguna Brava se registraron 33 especies/ msp con un solo individuo (singletons) y en El Perichón 32. Las especies/msp con dos individuos (doubletons) fueron 18 en ambas localidades (Cuadro 1). MATERIALES Y MÉTODOS Análisis estadísticos: para el análisis de datos se utilizó el programa estadístico Bio-Dap (Gordon y Douglas 1988: Bio-Dap, Ecological Diversity y its Measurement), se calculó la riqueza de especies (S), la diversidad mediante los índices de Shannon-Wienner (H´), de Simpson (D) y de equidad (E) y la similitud con el índice de Morisita-Horn (MH). Para determinar si el ambiente ha sido suficientemente muestreado se utilizó el esti- mador CHAO1 (Colwell y Coddington 1994): S1=Sobs + (a²/2b), donde Sobs corresponde al número de especies/msp observadas en la localidad, a es el número de especies/msp con un solo individuo y b es el número de especies/ msp con dos individuos en la localidad. Al comparar los dos bosques surge que Laguna Brava, si bien presenta menor abun- dancia de arañas, posee mayor riqueza espe- cífica y mayor diversidad y equidad (H´=4.23, D=0.023, E=0.86), que El Perichón (H´=4.08, D=0.029, E=0.84). La similitud entre ambos bosques fue MH=0.611. Las familias recolectadas en los dos bos- ques se organizaron en 8 gremios (Cuadro 2). Considerando la suma de los dos bosques, las arañas constructoras de telas orbiculares totali- zaron el mayor número de individuos (n=382) y el gremio de las arañas cazadoras al acecho presentó la mayor riqueza de especies (S=56). En Laguna Brava los gremios más abundantes correspondieron a constructoras de telas sába- nas (n=221), constructoras de telas orbiculares (n=170), cazadoras por emboscadas (n=149) y cazadoras al acecho (n=140), el resto de los gremios con un número menor a 130 indi- viduos; y los gremios con mayor número de especies fueron constructoras de telas orbicula- res y cazadoras al acecho (S=30), seguida por las constructoras de telas espaciales (S=19), cazadoras por emboscada (S=16) y vagabundas del suelo (S=15), los restantes con menos de 15 especies. En El Perichón los gremios más abundantes correspondieron a: constructoras de telas orbiculares (n=212), vagabundas de vege- tación (n=181), cazadoras al acecho (n=157) y constructoras de telas sábanas (n=137), el resto de los gremios representados por menos de 130 individuos; y los gremios con mayor riqueza específica fueron: cazadoras al acecho (S=29), RESULTADOS El total de arañas recolectadas en los dos bosques fue 2 067 individuos pertenecientes a 33 familias y 226 especies/msp. Los juveni- les (n=1 526) fueron más numerosos que los adultos y las hembras representaron el 68.6 % (371/541) de la población adulta. En Laguna Brava se hallaron 1 022 ejem- plares pertenecientes a 26 familias y 134 espe- cies/msp y en El Perichón 1 045, pertenecientes a 30 familias y 127 especies/msp, todos del Infraorden Araneomorphae (Cuadro 1). Tres familias (Dictynidae, Ctenidae y Scytodidae) se encontraron sólo en Laguna Brava y siete (Amaurobiidae, Deinopidae, Palpimanidae, Prodidomidae, Segestriidae, Theridiosomatidae y Zodariidae) sólo en El Perichón. Las familias más abundantes fueron: Araneidae (n=139), Linyphiidae (n=129), Anyphaenidae (n=121), Salticidae (n=90) y Thomisidae (n=85) en Laguna Brava; Anyphaenidae (n=166), Araneidae (n=161), Salticidae (n=118), Theridiidae (n=112) en El Perichón. El resto de las familias presentó Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 901 CUADRO 1 Familias y especies/morfoespecies de arañas asociadas a dos bosques del Chaco Húmedo (Corrientes, Argentina) (2001- 2002) Table 1 Table 1 Families and species/morphospecies of spiders associated with two forests of the Humid Chaco (Corrientes, Argentina)(2001-2002) Familia Espec Laguna Brava Amaurobiidae Anyphaenidae Araneidae Corinnidae Ctenidae Deinopidae Dictynidae Gnaphosidae Hahniidae Hersiliidae Linyphiidae Lycosidae Mimetidae Oonopidae Oxyopidae Palpimanidae Philodromidae Pholcidae Pisauridae Prodidomidae Salticidae Scytodidae Segestriidae Selenopidae Senoculidae Sparassidae Tetragnathidae Theridiidae Theridiosomatidae Thomisidae Titanoecidae Uloboridae Zodariidae ninguna Yessica sp.- Aysha sp.- Teudis sp. Wulfila sp.- 3 morfoespecies Acacesia sp.- Aculepeira sp.- Alpaida sp. Araneus sp.- Argiope argentata Cyclosa sp. - Eustala sp.- Mangora sp. Mecynogea sp.- Metepeira sp. Micrathena sp.- Neoscona sp. Ocrepeira sp.- Parawixia sp. Pronous sp.- 11 morfoespecies Trachelas sp.- Trachelopachys sp. Orthobula sp.- 3 morfoespecies 1 morfoespecie ninguna 2 morfoespecies 1 morfoespecie Austrohahnia praestans Tama sp. Microlinyphia sp.- 9 morfoespecies Aglaoctenus lagotis- 2 morfoespecies Gelanor altithorax- Gelanor sp. Mimetus sp.- 1 morfoespecie Gamasomorpha wasmanniae Neoxyphinus ogloblini- Oonops sp. Scaphiella sp. Hamataliwa sp. 1- Oxyopes sp. 1 Oxyopes sp. 2- Peucetia sp. ninguna 4 morfoespecies Guaranita munda- 4 morfoespecies 2 morfoespecies ninguna 22 morfoespecies Scytodes sp. ninguna Selenops maranhensis- Selenops sp. Senoculus sp. Polybetes rapidus Leucauge sp.- Tetragnatha sp. Argyrodes sp.- Episinus sp. Theridion sp.- 9 morfoespecies Ninguna 9 morfoespecies Goeldia sp. Conifaber guarani- Uloborus trilineatus Ninguna Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 902 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. Table 1 55 (3-4): 899-909, September-December 2007 CUADRO 2 Riqueza específica y abundancia de familias de arañas asociadas a dos bosques del Chaco Húmedo (Corrientes, Argentina, 2001-2002), agrupadas por gremio Table 2 Species richness and family abundance of spiders associated with two forests of the Humid Chaco (Corrientes, Argentina)(2001-2002), by guild Gremio Familia Especie/morfoespecie Total Laguna Brava El Perichón CTO Araneidae 26 (139) 18 (161) 35 (300) Tetragnathidae 2 (19) 2 (34) 2 (53) Uloboridae 2 (12) 2 (13) 2 (25) Deinopidae 0 (0) 1 (2) 1 (2) Theridiosomatidae 0 (0) 1 (2) 1 (2) Subtotal 30 (170) 24 (212) 41 (382) CTS Linyphiidae 10 (129) 8 (47) 18 (176) Amaurobiidae 0 (0) 3 (46) 3 (46) Titanoecidae 1 (66) 1 (9) 1 (75) Hahniidae 1 (15) 1 (35) 1 (50) Subtotal 12 (210) 13 (137) 23 (347) VV Anyphaenidae 7 (121) 8 (166) 11 (287) Sparassidae 1 (1) 4 (6) 4 (7) Selenopidae 2 (4) 2 (9) 2 (13) Subtotal 10 (126) 14 (181) 17 (307) VS Lycosidae 2 (9) 3 (35) 5 (44) Gnaphosidae 1 (36) 2 (26) 3 (62) Ctenidae 1 (3) 0 (0) 1 (3) Corinnidae 6 (27) 6 (24) 10 (51) Oonopidae 4 (19) 3 (16) 4 (35) Zodariidae 0 (0) 1 (7) 1 (7) Palpimanidae 0 (0) 1 (3) 1 (3) Scytodidae 1 (16) 0 (0) 1 (16) Prodidomidae 0 (0) 1 (3) 1 (3) Subtotal 15 (110) 17 (114) 27 (224) CA Salticidae 22 (90) 23 (118) 45 (208) Mimetidae 4 (16) 3 (24) 6 (40) Oxyopidae 4 (34) 3 (15) 5 (49) Subtotal 30 (140) 29 ( 157) 56 (297) CUADRO 2 Riqueza específica y abundancia de familias de arañas asociadas a dos bosques del Chaco Húmedo (Corrientes, Argentina, 2001-2002), agrupadas por gremio Table 2 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 903 Riqueza específica y abundancia de familias de arañas asociadas a dos bosques del Chaco Húmedo (Corrientes, Argentina, 2001-2002), agrupadas por gremio Riqueza específica y abundancia de familias de arañas asociadas a dos bosques del Chaco Húmedo (Corrientes, Argentina, 2001-2002), agrupadas por gremio Table 2 (Continued) Species richness and family abundance of spiders associated with two forests of the Humid Chaco (Corrientes, Argentina)(2001-2002), by guild Gremio Familia Especie/morfoespecie Total Laguna Brava El Perichón CE Thomisidae 9 (85) 5 (64) 14 (149) Philodromidae 4 (36) 4 (20) 8 (56) Pisauridae 2 (17) 2 (14) 4 (31) Hersiliidae 1 (11) 1 (9) 1 (20) Subtotal 16 (149) 12 (107) 27 (256) CTE Theridiidae 12 (73) 13 (112) 23 (185) Pholcidae 5 (36) 2 (11) 7 (47) Dictynidae 2 (3) 0 (0) 2 (3) Subtotal 19 (112) 15 (123) 32 (235) CTT Segestriidae 0 (0) 1 (3) 1 (3) Lycosidae (A. lagotis) 1 (4) 1 (2) 1 (6) Senoculidae 1 (1) 1 (9) 1 (10) Subtotal 2 (5) 3 (14) 3 (19) TOTAL 33 134 (1022) 127 (1045) 226 (2067) CTO= constructoras de telas orbiculares, CTS= constructoras de telas sábanas, VV= vagabundas de vegetación, VS= vagabundas del suelo, CA= cazadoras al acecho, CE= cazadoras por emboscada, CTE= constructoras de telas espaciales CTT= constructoras de telas tubulares. CTO= orb weavers, CTS= sheet web-builders, VV= foliage runners, VS= ground runners, CA= stalkers, CE= huntin ambushers, CTE= space web-builders, CTT= tangle weavers. Table 2 (Continued) Species richness and family abundance of spiders associated with two forests of the Humid Chaco (Corrientes, Argentina)(2001-2002), by guild CTO= constructoras de telas orbiculares, CTS= constructoras de telas sábanas, VV= vagabundas de vegetación, VS= vagabundas del suelo, CA= cazadoras al acecho, CE= cazadoras por emboscada, CTE= constructoras de telas espaciales, CTT= constructoras de telas tubulares. CTO= constructoras de telas orbiculares, CTS= constructoras de telas sábanas, VV= vagabundas de vegetación, VS= vagabundas del suelo, CA= cazadoras al acecho, CE= cazadoras por emboscada, CTE= constructoras de telas espaciales, CTT= constructoras de telas tubulares. CTO= orb weavers, CTS= sheet web-builders, VV= foliage runners, VS= ground runners, CA= stalkers, CE= hunting ambushers, CTE= space web-builders, CTT= tangle weavers. CTO= orb weavers, CTS= sheet web-builders, VV= foliage runners, VS= ground runners, CA= stalkers, CE ambushers, CTE= space web-builders, CTT= tangle weavers. Table 2 OLB y OPE: otoño, PLB y PPE: primavera, VLB y VPE: verano, ILB e IPE: invierno. Fig. 2. Cluster analisis of spider similarity (MH) and climatic stations in Laguna Brava (LB) and El Perichón (PE)(Corrientes, Argentina). OLB and OPE: autumn, SLB and SPE: spring, VLB and VPE: summer, ILB and IPE: winter. mediante un relevamiento de la araneofauna en la Reserva El Bagual, Formosa, encon- traron 23 familias al analizar diferentes hábitats con mues- treos intensivos. No obstante, debido al escaso estudio de la araneofauna en bosques natu- rales en Argentina, no sería conveniente sacar conclusio- nes sin un marco comparativo que permita hacer un análisis más exhaustivo. En coincidencia con lo observado por Costa et al. (1991), en la diversidad de familias recolectadas en ambos bosques, probable- mente hayan incidido facto- res como humedad relativa, luz u otros vinculados con la cobertura vegetal; esta rela- ción general quizás refleje la mayor cantidad y diversidad de nichos disponibles para las arañas en los hábitats más complejos y con menor fre- cuencia de disturbios. Esto es coincidente con lo que sostiene Desender et al. (1989). militud (MH) y PE) (Corrientes, y VPE: verano, ations in Laguna nd OPE: autumn, inter. En coincidencia con lo observado por Costa et al. (1991), en la diversidad de familias recolectadas en ambos bosques, probable- mente hayan incidido facto- res como humedad relativa, luz u otros vinculados con la cobertura vegetal; esta rela- ción general quizás refleje la mayor cantidad y diversidad de nichos disponibles para las arañas en los hábitats más complejos y con menor fre- urbios. Esto es coincidente con Desender et al. (1989). Fig. 2. Análisis de agrupamiento de arañas de acuerdo a la similitud (MH) y estaciones climáticas en Laguna Brava (LB) y El Perichón (PE) (Corrientes, Argentina). OLB y OPE: otoño, PLB y PPE: primavera, VLB y VPE: verano, ILB e IPE: invierno. Fig. 2. Cluster analisis of spider similarity (MH) and climatic stations in Laguna Brava (LB) and El Perichón (PE)(Corrientes, Argentina). OLB and OPE: autumn, SLB and SPE: spring, VLB and VPE: summer, ILB and IPE: winter. Fig. 2. Cluster analisis of spider similarity (MH) and climatic stations in Laguna Brava (LB) and El Perichón (PE)(Corrientes, Argentina). OLB and OPE: autumn, SLB and SPE: spring, VLB and VPE: summer, ILB and IPE: winter. unieron las estaciones otoño (OPE) con invier- no (IPE) y primavera (PPE) con verano (VPE). Table 2 25.1 %) y la mayor cantidad de especies en invierno (S=71), no obstante la diversidad (H´=3.72) y equitatividad (E=0.9) fueron supe- riores en verano. Asimismo, la mayor dominan- cia de especies (D=0.05) coincidió en invierno en ambos bosques (Fig. 1). La similitud más alta entre bosques se registró en otoño (MH=0.598) y la más baja en verano (MH=0.268). constructoras de telas orbiculares (S=24), vaga- bundas del suelo (S=17) y constructoras de telas espaciales (S=15), los restantes gremios con menos de 15 especies. El análisis por estación climática permitió evidenciar variaciones a lo largo del año. En Laguna Brava la mayor cantidad de individuos se observó en verano (n=287, 28.1 %) y en primavera (n=273, 26.7 %), correspondiéndole también a este último mes los mayores valo- res de riqueza específica (S=73), diversidad (H´=3.82) y equitatividad (E=0.89). En El Perichón la mayor abundancia se constató en invierno (n=315, 30.1 %) y en otoño (n=262, El análisis de agrupamiento de acuerdo con la similitud (MH) de ambas localidades y estaciones climáticas se representa en un feno- grama (Fig. 2). Se reconocen dos grupos, uno correspondiente a Laguna Brava (LB) y el otro a El Perichón (EP). En esta última localidad se Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 904 Fig. 1. Número de individuos (N), riqueza específica (S), diversidad (H’, D) y equidad (E), según localidad y estació climática (Corrientes, Argentina) (2001-2002). Fig. 1. Number of individual (N), specific richness (S), diversity (H´, D) and equity (E), according to locality and climat station (Corrientes, Argentina)(2001-2002). Fig. 1. Número de individuos (N), riqueza específica (S), diversidad (H’, D) y equidad (E), según localidad y estación climática (Corrientes, Argentina) (2001-2002). Fig. 1. Número de individuos (N), riqueza específica (S), diversidad (H’, D) y equidad (E), según localidad y estación climática (Corrientes, Argentina) (2001-2002). Fig. 1. Number of individual (N), specific richness (S), diversity (H´, D) and equity (E), according to locality and climatic station (Corrientes, Argentina)(2001-2002). Fig. 1. Number of individual (N), specific richness (S), diversity (H´, D) and equity (E), according to locality and climatic station (Corrientes, Argentina)(2001-2002). Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 905 Fig. 2. Análisis de agrupamiento de arañas de acuerdo a la similitud (MH) y estaciones climáticas en Laguna Brava (LB) y El Perichón (PE) (Corrientes, Argentina). Table 2 En Laguna Brava no se observan estas afini- dades y el verano (VLB) mostró baja similitud con las demás estaciones. El índice de Chao 1 indica que las especies halladas representan el 81 % y el 82 % de la riqueza estimada en Laguna Brava y El Perichón, respectivamente. Al comparar los resultados con otros traba- jos realizados en otros países, en ambientes con características parecidas, se observó que las familias halladas en ambos bosques, son simi- lares a las registradas por Silva (1996) en un bosque tropical del Perú, empleando técnicas de fumigación, agitación del follaje y barridos con red; esta autora obtiene una diversidad alta para las familias Araneidae, Theridiidae y Salticidae. En otro trabajo, Silva y Coddington (1996), realizado en Pakitza, Perú, obtienen resultados semejantes. DISCUSIÓN Desde el punto de vista taxonómico, las familias de arañas encontradas en este tra- bajo representan aproximadamente el 53 % del total de familias identificadas hasta el presente para Argentina (Platnick 2006). El total de familias recolectadas es significativo si lo comparamos con los resultados obtenidos por Corronca y Abdala (1994). Estos autores, Coincidiendo con Duffey (1962) y Breymeyer (1966) que sostienen que los adul- tos no superan el 48 % de las poblaciones natu- rales de Araneomorphae, en esta investigación la población adulta alcanzó el 26 % sumando las dos áreas de estudio. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 906 Los artrópodos se dispersan por medio de troncos u otra vegetación flotante sobre ambientes lóticos (Poi de Neiff 2003). El río Paraná atraviesa bosques de Brasil y Misiones (Argentina) transportando diversos elementos que sirven de medio de dispersión a la artropo- fauna. Este hecho podría justificar la presencia de familias, típicas de bosques más húmedos, como Theridiosomatidae, Deinopidae y espe- cies del género Amazonepeira (Araneidae) en el bosque de El Perichón, ubicado en cercanía de este río. El elevado número de taxones represen- tados por un solo individuo y dos individuos, coincide con la hipótesis de que la artropofauna tropical se caracteriza por presentar un alto número de especies, las cuales en su mayoría exhiben bajas densidades poblacionales o son raras (Flórez 1998). Resultados similares en bosques neotropicales han sido obtenidos por Silva (1996) y Silva y Coddington (1996). Según Ruzicka (1987) Lycosidae y Linyphiidae no superan juntas el 45 % de los individuos en áreas protegidas, alcanzando el 85 % en áreas deterioradas. Si bien no se uti- lizaron trampas de caída que sobrestiman los muestreos de licósidos (Dinter 1995), dichas familias alcanzan el 14 % y 8 % en Laguna Brava y El Perichón respectivamente. Los por- centajes presentados indicarían un bajo grado de deterioro en los bosques estudiados, en relación con los valores obtenidos (54.41 %) por Pérez-Miles et al. (1999) en ambientes urbanizados. La complejidad de las estructuras del bos- que parece ejercer un efecto directo sobre el emplazamiento de las telas, y en general, el aumento en la disponibilidad de sustratos donde fijarlas probablemente cause un aumen- to en la densidad de las arañas (Rypstra 1985, Samu y Szinetár 2002). Resumen Corronca, J.A. & C.S. Abdala. 1994. La fauna araneológica de la Reserva Ecológica “El Bagual”, Formosa, Argentina. Aracnología Supl. 9: 1-6. El importante avance de la degradación de bosques en la Provincia Biogeográfica del Chaco, Argentina, conlleva una pérdida importante de su poco conocida biodiversidad. Se realizó un estudio de la fauna de arañas en dos bosques de la provincia de Corrientes, Argentina, correspondientes al Distrito Oriental Húmedo del Chaco, en las localidades de Laguna Brava y El Perichón. Se realizaron muestreos estacionales entre los años 2001 y 2002. Las arañas reco- lectadas (2 067 individuos de 33 familias y 226 especies/ morfoespecies) fueron obtenidas por golpeteo del estrato arbustivo y tamizado de hojarasca. Las familias Araneidae, Anyphaenidae, Salticidae y Theridiidae fueron las más abundantes en los dos bosques. El gremio de arañas “cons- tructoras de telas orbiculares” presentó el mayor número de individuos (n=382) y el de “cazadoras al acecho” la mayor riqueza (S=56). En Laguna Brava se observó mayor abundancia en el verano (n=287) y primavera (n=273) y en el Perichón en invierno (n=315). La riqueza específica y el valor de los índices de diversidad, fueron mayores en Laguna Brava (S=134, H´=4.23, E=0.86, D=0.023) que en El Perichón (S=127, H´=4.08, E=0.84, D=0.029). La similitud entre ambos bosques fue MH=0.611. Costa, F.G., F. Pérez-Miles, E. Gudynas, L. Prandi & R.M. Capocasale. 1991. Ecología de los arácnidos criptozoicos, excepto ácaros, de Sierra de las Animas (Uruguay). Ordenes y familias. Aracnol. 13/15: 1-14. Desender, K., M. Alderweireldt & M. Pollet. 1989. Field edges y their importance for polyphagos predatory arthropods. Med. Fac. Landbouww. Rijksuniv. Gent. 54: 823-833. Dinter, A. 1995. Estimation of epigeic spider population densities using an intensive D-vac sampling tech- nique y comparison with pitfall trap catches in winter wheat. Arthropod natural enemies in arable land I. Acta Jutlandica 70: 23-32. Dippenaar-Schoeman, A.S., A.M. Van Den Berg & A. Van Den Berg. 1989. Species composition y rela- tive seasonal abundance of spiders from the field y tree layers of the Roodeplaat Dam Nature Reserve. Koedoe 32: 25-38. Duffey, E. 1962. A population study of spiders in limestone grassland, the fiel-layer fauna. Oikos 13: 15-34. Palabras clave: Araneae, biodiversidad, bosques húme- dos, Corrientes, Argentina. Duffey, E. 1978. Ecological Strategies in spiders including some characteristics of species in pioneer y nature habitats. Symp. Zool. Soc London 42: 109-123. DISCUSIÓN Esta postura parece coincidir con la gran densidad de arañas que pertenecen a los gremios constructoras de telas orbiculares y constructoras de telas sabanas, halladas en esta investigación. El análisis de agrupamiento demuestra una disposición de las estaciones climáticas en dos grupos (correspondientes a ambas localidades) lo que permite reflejar la araneofauna propia de cada bosque. El mayor número de individuos registrado en invierno en El Perichón y el número más o menos constantes de arañas halladas en las otras estaciones climáticas, indicaría que los bosques funcionan como hábitats de micro- climas estables y sitio de preferencia para el establecimiento de la fauna. Además, la per- manente cobertura del suelo, la cual provee refugio y disponibilidad de presas, facilitaría la aireación y la regulación de la temperatura ambiente, haciendo a estas áreas más propicias para el desarrollo de las arañas (Duffey 1978, Gravesen y Toft 1987, Desender et al. 1989). Asimismo, la abundancia de arañas en el vera- no del bosque Laguna Brava puede asociarse a la disponibilidad de presas en esta época del año (Uetz 1976, Riechert y Luczak 1982). Si bien no responde a lo observado en Laguna Brava, la similitud entre otoño e invier- no (OPE-IPE) y primavera y verano (PPE- VPE) en El Perichón podría explicarse por la estacionalidad seca-lluviosa propia de esta región del país. Teniendo en cuenta la abundancia y la riqueza de arañas halladas en las áreas estu- diadas, son necesarias investigaciones futuras en bosques naturales que permitan tener un panorama más amplio y hacer comparacio- nes objetivas. Además, estudios realizados en diferentes ecosistemas naturales de Europa han demostrado que las arañas alcanzan alta densidad cuando los disturbios causados por la actividad humana son mínimos (Kajak 1971, Lohmeyer y Pretscher 1979). Por ello es fun- damental la conservación de estos ambientes naturales, actualmente desprotegidos. Los valores más altos registrados con el índice de Simpson (D) en invierno en ambas localidades están dados por un incremento en el número de individuos de Tmarus sp.1 y Aysha sp. en Laguna Brava y Aysha sp. y msp nº2 (Theridiidae) en El Perichón. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 55 (3-4): 899-909, September-December 2007 907 REFERENCIAS Barnes, B.V., D.R. Zak, S.R. Denton & S.H. Spurr. 1998. 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https://openalex.org/W2803580854	https://zenodo.org/records/1295545/files/46-50.pdf	Ukrainian	null	Daily profile and variability of blood pressure in patients with arterial hypertension (AH) in combination with COPD.	Medičnì perspektivi	2,018	cc-by	3,200	УДК 616.12-008.331.1-047.36:616.24-007.272-036.1 https://doi.org/10.26641/2307-0404.2018.2(part1).129515 https://doi.org/10.26641/2307-0404.2018.2(p В.А. Потабашній, В.І. Фесенко, Т.З. Буртняк ДЗ «Дніпропетровська державна медична академія МОЗ України» кафедра терапії, кардіології та сімейної медицини ФПО (зав. – д. мед. н., проф. В.А. Потабашній) пл. 30-ти річчя Перемоги, 2, Кривий Ріг, Дніпропетровська область, 50000 Україна SE «Dnipropetrovsk medical academy of Health Ministry of Ukraine» Department of therapy, cardiology and family medicine of FPE 30-ty richchia Peremogy, 2, Kryvyi Rih, Dnipropetrovsk region, 50000, Ukraine e-mail: burtniak@i.ua ДЗ «Дніпропетровська державна медична академія МОЗ України» кафедра терапії, кардіології та сімейної медицини ФПО (зав. – д. мед. н., проф. В.А. Потабашній) ДЗ «Дніпропетровська державна медична академія МОЗ України» кафедра терапії, кардіології та сімейної медицини ФПО (зав. – д. мед. н., проф. В.А. Потабашній) пл. 30-ти річчя Перемоги, 2, Кривий Ріг, Дніпропетровська область, 50000 Україна SE «Dnipropetrovsk medical academy of Health Ministry of Ukraine» Department of therapy, cardiology and family medicine of FPE 30-ty richchia Peremogy, 2, Kryvyi Rih, Dnipropetrovsk region, 50000, Ukraine e-mail: burtniak@i.ua ДЗ «Дніпропетровська державна медична академія МОЗ України» кафедра терапії, кардіології та сімейної медицини ФПО (зав. – д. мед. н., проф. В.А. Потабашній) пл. 30-ти річчя Перемоги, 2, Кривий Ріг, Дніпропетровська область, 50000 Україна SE «Dnipropetrovsk medical academy of Health Ministry of Ukraine» Department of therapy, cardiology and family medicine of FPE 30-ty richchia Peremogy, 2, Kryvyi Rih, Dnipropetrovsk region, 50000, Ukraine e-mail: burtniak@i.ua ( р ф ) пл. 30-ти річчя Перемоги, 2, Кривий Ріг, Дніпропетровська область, 50000 Україна SE «Dnipropetrovsk medical academy of Health Ministry of Ukraine» Department of therapy, cardiology and family medicine of FPE 30-ty richchia Peremogy, 2, Kryvyi Rih, Dnipropetrovsk region, 50000, Ukraine e-mail: burtniak@i.ua Ключові слова: артеріальна гіпертензія, хронічне обструктивне захворювання легень, добовий профіль і Ключові слова: артеріальна гіпертензія, хронічне обструктивне захворювання легень, добовий профіль артеріального тиску р р у Key words: arterial hypertension, chronic obstructive pulmonary desease, daily blood pressure profile Реферат. Суточный профиль и вариабельность артериального давления у пациентов с АГ в сочетании с ХОБЛ. Потабашний В.А., Фесенко В.И., Буртняк Т.З. На сегодняшний день, несмотря на усовершенство- ванный поход к ведению больных с АГ, у отдельных категорий пациентов остается неудовлетворительным контроль артериального давления. Особую актуальность эта проблема имеет при сочетании АГ и ХОБЛ в связи с особенностями течения и сложностью в их диагностике. Благодаря использованию метода суточного мониторирования возможна объективизация истинного профиля АД в течение суток и диагностика АГ на качественно новом уровне. У пациентов с сочетанием АГ и ХОБЛ существуют определенные особенности клинического течения, которые можно определить с помощью суточного мониторирования АД. В.А. Потабашній, В.І. Фесенко, Т.З. Буртняк Выявлена прямая зависимость между профилем АД, клиническими группами ХОБЛ, а также временем ее возникновения относительно АГ. Abstract. Daily profile and variability of blood pressure in patients with arterial hypertension (AH) in combination with COPD. Potabashniy V.A., Fesenko V.I., Burtniak T.Z. To date, despite the improved approach to managing patients with AH, control of blood pressure remains unsatisfactory in some categories of patients. This problem is particularly topical in AH combined with COPD depending on the peculiarities of the course and difficulty in their diagnosis. Thanks to the use of 24-hour monitoring method, it is possible to objectify the true blood pressure profile during the day and diagnose AH at a qualitatively new level. In patients with a combination of AH and COPD, there are certain features of the clinical course that can be determined with the use of 24-hour BP monitoring. A direct relationship between the profile of blood pressure, the clinical groups of COPD, as well as the time of its onset regarding hypertension was revealed. В Україні на хвороби системи кровообігу страждають близько 22,3 млн осіб, що становить 52,4% усього населення [2]. Артеріальна гіпер- тензія (АГ) – одна з найпоширеніших хвороб у світі та в Україні (43,9% дорослого населення), яка часто поєднується з іншими захворюваннями (цукровий діабет, хронічні ниркові захворюван- ня, бронхіальна астма та хронічне обструктивне захворювання легень (ХОЗЛ)), які впливають на вибір антигіпертензивного препарату. Поши- реність АГ також зростає з віком та діагно- стується в більшої половини людей у віці 60-69 років і приблизно у три чверті віком 70 років і старше [2, 6]. половина пацієнтів з обмеженням потоку повітря безсимптомні, що призводить до проблеми вчасної діагностики. Поширеність ХОЗЛ з віком зростає з 3,2% у віці 18-44 років до 11,7% у віці 65 років [8]. Все частіше зустрічається поєднання АГ з ХОЗЛ, що становить близько 35% [1]. Вперше зв'язок АГ з бронхообструктивним синдромом відзначено Н.М. Мухарлямовим, який описав підйом АТ після наростання явищ бронхо- обструкції. В основу було покладено роль гі- поксії та порушення функції легень у мета- болізмі вазоактивних речовин [3]. Протягом 40 років ведуться дискусії щодо відокремлення окремої пульмоногенної АГ. Останнім часом приділяється увага визначенню фенотипів ХОЗЛ, метою яких є стратифікація пацієнтів на групи залежно від прогнозу та відповіді на терапію, що сприяє покращенню клінічного ведення ХОЗЛ на цей час є четвертою причиною смерті, та прогнозується, що до 2020 року вона посяде 3 місце. ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ МАТЕРІАЛИ ТА МЕТОДИ ДОСЛІДЖЕНЬ Всього обстежено 65 хворих: І група (n=32) включала пацієнтів з АГ, яка виникала до роз- витку ХОЗЛ. До ІІ групи (n=33) відповідно ввійшли пацієнти, в яких ХОЗЛ виникало раніше, ніж АГ. АГ розподіляли залежно від сту- пеня підвищення артеріального тиску відповідно до Наказу МОЗ України № 384 від 24.05.2012 року та рекомендацій Європейського товариства кардіологів та Української асоціації кардіологів (2013) [4, 7]. За тяжкістю ХОЗЛ пацієнтів роз- поділено на клінічні групи згідно з Керівництвом GOLD 2017 та Наказом МОЗ України № 555 від 27.06. 2013 року [5, 8]. Однією з ключових змін у новому документі Керівництва GOLD 2017 [8] є відокремлення оцінки симптомів від спіромет- ричної оцінки. Незважаючи на те, що досліджен- ня функції зовнішнього дихання залишається необхідним для постановки діагнозу, основними цілями при обстеженні є оцінка симптомів, ризику загострень, а також ступеня впливу захворювання на загальний стан здоров'я пацієнтів. Проведений кореляційний аналіз показав, що ступінь АГ має прямий кореляційний зв’язок (+0,48) із кількістю загострень на рік у пацієнтів з АГ у поєднанні з ХОЗЛ. У пацієнтів з АГ 1 ст. у поєднанні з ХОЗЛ скарги за Модифікованою шкалою задишки (мМДР) становили 1,0±0,5 бала. Тест оцінки ХОЗЛ (ТОХ) становив 6,4±1,15 бала. У пацієнтів з АГ 2 ст. у поєднанні з ХОЗЛ скарги за шкалою мМДР відповідали 2,5±0,8 бала, а ТОХ – 18,3±3,65 бала. У пацієнтів з АГ 3 ст. у поєднанні з ХОЗЛ наявні скарги відповідали 3,4±1,0 балів за шка- лою мМДР, ТОХ – 31,2±4,15 балів. Встановлено, що ступінь АГ має прямий коре- ляційний зв'язок (+0,50) з вираженістю симпто- матики ХОЗЛ. Встановлено, що ступінь АГ має прямий коре- ляційний зв'язок (+0,50) з вираженістю симпто- матики ХОЗЛ. При аналізі даних хворих ІІ групи визначено, що АГ 1 ступеня виявлено в 7 осіб (21,2%), 2 сту- пеня – 22 (66,7%), 3 ступеня – 4 (12,1%). І група включала 23 чоловіків та 9 жінок, се- редній вік яких становив 53 [44-76] роки, трива- лість захворювання на АГ становила в середньо- му 11 [5-17] років, на ХОЗЛ відповідно – 6 [2- 10] років. Систолічний АТ становив 163 [140- 185] мм рт. ст., діастолічний АТ – 103 [96- 110] мм рт. ст. ЧСС – 95 [80-110] уд./хв., ЧД – 22 [18-26] за хв., ОФВ1 – 52 [38-62]%, ФЖЄЛ – 56 [47-65]%, ОФВ1/ФЖЄЛ – 0,62 [0,57-0,68]. У ІІ групі за поширеністю рейтинг клінічних груп за GOLD 2017 показав, що на І місці кл. гр. В.А. Потабашній, В.І. Фесенко, Т.З. Буртняк Більше 3 мільйонів людей по- мерли від ХОЗЛ у 2012 році, що становить 6% усіх смертей у світі [8]. Однак дійсна поши- реність може бути набагато більшою, оскільки МЕДИЧНІ ПЕРСПЕКТИВИ / MEDICNI PERSPEKTIVI 46 пацієнтів з АГ у поєднанні з ХОЗЛ та підходу до діагностики та лікування. пацієнтів з АГ у поєднанні з ХОЗЛ та підходу до діагностики та лікування. пацієнтів з АГ у поєднанні з ХОЗЛ та підходу до діагностики та лікування. Методи дослідження: клінічний, спірометрія, електрокардіографія, ЕхоКГ, ліпіди крові, гліке- мія. Показники добового моніторування АТ ви- значалися за допомогою портативного монітора «Кардиотехника - 4000 АД» фірми «Инкарт», Ст- Петербург. Аналіз та статистичну обробку ма- теріалу проводили за допомогою програми «Ex- cel» (Microsoft Office 2010), пакета статистичних програм Statistica 6.0. За даними дослідження Marie Fisk et al., ХОЗЛ слід вважати незалежним серцево-судин- ним фактором ризику, оскільки підтверджено зв'язок між ХОЗЛ та підвищеною жорсткістю інтими-медіа і швидкістю пульсової хвилі [9]. У практичній діяльності необхідні швидкі, прості та неінвазивні методи, які дозволять про- гнозувати серцево-судинний ризик у пацієнтів з поєднаною патологією. У практичній діяльності необхідні швидкі, прості та неінвазивні методи, які дозволять про- гнозувати серцево-судинний ризик у пацієнтів з поєднаною патологією. МАТЕРІАЛИ ТА МЕТОДИ ДОСЛІДЖЕНЬ В (n=12; 36,4%) та D (n=11; 33,3%), на ІІ – А (n=7; 21,2%), на ІІІ – С (n=3; 9,1%). Частота за- гострень становила 1 раз на рік у 7 (21,2%) па- цієнтів, 2 рази на рік – у 10 (30,3%), 3-4 рази на рік – у 16 (48,5%) пацієнтів. ІІ група включала 25 чоловіків та 8 жінок, се- редній вік яких становив 58 [45-80] років, три- валість захворювання на АГ становила в серед- ньому 7 [4-9] років, на ХОЗЛ відповідно – 12 [7- 17] років. Систолічний АТ становив 166 [145- 190] мм рт. ст., діастолічний АТ – 105 [95- 115] мм рт. ст. ЧСС – 98 [82-115] за хв., ЧД – 25 [13-32] за хв., ОФВ1 – 43 [26-60]%, ФЖЄЛ – 51 [39-63]%, ОФВ1/ФЖЄЛ – 0,56 [0,55-0,65]. Встановлено прямий кореляційний зв’язок (+0,62) між ступенем АГ та кількістю загострень за рік у пацієнтів з АГ у поєднанні з ХОЗЛ. У пацієнтів з АГ 1 ст. у поєднанні з ХОЗЛ скарги за мМДР становили 2,0±0,7 бала. ТОХ становив 8,5±1,23 бала. У пацієнтів з АГ 2 ст. у поєднанні з ХОЗЛ скарги за шкалою мМДР відповідали 3,0±0,8 бала, а ТОХ – 24,1±5,71 бала. У пацієнтів з АГ 3 ст. у поєднанні з ХОЗЛ наявні РЕЗУЛЬТАТИ ТА ЇХ ОБГОВОРЕННЯ У хворих І групи АГ 1 ступеня виявлено у 8 осіб (25,0%), 2 ступеня – у 20 (62,5%), 3 ступеня – у 4 (12,5%). Згідно з Керівництвом GOLD 2017, проведена стратифікація пацієнтів на клінічні групи A, B, C, D, яка показала, що в І групі за по- ширеністю 1-е місце посіла кл. гр. В (n=14; 43,8%), 2-е – С (n=8; 25,0%) та 3-є – А і D (по n=5; у кожній по 15,6%). Частота загострень становила 1 раз на рік у 6 (18,8%) пацієнтів, 2 рази на рік – у 17 (53,1%), 3-4 рази на рік – у 9 (28,1%). Мета роботи – встановити особливості до- бового профілю і варіабельності АТ у пацієнтів з АГ у поєднанні з ХОЗЛ залежно від клінічних груп та часу виникнення ХОЗЛ відносно АГ. 18/ Том XXIІI / 2 ч. 1 47 ССП ПИ ИССО ОКК ЛЛІІТТЕЕРРААТТУУРРИ И допомоги при хронічному обструктивному захворю- ванні легень: Наказ МОЗ України від 27.06.2013 № 555. 1. Долгушева Ю.А. β-агонисты различной дли- тельности действия у пациентов с сочетанием сер- дечно-сосудистой патологии и бронхообструктивных заболеваний / Ю.А. Долгушева, К.А. Зыков, И.Е. Чазова // Практ.пульмонология. – 2015. – № 2. – С. 41-47. 1. Долгушева Ю.А. β-агонисты различной дли- тельности действия у пациентов с сочетанием сер- дечно-сосудистой патологии и бронхообструктивных заболеваний / Ю.А. Долгушева, К.А. Зыков, И.Е. Чазова // Практ.пульмонология. – 2015. – № 2. – С. 41-47. 6. Chandy Dipak. Current perspectives on treatment of hypertensive patients with chronic obstructive pul- monary disease / Dipak Chandy, Wilbert S. Aronow, Ma- ciej Banach // Integrated Blood Pressure Control. – 2013. –N 6. – P. 101-109. 6. Chandy Dipak. Current perspectives on treatment of hypertensive patients with chronic obstructive pul- monary disease / Dipak Chandy, Wilbert S. Aronow, Ma- ciej Banach // Integrated Blood Pressure Control. – 2013. –N 6. – P. 101-109. 2. Коваленко В.М. Проблеми здоров’я і трива- лості життя в сучасних умовах: посібник / В.М. Кова- ленко, В.М. Корнацький. – Київ, 2017. – 24-28. 7. ESH/ESC Guidelines For The Management Of Arterial Hypertension. – 2013. 3. Мухарлямов Н.М. Системная артериальная ги- пертензия у больных с хроническими обструктив- ными заболеваниями легких / Н.М. Мухарлямов, Ж.С. Сатбеков, В.В. Сучков // Кардиология. – 1974. – № 12. – С. 55-58. 8. Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for the diagnosis, mana- gement and prevention of chronic obstructive pulmonary disease // Medical Communications Resources. – 2017. 4. Про затвердження та впровадження медико- технологічних документів зі стандартизації медичної допомоги при артеріальній гіпертензії: Наказ МОЗ України від 24.05.2012 № 384. 4. Про затвердження та впровадження медико- технологічних документів зі стандартизації медичної допомоги при артеріальній гіпертензії: Наказ МОЗ України від 24.05.2012 № 384. 9. Surrogate Markers of Cardiovascular Risk and Chronic Obstructive Pulmonary Disease. A Large Case- Controlled Study / Fisk Marie, McEniery Carmel M., Gale Nichola // Hypertension. – 2018. – 71. – 499-506. 5. Про затвердження та впровадження медико- технологічних документів зі стандартизації медичної ВИСНОВКИ 3. Доведено, що кардіоваскулярний ризик у пацієнтів з АГ у поєднанні з ХОЗЛ зростає при більш тяжкому перебігу ХОЗЛ. 1. Виявлено пряму залежність між профілем АТ, клінічними групами ХОЗЛ та часом ви- никнення ХОЗЛ відносно АГ. 4. Встановлено, що в пацієнтів, у яких ХОЗЛ виникло раніше, ніж АГ, тяжкість бронхіальної обструкції більш виражена. 4. Встановлено, що в пацієнтів, у яких ХОЗЛ виникло раніше, ніж АГ, тяжкість бронхіальної обструкції більш виражена. 2. Встановлено прямий зв’язок між ступенем АГ та клінічними групами ХОЗЛ. Рис. 2. Ступінь бронхіальної обструкції залежно від групи дослідження Рис. 2. Ступінь бронхіальної обструкції залежно від групи дослідження ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ При аналізі співвідношення ступеня АГ до клінічної групи ХОЗЛ (рис. 1) виявлено пряму залежність між ступенем АГ та клінічними групами ХОЗЛ, оскільки чим більший ступінь АГ, тим частіша госпіталізація пацієнтів. скарги відповідали 3,4±1,25 бала за шкалою мМДР, ТОХ – 36,2±7,41 бала. скарги відповідали 3,4±1,25 бала за шкалою мМДР, ТОХ – 36,2±7,41 бала. Відповідно, ступінь АГ має прямий кореля- ційний зв'язок (+0,61) з вираженістю симптома- тики ХОЗЛ. 0 2 4 6 8 10 12 14 16 18 20 A B C D 1 ст. 2 ст. 3 ст. Рис. 1. Співвідношення ступеня АГ до клінічної групи ХОЗЛ Рис. 1. Співвідношення ступеня АГ до клінічної групи ХОЗЛ З'ясовано, що при більш тяжкому перебігу ХОЗЛ зростає кардіоваскулярний ризик у па- цієнтів з АГ у поєднанні з ХОЗЛ (табл.). При АГ 1 ст. та 2 ст. у пацієнтів з клінічними групами А та В встановлюється додатковий помірний сер- цево-судинний ризик (ССР). При АГ 3 ст. у клін. гр. А та при АГ 1 ст. та 2 ст. у клін. групі С – до- датковий високий ССР. Відповідно при клін. групі D та АГ 3 ст. при клін. групах В, С – до- датковий дуже високий ССР. Існує ряд меха- нізмів, які пояснюють взаємовідношення АГ та ХОЗЛ, включаючи системне запалення, низьку фізичну активність, нейрогуморальну активацію, аритмії, ендотеліальну дисфункцію та значне падіння плеврального тиску під час вдиху. групі D та АГ 3 ст. при клін. групах В, С – до- датковий дуже високий ССР. Існує ряд меха- нізмів, які пояснюють взаємовідношення АГ та ХОЗЛ, включаючи системне запалення, низьку фізичну активність, нейрогуморальну активацію, аритмії, ендотеліальну дисфункцію та значне падіння плеврального тиску під час вдиху. МЕДИЧНІ ПЕРСПЕКТИВИ / MEDICNI PERSPEKTIVI 48 Сумарний серцево-судинний ризик за клінічними групами ХОЗЛ АГ ХОЗЛ АГ 1 ступінь САТ 140-159 ДАТ 90-99 АГ 2 ступінь САТ 160-179 ДАТ 100-109 АГ 3 ступінь САТ> 180 ДАТ> 110 A Додатковий помірний Додатковий помірний Додатковий високий B Додатковий помірний Додатковий помірний Додатковий дуже високий C Додатковий високий Додатковий високий Додатковий дуже високий D Додатковий дуже високий Додатковий дуже високий Додатковий дуже високий Сумарний серцево-судинний ризик за клінічними групами ХОЗЛ МЕДИЧНІ ПЕРСПЕКТИВИ / MEDICNI PERSPEKTIVI 48 МЕДИЧНІ ПЕРСПЕКТИВИ / MEDICNI PERSPEKTIVI 48 У результаті дослідження виявлено, що в ІІ групі тяжкість бронхіальної обструкції більш виражена порівняно з І групою, що свідчить про більшу тривалість ХОЗЛ, ніж АГ (рис. 2). ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ 0 10 20 30 40 50 60 70 ОФВ1 ФЖЄЛ ОФВ1/ФЖЄЛ І група ІІ група 0 10 20 30 40 50 60 70 ОФВ1 ФЖЄЛ ОФВ1/ФЖЄЛ І група ІІ група Р 2 С і б і ї б ії і і Рис. 2. Ступінь бронхіальної обструкції залежно від групи дослідження RREEFFEERREENNCCEESS of Medical-Technological Documents for the Standardization of Medical Assistance in Chronic Ob- structive Pulmonary Disease"]. Kyiv; 2013. Ukrainian. of Medical-Technological Documents for the Standardization of Medical Assistance in Chronic Ob- structive Pulmonary Disease"]. Kyiv; 2013. Ukrainian. 1. Dolhusheva Yu.A. [β-agonists of different dura- tion of action in patients with combination of cardio- vascular pathology and broncho-obstructive illnesses]. Prakt. pulmonolohyia. 2015;2:41-47. Russian. 6. Chandy Dipak, Aronow S Wilbert, Banach Ma- ciej. Current perspectives on treatment of hypertensive patients with chronic obstructive pulmonary disease. Integrated Blood Pressure Control. 2013;6:101-9. p y 2. Kovalenko VM, Kornatskyi VM. [Problems of health and life expectancy in modern conditions]. Po- sibnyk. 2017;24-28. Ukrainian. 2. Kovalenko VM, Kornatskyi VM. [Problems of health and life expectancy in modern conditions]. Po- sibnyk. 2017;24-28. Ukrainian. g ; 7. ESH/ESC Guidelines For The Management Of Arterial Hypertension; 2013. 3. Mukharliamov NM, Satbekov ZhS, Suchkov VV. [Systemic arterial hypertension in patients with chronic obstructive pulmonary disease]. Kardiologiya. 1974;12:55-58. Russian. 3. Mukharliamov NM, Satbekov ZhS, Suchkov VV. [Systemic arterial hypertension in patients with chronic obstructive pulmonary disease]. Kardiologiya. 1974;12:55-58. Russian. 8. Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for the diagnosis, ma- nagement and prevention of chronic obstructive pulmo- nary disease. Medical Communications Resources; 2017. 4. [Order of the Ministry of Health of Ukraine dated May 24, 2012 No. 384 "On Approval and Implementation of Medical-Technological Documents for the Stan- dardization of Medical Assistance in Arterial Hyper- tension"]. Kyiv; 2012. Ukrainian. 4. [Order of the Ministry of Health of Ukraine dated May 24, 2012 No. 384 "On Approval and Implementation of Medical-Technological Documents for the Stan- dardization of Medical Assistance in Arterial Hyper- tension"]. Kyiv; 2012. Ukrainian. y 9. Marie Fisk, Carmel M. McEniery, Nichola Gale. Surrogate Markers of Cardiovascular Risk and Chronic Obstructive Pulmonary Disease. A Large Case-Controlled Study. Hypertension. 2018;71:499-506. ] y 5. [Order of the Ministry of Health of Ukraine dated June 27, 2013 № 555 "On Approval and Implementation 5. [Order of the Ministry of Health of Ukraine dated June 27, 2013 № 555 "On Approval and Implementation https://doi.org/10.26641/2307-0404.2018.2(part1).129516 УДК 612.1:577.15:616.24.-002.17 18/ Том XXIІI / 2 ч. 1 49 ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ ВВИ ИККО ОРРИ ИССТТААН НН НЯЯ П ПО ОККААЗЗН НИ ИККАА ТТРРИ ИП ПССИ ИН НО ОП ПО ОДДІІББН НО ОЇЇ ААККТТИ ИВВН НО ОССТТІІ ККРРО ОВВІІ ЯЯКК М МААРРККЕЕРРАА ТТЯЯЖ ЖККО ОССТТІІ П ПЕЕРРЕЕББІІГГУУ ЛЛЕЕГГЕЕН НЕЕВВО ОГГО О Ф ФІІББРРО ОЗЗУУ В.В. Родіонова, О.В. Карасьова ЗД «Дніпропетровська медична академія МОЗ України» кафедра професійних хвороб та клінічної імунології (зав. – д. мед. н., доц. К.Ю. Гашинова) вул. В. Вернадського, 9, Дніпро, 49044, Україна SE «Dnipropetrovsk medical academy of Health Ministry of Ukraine» Department of Occupational Diseases and Clinical Immunology V. Vernadsky str., 9, Dnipro, 49044, Ukraine e-mail: v.rodionova@i.ua Ключові слова: легеневий фіброз, біомаркери, трипсиноподібна активність крові Key words: pulmonary fibrosis (PF), biomarkers, trypsin-like activity of blood Реферат. Использование показателя трипсиноподобной активности крови как маркера тяжести течения легочного фиброза. Родионова В.В., Карасева О.В. Цель – определить изменения показателя трипсинопо- добной активности крови и его взаимосвязь с острофазовыми показателями воспаления у больных с легочным фиброзом как маркера тяжести течения и прогноза заболевания. Материалы и методы: в исследование было включено 18 пациентов: 15 (83%) женщин и 3 (17%) мужчин, средний возраст – 53±2,5 года. В контрольную группу вошли 15 практически здоровых лиц. Все обследованные пациенты (n=18) были разделены на две группы: с легким или умеренно тяжелым течением легочного фиброза (ЛФ) – 8 (44,4%) пациентов (І группа), МЕДИЧНІ ПЕРСПЕКТИВИ / MEDICNI PERSPEKTIVI 50
https://openalex.org/W2531367136	https://figshare.le.ac.uk/articles/journal_contribution/Concomitant_Carboxylate_and_Oxalate_Formation_From_the_Activation_of_CO2_by_a_Thorium_III_Complex_/10209737/1/files/18406973.pdf	English	null	Concomitant Carboxylate and Oxalate Formation From the Activation of CO<sub>2</sub> by a Thorium(III) Complex	Chemistry	2,016	cc-by	3,242	Communication Communication DOI: 10.1002/chem.201604622 DOI: 10.1002/chem.201604622 Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] Kerridge Department of Chemistry, Lancaster University, Lancaster, LA1 4YB (UK) [d] Dr. L. Castro, Prof. L. Maron LPCNO, CNRA and INSA, Universit8 Paul Sabatier, 135 Avenue de Rangeuil, Toulouse 31077 (France) E-mail: laurent.maron@irsamc.ups-tlse.fr Supporting information for this article and authors’ ORCIDs are available on the WWW under http://dx.doi.org/10.1002/chem.201604622. T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons At- tribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. [a] A. Formanuik, Dr. F. Ortu, Dr. D. P. Mills School of Chemistry, The University of Manchester Manchester, M13 9PL (UK) E-mail: david.mills@manchester.ac.uk Scheme 1. Synthesis of 2 and 3 from 1. [b] C. J. Inman Department of Chemistry and Biochemistry, School of Life Sciences University of Sussex, Brighton, BN1 9QL (UK) [b] C. J. Inman Department of Chemistry and Biochemistry, School of Life Sciences University of Sussex, Brighton, BN1 9QL (UK) UIII chemistry as the double reduction of CS2 by [U(Cp’)3] (Cp’= C5H4SiMe3) yields [{U(Cp’3)}2(m-k1:k2-CS2)].[12] However, 1 reacts with excess CO2 to give [{Th(Cp’’)2[h2-O2C{C5H3-3,3’- (SiMe3)2}]}2(m-k2:k2-C2O4)] (3) in 65% yield (Scheme 1), in con- trast to the UIII reduction of CO2 by [U(Cp’)3] to afford [{U(Cp’)3}2(m-O)] and CO.[13] The FTIR spectrum of 3 has absorp- tions at 1653 cm@1 and 1560 cm@1 that can be attributed to asymmetric C@O stretches of the oxalate and carboxylate groups respectively.[14] The reaction of 2 with CO2 gave a mix- ture of products including carboxylate (see the Supporting In- formation). UIII chemistry as the double reduction of CS2 by [U(Cp’)3] (Cp’= C5H4SiMe3) yields [{U(Cp’3)}2(m-k1:k2-CS2)].[12] However, 1 reacts with excess CO2 to give [{Th(Cp’’)2[h2-O2C{C5H3-3,3’- (SiMe3)2}]}2(m-k2:k2-C2O4)] (3) in 65% yield (Scheme 1), in con- trast to the UIII reduction of CO2 by [U(Cp’)3] to afford [{U(Cp’)3}2(m-O)] and CO.[13] The FTIR spectrum of 3 has absorp- tions at 1653 cm@1 and 1560 cm@1 that can be attributed to asymmetric C@O stretches of the oxalate and carboxylate groups respectively.[14] The reaction of 2 with CO2 gave a mix- ture of products including carboxylate (see the Supporting In- formation). [c] Dr. A. Kerridge Department of Chemistry, Lancaster University, Lancaster, LA1 4YB (UK) [d] Dr. L. Castro, Prof. L. [a] A. Formanuik, Dr. F. Ortu, Dr. D. P. Mills School of Chemistry, The University of Manchester Manchester, M13 9PL (UK) E-mail: david.mills@manchester.ac.uk Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] plexes have received most attention for CO2 activation as their inherent oxophilicity is advantageous in overcoming the con- siderable thermodynamic and kinetic barriers in this process.[5] Similarly, actinides are highly oxophilic, so CO2 activation by UIII complexes is also developing rapidly[6] and proof of concept catalytic processes have been disclosed.[7] The mapping of UIII- mediated CO2 activation by DFT calculations has provided key insights into possible mechanistic pathways.[8] In contrast, Cloke reported the only example of CO2 activation by a puta- tive ThIII intermediate[9] as ThIII small molecule activation is in its infancy.[10,11] Herein we report the first reaction of an isolat- ed ThIII complex with CO2, and CS2 for comparative studies. Abstract: Improving our comprehension of diverse CO2 activation pathways is of vital importance for the wide- spread future utilization of this abundant greenhouse gas. CO2 activation by uranium(III) complexes is now relatively well understood, with oxo/carbonate formation predomi- nating as CO2 is readily reduced to CO, but isolated thoriu- m(III) CO2 activation is unprecedented. We show that the thorium(III) complex, [Th(Cp’’)3] (1, Cp’’={C5H3(SiMe3)2- 1,3}), reacts with CO2 to give the mixed oxalate-carboxyl- ate thorium(IV) complex [{Th(Cp’’)2[k2-O2C{C5H3-3,3’- (SiMe3)2}]}2(m-k2:k2-C2O4)] (3). The concomitant formation of oxalate and carboxylate is unique for CO2 activation, as in previous examples either reduction or insertion is fa- vored to yield a single product. Therefore, thorium(III) CO2 activation can differ from better understood uranium(III) chemistry. [Th(Cp’’)3] (1, Cp’’={C5H3(SiMe3)2-1,3}) reacts with 0.5 to 10 equivalents of CS2 to give [{Th(Cp’’3)}2(m-k1:k2-CS2)] (2) as the only isolable product in 45% yield (Scheme 1; see the Support- ing Information for full details). This reaction is consistent with Scheme 1. Synthesis of 2 and 3 from 1. There has been an international drive to reduce emissions of CO2 through cleaner energy generation since its identification as a key contributor to global warming.[1] In tandem the em- ployment of CO2 as a C1 feedstock for fine chemical (by direct insertion into organic molecules)[2] and liquid fuel (via reduc- tion to CO for Fischer–Tropsch processes)[3] synthesis have rap- idly expanded to complement the optimized photosynthetic pathways employed by nature.[4] Early d-transition metal com- [a] A. Formanuik, Dr. F. Ortu, Dr. D. P. Mills School of Chemistry, The University of Manchester Manchester, M13 9PL (UK) E-mail: david.mills@manchester.ac.uk [b] C. J. Inman Department of Chemistry and Biochemistry, School of Life Sciences University of Sussex, Brighton, BN1 9QL (UK) [c] Dr. A. Chem. Eur. J. 2016, 22, 17976 – 17979 Concomitant Carboxylate and Oxalate Formation From the Activation of CO2 by a Thorium(III) Complex Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 17976 Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] Figure 2 shows the calculated enthalpy reaction profile for the formation of 3, with the double reduction of CO2 to give a m-k1:k2-CO2 dinuclear ThIV complex the proposed first step based on the analogous CS2 reaction as well as CO2 reactivity reported with other actinide complexes.[8,12,15,17] The oxalate formation invokes nucleophilic attack of a CO2 mole- cule by a dimetalloxycarbene intermediate [{Th(Cp’’3)}2(m-k1:k1- CO2)] (C2) in a carbenic fashion, which has previously only been seen in d-block CO2 activation for TiIV.[19] No pre-interac- tion is required between the ThIV centers and the second CO2 molecule, which is in contrast with all previous examples of SmII[17a–c] and UIII[17d] oxalate formation. The resultant cis-m-k1:k1- C2O4 transition state (TS1) is one of several possible conformers that have DrH8 values within the estimated error of the calcula- tion (ca. 3 kcalmol@1) of each other, thus we do not comment on this further. Rearrangement of the oxalate to a trans-m- k1:k1-binding mode increases the steric demands about the ThIV centers (C3). The potential energy surface for these rear- rangements is very flat and despite our efforts it was not possi- ble to locate a transition state. This leads to insertion of CO2 at a single position of a Th–Cp’’ moiety at each ThIV center (TS2) as the silicon centers stabilize negative charge at the beta po- sition, allowing the best overlap with the empty orbital of CO2. These insertions are accompanied by the rearrangement of the oxalate to a m-k2:k2-binding mode. Subsequent proton and silyl group migrations in the dearomatized Cp’’ rings give the observed product 3 at an energetic minimum. Figure 1. Molecular structure of a) 2 and b) 3.2C7H8 with selected atom la- belling and displacement ellipsoids set to 30% probability level. Hydrogen atoms, minor disorder components and lattice solvent omitted for clarity. The solid state structures of 2 and 3·2C7H8 were determined by single crystal XRD (Figure 1. A polymorph 2b·2C6H14 was also obtained; see the Supporting Information). The (m-CS2)2@ unit in 2 was disordered over two positions so only the major component is discussed here. Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] Maron LPCNO, CNRA and INSA, Universit8 Paul Sabatier, 135 Avenue de Rangeuil, Toulouse 31077 (France) E-mail: laurent.maron@irsamc.ups-tlse.fr Supporting information for this article and authors’ ORCIDs are available on the WWW under http://dx.doi.org/10.1002/chem.201604622. T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons At- tribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Chem. Eur. J. 2016, 22, 17976 – 17979 T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 17976 Communication Figure 1. Molecular structure of a) 2 and b) 3.2C7H8 with selected atom la- belling and displacement ellipsoids set to 30% probability level. Hydrogen atoms, minor disorder components and lattice solvent omitted for clarity. insertion of CO2 into a Th–Cp’’ moiety and silyl/proton migra- tion yields 3. The insertion of CO2 into lanthanide-CpR bonds to form carboxylates has been postulated not to require steric strain to proceed.[18] Additional experiments were performed to probe the mechanism of formation of 3 (see the Supporting Information for full details). A Toepler pump was used to react 1 with 1 or 2 equivalents of CO2 or 13CO2 at @788C, and 3/3- 13C was the only identifiable product by 1H and 13C{1H} NMR spectroscopy in all cases. The reaction of 1 with supercritical CO2 was monitored by 1H NMR spectroscopy, and comparison with an authentic sample showed the formation of 3. Minor products in all reaction mixtures could not be identified. In situ FTIR spectroscopy was used to monitor the conversion of 1 to 3 at @788C in methylcyclohexane with stoichiometric CO2. No intermediates could be detected but the CO2(g) absorption at 2338 cm@1 diminished on slow warming to room temperature, coincident with the ingress of an oxalate absorption at 1653 cm@1 that is seen in the FTIR spectrum of crystalline 3. The experiment was repeated with 13CO2 and the oxalate ab- sorbance of 3-13C was observed at 1609 cm@1, consistent with reduced mass considerations. Given that no intermediates could be detected experimen- tally, we performed DFT studies to rationalize this unusual mechanism. T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 17977 Chem. Eur. J. 2016, 22, 17976 – 17979 www.chemeurj.org Chem. Eur. J. 2016, 22, 17976 – 17979 Experimental Section Full synthetic details, characterization data and computational data for 2 and 3 is available in the Supporting Information. Additional research data supporting this publication are available from The University of Manchester eScholar repository at DOI: 10.15127/ 1.302780. [7] a) D. P. Halter, F. W. Heinemann, J. Bachmann, K. Meyer, Nature 2016, 530, 317–321; b) E. Lu, S. T. Liddle, Dalton Trans. 2015, 44, 12924– 12941; c) B. M. Gardner, J. C. Stewart, A. L. Davis, J. McMaster, W. Lewis, A. J. Blake, S. T. Liddle, Proc. Natl. Acad. Sci. USA 2012, 109, 9265–9270; d) A.-C. Schmidt, A. V. Nizovtsev, A. Scheurer, F. W. Heinemann, K. Meyer, Chem. Commun. 2012, 48, 8634–8636; e) A. R. Fox, S. C. Bart, K. Meyer, C. C. Cummins, Nature 2008, 455, 341–349. Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] a mechanism only seen previously in d-block chemistry, where- as the carboxylate forms via a route only seen before in f-ele- ment chemistry for lanthanide complexes. This shows that ThIII small molecule activation can furnish results that complement and contrast with uranium, lanthanide and d-transition metals. Future studies will target heteroallene activation by ThIII com- plexes supported by different ligand systems to test the gener- ality or divergence of these processes. [2] a) T. Sakakura, J. C. Choi, H. Yasuda, Chem. Rev. 2007, 107, 2365–23 b) Q. Liu, L. Wu, R. Jackstell, M. Beller, Nat. Commun. 2015, 6, 5933. b) Q. Liu, L. Wu, R. Jackstell, M. Beller, Nat. Commun. 2015, 6, 5933. [3] N. M. West, A. J. Miller, J. A. Labinger, J. E. Bercaw, Coord. Chem. Rev. 2001, 255, 881–898. [4] a) M. M. Halmann, Chemical Fixation of Carbon Dioxide: Methods for Re- cycling CO2 into Useful Products, CRC, Boca Raton, 1993; b) M. Aresta, Carbon Dioxide as a Chemical Feedstock, Wiley-VCH, Weinheim, 2010. Carbon Dioxide as a Chemical Feedstock, Wiley-VCH, Weinheim, 2010 Future studies will target heteroallene activation by ThIII com- plexes supported by different ligand systems to test the gener- ality or divergence of these processes. [5] a) W. B. Tolman, Activation of Small Molecules: Organometallic and Bioin- organic Perspectives, Wiley-VCH, Weinheim, 2006; b) A. Paparo, J. Okuda, Coord. Chem. Rev. 2016, doi: 10.1016/j.ccr.2016.06.005. [6] a) S. T. Liddle, Angew. Chem. Int. Ed. 2015, 54, 8604–8641; Angew. Chem. 2015, 127, 8726–8764; b) H. S. La Pierre, K. Meyer, Prog. Inorg. Chem. 2014, 58, 303–416; c) B. M. Gardner, S. T. Liddle, Eur. J. Inorg. Chem. 2013, 3753–3770; d) O. P. Lam, K. Meyer, Polyhedron 2012, 31, 1–9; e) P. L. Arnold, Chem. Commun. 2011, 47, 9005–9010; f) O. T. Summer- scales, F. G. N. Cloke, Struct. Bonding (Berlin) 2008, 127, 87–117; g) I. Castro-Rodr&guez, K. Meyer, Chem. Commun. 2006, 1353–1368; h) W. J. Evans, S. A. Kozimor, Coord. Chem. Rev. 2006, 250, 911–935; i) M. Ephriti- khine, Dalton Trans. 2006, 2501–2516; j) I. Korobkov, S. Gambarotta, Prog. Inorg. Chem. 2005, 54, 321–348. Alasdair Formanuik,[a] Fabrizio Ortu,[a] Christopher J. Inman,[b] Andrew Kerridge,[c] Ludovic Castro,[d] Laurent Maron,[d] and David P. Mills[a] This fragment binds in an asym- metrical m-k1:k2-fashion [S@C: 1.644(11) and 1.717(10) a; S-C-S: 124.4(7)8], in common with the motif seen for [{U(Cp’3)}2(m- k1:k2-CS2)] [S@C: 1.464(19) and 1.831(19) a; S-C-S: 131.7(13)8][12] and similar to that seen for [{U[OSi(OtBu)3]3}2(m-k2:k2-CS2)] [S@C: 1.594(12) and 1.748(11) a; S-C-S: 131.6(8)8].[15] The oxalate of 3 has similar metrical parameters to those seen in [{Th(COTTIPS2)(Cp*)}2(m-k2:k2-C2O4)].[9] The carboxylate ligand ex- hibits both C@C and C=C lengths in the C5 ring and a geminal 3,3’-disilane. The C@Ocarboxylate [1.284(5) and 1.263(5) a] lengths evidence delocalization about the carboxylate framework, al- though the binding is asymmetric due to sterics [Th@Ocarboxylate 2.400(3) and 2.484(2) a]. The electronic structures of 2 and 3 were characterized at the DFT level, employing the B3LYP ex- change-correlation functional and a polarized split-valence basis set for structural optimizations. Structural parameters of 3 were in good agreement with experiment (see the Support- ing Information for full details). We postulated that 3 forms via a [{Th(Cp’’)3}2(m-CO2)] inter- mediate that is analogous to 2. The bulky Cp’’ ligands hinder the elimination of CO and the formation of [{Th(Cp’’)3}2(m- O)],[16] so a second molecule of CO2 reacts with the (m-CO2)2@ fragment to give an oxalate. There are many examples of steri- cally demanding ligands promoting oxalate formation over a m-oxo or carbonate in f-block CO2 activation.[8,17] Subsequent To conclude we have shown that although CS2 activation by [Th(Cp’’)3] is analogous to that seen for a similar UIII system, the mechanism by which it reacts with CO2 to form a mixed oxalate/carboxylate product has no precedent in UIII chemistry, in which CO2 reduction (and subsequent carbonate formation depending on the supporting ligands) predominates. We probed this reaction to show that the oxalate is generated by www.chemeurj.org Figure 2. Computed enthalpy reaction profile for the formation of 3. Commu Figure 2. Computed enthalpy reaction profile for the formation of 3. [2] a) T. Sakakura, J.-C. Choi, H. Yasuda, Chem. Rev. 2007, 107, 2365–2387; [2] a) T. Sakakura, J.-C. Choi, H. Yasuda, Chem. Rev. 2007, 107, 2365–2387 b) Q. Liu, L. Wu, R. Jackstell, M. Beller, Nat. Commun. 2015, 6, 5933. a mechanism only seen previously in d-block chemistry, where- as the carboxylate forms via a route only seen before in f-ele- ment chemistry for lanthanide complexes. This shows that ThIII small molecule activation can furnish results that complement and contrast with uranium, lanthanide and d-transition metals. [1] P. Falkowski, R. J. Scholes, E. Boyle, J. Canadell, D. Canfield, J. Elser, N. Gruber, K. Hibbard, P. Hogberg, S. Linder, F. T. Mackenzie, B. Moore III, T. Pedersen, Y. Rosenthal, S. Seitzinger, V. Smetacek, W. Steffen, Science 2000, 290, 291–296. T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 17978 Chem. Eur. J. 2016, 22, 17976 – 17979 Chem. Eur. J. 2016, 22, 17976 – 17979 Acknowledgements This work was supported by the EPSRC (grant numbers EP/ L014416/1, EP/M023885/1 and EP/J002208/2), the Nuclear FiRST DTC and the University of Manchester. 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https://openalex.org/W4386903378	https://resources.today/PDF/50ECOR422.pdf	Russian	null	Global ESG transformation and sustainable development trends of Russian companies under sanctions pressure	Othody i resursy	2,022	cc-by	25,486	Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 Интернет-журнал «Отходы и ресурсы» https://resources.today Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 / 2022, Vol 9, No 4 https://resources.today/issue-4-2022.html URL статьи: https://resources.today/PDF/50ECOR422.pdf DOI: 10.15862/50ECOR422 (https://doi.org/10.15862/50ECOR422) Ссылка для цитирования этой статьи: Кудряшов, А. Л. Глобальная ESG-трансформация и тенденции устойчивого развития российских компаний в условиях санкционного давления / А. Л. Кудряшов // Отходы и ресурсы. — 2022. — Т. 9. — № 4. — URL: https://resources.today/PDF/50ECOR422.pdf DOI: 10.15862/50ECOR422 For citation: Глобальная ESG-трансформация и тенденции устойчивого развития российских компаний в условиях санкционного давления Аннотация. В статье рассматривается концепция экологических, социальных и управленческих факторов (ESG) и их значение для устойчивого развития. В последние годы данная концепция приобрела значительную популярность в качестве основы для оценки нефинансовой деятельности компаний, при этом инвесторы и стейкхолдеры все больше осознают важность факторов ESG в создании долгосрочной стоимости, управлении рисками и взаимодействии с заинтересованными сторонами. В работе исследуются ключевые тенденции и проблемы в текущей бизнес-среде, такие как распространение форм отчетности, потребность в более стандартизированных и сопоставимых данных о факторах ESG, а также важность участия стейкхолдеров в принятии решений по реализации политик ESG. Автор подчеркивает соответствие между целями устойчивого развития и ESG факторов в деятельности компаний. Исследуется теоретическая основа концепции ESG с изложением ключевых принципов, а также связь с корпоративной эффективностью. Факторы ESG определены как ключевые движущие силы процесса устойчивого развития, и в статье представлен углубленный анализ каждого из этих факторов с выделением проблем и возможностей, которые они представляют для реализации данной концепции. В статье исследуется вопрос создания санкциями рисков и возможностей для компаний и инвесторов, стремящихся внедрять ESG в бизнес-практику. Страница 1 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Приводится анализ практик внедрения ESG в российских организациях на основе некоторых тематических исследований и эмпирических данных, чтобы проиллюстрировать текущее состояние внедрения ESG в стране. Автор определил основные проблемы и возможности для компаний в России. В целом, в статье представлены основанные на фактических данных выводы и предложения для компаний, инвесторов, политиков и общества для обоснования их усилий в создании более устойчивого будущего. Ключевые слова: устойчивое развитие; концепция ESG; внедрение ESG; санкции; трансформация ESG; российская экономика; устойчивый бизнес; стейкхолдеры; факторы ESG Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Введение Устойчивое развитие стало насущной глобальной проблемой, поскольку организации и политики признают важность баланса экономического роста с социальными и экологическими проблемами. Перед миром стоит серьезная задача — как добиться устойчивого развития, обеспечив при этом экономический рост и процветание. В результате концепция «Экология, социальная ответственность и управление» (ESG) стала основой для оценки устойчивости компаний и поощрения ответственной деловой практики. Концепция ESG выходит за рамки традиционного финансового анализа, включая экологические и социальные проблемы, а также корпоративное управление в процессы принятия решений в организациях. Эта статья направлена на изучение концепции ESG и ее последствий для устойчивого развития. Мы рассматриваем важность ESG в продвижении устойчивого развития, проблемы внедрения практики ESG, и даем рекомендации организациям и политикам по эффективному включению ESG в свою деятельность. Предоставляя всесторонний анализ концепции ESG, это исследование призвано внести вклад в продолжающийся диалог об устойчивом развитии и ответственной деловой практике. Факторы ESG стали важной основой для оценки показателей устойчивого развития компании, поэтому актуальным является всесторонний обзор концепции ESG и оценка ее важности в содействии устойчивому развитию. Одной из проблем внедрения практики ESG в деятельность бизнеса мы выделяем такие как отсутствие стандартизированной отчетности и сложность измерения воздействия инициатив ESG. Интеграция экологических, социальных и управленческих факторов стала основной тенденцией последних лет в области устойчивого инвестирования. От компаний все чаще ожидают, что они будут работать ответственно и устойчиво, а инвесторы требуют прозрачности в своей практике ESG. Эта статья направлена на изучение последних тенденций и разработок в области ESG, изучение того, как компании учитывают аспекты ESG в своих процессах принятия решений и как инвесторы включают факторы ESG в свои инвестиционные стратегии. В статье также будет оцениваться влияние тенденций ESG на корпоративную эффективность и финансовую отдачу, а также обсуждаются проблемы и возможности для компаний и инвесторов в навигации в этой быстро развивающейся области. Цели Организации Объединенных Наций в области устойчивого развития (ЦУР) и экологические, социальные и управленческие критерии (ESG) стали важными критериями для оценки показателей устойчивого развития компаний. Многие компании осознали важность устойчивого развития и начали приводить свою деятельность в соответствие с ЦУР и критериями ESG. Такое согласование может принести много преимуществ, таких как повышение репутации компании, привлечение инвесторов и улучшение ее долгосрочных финансовых показателей. Интегрируя факторы ESG в свои бизнес-стратегии, компании могут снижать риски, выявлять возможности и способствовать устойчивому развитию. Приведение Страница 1 из 49 Страница 1 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Страница 2 из 49 Страница 2 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today бизнес-процессов в соответствие с критериями ЦУР и ESG является важным шагом на пути к продвижению устойчивого развития и ответственной деловой практики. Хоть и существуют проблемы с внедрением этих критериев, компании могут преодолеть их, приняв эффективные стратегии и работая с заинтересованными сторонами для достижения своих целей в области устойчивого развития. Таким образом, компании могут создавать долгосрочную ценность, способствуя более устойчивому будущему. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling бизнес-процессов в соответствие с критериями ЦУР и ESG является важным шагом на пути к продвижению устойчивого развития и ответственной деловой практики. Хоть и существуют проблемы с внедрением этих критериев, компании могут преодолеть их, приняв эффективные стратегии и работая с заинтересованными сторонами для достижения своих целей в области устойчивого развития. Таким образом, компании могут создавать долгосрочную ценность, способствуя более устойчивому будущему. бизнес-процессов в соответствие с критериями ЦУР и ESG является важным шагом на пути к продвижению устойчивого развития и ответственной деловой практики. Хоть и существуют проблемы с внедрением этих критериев, компании могут преодолеть их, приняв эффективные стратегии и работая с заинтересованными сторонами для достижения своих целей в области устойчивого развития. Таким образом, компании могут создавать долгосрочную ценность, способствуя более устойчивому будущему. Мы отмечаем, что концепция ESG привлекла значительное внимание в корпоративном мире, поскольку компании начали осознавать необходимость работать устойчиво и ответственно. ESG охватывает ряд вопросов, таких как изменение климата, права человека и ряд других, а инвесторы все чаще используют критерии ESG для оценки долгосрочной жизнеспособности бизнеса. Чтобы проиллюстрировать влияние политик ESG, мы рассмотрели некоторые тематические исследования из различных отраслей и регионов, таких как устойчивая цепочка поставок Patagonia, обязательство Microsoft по углеродной нейтральности, план устойчивого образа жизни Unilever и др. В этих исследованиях будет показано, как компании интегрировали аспекты ESG в свои бизнес-стратегии, операции и взаимодействие с заинтересованными сторонами и как это привело к положительным результатам, таким как улучшение репутации, увеличение лояльности сотрудников и снижение воздействия на окружающую среду. Приводя практические примеры успешного внедрения ESG, эта статья призвана обозначить направление развития бизнесу, стремящемуся обеспечить устойчивый рост и оказать положительное влияние. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 2 из 49 Термин ESG стал широко признанным термином в инвестиционном и корпоративном мире, но его теоретические основы не всегда ясны. ESG относится к набору нефинансовых факторов, которые используются для оценки долгосрочной устойчивости компании и ее влияния на общество и окружающую среду. Интеграция ESG в процесс принятия инвестиционных решений и корпоративных стратегий привлекла значительное внимание, поскольку рассматривается как способ снижения рисков, создания долгосрочной ценности и вклада в устойчивое развитие. Теоретическая основа ESG опирается на различные точки зрения и теории из различных областей, включая экономику, финансы, менеджмент. Одной из точек зрения является теория заинтересованных сторон, которая предполагает, что компании должны учитывать интересы всех стейкхолдеров, включая клиентов, сотрудников, поставщиков, сообщества и окружающую среду, в дополнение к акционерам, для создания долгосрочной ценности бизнеса. Другая точка зрения — это агентская теория, которая подчеркивает потенциальные конфликты интересов между акционерами и руководством и необходимость эффективных механизмов управления для согласования их интересов. Следует отметить, что интеграция факторов ESG в процесс принятия инвестиционных решений и корпоративных стратегий становится все более важной. Теоретические основы ESG основаны на различных точках зрения и теориях из различных областей, а структуры и стандарты ESG предоставляют набор руководств и принципов для оценки и измерения эффективности ESG. Тем не менее, существуют некоторые проблемы и ограничения, связанные с ESG, и необходимы дальнейшие исследования и разработки, для повышения эффективности и решения возникающих проблем. Включение факторов ESG в процесс принятия корпоративных решений в последние годы становится все более важным, поскольку компании сталкиваются с растущим давлением со стороны инвесторов, клиентов и регулирующих органов, требующих внедрения устойчивых методов и демонстрации своей приверженности вопросам ESG. Несмотря на растущее внимание к ESG, точный характер связи между ESG и корпоративной эффективностью остается Страница 3 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today предметом пристального интереса и споров среди исследователей, политиков и бизнес-лидеров. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today предметом пристального интереса и споров среди исследователей, политиков и бизнес-лидеров. предметом пристального интереса и споров среди исследователей, политиков и бизнес-лидеров. Эта научная статья призвана внести свой вклад в этот продолжающуюся дискуссию, исследуя связь между ESG и корпоративной эффективностью. Страница 2 из 49 На основе обзора существующей литературы по этому вопросу, статья призвана дать представление о том, в какой степени проблемы ESG влияют на финансовые показатели фирмы, инновации и общее конкурентное преимущество. Статья направлена на детальный и основанный на фактических данных анализ взаимосвязи между ESG и корпоративной эффективностью. К сожалению, Россия в 2022 году столкнулась с беспрецедентным санкционным давлением. Использование санкций в качестве инструмента международной дипломатии стало популярным методом оказания давления на страны, которые, как считается, действуют в нарушение международных норм. В 2022 году Россия оказалась в сложной ситуации из-за роста проблемам, связанными с ESG, и продолжающимся введением санкций со стороны США и других стран. Мы исследуем влияние ESG и санкционного давления на экономику и общество и предлагаем понимание потенциальной будущей траектории развития страны, выделяя драйверы продвижения политик ESG. Важность ESG при оценке экономической и социальной стабильности страны возрастает в геометрической прогрессии. В этом контексте Россия, страна с огромными природными ресурсами и значительным геополитическим влиянием, оказывается под растущим давлением со стороны как проблем ESG, так и международных санкций. Эти многогранные факторы создали существенные проблемы для экономического развития России и международных отношений. Благодаря этому всестороннему анализу мы надеемся прояснить сложное взаимодействие между критериями ESG, санкционным давлением, экономической и социальной структурой России. Мы стремимся предоставить важные рекомендации инвесторам, которые стремятся разобраться в нюансах постоянно меняющейся внешне- и внутриполитической ситуации в России. Эта работа стремится предоставить всесторонний и убедительный анализ ESG и его последствий для устойчивого развития. Мы верим, что ESG — это мощная концепция, способная изменить будущее бизнеса и общества. Мы стремимся внести свой вклад в продолжающийся глобальный диалог о том, как сбалансировать экономический рост с устойчивыми и ответственными методами ведения бизнеса, а также способствовать созданию более устойчивого и справедливого мира для всех. Таким образом, цель исследования — анализ ESG-трансформации в условиях санкционного давления. Объектом исследования является концепция устойчивого развития. Предметом исследования является ESG-трансформация российских компаний в условиях санкционного давления. Методы и материалы При написании научной публикации авторами использовались следующие методы: сравнительный, статистический, математический анализы, анализ и обобщение нормативно-правовых актов и документов, научных исследований и статей. Страница 4 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Для достижения данной цели в работе были поставлены следующие задачи: • рассмотреть особенности концепции устойчивого развития; • выявить тенденции развития и распространения концепции устойчивого развития; • проанализировать примеры успешного внедрения ESG компаниями; • обосновать связь между ESG и корпоративной эффективностью; • рассмотреть особенности реализации ESG-принципов в России. • рассмотреть особенности реализации ESG-принципов в России. Исследование основывается на теоретических и методологических положениях, разработанных отечественными авторами, нормативно-правовых документах стратегического планирования, предложениях, раскрывающих перспективы реализации принципов ESG в российских компаниях. Различные аспекты ESG-повестки, а также тенденции ее развития рассмотрены в работах Соболевой Г.В. [1], Андреевой О.В. [2], Баторшиной Г.Д. [3], Захаровой А.А. [4], Цыгаловым Ю.М. [5] и других. Информационной базой исследования послужили научные труды отечественных ученых, Интернет-ресурсы (официальные сайты) и зарубежные источники по тематике работы. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Результаты и обсуждения Международные исследования показали, что компании с высокими показателями ESG имеют более высокую отдачу от инвестиций, более низкую стоимость капитала и более низкий уровень финансового риска. Подходы ESG также могут помочь организациям определить возникающие риски и возможности, что позволит им адаптироваться и внедрять инновации в ответ на меняющиеся рыночные условия. Принципы ESG также помогают компаниям укрепить отношения с заинтересованными сторонами, включая сотрудников, клиентов, поставщиков и инвесторов. Принимая во внимание социальные и экологические последствия своей деятельности, компании могут завоевать доверие и улучшить свою репутацию. Это способствует повышению лояльности клиентов, более высокому уровню сохранения кадрового потенциала и повышению доверия инвесторов. Однако внедрение принципов ESG также может быть сопряжено с трудностями. Одной из основных проблем является доступность и качество данных. Отчетность ESG все еще находится в зачаточном состоянии, и многим компаниям не хватает систем и процессов для эффективного сбора, анализа и представления данных ESG. Еще одной проблемой является отсутствие стандартизации, из-за чего инвесторам и другим заинтересованным сторонам сложно сравнивать и оценивать эффективность ESG в разных организациях. Соблюдение требований также является проблемой, поскольку компании все чаще подвергаются регулированию и контролю со стороны инвесторов и общественных организаций. Растущее значение ESG отражено в том факте, что он стал основным подходом к устойчивому развитию, принятым организациями по всему миру [7]. Концепция ESG претерпела значительные изменения за последние несколько десятилетий. Со временем концепция ESG расширилась, включив в себя более широкий круг вопросов, включая методы управления, этичное деловое поведение и взаимодействие с заинтересованными сторонами. Сегодня ESG — это основной подход к устойчивому развитию, обусловленный повышением осведомленности об экологических и социальных рисках и необходимостью более ответственной деловой практики. ESG — важный инструмент для обеспечения устойчивого развития. Это помогает организациям снизить риски, связанные с экологическими, социальными и управленческими факторами, которые могут повлиять на их долгосрочный рост и финансовые показатели. Практика ESG также может помочь улучшить репутацию организации, привлечь инвесторов и повысить вовлеченность сотрудников, а также позволит организациям определить новые возможности для роста, такие как разработка новых продуктов или услуг, отвечающих потребностям потребителей, которые все больше обеспокоены экологическими и социальными проблемами. Хотя принятие принципов ESG предлагает много потенциальных преимуществ для организаций, существует несколько проблем, связанных с их внедрением. Одной из основных является отсутствие стандартизации показателей ESG и отчетности. Это может затруднить для организаций оценку их эффективности, а для инвесторов — сравнение различных компаний. Еще одной проблемой является потребность в адекватных ресурсах, включая квалифицированный персонал и технологии, для эффективного внедрения практики ESG. Результаты и обсуждения Концепция экологических, социальных и управленческих факторов (ESG) становится все более распространенной в корпоративном мире благодаря своему потенциалу для содействия устойчивому развитию. Концепция ESG претерпела значительные изменения за прошедшие годы, начиная с ее истоков в корпоративной социальной ответственности (КСО) и заканчивая ее нынешним статусом основного подхода к устойчивому развитию. Сегодня ESG является важным фактором для бизнеса, инвесторов и политиков и играет решающую роль в формировании будущего устойчивого развития. Устойчивое развитие стало приоритетом для правительств государств, бизнеса и организаций гражданского общества во всем мире. По своей сути устойчивое развитие стремится сбалансировать экономические, социальные и экологические проблемы, чтобы обеспечить лучшее будущее для всего человечества. Для достижения этой цели растет признание важности экологических, социальных и управленческих подходов в процессах принятия решений. ESG относится к набору факторов, которые предприятия и инвесторы должны учитывать при принятии решений, влияющих на окружающую среду, общество и управление. Эти факторы больше не считаются необязательными, но теперь считаются важными для долгосрочного процветания бизнеса и общественного благополучия. Концепция ESG развивалась с течением времени, начиная с первых дней корпоративной социальной ответственности и заканчивая ее нынешним статусом основного подхода к устойчивому развитию [6]. ESG является важным компонентом устойчивого развития, поскольку позволяет предприятиям и инвесторам интегрировать экологические, социальные и управленческие практики в свои процессы принятия решений. Эта интеграция позволяет организациям создавать долгосрочную ценность, управлять рисками и укреплять доверие стейкхолдеров. Применение подходов ESG в деятельности организаций также могут помочь компаниям улучшить свою репутацию, привлечь новых клиентов и инвесторов, расширить социальное одобрение своей деятельности. Одним из наиболее значительных преимуществ ESG является Страница 5 из 49 Страница 5 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling возможность повышения финансовых показателей. Международные исследования показали, что компании с высокими показателями ESG имеют более высокую отдачу от инвестиций, более низкую стоимость капитала и более низкий уровень финансового риска. Подходы ESG также могут помочь организациям определить возникающие риски и возможности, что позволит им адаптироваться и внедрять инновации в ответ на меняющиеся рыночные условия. возможность повышения финансовых показателей. Результаты и обсуждения Наконец, эффективное внедрение ESG требует поддержки со стороны высшего руководства и взаимодействия с заинтересованными сторонами, что может быть непросто в некоторых организациях [8]. Тенденции ESG — важный инструмент для достижения устойчивого развития. Принимая во внимание экологические, социальные и управленческие факторы, компании могут помочь в решении ряда глобальных проблем, включая изменение климата, социальное неравенство и Страница 6 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 6 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today коррупцию. Цели Организации Объединенных Наций в области устойчивого развития (ЦУР) обеспечивают основу для решения этих проблем, и многие компании в настоящее время используют ESG для поддержки достижения этих целей. Будущее ESG многообещающе, поскольку ряд тенденций и инноваций способствуют его масштабному внедрению. Одной из ключевых тенденций является усиление внимания к изменению климата и роли ESG в поддержке перехода к низкоуглеродной экономике. Это включает в себя разработку новых финансовых продуктов и услуг, связанных с ESG, таких как «зеленые» облигации и устойчивые инвестиционные фонды. Другой тенденцией является растущее внимание к социальным факторам, включая разнообразие и инклюзивность, права человека и трудовые отношения. Это нашло отражение в появлении новых рамок и стандартов отчетности ESG, которые сосредоточены на социальных проблемах. Анализ компаний, применяющих подходы устойчивого развития, показал, что ESG становится важным фактором для инвесторов, поскольку все больше институциональных и розничных инвесторов включают критерии ESG в свои инвестиционные стратегии. Эта тенденция, вероятно, сохранится и в будущем благодаря растущему осознанию важности вопросов устойчивого развития, регуляторного давления и растущей доступности данных и инструментов ESG. Исследование выявило несколько новых тенденций в области ESG, которые, вероятно, определят будущее устойчивого инвестирования. Одной из наиболее значимых тенденций является интеграция искусственного интеллекта (ИИ) и аналитики больших данных в анализ ESG [9]. Эти технологии могут повысить качество и глубину анализа ESG, позволяя инвесторам выявлять риски и возможности, которые могут быть незаметны при использовании традиционных методов. Другой важной тенденцией является развитие импакт-инвестирования, которое направлено на достижение как финансовой отдачи, так и положительных социальных и экологических результатов. Этот подход набирает обороты среди инвесторов, которые хотят изменить ситуацию к лучшему, получая прибыль. Другие тенденции, возникшие в последние годы, включают использование блокчейна и цифровых платформ для обеспечения прозрачности отчетности ESG, растущее значение рисков и возможностей, связанных с климатом, в анализе ESG, а также повышенное внимание к социальным вопросам, таким как разнообразие и инклюзия. Эти тенденции, вероятно, будут иметь серьезные последствия для будущего ESG, определяя подход инвесторов к устойчивому инвестированию и то, как компании отчитываются о своей эффективности ESG. Мы отмечаем некоторый прогресс, достигнутый в последние годы, однако все еще существует ряд проблем, которые необходимо решить, чтобы обеспечить дальнейшее развитие и рост ESG. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 6 из 49 Эти проблемы включают необходимость большей стандартизации и гармонизации отчетности ESG, разработку надежных и непротиворечивых данных ESG и интеграцию анализа ESG в процессы принятия инвестиционных решений. Решение этих проблем потребует сотрудничества между инвесторами, компаниями, регулирующими органами и другими стейкхолдерами. В целом можно сказать, что ESG является быстро развивающейся областью, в которой постоянно появляются новые тенденции и разработки. Будущее ESG, вероятно, будет зависеть от множества факторов, включая технологические инновации, изменение предпочтений инвесторов и изменения в нормативно-правовой базе. Несмотря на наличие проблем, которые необходимо решить, потенциальные преимущества ESG для устойчивого развития значительны, что делает их важной областью для дальнейших исследований и анализа. Интеграция технологий в ESG-отчетность может революционизировать подход компаний и инвесторов к устойчивому развитию. Используя преимущества машинного обучения, искусственного интеллекта и технологии блокчейн, отчетность ESG может стать более точной, прозрачной и эффективной, тем самым создавая новые возможности для Страница 7 из 49 Страница 7 из 49 Страница 7 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today устойчивого развития. Преимущества интеграции технологий в отчеты ESG очевидны. Автоматизируя сбор и анализ данных, предприятия могут снизить риск ошибок и упущений в отчетности по устойчивому развитию, что может повысить точность и своевременность отчетности, позволяя инвесторам и стейкхолдерам принимать более обоснованные решения в отношении устойчивых инвестиций и корпоративного управления. Кроме того, интеграция технологий может повысить прозрачность отчетности ESG, позволяя предприятиям раскрывать данные более последовательным и стандартизированным образом. Это поможет укреплению доверия между предприятиями и их стейкхолдерами, что имеет решающее значение для долгосрочной устойчивости. Однако интеграция технологий также создает проблемы, которые необходимо решать. Стандартизированная отчетность имеет решающее значение для сравнения данных по отраслям и регионам, и необходимо гарантировать качество данных. Кроме того, безопасность данных является критическим фактором, который необходимо учитывать при интеграции технологий в отчеты ESG, особенно с учетом конфиденциального характера некоторых данных об устойчивом развитии. Мы рекомендуем крупному бизнесу и регулирующим органам инвестировать в технологии для улучшения отчетности ESG, особенно в таких областях, как машинное обучение, искусственный интеллект и блокчейн. Крайне важно, чтобы предприятия и государственные органы инвестировали в технологии, что могло бы гарантировать соответствие отчетности в области устойчивого развития самым высоким стандартам точности и прозрачности. Согласование ЦУР и ESG в деятельности компаний Цели устойчивого развития Организации Объединенных Наций были приняты в 2015 году, чтобы обеспечить основу для достижения более устойчивого будущего. 17 ЦУР охватывают ряд вопросов, включая бедность, голод, здоровье, образование, гендерное равенство, чистую воду и санитарию, возобновляемые источники энергии, ответственное потребление и производство, а также меры по борьбе с изменением климата [10]. Для достижения ЦУР потребуются согласованные усилия правительств государств, общественных организаций и бизнеса. Роль компаний в продвижении ЦУР имеет решающее значение. Благодаря своей финансовой и хозяйственной деятельности компании могут оказывать значительное влияние на решение социальных и экологических проблем, решаемых ЦУР. В результате многие организации сделали экологические, социальные и управленческие (ESG) соображения ключевым элементом своей бизнес-стратегии. Практика ESG соответствует нескольким ЦУР и может способствовать их достижению. Например, сокращение выбросов парниковых газов и продвижение возобновляемых источников энергии могут помочь в достижении ЦУР 7 (Доступная и чистая энергия) и ЦУР 13 (Борьба с изменением климата). Точно так же продвижение гендерного равенства и разнообразия на рабочем месте может способствовать достижению ЦУР 5 (Гендерное равенство). Надлежащая практика корпоративного управления также может помочь в достижении ЦУР 16 (Мир, справедливость и сильные институты). Однако внедрение практик ESG не лишено проблем. Отсутствие стандартизации в отчетности ESG, например, затрудняет сравнение и оценку влияния различных практик ESG [11]. Примеры успешного внедрения ESG компаниями Тематические исследования показали, что для успешного внедрения принципов и практик ESG требуются твердая приверженность руководства, взаимодействие с Страница 8 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today заинтересованными сторонами, а также эффективные механизмы управления, отчетность и прозрачность. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling заинтересованными сторонами, а также эффективные механизмы управления, отчетность и прозрачность. Экологические, социальные и управленческие (ESG) принципы и практики становятся все более популярными среди организаций благодаря их способности формировать устойчивое развитие. Однако внедрение практики ESG остается проблемой для многих организаций. В качестве конкретных примеров мы остановились на нескольких компаниях, которые успешно внедрили принципы и практики ESG, с целью предоставления информации, которая может быть использована другими организациями, стремящимися к внедрению ESG принципов. Unilever — многонациональная компания по производству потребительских товаров, которая успешно интегрировала принципы и практики ESG в свою бизнес-модель. В Плане устойчивого развития компании, запущенном в 2010 году, изложены конкретные цели по снижению воздействия ее продуктов на окружающую среду и повышению социального воздействия ее бизнес-операций. Unilever достигла ряда важных результатов в рамках плана, в том числе сократила выбросы парниковых газов на 50 % и достигла статуса нулевых отходов на свалках на более чем 600 своих объектах. Компания также расширила использование методов устойчивого снабжения: теперь 80 % ее сельскохозяйственного сырья поступает из экологически чистых источников. Приверженность Unilever принципам и практикам ESG не только способствовала устойчивому развитию, но и стала ключевым фактором ее финансового успеха. Устойчивые бренды компании, в том числе Ben & Jerry's и Dove, превзошли другие бренды с точки зрения роста и прибыльности. Кроме того, компания получила признание за свои усилия в области ESG и заняла первое место в рейтинге 100 самых устойчивых корпораций мира 2017 года [12]. Patagonia — компания по производству верхней одежды из Калифорнии, активно выступает за экологическую устойчивость и социальную ответственность. Компания предприняла ряд шагов для уменьшения своего воздействия на окружающую среду, например, используя переработанные материалы в своих продуктах и сокращая использование вредных химических веществ в своих производственных процессах. Patagonia также активно продвигает экологическую активность, например, поддерживает массовые кампании по защите общественных земель и сокращению выбросов углерода. В 2012 году компания получила статус B Corp — сертификат, который присуждается компаниям за соблюдение строгих стандартов социальной и экологической деятельности, подотчетности и прозрачности. Датская компания Orsted, занимающаяся возобновляемыми источниками энергии, в последние годы претерпела существенные изменения, сместив акцент с ископаемых видов топлива на возобновляемые источники энергии. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling р ур р ур Russian Journal of Resources, Conservation and Recycling Interface — мировой производитель модульной ковровой плитки, придерживающийся принципов устойчивого развития и ESG. Обязательство компании Mission Zero, принятое в 1994 году, направлено на устранение негативного воздействия на окружающую среду к 2020 году. Interface добился значительного прогресса в достижении этой цели, сократив выбросы парниковых газов на 96 %, потребление воды на 87 % и количество отходов на свалках. на 91 %. Компания также внедрила ряд практик ESG, которые способствовали ее финансовому успеху. Например, внимание Interface к устойчивому развитию позволило ей выделиться среди конкурентов, что привело к увеличению доли рынка и лояльности клиентов. Кроме того, инициативы компании в области устойчивого развития позволили сократить расходы и повысить эффективность, что способствовало увеличению ее прибыли [14]. Tesla — компания, которая занимается экологически безопасным транспортом посредством разработки и производства электромобилей. Компания успешно интегрировала принципы ESG в свою бизнес-модель, сосредоточив внимание на снижении воздействия транспорта на окружающую среду и продвижении методов устойчивой энергетики. Tesla достигла ряда значительных успехов в этой области, включая разработку доступных высокопроизводительных электромобилей и строительство крупнейшего в мире хранилища литий-ионных аккумуляторов. Приверженность компании принципам ESG также стала движущей силой ее финансового успеха. Инновационные продукты и методы устойчивого развития Tesla отличают ее от конкурентов и способствуют ее быстрому росту. Кроме того, внимание компании к устойчивому развитию привлекло инвестиции и положительное внимание средств массовой информации, что еще больше способствовало ее финансовому успеху. Представленные примеры, демонстрируют, что успешное внедрение принципов и практик ESG может способствовать устойчивому развитию и финансовому росту. Организации, приверженные принципам ESG, смогли выделиться среди конкурентов, привлечь клиентов и инвесторов и сократить расходы за счет устойчивых методов управления бизнесом. Принципы и практика ESG становятся все более важными для потребителей, и опросы показывают, что они готовы платить больше за устойчивые продукты и услуги. Однако внедрение практики ESG остается проблемой для многих организаций. Чтобы успешно интегрировать принципы ESG в бизнес-модели, организации должны разработать четкие и измеримые цели, привлечь стейкхолдеров и разработать стратегии для преодоления препятствий на пути реализации. Кроме того, организации должны стремиться к прозрачности и раскрытию информации, предоставляя заинтересованным сторонам исчерпывающую информацию о своей деятельности. Как показывают примеры, организации, приверженные ESG, могут создавать ценность как для своих стейкхолдеров, так и для общества в целом [15]. Выводы и рекомендации Можно сделать несколько ключевых выводов, связанных с ESG и устойчивостью. Во-первых, растет осознание важности ESG для инвесторов и компаний. Инвесторы все чаще учитывают факторы ESG при принятии инвестиционных решений, а компании осознают ценность устойчивых методов для долгосрочного развития. Во-вторых, существует потребность в стандартизированной отчетности ESG, которая позволит инвесторам принимать обоснованные решения, а компаниям — улучшать свои показатели устойчивого развития. В-третьих, есть достоверные данные компаний, о том, что ESG может оказать положительное влияние на эффективность бизнеса и репутацию, а также на социальные и экологические последствия. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 50ECOR422 Компания поставила перед собой амбициозную цель стать углеродно-нейтральной к 2025 году и добилась значительного прогресса в достижении этой цели, инвестируя значительные средства в ветряную энергию и постепенно отказываясь от угольных электростанций. Помимо внимания к экологической устойчивости, Orsted также получила признание за свою приверженность социальной ответственности, например, за усилия по поощрению разнообразия и интеграции своего персонала. Natura & Co — бразильская компания, владеющая несколькими ведущими брендами средств личной гигиены, такими как Natura, The Body Shop и Aesop. Компания твердо привержена принципам устойчивого развития и поставила перед собой амбициозные цели по снижению воздействия на окружающую среду и повышению социальной ответственности. Например, компания Natura & Co взяла на себя обязательство стать углеродно-нейтральным к 2030 году и поставила перед собой цель сократить потребление воды на 50 % к 2030 году, а также его приверженность защите животных [13]. Страница 9 из 49 Страница 9 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Нами представлено несколько тематических исследований, чтобы проиллюстрировать, как организации успешно внедрили принципы и практики ESG. Эти тематические исследования охватывают ряд секторов экономики. Ключевые темы этих тематических исследований включают важность взаимодействия с заинтересованными сторонами, необходимость четкой стратегии ESG и преимущества интеграции ESG в бизнес-операции. В исследованиях по ESG и устойчивому развитию выделяются несколько важнейших тенденций и проблем, с которыми сегодня сталкиваются организации. Одной из наиболее важных тенденций является необходимость более комплексного подхода к ESG и устойчивому развитию. Многие компании по-прежнему рассматривают ESG и устойчивое развитие отдельно от других видов деятельности, а не интегрируют их в свои общие бизнес-стратегии. Это может привести к фрагментарному подходу к устойчивому развитию, который не принесет ожидаемых выгод. Необходимы более всесторонние исследования интеграции ESG и устойчивого развития в корпоративные процессы принятия решений, чтобы понять, как лучше всего добиться этой интеграции. Наше исследование также определяет роль технологий в отчетности ESG как важнейшую область для будущих исследований. Многие компании изо всех сил пытаются эффективно собирать, анализировать и сообщать данные ESG, и существует потребность в инновационных подходах для повышения точности и надежности данных ESG [16]. Использование новых технологий, таких как блокчейн, искусственный интеллект и машинное обучение, может изменить отчетность ESG и повысить прозрачность и ответственность организаций в этой области. Еще одним ключевым выводом является важность участия стейкхолдеров в разработке стратегий ESG. Привлечение заинтересованных сторон, включая сотрудников, клиентов и местные сообщества, может помочь заручиться поддержкой инициатив ESG и повысить доверие к показателям устойчивого развития организации. Необходимо провести более всесторонние исследования лучших практик взаимодействия со стейкхолдерами, чтобы понять, как лучше всего их можно вовлечь в разработку и реализацию стратегий ESG. Мы подготовили некоторые рекомендации для будущих исследований в области ESG и устойчивого развития. Во-первых, необходимо больше эмпирических исследований для оценки влияния ESG на эффективность и репутацию компании. Во-вторых, необходимо больше исследований роли ESG в конкретных секторах, таких как энергетика и транспорт. В-третьих, необходимо привлечь более широкий круг заинтересованных сторон, таких как клиенты и сотрудники, к процессу принятия решений в области ESG. В-четвертых, необходимо изучить влияние технологий на отчетность ESG и практику устойчивого развития. Наконец, необходимо провести дополнительные исследования влияния ESG на достижение Целей устойчивого развития Организации Объединенных Наций (ЦУР) [17]. Концепция ESG становится все более важным подходом к продвижению устойчивого развития в корпоративном мире. Страница 10 из 49 Страница 10 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today 50 CO Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ESG стала популярной основой для компаний, чтобы измерять и сообщать о своих показателях устойчивого развития. Основополагающие принципы ESG базированы на концепции корпоративной социальной ответственности (КСО), которая относится к ответственности компаний за рассмотрение воздействия своей деятельности на общество и окружающую среду. Концепция КСО развивалась с течением времени, и ESG стала последней итерацией, отражающей более целостный и интегрированный подход к устойчивому развитию. Связь между теорией заинтересованных сторон и ESG можно понять в контексте теоретической основы ESG [18]. ESG отражает убеждение в том, что компании обязаны учитывать интересы всех стейкхолдеров, а не только акционеров. Это согласуется с теорией заинтересованных сторон, которая утверждает, что компании несут социальную ответственность за учет интересов всех заинтересованных сторон, включая сотрудников, клиентов, поставщиков и общество. Существует несколько способов, с помощью которых включение теории заинтересованных сторон в практику ESG может повысить устойчивость компаний. Во-первых, включение теории заинтересованных сторон в практику ESG может помочь компаниям определить и расставить приоритеты интересов всех заинтересованных сторон. Это может привести к более эффективному взаимодействию с заинтересованными сторонами, что, в свою очередь, может повысить устойчивость компаний. Во-вторых, включение теории заинтересованных сторон в практику ESG может помочь компаниям интегрировать соображения устойчивого развития в свои процессы принятия решений. Принимая во внимание интересы всех заинтересованных сторон, компании могут принимать более обоснованные решения, которые уравновешивают потребности всех заинтересованных сторон и с большей вероятностью будут устойчивыми в долгосрочной перспективе. В-третьих, включение теории заинтересованных сторон в практику ESG может помочь компаниям завоевать доверие со стороны заинтересованных сторон. Демонстрируя приверженность учету интересов всех заинтересованных сторон, компании могут построить более прочные отношения с ними, что, в свою очередь, может повысить устойчивость компаний [19]. Есть несколько направлений для будущих исследований связи между теорией заинтересованных сторон и ESG. Во-первых, будущие исследования могут быть сосредоточены на влиянии включения теории заинтересованных сторон в практику ESG на финансовые результаты компаний. Это может помочь создать более четкое экономическое обоснование для включения теории заинтересованных сторон в практику ESG. Во-вторых, будущие исследования могут изучить взаимосвязь между теорией заинтересованных сторон и различными аспектами ESG, включая экологические, социальные и управленческие факторы. Это может помочь определить, какие аспекты ESG наиболее тесно связаны с теорией заинтересованных сторон, а какие менее. В-третьих, будущие исследования могут изучить влияние различных групп заинтересованных сторон на связь между теорией заинтересованных сторон и ESG. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Это может помочь определить, какие группы заинтересованных сторон наиболее важны для формирования связи между теорией заинтересованных сторон и ESG, а какие менее [20]. Теория агентства и практика ESG имеют некоторые общие элементы, особенно в их ориентации на заинтересованные стороны. Теория агентства подчеркивает роль менеджеров и директоров в максимизации благосостояния акционеров, в то время как практика ESG фокусируется на более широких социальных и экологических последствиях деятельности компании. Теория агентства предполагает возможный конфликт интересов между менеджерами и акционерами, что приведет к краткосрочному принятию решений и невниманию к проблемам ESG. Этот конфликт интересов можно смягчить за счет использования вознаграждения, основанного на результатах деятельности, и назначения независимых директоров, обладающих соответствующим опытом в области ESG. Таким Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Теоретическая основа ESG Теоретическая основа ESG имеет связи с несколькими различными дисциплинами, включая экономику, менеджмент, социологию, политологию и этику. Основные теории, лежащие в основе ESG, включают теорию заинтересованных сторон, теорию агентства, институциональную теорию, теорию социального капитала и теорию легитимности. Рассмотрим их подробнее. Существует тесная связь между теорией заинтересованных сторон и ESG. Включение этой теории в практику ESG может повысить устойчивость компаний и создать долгосрочную ценность для всех заинтересованных сторон. Страница 11 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Страница 12 из 49 Страница 12 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today образом, существует потребность более тесного взаимодействия между компаниями и их заинтересованными сторонами, особенно в области воздействия на окружающую среду и социальную сферу. Теория агентства может создать ощущение дистанции между компаниями и их заинтересованными сторонами, которое можно преодолеть с помощью таких инициатив, как программы взаимодействия с заинтересованными сторонами и программами инвестиций в сообщества. Теория агентства предполагает, что механизмы прозрачности и подотчетности необходимы для решения проблемы принципала-агента. Это означает, что практика ESG должна быть прозрачной и достоверной перед заинтересованными сторонами, включая инвесторов, клиентов и сотрудников. Эффективные механизмы отчетности, такие как отчеты об устойчивом развитии и рейтинги ESG, могут помочь повысить прозрачность и достоверность практик исполнения ESG. Будущие исследования этого теоретического вопроса могут быть сосредоточены на разработке более эффективных методов измерения влияния практики ESG на эффективность компании, а также на изучении роли практики ESG в смягчении агентских проблем в компаниях [21]. образом, существует потребность более тесного взаимодействия между компаниями и их заинтересованными сторонами, особенно в области воздействия на окружающую среду и социальную сферу. Теория агентства может создать ощущение дистанции между компаниями и их заинтересованными сторонами, которое можно преодолеть с помощью таких инициатив, как программы взаимодействия с заинтересованными сторонами и программами инвестиций в сообщества. Теория агентства предполагает, что механизмы прозрачности и подотчетности необходимы для решения проблемы принципала-агента. Это означает, что практика ESG должна быть прозрачной и достоверной перед заинтересованными сторонами, включая инвесторов, клиентов и сотрудников. Эффективные механизмы отчетности, такие как отчеты об устойчивом развитии и рейтинги ESG, могут помочь повысить прозрачность и достоверность практик исполнения ESG. Будущие исследования этого теоретического вопроса могут быть сосредоточены на разработке более эффективных методов измерения влияния практики ESG на эффективность компании, а также на изучении роли практики ESG в смягчении агентских проблем в компаниях [21]. Институциональная теория обеспечивает теоретическую основу для понимания внедрения ESG-практик организациями. Институциональное давление, включая регулятивное и нормативное давление, играет важную роль в стимулировании внедрения практик ESG. Стейкхолдеры могут более благосклонно относиться к организациям, применяющим практику ESG, что может привести к положительным репутационным (имиджевым) и финансовым результатам. Однако внедрение практик ESG может оказаться сложной задачей, поскольку требует изменений в организационной культуре и поведении. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 12 из 49 Анализируя механизмы принятия и распространения ESG, автор подчеркивает важность институциональных факторов в формировании практики ESG в организациях [23]. влияние практики ESG на организационную эффективность и финансовые результаты. Наконец, будущие исследования должны изучить эффективность различных институциональных механизмов, таких как кодексы поведения, сертификаты соответствия производств и отраслевые правила, в стимулировании внедрения ESG-практик организациями. Анализируя механизмы принятия и распространения ESG, автор подчеркивает важность институциональных факторов в формировании практики ESG в организациях [23]. ESG — это набор экологических, социальных и управленческих показателей, которые все чаще используются для оценки устойчивости бизнеса и инвестиций. Теория социального капитала, с другой стороны, предполагает, что социальные отношения и социальные связи могут оказывать положительное влияние на широкий спектр результатов, включая экономическое развитие, участие в политической жизни и благосостояние сообщества. Исследования по теории социального капитала предполагает наличие нескольких аспектов социального капитала, включая структурный, когнитивный и реляционный. Структурный социальный капитал относится к формальным отношениям и общественным связям, существующим в сообществе или обществе, таким как членство в организациях или участие в общественной жизни. Когнитивный социальный капитал, с другой стороны, относится к общим ценностям и убеждениям, которые лежат в основе социальных отношений, в то время как реляционный социальный капитал относится к качеству самих отношений [23]. В последние годы растет интерес к исследованиям взаимодействия теории социального капитала и ESG. Некоторые ученые утверждают, что социальный капитал может быть ключевым фактором устойчивости, поскольку он может способствовать сотрудничеству, обмену информацией и коллективным действиям. Например, социальный капитал может позволить сообществам объединиться для решения экологических проблем, таких как загрязнение или изменение климата. Точно так же социальный капитал может способствовать этичному поведению и ответственному принятию решений, которые играют центральную роль в управленческом аспекте ESG. Однако существуют также проблемы, связанные со связью между теорией социального капитала и ESG. Одна из проблем заключается в том, что сосредоточение внимания на социальных отношениях и сетях может отвлечь внимание от более ощутимых результатов, связанных с ESG. Существует и риск того, что использование социального капитала в качестве показателя устойчивости может привести к фрагментарному вниманию к интересам конкретных групп или сообществ, а не к более широкой заботе о социальной и экологической справедливости [24]. Теория социального капитала может предоставить актуальную основу для понимания связи между ESG и эффективностью организации. Она может помочь объяснить, как методы ESG могут улучшить социальные отношения, укрепить доверие и создать общие ценности внутри организации, что, в свою очередь, может привести к высоким финансовым и нефинансовым результатам. Страница 12 из 49 Институциональная теория обеспечивает полезную основу для понимания того, как организации внедряют практики ESG. Однако внедрение практик ESG также может оказаться сложной задачей. Институциональное давление, включая регулятивное, нормативное и когнитивное давление, играет решающую роль в стимулировании внедрения ESG-практик организациями. В частности, регуляторное давление в форме законов и правил, нормативное давление в форме социальных норм и ценностей и когнитивное давление в форме общих убеждений и предположений оказались наиболее влиятельными в стимулировании внедрения практик ESG в компаниях. Можно сделать вывод о важности роли институциональной среды в формировании внедрения практик ESG. Институциональный контекст, такой как политическая и экономическая системы, культурные ценности и социальные нормы, может либо способствовать, либо препятствовать внедрению ESG-практик организациями [22]. Наше исследование имеет несколько практических выводов для организаций, политиков и инвесторов. Например, организации могут использовать институциональную теорию для определения институционального давления, которое стимулирует внедрение методов ESG, и разрабатывать стратегии для их устранения. Государственные органы могут использовать результаты этого исследования для разработки правил и политик, способствующих внедрению практики ESG. Инвесторы могут использовать институциональную теорию для выявления организаций, которые с большей вероятностью примут практику ESG и интегрируют факторы ESG в свои инвестиционные решения. Важно определить несколько областей для будущих исследований, в том числе контекстуальные факторы, влияющие на внедрение практик ESG, влияние практик ESG на организационные показатели и финансовые результаты, а также эффективность различных институциональных механизмов, стимулирующих внедрение практик ESG компаниями. Эти области исследований будут способствовать более полному пониманию взаимосвязи между институциональной теорией и ESG, послужат руководством для организаций, политиков и инвесторов, стремящихся продвигать устойчивые и ответственные методы ведения бизнеса. Автором определены несколько рекомендаций для будущих исследований. Во-первых, будущие исследования должны быть сосредоточены на контекстуальных факторах, влияющих на внедрение ESG-практик организациями. Во-вторых, будущие исследования должны изучить Страница 13 из 49 Страница 13 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today влияние практики ESG на организационную эффективность и финансовые результаты. Наконец, будущие исследования должны изучить эффективность различных институциональных механизмов, таких как кодексы поведения, сертификаты соответствия производств и отраслевые правила, в стимулировании внедрения ESG-практик организациями. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 12 из 49 Также теорию социального капитала можно использовать для выявления ключевых стейкхолдеров, которые имеют решающее значение для развития инициатив ESG, и для понимания того, как они могут быть эффективно вовлечены в процесс развития инициатив ESG. Существует несколько областей, в которых необходимы дальнейшие исследования. Во-первых, автор указывает на необходимость дополнительных эмпирических исследований, в которых изучается взаимосвязь между ESG и социальным капиталом в различных организационных контекстах. Во-вторых, будущие исследования должны изучить, как теория социального капитала может быть применена для разработки более эффективных стратегий ESG и для измерения влияния практики ESG на социальные и экологические результаты. В-третьих, существует потребность в дополнительных исследованиях того, как организации могут эффективно сообщать о своей практике ESG своим стейкхолдерам. Теория социального Страница 14 из 49 Страница 14 из 49 50 CO Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today капитала обладает значительным потенциалом для содействия развитию более устойчивых и справедливых экономических систем [25]. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling капитала обладает значительным потенциалом для содействия развитию более устойчивых и справедливых экономических систем [25]. Теория легитимности является одним из важнейших теоретических направлений в области организационных исследований. Это относится к тому, как организации стремятся получить и сохранить поддержку и одобрение своих заинтересованных сторон, включая инвесторов, клиентов, сотрудников и регулирующие органы. Теория легитимности предполагает, что организации будут более успешными, если их стейкхолдеры будут считать их законными, и что у них будет больше шансов выжить и развиться, если они смогут поддерживать эту легитимность в течение долгого времени. В контексте ESG теория легитимности особенно актуальна, поскольку она обеспечивает основу для понимания того, как организации могут получить и сохранить легитимность в отношении своего экологического и социального воздействия, а также своей практики управления [7]. В ряде исследований изучалась связь между теорией легитимности и ESG. Например, некоторые исследователи предположили, что организации, которые считаются более законными в глазах своих заинтересованных сторон, с большей вероятностью примут методы, связанные с ESG, и добьются успеха в их реализации. В других исследованиях изучались способы, с помощью которых организации могут использовать свою легитимность для формирования дискурса вокруг ESG и влияния на восприятие своих заинтересованных сторон. Исследование теории легитимности и ее связи с ESG дает ценную информацию о роли ESG в повышении корпоративной легитимности. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 12 из 49 Понятие легитимности относится к восприятию действий и решений компании как уместных, желательных и правильных в контексте социальных и культурных норм и ценностей общества, в котором она работает. Теория легитимности использовалась для объяснения того, почему компании применяют практики ESG, факторов, влияющих на восприятие легитимности заинтересованными сторонами, и последствий утраты легитимности. Практика ESG может повысить легитимность компании за счет улучшения ее репутации и доверия среди заинтересованных сторон, снижения социальных и экологических рисков и содействия достижению целей устойчивого развития. Практика ESG также помогает компаниям привести свою деятельность в соответствие с ожиданиями общества, что может укрепить их социальный контракт и повысить их долгосрочный успех. Стоит отметить важность прозрачности и раскрытия информации для повышения легитимности. Компании, которые достоверно сообщают о своей практике ESG и раскрывают информацию о своем влиянии на общество и окружающую среду, с большей вероятностью будут восприниматься стейкхолдерами как законные. Напротив, компании, которым не хватает прозрачности или которые занимаются гринвошингом, скорее всего, столкнутся с репутационным ущербом и потерей легитимности. Теорию легитимности можно использовать для руководства будущими исследованиями ESG. В частности, теорию можно использовать для выявления факторов, влияющих на восприятие заинтересованными сторонами легитимности, и механизмов, с помощью которых практика ESG может повысить или подорвать легитимность. Это поможет компаниям и политикам разработать эффективные стратегии для продвижения внедрения практик ESG и повышения их легитимности [26]. Наше исследование продемонстрировало актуальность и важность теории легитимности для понимания связи между ESG и корпоративной эффективностью. Практика ESG может повысить легитимность компании и способствовать ее долгосрочному развитию. Однако достижение легитимности требует прозрачности, раскрытия информации и приверженности социальной и экологической устойчивости. Теория легитимности обеспечивает полезную основу для понимания факторов, влияющих на восприятие легитимности заинтересованными сторонами, и роли ESG в укреплении корпоративной легитимности. Будущие исследования Страница 15 из 49 Выводы и рекомендации Автором были изучены теоретические основы ESG, выявлены и рассмотрены различные теоретические точки зрения, лежащие в основе концепции ESG. Исследование показало, что ESG охватывает экологические, социальные и управленческие факторы, которые имеют решающее значение для долгосрочной устойчивости бизнеса и общества в целом. Сделан акцент на различных теоретических подходах, включая теорию заинтересованных сторон, агентскую теорию, институциональную теорию, теорию социального капитала и теорию легитимности, а также проведен анализ их взаимосвязи с ESG. Результаты показывают, что теория заинтересованных сторон обеспечивает основу для понимания важности ESG в построении прочных и устойчивых отношений с заинтересованными сторонами, включая клиентов, сотрудников, поставщиков и более широкое сообщество. Агентская теория выявляет потенциальные конфликты интересов между акционерами и руководством и утверждает, что ESG можно использовать для согласования их интересов и содействия созданию долгосрочной стоимости. Институциональная теория указывает на роль норм, ценностей и институционального давления в формировании организационного поведения и утверждает, что ESG может повысить легитимность организации и повысить ее эффективность. Теория социального капитала подчеркивает роль социальных отношений в создании ценности и предполагает, что ESG может способствовать созданию и поддержанию социального капитала. Теория легитимности говорит о важности социального и экологического воздействия организаций и утверждает, что ESG может повысить легитимность организации и укрепить доверие заинтересованных сторон. Автор подчеркивает необходимость более комплексного и междисциплинарного подхода к ESG, который включает в себя различные теоретические точки зрения для более полного понимания концепции. Результаты также показывают, что ESG становится все более важным для инвесторов, регулирующих органов и общества в целом, и существует необходимость в дополнительных исследованиях для более глубокого понимания этой концепции. Отметим, что теоретическая основа ESG имеет решающее значение для понимания факторов, лежащих в основе концепции, и их значимости для долгосрочной устойчивости организаций. Страница 15 из 49 Страница 15 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling должны основываться на этих выводах для разработки более эффективных стратегий продвижения внедрения практики ESG и повышения корпоративной устойчивости. Теория легитимности — фундаментальная теоретическая точка зрения, имеющая важные последствия для изучения ESG [27]. Предоставляя основу для понимания того, как организации могут получить и сохранить легитимность в отношении своей деятельности, связанной с ESG, теория легитимности предлагает ценную информацию о факторах, которые стимулируют принятие и внедрение практик ESG. Таким образом, теория легитимности является важным инструментом для всех, кто желает понять роль ESG в современном бизнесе и финансах. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Учитывая важность факторов ESG как для компаний, так и для инвесторов, понимание взаимосвязи между ESG и корпоративной эффективностью стало важной областью исследований. Еще одной примечательной тенденцией в исследованиях ESG является растущее признание взаимосвязи между экологическими, социальными и управленческими факторами. Методы управления компании могут играть решающую роль в ее способности эффективно управлять экологическими и социальными рисками, в то время как экологические и социальные факторы также могут влиять на методы управления посредством взаимодействия с заинтересованными сторонами и общественным мнением [28]. На самом деле, исследование, проведенное Гарвардской школой бизнеса, показало, что компании с твердой приверженностью принципам устойчивого развития и социальной ответственности в долгосрочной перспективе превосходят своих конкурентов как с точки зрения фондового рынка, так и с точки зрения управления бизнесом. Фирмы с высокими рейтингами ESG имеют более низкую стоимость капитала, что указывает на то, что инвесторы рассматривают показатели ESG как положительный сигнал долгосрочной финансовой устойчивости. Однако другие исследования показали, что взаимосвязь между ESG и корпоративной эффективностью более тонкая и зависит от конкретных рассматриваемых факторов ESG. Например, исследование, опубликованное в Global Finance Journal, показало, что экологические показатели положительно влияют на финансовые показатели, в то время как социальные показатели не имеют существенной связи (Jo Hoje и др., 2015). И наоборот, исследование Гамбургского университета показало, что корпоративное управление оказывает более сильное влияние на финансовые показатели, чем экологические или социальные показатели (Sila Ismail, Cek Kemal, 2017) [29]. Имеются данные, свидетельствующие о том, что компании, которые отдают приоритет факторам ESG, с большей вероятностью будут иметь лучшие долгосрочные финансовые показатели, более низкие профили рисков и более высокую удовлетворенность сотрудников и клиентов. Например, компании с жесткой экологической политикой с большей вероятностью смогут избежать высоких штрафов и судебных исков, связанных с загрязнением окружающей среды и другими экологическими проблемами, а также будут иметь более низкие затраты на энергию благодаря энергоэффективным методам ведения бизнеса. Точно так же компании с сильной социальной политикой, поощряющей разнообразие и инклюзивность, как правило, имеют более вовлеченных сотрудников и оказываются более привлекательными для потенциальных работников. Такие компании также с большей вероятностью избегают репутационного ущерба, связанного с дискриминацией и домогательствами. Наконец, компании с сильной политикой корпоративного управления, как правило, имеют лучшие методы управления рисками, более прозрачны и честны к стейкхолдерам, что может привести к повышению доверия и созданию долгосрочной ценности [30]. Несмотря на растущее число исследований, предлагающих преимущества интеграции ESG, все еще есть некоторые скептики, которые сомневаются в действенности этих факторов для корпоративной эффективности. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Тем не менее, тенденция к отчетности и интеграции ESG только усиливается, поскольку как компании, так и инвесторы признают важность устойчивого развития и управления нефинансовыми рисками. Остановимся на каждом факторе детально. Тщательная оценка корпоративной деятельности зависит от множества факторов, охватывающих экономическую, социальную и экологическую области. Исторически сложилось так, что экономические факторы, особенно рост выручки и прибыльности, оставались в центре внимания при оценке корпоративной эффективности. Однако в последнее время значение социальных и экологических факторов приобрело все большее значение, и они заняли незаменимую позицию в измерении корпоративной эффективности. В частности, Страница 17 из 49 Связь между ESG и корпоративной эффективностью За последние несколько лет было проведено значительное количество исследований для изучения связи между факторами ESG и корпоративной эффективностью. Многочисленные исследования изучали влияние показателей ESG на различные финансовые показатели, но результаты оказались неоднозначными. Некоторые исследования обнаружили положительную корреляцию между эффективностью ESG и финансовыми показателями, в то время как другие не обнаружили существенной связи или даже отрицательной связи. Страница 16 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Экологические факторы Влияние экологических факторов на корпоративное управление становится более очевидным при рассмотрении экологических проблем. Корпоративный сектор вносит большой вклад в решение экологических проблем, включая изменение климата, загрязнение окружающей среды и истощение природных ресурсов. Одним из важных экологических факторов, влияющих на корпоративное управление, является государственное регулирование. Правительства могут вводить экологические нормы, которые требуют от корпораций принятия экологически безопасных методов ведения бизнеса. Например, они могут потребовать от корпораций сокращения выбросов парниковых газов или использования возобновляемых источников энергии. В таких случаях практика корпоративного управления должна быть адаптирована для соответствия этим правилам. Еще одним экологическим фактором, влияющим на корпоративное управление, является потребительский спрос на экологически чистые товары и услуги. Потребители все больше узнают об экологических проблемах и с большей вероятностью будут поддерживать компании, демонстрирующие ответственное отношение к окружающей среде [32]. Эта тенденция побуждает корпорации внедрять устойчивые методы управления, направленные на решение экологических проблем. Доступность природных ресурсов также является важным экологическим фактором. Компании, которые полагаются на природные ресурсы в своей деятельности, с большей вероятностью столкнутся с проблемами, когда эти ресурсы станут дефицитными и дорогими. Чтобы решить эту проблему, компаниям может потребоваться внедрить методы устойчивого управления, которые уменьшат их зависимость от природных ресурсов. Наконец, экологические риски, такие как стихийные бедствия, могут повлиять на корпоративное управление. Компании должны иметь эффективные методы управления рисками, чтобы смягчить влияние этих рисков на свою деятельность. Следует отметить, что факторы внешней среды оказывают значительное влияние на корпоративное управление. Корпорациям необходимо внедрить устойчивые и ответственные методы управления, учитывающие экологические проблемы. Правительства государств, потребители и другие заинтересованные стороны должны сыграть свою роль в поощрении корпораций к внедрению этих методов. Поступая таким образом, мы можем создать более устойчивое будущее для всех. Рассмотрим пример компании Walmart, которая успешно интегрировала экологические подходы в свою деятельность и добилась положительных результатов с точки зрения финансовых показателей. Walmart — компания, которая в последние годы добилась значительных успехов в снижении своего воздействия на окружающую среду. Помимо финансовой выгоды, экологические инициативы Walmart также имели положительные социальные последствия. Например, стремление компании сократить количество отходов привело к разработке инновационных упаковочных решений, таких как многоразовые пакеты и биоразлагаемая упаковка. Эти инициативы помогли сократить количество отходов, отправляемых на свалки, и оказали положительное влияние на местные сообщества. Кроме того, экологические инициативы Walmart помогли улучшить репутацию компании и имидж бренда. Продвигая устойчивые методы, компания смогла выделиться среди конкурентов и позиционировать себя как ответственный корпоративный бизнес. Это помогло завоевать доверие клиентов и стейкхолдеров, а также укрепить бренд Walmart в долгосрочной перспективе. Страница 17 из 49 Страница 17 из 49 50ECOR422 50 CO Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today факторы окружающей среды привлекли значительное внимание из-за их многовекторного воздействия на бизнес-операции, финансовые показатели и долгосрочную устойчивость организаций [31]. факторы окружающей среды привлекли значительное внимание из-за их многовекторного воздействия на бизнес-операции, финансовые показатели и долгосрочную устойчивость организаций [31]. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Экологические факторы В целом опыт Walmart демонстрирует важность интеграции экологических подходов в бизнес-операции. Таким образом, компании могут получить финансовую выгоду, Страница 18 из 49 Страница 18 из 49 Страница 18 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today 2022, Том 9, № 4 2022, Vol 9, No 4 Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today улучшить свою репутацию и имидж бренда, а также оказать положительное влияние на общество и окружающую среду. По мере того, как мир становится все более экологически сознательным, предприятия, которые не внедряют устойчивые методы управления, могут остаться позади. Более того, уменьшая воздействие на окружающую среду, компании также могут снизить свои операционные расходы, что может привести к увеличению прибыльности. Например, Walmart внедрил устойчивые методы в своей цепочке поставок, что привело к снижению транспортных расходов и сокращению отходов. Это позволило компании сэкономить деньги и повысить эффективность и способствовало ее высокому финансовому уровню. Помимо финансовых выгод, инициативы Walmart в области устойчивого развития также оказали положительное влияние на окружающую среду и общество. Компания сократила выбросы парниковых газов, уменьшила количество отходов и инвестировала в возобновляемые источники энергии. Принимая эти меры, Walmart помогает создать более устойчивое будущее для всего человечества. Опыт Walmart в интеграции экологических подходов в свою деятельность демонстрирует потенциальные преимущества для компаний, приверженных принципам устойчивого развития. Снижая свое воздействие на окружающую среду, компании могут улучшить свои финансовые показатели, повысить лояльность клиентов, привлечь долгосрочных инвесторов и внести свой вклад в более устойчивое будущее. Поскольку мир по- прежнему уделяет первостепенное внимание экологическим проблемам, компании, отдающие предпочтение устойчивому развитию, вероятно, будут иметь конкурентное преимущество в долгосрочной перспективе [19]. Тенденции бизнеса в использовании принципов ESG в своей деятельности, в том числе экологических факторов, находит подтверждение в различных отчетах международных организаций. Например, согласно отчету Института управления и отчетности (IMA), доля компаний из списка S&P 500, публикующих отчеты об устойчивом развитии, увеличилась с 20 % в 2011 году до 90 % в 2020 году. Исследование, проведенное Глобальным договором Организации Объединенных Наций, показало, что 96 % руководителей, опрошенных в 2020 году, считают, что устойчивость важна для будущего успеха их бизнеса. Глобальная инициатива по отчетности (GRI) сообщила, что по состоянию на 2020 год более 80 % из 250 крупнейших компаний мира отчитываются о своих показателях устойчивого развития, и 71 % этих компаний отчитываются с использованием стандартов отчетности GRI в области устойчивого развития. Страница 18 из 49 CDP (бывший Carbon Disclosure Project) сообщило, что по состоянию на 2020 год более 9600 компаний по всему миру раскрывают свои экологические данные через платформу CDP, что на 14 % больше, чем в предыдущем году. Данные свидетельствуют об устойчивом росте внедрения компаниями по всему миру политик ESG [33]. Социальные факторы Социальные факторы, влияющие на корпоративную деятельность, разнообразны и могут включать изменения в социальных ценностях, демографии, человеческом капитале, трудовых отношениях, отношениях со стейкхолдерами и поведении потребителей. Способность компании выявлять, понимать и управлять этими социальными факторами имеет решающее значение для ее процветания. Социальные факторы, влияющие на корпоративную деятельность. Изменения в социальных ценностях. Социальные ценности постоянно меняются и могут существенно повлиять на развитие бизнеса. Компании, которые не могут адаптироваться к изменяющимся общественным ценностям, могут столкнуться с негативными последствиями, такими как снижение продаж и ущерб репутации. Например, компании, практикующие неэтичные методы, такие как трудовая эксплуатация или дискриминация, могут столкнуться с Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Человеческий капитал. Процветание компании тесно связано с навыками и знаниями ее сотрудников. Такие факторы, как уровень образования, профессиональные навыки и обучение в процессе работы, могут повлиять на способность компании конкурировать на рынке. Компании, которые инвестируют в развитие своих сотрудников и предоставляют возможности для продвижения по службе, с большей вероятностью сохранят квалифицированных работников и привлекут новых [34]. Трудовые отношения. Страница 19 из 49 Страница 19 из 49 Страница 19 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today негативной рекламой, бойкотами и регулятивными мерами, которые могут негативно повлиять на их деятельность. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling негативной рекламой, бойкотами и регулятивными мерами, которые могут негативно повлиять на их деятельность. негативной рекламой, бойкотами и регулятивными мерами, которые могут негативно повлиять на их деятельность. Демография. Изменения в демографии могут повлиять на стратегии продаж и маркетинга компании. Например, если основная клиентская база компании стареет, ей может потребоваться адаптировать свой маркетинг, чтобы привлечь более молодую аудиторию для поддержания роста продаж. Кроме того, компаниям может потребоваться скорректировать свои предложения продуктов, чтобы отразить меняющуюся демографию, например, предлагая более экологически чистые продукты, чтобы привлечь внимание потребителей, обеспокоенных изменением климата. Человеческий капитал. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Идея интеграции социальных факторов в бизнес-операции широко пропагандировалась как эффективный способ не только создать долгосрочную ценность, но и улучшить финансовые показатели. Яркой иллюстрацией этого являются компании Danone и Salesforce. Компания Danone, мировой лидер в производстве продуктов питания и напитков, всегда выступала за социальную ответственность, ставя перед собой высокие цели по сокращению воздействия на окружающую среду, пропаганде здорового питания и оказанию поддержки местным сообществам. Эти совместные усилия позволили компании укрепить имидж бренда и повысить лояльность клиентов. Среди его успешных инициатив — инициатива «Одна планета». Программа One Health, направленная на содействие устойчивому развитию сельского хозяйства при одновременном снижении углеродного следа компании, оказалась особенно эффективной в повышении экологических показателей и репутации Danone [7]. Salesforce, компания по разработке облачного программного обеспечения, также включила социальную ответственность в качестве жизненно важного аспекта своей бизнес- модели. Она реализовала несколько социальных инициатив, в том числе твердую приверженность гендерному равенству, поддержку малого бизнеса и направление на устойчивость. Усилия Salesforce не только помогли ей достичь своих социальных целей, но и в значительной степени способствовали ее финансовому успеху. В 2020 году выручка Salesforce составила 17,1 миллиарда долларов, что на 29 % больше, чем годом ранее [36]. Примеры Danone и Salesforce служат свидетельством потенциального влияния социальных факторов на корпоративную эффективность. Интегрируя социальную ответственность в свою деятельность, компании могут повысить репутацию своего бренда, привлечь и удержать клиентов, а также сократить расходы, тем самым создавая долгосрочную ценность. Ключевые факторы успеха включают постановку амбициозных целей, взаимодействие с заинтересованными сторонами и реализацию эффективных инициатив по решению социальных и экологических проблем. Трудовые отношения. Отношения компании со своими сотрудниками могут оказать существенное влияние на ее производительность. Плохие трудовые отношения, такие как споры по поводу заработной платы или условий труда, могут привести к забастовкам, снижению производительности и ущербу для репутации. Напротив, компании, которые поддерживают позитивные трудовые отношения и предлагают справедливую заработную плату и льготы, с большей вероятностью имеют мотивированных и вовлеченных сотрудников, которые привержены развитию компании. Отношения с заинтересованными сторонами. Отношения компании с заинтересованными сторонами, такими как инвесторы, поставщики и местные сообщества, также могут влиять на ее деятельность. Компании, которые отдают приоритет взаимодействию со стейкхолдерами и общению с ними, с большей вероятностью будут строить прочные отношения и смогут завоевать доверие и поддержку со стороны этих групп. Напротив, компании, которые игнорируют заинтересованные стороны или плохо обращаются с ними, могут столкнуться с негативными последствиями, такими как ограничение доступа к капиталу или негативной оценкой [35]. Поведение потребителей. Изменения в поведении потребителей могут повлиять на стратегии продаж и маркетинга компании. Например, если потребители станут больше беспокоиться о воздействии продукции на окружающую среду, компании, предлагающие устойчивую продукцию, могут получить конкурентное преимущество. Кроме того, компании, которые прислушиваются к потребностям и предпочтениям своих клиентов и предлагают продукты и услуги, отвечающие этим потребностям, с большей вероятностью будут формировать лояльность к бренду и поддерживать устойчивый рост продаж. Таким образом, социальные факторы имеют значительное влияние на деятельность компании. Компании, способные выявить эти факторы и отреагировать на них, с большей вероятностью смогут увеличить прибыль и обеспечить долгосрочную устойчивость. Страница 20 из 49 Страница 20 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling р ур д р ур Russian Journal of Resources, Conservation and Recycling Вознаграждение руководителей — еще один важный фактор корпоративного управления, который может повлиять на результаты деятельности компании. Компенсационный пакет предназначен для привлечения и удержания лучших специалистов и согласования интересов руководителей с интересами акционеров компании. Исследования показали, что существует положительная корреляция между вознаграждением руководителей и корпоративной эффективностью. Однако чрезмерное вознаграждение руководителей может нанести ущерб финансовому состоянию и репутации компании [38]. Права акционеров являются еще одним важным фактором корпоративного управления, который может повлиять на результаты деятельности компании. Акционеры заинтересованы в успехе компании и могут влиять на процессы принятия решений в компании. Компании с более сильными правами акционеров, как правило, имеют лучшие финансовые показатели. Компании, которые более прозрачны и подотчетны своим акционерам, как правило, более успешны в долгосрочной перспективе. Прозрачность является критическим фактором управления, который может повлиять на корпоративную эффективность. Это относится к степени, в которой компания открыта и честна в своей деятельности и процессах принятия решений. Открытые компании, как правило, заслуживают большего доверия и имеют более высокий уровень одобрения стейкхолдеров. Исследования показали, что существует положительная корреляция между прозрачностью и корпоративной эффективностью. Компании, которые более прозрачны, как правило, имеют лучшие финансовые показатели и более высокий уровень участия [28]. Таким образом, корпоративное управление имеет решающее значение для построения успешного бизнеса. Эффективные методы управления могут улучшить финансовые показатели, снизить риск мошенничества и коррупции и повысить доверие заинтересованных сторон. Состав совета директоров, вознаграждение руководителей, права акционеров и открытость являются важными факторами, которые могут повлиять на корпоративную деятельность. Сосредоточившись на этих элементах, компании могут заложить прочный фундамент для долгосрочного развития. Компании, которые отдают приоритет эффективной практике управления, лучше способны управлять рисками, адаптироваться к изменениям и создавать долгосрочную акционерную стоимость. Есть несколько примеров компаний, которые успешно интегрировали факторы корпоративного управления в свою деятельность, что привело к положительным финансовым результатам. Unilever, компания по производству потребительских товаров, поставила устойчивость и эффективное управление на первое место в своей повестке развития. Компания получила признание за исключительную практику управления, в том числе за приверженность прозрачности и открытости. В 2020 году Unilever заняла первое место в индексе устойчивости Доу-Джонса, который оценивает компании на основе их экологических, социальных и управленческих показателей (ESG). Выдающиеся показатели ESG компании Unilever также привели к высоким финансовым результатам и высокой доходности акций [39]. Microsoft, технологическая компания, в последние годы добилась значительных успехов в совершенствовании методов управления. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Компания получила признание за приверженность этическим методам ведения бизнеса, в том числе за инициативы по продвижению прозрачности и подотчетности в своей деятельности. В 2020 году Microsoft была названа самой дорогой компанией в мире по рыночной капитализации, продемонстрировав успех своей бизнес-стратегии и методов управления [40]. Управленческие факторы. Управленческие факторы. Корпоративное управление является важнейшим аспектом управления и контроля компанией. Это относится к политике, процедурам и практикам, которые регулируют поведение корпораций и их агентов по отношению к различным заинтересованным сторонам, включая акционеров, сотрудников, клиентов и более широкое сообщество. Эффективное корпоративное управление может положительно повлиять на финансовые показатели компании, снизить риск мошенничества и коррупции и повысить доверие заинтересованных сторон. Практика надлежащего управления способствует этичному поведению, прозрачности и подотчетности, что, в свою очередь, может улучшить работу организации и способствовать ее долгосрочному успеху. С другой стороны, плохая практика управления может привести к убыткам и репутационному ущербу. Есть несколько управленческих факторов, которые могут повлиять на корпоративную деятельность, такие как состав совета директоров, вознаграждение руководителей, права акционеров и прозрачность [37]. Совет директоров является важнейшим элементом корпоративного управления. Он отвечает за обеспечение надзора и руководства управленческой командой компании. Эффективный совет директоров должен обладать разнообразными навыками и опытом, чтобы помочь компании справиться с возникающими трудностями. Исследования показали, что компании с более разнообразным советом директоров, как правило, имеют лучшие финансовые показатели. Советы директоров, в состав которых входит больше женщин, могут помочь улучшить социальные и экологические показатели компании. Страница 21 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» р ур д р ур Russian Journal of Resources, Conservation and Recycling Russian Journal of Resources, Conservation and Recycling JPMorgan Chase, компания, предоставляющая финансовые услуги, сделала корпоративное управление важнейшим приоритетом. Компания реализовала ряд инициатив, чтобы гарантировать прозрачность и открытость своей деятельности, в том числе создала комитет по рискам на уровне совета директоров и назначила директора по соблюдению нормативных требований. Эффективная практика управления JPMorgan помогла ей противостоять финансовым кризисам и адаптироваться к рыночным изменениям, способствуя ее долгосрочному успеху [41]. Coca-Cola, компания по производству напитков, реализовала несколько инициатив по снижению воздействия на окружающую среду, включая инвестиции в устойчивую упаковку и сокращение потребления воды. Высокие показатели ESG Coca-Cola также привели к финансовым победам: компания продемонстрировала впечатляющий рост выручки и высокие доходы акционеров [42]. Существуют обширные данные о корпоративном управлении, представленные организациями, занимающимися анализом корпоративной отчетности различных компаний. Согласно отчету Института управления и отчетности (IVA), доля компаний из списка S&P 500, публикующих отчеты об устойчивом развитии с акцентом на факторы управления, увеличилась с 26 % в 2011 году до 93 % в 2020 году. Отчет Глобальной инициативы по отчетности (GRI) показал, что по состоянию на 2020 год 76 % из 250 крупнейших компаний мира отчитываются о своей эффективности управления, и 67 % этих компаний отчитываются, используя для этого Стандарты отчетности GRI в области устойчивого развития. Что касается данных на уровне стран, исследование Всемирного банка, проведенное в 2019 году, показало, что страны с более сильными системами корпоративного управления, как правило, имеют более высокий уровень отчетности в области устойчивого развития. Исследование, проведенное Boston Consulting Group, показало, что 79 % компаний со стратегией устойчивого развития также имеют специальную структуру управления. Что касается данных на уровне стран, отчет Vigeo Eiris показал, что в Соединенном Королевстве, Франции и Швеции был самый высокий процент компаний, сообщающих о факторах управления в своих отчетах об устойчивом развитии в Европе по состоянию на 2020 год. Барометр доверия Edelman 2021 года показал, что 60 % сотрудников во всем мире считают, что компании должны уделять первоочередное внимание этике и открытости, чтобы построить доверие со своими заинтересованными сторонами [43]. Вышеупомянутые компании (Unilever, Microsoft, JPMorgan Chase и Coca-Cola) — лишь несколько примеров, демонстрирующих, как разумные принципы управления могут способствовать финансовому процветанию в долгосрочной перспективе. Установив открытость и устойчивость в качестве приоритетов, эти компании смогли управлять рисками, справиться с трансформацией и создать ценность для своих акционеров. Компании, которые уделяют первоочередное внимание практике эффективного управления, лучше ориентируются в сложной и быстро меняющейся деловой среде 21-го века [44]. Связь между ESG и корпоративной эффективностью сложна и многогранна. р ур д р ур Russian Journal of Resources, Conservation and Recycling Хотя корреляция не всегда прямая, существуют доказательства того, что компании, которые обращают внимание на факторы ESG, как правило, превосходят тех, кто этого не делает. Автор учел различные компоненты ESG и их влияние на различные аспекты корпоративной деятельности. Поскольку компании сталкиваются с растущим давлением в отношении решения проблем ESG, для них крайне важно осознать значение ESG и то, как его можно использовать для обеспечения долгосрочной устойчивости и прибыльности. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» ISSN 2500-0659 https://resources.today Страница 22 из 49 Страница 22 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com ESG в странах с развивающейся экономикой Вопросы ESG привлекают все большее внимание в последние годы, а растущее осознание влияния факторов ESG привело к повышению интереса к интеграции этих факторов в инвестиционные решения. В то время как вопросы ESG широко обсуждались в развитых странах, их значение и реализация в странах с развивающейся экономикой остаются неясными. На внедрение практики ESG в странах с развивающейся экономикой влияют различные факторы, в том числе местное управление, культурные различия и ограниченность ресурсов. Регуляторная среда, давление заинтересованных сторон и глобальный спрос на интеграцию ESG также играют важную роль в принятии практики ESG в странах с развивающейся экономикой. Страны с развивающейся экономикой сталкиваются с уникальными проблемами, включая деградацию окружающей среды, социальное неравенство и слабые структуры управления, которые можно решить путем внедрения практики ESG. Внедрение практики ESG в странах с развивающейся экономикой может обеспечить конкурентное преимущество для предприятий за счет улучшения их репутации, снижения затрат и привлечения инвесторов. Однако предприятия в странах с развивающейся экономикой сталкиваются с рядом проблем при внедрении практики ESG, включая нехватку ресурсов, неадекватную нормативно- правовую базу и культурные различия. Интеграция практики ESG в стратегии бизнеса может предоставить значительные возможности для роста и развития в странах с развивающейся экономикой. Инвесторы в странах с развивающейся экономикой сталкиваются с рядом проблем, включая политическую нестабильность, валютный риск и недостаточность данных. Интеграция ESG может обеспечить основу для выявления и управления рисками, связанными с инвестициями в страны с развивающейся экономикой [27]. Принятие инвесторами методов ESG также может побудить предприятия интегрировать методы ESG в свою деятельность, что приведет к положительному влиянию на окружающую среду и общество. Внедрение практики ESG в странах с развивающейся экономикой имеет последствия для других заинтересованных сторон, включая правительства стран, общественные организации и местные сообщества. Практика ESG может способствовать устойчивому экономическому росту, социальному прогрессу и защите окружающей среды. Участие этих стейкхолдеров может сыграть важную роль в формировании практики внедрения ESG в странах с развивающейся экономикой [45]. Страны с формирующимся рынком все больше осознают важность факторов ESG для устойчивого развития. Повышение осведомленности о проблемах ESG обусловлено несколькими факторами, в том числе растущими экологическими и социальными рисками, изменением ожиданий заинтересованных сторон и необходимостью привлечения международных инвестиций. Мы обнаружили, что многие страны с развивающейся экономикой предприняли значительные шаги для продвижения практики ESG, такие как разработка нормативно-правовой базы, установление требований к отчетности ESG и запуск инициатив по зеленому финансированию. Однако интеграция ESG в странах с развивающейся экономикой все еще находится на ранних стадиях, и между развитыми и развивающимися странами существуют значительные пробелы в практике ESG [46]. Страница 23 из 49 Страница 23 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2022, Том 9, № 4 2022, Vol 9, No 4 Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ESG в странах с развивающейся экономикой Одним из ключевых факторов, способствующих внедрению практики ESG в странах с развивающейся экономикой, является роль правительств. Правительства могут сыграть решающую роль в продвижении практики ESG, создав благоприятную среду, предоставив стимулы для устойчивой практики и обеспечив соблюдение правил ESG. Некоторые страны с развивающейся экономикой, такие как Китай и Бразилия, осознали важность факторов ESG и внедрили политику и инициативы, направленные на содействие устойчивому развитию. Институциональные рамки также важны для внедрения практики ESG в странах с развивающейся экономикой. Наличие сильной правовой базы, независимых регулирующих органов и надежных организаций гражданского общества может создать необходимые условия Страница 24 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 24 из 49 Страница 24 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today для интеграции ESG. Кроме того, существуют опасения по поводу качества и последовательности отчетности ESG в странах с развивающейся экономикой, что может подорвать эффективность практики ESG [47]. для интеграции ESG. Кроме того, существуют опасения по поводу качества и последовательности отчетности ESG в странах с развивающейся экономикой, что может подорвать эффективность практики ESG [47]. Взаимодействие с заинтересованными сторонами является еще одним важным фактором для внедрения практики ESG в странах с развивающейся экономикой. Взаимодействие с заинтересованными сторонами может помочь организациям понять местный контекст, выявить риски и возможности ESG и укрепить доверие местных сообществ. В ряде стран с развивающейся экономикой отсутствуют эффективные механизмы взаимодействия с заинтересованными сторонами, что может препятствовать интеграции практики ESG. Существуют некоторые проблемы и ограничения, которые могут помешать внедрению практики ESG в странах с развивающейся экономикой. Эти проблемы включают отсутствие осведомленности об ESG, ограниченные ресурсы и конкурирующие приоритеты. Также существуют опасения по поводу затрат компаний на соблюдение ESG, что может стать серьезным препятствием для малых и средних предприятий. В настоящее время компании на развивающихся рынках осознают ценность факторов ESG для создания устойчивой долгосрочной стоимости, улучшения своей репутации и снижения рисков, ориентируясь на ряд факторов, которые способствуют более широкому внедрению ESG на развивающихся рынках, в том числе на растущее значение рейтингов компаний по оценке ESG, спрос инвесторов на информацию об ESG, а также нормативные и юридические требования к отчетности ESG [48]. Ряд тематических исследований подчеркивают различные подходы к внедрению ESG в развивающихся странах. Внедрение ESG в развивающихся странах обусловлено целым рядом факторов, в том числе регуляторным давлением, давлением заинтересованных сторон и конкурентными преимуществами. В некоторых странах государственная политика стимулирует компании внедрять методы ESG. Например, правительство Индии обязало 500 ведущих компаний, зарегистрированных на бирже, отчитываться о своих показателях ESG. Точно так же Китай ввел ряд политик, которые стимулируют компании улучшать свои показатели ESG. В других случаях давление заинтересованных сторон побуждает компании внедрять методы ESG. Например, потребительский спрос на экологически чистые продукты побуждает компании сокращать свое воздействие на окружающую среду. Страница 24 из 49 Кроме того, компании признают конкурентное преимущество внедрения ESG. Приведем некоторые отраслевые примеры. В Индии ведущая фармацевтическая компания внедрила стратегию устойчивого развития, направленную на сокращение отходов, повышение энергоэффективности и укрепление здоровья и безопасности сотрудников. Компания разработала всеобъемлющий отчет об устойчивом развитии, который предоставил заинтересованным сторонам подробную информацию о ее методах ESG, и взаимодействовала с заинтересованными сторонами по различным каналам, включая общие собрания, социальные сети и опросы заинтересованных сторон. В Южной Африке крупная горнодобывающая компания внедрила программу устойчивого развития, направленную на обеспечение экологической устойчивости, социальной ответственности и надлежащего корпоративного управления. Компания взаимодействовала с местными сообществами по различным каналам, включая консультации с населением, форумы по взаимодействию с заинтересованными сторонами и местные партнерские отношения. Компания также внедрила комплексную систему мониторинга и оценки для отслеживания прогресса в достижении целей устойчивого развития [49]. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Сельскохозяйственная отрасль вносит значительный вклад в экономику многих развивающихся стран. Сельскохозяйственные компании в развивающихся странах реализовали различные инициативы ESG для повышения производительности и снижения воздействия на окружающую среду. Например, в Бразилии рабочая группа по производству сои Мату-Гросу (GTS) предприняла шаги по содействию устойчивому производству сои путем создания системы мониторинга для предотвращения вырубки лесов и продвижения устойчивых методов среди производителей сои [50]. Энергетический сектор имеет решающее значение для экономического роста развивающихся стран. Однако энергетические компании сталкиваются со значительными экологическими и социальными рисками, связанными с их деятельностью. Энергетические компании в развивающихся странах реализовали различные инициативы ESG, чтобы уменьшить свое воздействие на окружающую среду и улучшить социальные результаты. Например, в Индии Tata Power реализовала различные инициативы ESG, включая проекты по возобновляемым источникам энергии, меры по повышению энергоэффективности и программы развития сообщества. Примеры внедрения ESG в развивающихся странах показали, что существует несколько потенциальных преимуществ интеграции ESG для бизнеса. Эти преимущества включают улучшение экологических и социальных показателей, повышение репутации бренда и снижение юридических и нормативных рисков. Примеры внедрения ESG также показывают, что интеграция ESG может привести к расширению доступа к капиталу и улучшению кредитных рейтингов. Однако внедрение принципов ESG в развивающихся странах сопряжено с трудностями. Одной из серьезных проблем является отсутствие стандартизации и регулирования. Без четких стандартов и правил компаниям и инвесторам сложно понять, какие факторы ESG следует учитывать, как их измерять и составлять отчеты. Еще одной проблемой является отсутствие осведомленности и знаний о принципах ESG среди предприятий, инвесторов и политиков [51]. Чтобы преодолеть эти проблемы, правительствам, обществу и другим заинтересованным сторонам необходимо работать вместе, чтобы создать благоприятную среду для реализации принципов ESG. Для этого необходимо разработать четкие стандарты и правила ESG, учитывающие особенности развивающихся стран. Правительствам и регулирующим органам также следует разработать политику, стимулирующую ESG и создающую стимулы для предприятий, чтобы они применяли устойчивые методы. Более того, обществу, бизнесу и инвесторам необходимо повысить осведомленность и понимание принципов ESG, чтобы повысить их эффективность. Отметим, что интеграция ESG в странах с развивающейся экономикой все еще находится на ранней стадии. Тем не менее, в последние годы наблюдается растущее осознание и принятие факторов ESG. Инвесторы все чаще требуют большей прозрачности и раскрытия информации о факторах ESG от компаний на развивающихся рынках. Кроме того, регулирующие органы и фондовые биржи вводят требования и рекомендации по отчетности, связанные с ESG. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Одной из основных проблем интеграции ESG в странах с развивающейся экономикой является отсутствие доступности и качества данных, связанных с ESG. Многие компании в этих странах не раскрывают информацию о применении и эффективности ESG, что затрудняет оценку инвесторами рисков и возможностей ESG. Кроме того, отчетность ESG часто не стандартизирована, что затрудняет сравнение показателей ESG компаний на разных развивающихся рынках. Несмотря на проблемы, существуют значительные возможности, связанные с интеграцией ESG в странах с развивающейся экономикой. Одной из ключевых возможностей является потенциал для обеспечения устойчивого экономического роста и развития. Страница 25 из 49 Страница 25 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Интегрируя факторы ESG в бизнес-стратегии, компании могут создать долгосрочную ценность для всех заинтересованных сторон и внести свой вклад в устойчивое развитие своих стран. Также появляется все больше данных того, что компании, которые хорошо справляются с факторами ESG, как правило, превосходят своих конкурентов с точки зрения финансовых показателей. Компании, способные эффективно управлять рисками и возможностями ESG, имеют больше возможностей для обеспечения стабильной прибыли своим инвесторам. Существует несколько примеров компаний в странах с развивающейся экономикой, которые успешно интегрировали факторы ESG в свои бизнес-стратегии. Например, бразильская горнодобывающая компания Vale внедрила комплексную систему управления ESG, которая включает оценку социального и экологического воздействия, взаимодействие с заинтересованными сторонами и отчеты об устойчивом развитии. Компания также поставила амбициозные цели в области устойчивого развития, такие как сокращение выбросов парниковых газов на 36 % к 2023 году. Другим примером является индийская компания Tata Steel, которая реализовала ряд инициатив ESG, включая повышение энергоэффективности, сокращение отходов и программы развития сообщества. Компания также внедрила систему отчетности в области устойчивого развития, которая включает оценку существенности и взаимодействие с заинтересованными сторонами [52]. ESG в России Вопросы, связанные с окружающей средой, обществом и управлением, стали важными факторами, определяющими инвестиционные решения, так как инвесторы все больше отдают приоритет социальному и экологическому воздействию корпораций. Поскольку Россия является заслуживающим внимания развивающимся рынком, страна привлекла инвесторов, уделяющих особое внимание ESG. Диверсифицированная экономика России, обширные природные ресурсы и огромные потенциальные возможности делают ее привлекательным местом для иностранных инвестиций. Однако в последние годы России пришлось столкнуться с вызовами, связанными с управлением, социальными и экологическими проблемами, включая коррупцию, низкие трудовые стандарты и слабое корпоративное управление. Экологические аспекты оказывают существенное влияние на деятельность компаний в России. Страна обладает богатыми природными ресурсами, такими как нефть, газ и полезные ископаемые, и является крупным производителем этих ресурсов. Однако эксплуатация этих ресурсов привела к ущербу и загрязнению окружающей среды, тем самым создав риск для здоровья местных жителей. Кроме того, несмотря на то, что Россия является одним из крупнейших источников выбросов парниковых газов в мире, она не смогла уменьшить свой углеродный след и перейти к низкоуглеродной экономике. Чтобы смягчить эти негативные факторы, многие фирмы, ведущие бизнес в России, внедрили несколько экологических, социальных и управленческих политик (ESG), таких как сокращение углеродного следа и внедрение устойчивых методов производства. Одна из таких компаний, «Роснефть», крупная нефтегазовая компания в России, поставила перед собой цели по сокращению выбросов парниковых газов и повышению энергоэффективности. Кроме того, компания инвестировала в проекты устойчивой энергетики, такие как ветровая и солнечная энергия, чтобы расширить свои источники энергии и ограничить воздействие на окружающую среду [45]. Помимо экологических факторов, социальные факторы также оказывают значительное влияние на деятельность фирм в России. Страна столкнулась с проблемами, связанными с нарушениями трудовых прав, коррупцией и неразвитыми программами социального обеспечения, которые отрицательно сказались на компаниях, работающих в стране, подвергая Страница 27 из 49 Страница 27 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 26 из 49 Страница 26 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Страница 27 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today их юридическим санкциям и репутационным рискам. Чтобы противостоять этим вызовам, компании, ведущие бизнес в России, внедрили различные меры ESG для улучшения трудовых стандартов и снижения коррупции в бизнесе. Например, X5 Retail Group, один из ведущих российских ритейлеров, запустил несколько программ по улучшению условий труда своих сотрудников. X5 внедрила кодекс поведения, отражающий приверженность компании продвижению прав человека и трудовых норм, а также программы обучения для обеспечения соблюдения этих политик. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling их юридическим санкциям и репутационным рискам. Чтобы противостоять этим вызовам, компании, ведущие бизнес в России, внедрили различные меры ESG для улучшения трудовых стандартов и снижения коррупции в бизнесе. Например, X5 Retail Group, один из ведущих российских ритейлеров, запустил несколько программ по улучшению условий труда своих сотрудников. X5 внедрила кодекс поведения, отражающий приверженность компании продвижению прав человека и трудовых норм, а также программы обучения для обеспечения соблюдения этих политик. В России практика ответственного управления играет важную роль в корпоративном успехе компаний. Страна борется с коррупцией, неэффективной нормативно-правовой базой и отсутствием эффективной практики корпоративного управления. В ответ на эти вызовы компании, ведущие бизнес в России, предприняли согласованные усилия по внедрению различных мер ESG, направленных на совершенствование корпоративного управления и повышение прозрачности. Возьмем, к примеру, Сбербанк, гиганта российского банковского сектора. Финансовое учреждение предприняло решительные шаги для укрепления своей практики корпоративного управления. Банк внедрил кодекс поведения, в котором особое внимание уделяется этичному ведению бизнеса и прозрачности. Сбербанк также принял другие меры, направленные на повышение прозрачности, в том числе публикацию годового отчета об устойчивом развитии и внедрение программы информирования о нарушении. В соответствии с нашим исследованием общедоступных данных об экологических, социальных и управленческих показателях (ESG) и отчетности предприятий, котирующихся на Московской бирже, мы пришли к выводу, что ESG приобретает все большее значение в России. Мы отмечаем, что множество крупнейших корпораций, расположенных в России, приняли политику и практику ESG, и наблюдается восходящая траектория с точки зрения всеобъемлющей отчетности ESG. Несмотря на то, что за последние несколько лет Россия добилась значительных успехов в деятельности, связанной с ESG, она по-прежнему отстает от других развитых стран. Страница 27 из 49 Исследование, проведенное российской государственной корпорацией развития ВЭБ.РФ, получило данные, что по состоянию на 2019 год 75 % российских компаний не имели плана ESG или механизма отчетности. Тем не менее, число корпораций, раскрывающих свои показатели ESG, неуклонно растет. В том же исследовании сообщается, что 30 % крупнейших российских компаний публиковали отчеты по ESG в 2019 году, что представляет собой значительный рост по сравнению с 7 % в 2016 году. Ожидается, что эта тенденция сохранится, поскольку инвесторы все чаще требуют раскрытия ESG от компаний, работающих в России. Поскольку глобальные инвесторы все больше внимания уделяют устойчивому развитию и ответственному инвестированию, российские компании осознают важность интеграции практики ESG в свою деятельность, для сохранения конкурентоспособности [53]. Еще одним фактором, стимулирующим внедрение ESG в России, является повышенное внимание со стороны местных регуляторов. В последние годы Центральный банк России выпустил руководство по устойчивому финансированию и призвал банки учитывать риски ESG при принятии решений о кредитовании. Правительство России также разработало национальную стратегию устойчивого развития, которая включает меры по содействию устойчивому развитию и сокращению выбросов парниковых газов. Кроме того, пандемия COVID-19 подчеркнула важность факторов ESG для снижения рисков и повышения устойчивости. Компании, которые отдают приоритет практикам ESG, лучше подготовлены к преодолению кризисов и защите своих стейкхолдеров, включая сотрудников, клиентов и сообщества. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» В целом внедрение ESG в России является позитивным событием, поскольку оно может способствовать устойчивому экономическому росту и улучшению социальных и экологических результатов. По мере того, как все больше российских компаний внедряют методы ESG, они могут привлекать новых инвесторов, улучшать свою репутацию и вносить свой вклад в более устойчивое будущее. Экологические нормы и правоприменение в России не такие строгие, как в некоторых других развитых странах. Это ставит перед компаниями задачу сбалансировать экономический рост и экологическую ответственность. Однако есть некоторые признаки прогресса, такие как внедрение системы торговли квотами на выбросы и введение налога на выбросы углерода. Таким образом, несмотря на то что некоторые российские компании добились определенного прогресса в снижении своего воздействия на окружающую среду, еще есть необходимость для улучшения экологической отчетности, стандартизации и регулирования. Сильная зависимость страны от энергетического сектора и медленный прогресс в сокращении выбросов углерода подчеркивают необходимость увеличения инвестиций в возобновляемые источники энергии и более строгой экологической политики. В качестве примера приверженности политики ESG, можно выделить компании, которые обязались сократить выбросы парниковых газов в соответствии с Парижским соглашением. «Газпром» поставил цель сократить свои выбросы на 20 % к 2030 г., а «Роснефть» стремится снизить интенсивность выбросов парниковых газов на 10 % к 2022 г. Однако, несмотря на эти усилия, существуют опасения по поводу того, насколько эти компании действительно привержены сокращению своего воздействия на окружающую среду. Некоторые экологические организации критикуют «Газпром» и «Роснефть» за то, что они недостаточно делают для перехода на возобновляемые источники энергии и продолжают инвестировать в новые нефтегазовые проекты. Также есть вопросы к прозрачности и точности данных о выбросах компаний, а также к их соответствию природоохранным нормам. В целом, несмотря на то что «Газпром» и «Роснефть» предприняли некоторые шаги по снижению своего воздействия на окружающую среду, еще многое можно сделать для перехода к более устойчивой энергетической системе. Этим компаниям будет важно продолжать инвестировать в низкоуглеродные технологии и уделять первоочередное внимание сокращению выбросов парниковых газов, чтобы помочь смягчить последствия изменения климата [54]. Важным социальным фактором является важность социальных сетей и личных отношений в российской деловой культуре. Создание и поддержание прочных отношений с деловыми партнерами и государственными чиновниками имеет важное значение для процветания бизнеса. Иногда это может привести к отсутствию прозрачности и этическим проблемам, таким как взяточничество и коррупция. Гендерное неравенство также является серьезной социальной проблемой в России. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Согласно Докладу Всемирного экономического форума о глобальном гендерном разрыве за 2021 год, Россия занимает 81-е место из 156 стран по уровню гендерного равенства (6). Женщины в России сталкиваются с дискриминацией на рабочем месте и часто имеют ограниченный доступ к экономическим и политическим возможностям. Растущий средний класс в России вызывает изменения в поведении и ожиданиях потребителей [55]. Они требуют от компаний более качественных продуктов и услуг, а также более этичного и устойчивого производства. Это создает как проблемы, так и возможности для бизнеса, работающего в России. Еще одной социальной проблемой в России является состояние системы здравоохранения. Хотя в последние годы произошли улучшения, система по- прежнему недофинансируется, а доступ к качественному медицинскому обслуживанию во многих регионах ограничен. Это привело к высокому уровню недовольства населения и представляет собой серьезную проблему для правительства. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 28 из 49 Страница 28 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Наконец, факторы окружающей среды также являются важной частью ESG. Россия является крупным производителем нефти и газа, и эта отрасль оказывает значительное влияние на окружающую среду. Россия также сталкивается с проблемами загрязнения воздуха и воды, обезлесения и изменения климата. В целом, несмотря на то что Россия добилась прогресса в решении некоторых из этих социальных и экологических проблем, предстоит еще много работы для улучшения показателей ESG. Инвесторы и заинтересованные стороны все больше внимания уделяют вопросам ESG, и компаниям, работающим в России, необходимо будет уделять этим факторам приоритетное внимание, чтобы оставаться конкурентоспособными и привлекать инвестиции. Одним из основных препятствий для интеграции ESG в России является отсутствие четких и последовательных правил. Хотя существуют законы, затрагивающие экологические и социальные вопросы, они часто слабо соблюдаются, и их соблюдение не всегда является приоритетом. Кроме того, существует культурное мышление, которое ставит краткосрочную прибыль выше долгосрочной устойчивости. Однако есть компании, которые добиваются успехов в области ESG. Например, нефтегазовая компания «Газпром» предприняла усилия по снижению своего воздействия на окружающую среду и получила признание за свою отчетность об устойчивом развитии. Горнодобывающая компания «Норильский никель» также приняла меры по снижению своего воздействия на окружающую среду, такие как использование более чистых технологий и сокращение потребления воды. Другие компании обнаружили, что внедрение практики ESG может принести финансовую выгоду. Например, компания розничной торговли X5 Retail Group внедрила меры по обеспечению устойчивого развития, такие как сокращение пищевых отходов и потребления энергии, что привело к экономии средств и повышению удовлетворенности клиентов. Аналогичным образом Сбербанк обнаружил, что сосредоточение внимания на экологических и социальных проблемах привело к повышению лояльности клиентов и улучшению репутации. Чтобы преодолеть проблемы интеграции практик ESG, компаниям необходимо уделить первоочередное внимание обучению и повышению осведомленности своих сотрудников и стейкхолдеров. Кроме того, необходимо добиваться более четких правил и стимулов для компаний, которые отдают приоритет устойчивому развитию. В конечном счете, успешная интеграция практик ESG в России потребует культурного сдвига в сторону долгосрочного и устойчивого мышления [56]. Драйверы ESG в России. Россия является одним из крупнейших в мире производителей энергии, и воздействие ее деятельности на окружающую среду в последние годы находится в центре внимания. Правительство России предприняло значительные усилия для решения экологических проблем и сокращения выбросов углерода в ответ на международное давление и продемонстрировало большую заинтересованность в содействии устойчивому развитию, а также предприняло шаги для поощрения внедрения практики ESG в бизнес-сообществе. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 29 из 49 Страница 29 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Благодаря этому компании, которые уделяют большее внимание вопросам ESG, получают преимущество перед конкурентами и обеспечивают свою долгосрочную устойчивость. Также стоит отметить, что российские компании, особенно в энергетическом секторе, все еще сталкиваются с рядом проблем, связанных с экологией и социальной ответственностью. Но благодаря активным действиям правительства и росту интереса со стороны инвесторов, можно ожидать дальнейшего развития практики ESG в России. Драйверы ESG в России включают в себя следующие факторы: Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Для того чтобы соответствовать требованиям ESG, компании в России должны не только уделять большее внимание экологической и социальной ответственности, но и развивать корпоративное управление. Это включает в себя улучшение прозрачности и отчетности, сокращение коррупционных рисков и установление этичных стандартов поведения. На российском рынке существует ряд драйверов, которые способствуют развитию практики ESG. Один из них — внедрение рыночных механизмов, таких как торговля квотами на выбросы парниковых газов. Это позволяет компаниям стимулировать уменьшение своих выбросов и получать дополнительные доходы за продажу ненужных квот. Другой драйвер — повышение уровня осведомленности и образованности инвесторов и общества в целом. Страница 30 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Социальные инициативы: «Норникель» реализовал ряд инициатив, направленных на повышение безопасности и благополучия своих сотрудников и местного населения, в регионе ведения бизнеса. Компания разработала программу «Культура безопасности» для повышения осведомленности о безопасности и предотвращения несчастных случаев, а также внедрила ряд программ в области здоровья для повышения благополучия своих сотрудников. «Норникель» предпринял ряд инициатив по поддержке сообществ, в которых он работает, включая строительство школ и больниц, а также поддержку местного бизнеса. Инициативы в области управления: «Норникель» предпринял шаги по совершенствованию своей практики корпоративного управления, включая повышение прозрачности своей отчетности и усиление внутреннего контроля. Компания создала Совет директоров и Комитет по аудиту для обеспечения надзора за своей деятельностью, а также внедрила ряд политик и процедур для обеспечения соблюдения законодательных и нормативных требований. Сбербанк, крупнейший банк в России, является еще одним примером компании, успешно внедрившей принципы ESG в свою деятельность. Банк реализовал ряд инициатив ESG, в том числе поощрение доступа к финансовым услугам, улучшение экологических показателей и укрепление практики корпоративного управления. Экологические инициативы: Сбербанк предпринял ряд шагов для снижения своего воздействия на окружающую среду, в том числе инвестировал в возобновляемые источники энергии и технологии повышения энергоэффективности, сократил использование бумаги и рекомендовал устойчивые методы ведения бизнеса среди своих клиентов. Банк также внедрил систему управления экологическими и социальными рисками для выявления и снижения экологических и социальных рисков, связанных с его кредитной деятельностью. Социальные инициативы: Сбербанк предпринял ряд инициатив, направленных на расширение доступа к финансовым услугам и улучшение благосостояния сообществ, в которых он работает. Банк создал Центр микрофинансирования для предоставления доступа к кредитам и другим финансовым услугам малым и средним предприятиям, а также реализовал ряд программ по повышению финансовой грамотности среди своих клиентов. Кроме того, Сбербанк реализовал ряд программ поддержки сообществ, в которых он работает, включая поддержку социальных и культурных инициатив. Инициативы в области управления: Сбербанк предпринял шаги по совершенствованию своей практики корпоративного управления, включая повышение прозрачности своей отчетности и усиление внутреннего контроля. Банк создал Совет директоров и Комитет по аудиту для обеспечения надзора за его операциями, а также внедрил ряд политик и процедур для обеспечения соблюдения законодательных и нормативных требований. «Газпром», одна из крупнейших энергетических компаний мира, реализовал ряд инициатив по решению проблем ESG. В 2010 году «Газпром» запустил программу «Газпром нефть — детям», направленную на улучшение здоровья и благополучия детей в России. Программа оказывает финансовую поддержку больницам и школам, а также финансирует ряд образовательных и культурных инициатив. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling В 2019 году «Газпром» объявил, что за предыдущие пять лет инвестировал более 12,5 млрд рублей в природоохранные инициативы. Эти инициативы включали сокращение выбросов парниковых газов, защиту биоразнообразия и повышение энергоэффективности [53]. Экологические, социальные и управленческие (ESG) факторы все больше привлекают внимание компаний по всему миру. Россия, как ведущая мировая экономика, не является исключением. Вопросы ESG стали более важными в России, поскольку страна продолжает развиваться в экономическом и политическом плане. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Драйверы ESG в России включают в себя следующие факторы: Законодательство и регулирование: Российское правительство принимает регламенты и положения, которые способствуют устойчивому развитию и привлекают внимание инвесторов к ESG. Например, в 2019 году был принят ФЗ № 225 об ответственном обращении с отходами, который обязывает компании соблюдать строгие правила утилизации отходов. Растущий интерес инвесторов: Инвесторы все больше уделяют внимание практике устойчивого инвестирования и требуют, чтобы компании, в которые они инвестируют, учитывали принципы ESG. В России также появились фонды, которые специализируются на инвестировании в компании, которые соблюдают принципы ESG. Внедрение практики ESG в бизнес-сообществе: Российские компании начинают все больше учитывать в своей деятельности принципы ESG. Некоторые из них внедряют программы сокращения выбросов углерода, повышения энергоэффективности и улучшения условий труда для сотрудников. Рост экологических проблем: Россия сталкивается с серьезными экологическими проблемами, такими как загрязнение водоемов, воздуха и почвы. Эти проблемы приводят к ухудшению здоровья людей и животных, а также угрожают экономике страны. Поэтому, российские компании осознают важность решения этих проблем и стараются сократить негативное воздействие на окружающую среду. В целом, драйверы ESG в России указывают на то, что практика ответственного бизнеса становится все более важной как для самой России, так и для ее бизнес-сообщества. Компании, которые не принимают принципы ESG, рискуют остаться в состоянии отстающих на рынке и потерять доступ к финансированию и инвестиционным возможностям. Для иллюстрации позитивных практик внедрения ESG в российские компании, стоит привести несколько кейсов, которые дают понимание факторов, способствующих укреплению бизнеса. «Норникель», российская горно-металлургическая компания, является ярким примером компании, успешно внедрившей принципы ESG в свою деятельность. В 2020 году компания впервые была включена в индекс устойчивого развития Доу-Джонса (DJSI), что подчеркивает ее приверженность устойчивому развитию. «Норникель» реализовал ряд ESG-инициатив, включая снижение воздействия на окружающую среду, усиление мер безопасности для своих сотрудников и совершенствование практики корпоративного управления. Экологические инициативы: «Норникель» предпринял ряд шагов для снижения своего воздействия на окружающую среду, включая инвестиции в технологии контроля загрязнения, совершенствование методов обращения с отходами и продвижение устойчивых методов добычи полезных ископаемых. Компания внедрила Систему экологического менеджмента (EMS), чтобы обеспечить соблюдение экологических норм и постоянно улучшать свои экологические показатели. Также «Норникель» поставил амбициозные цели по сокращению выбросов парниковых газов и предпринял шаги по увеличению использования возобновляемых источников энергии [53]. Страница 31 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Сравнение практики ESG в России с практикой применения на развитых рынках показывает резкие различия в отчетности по устойчивому развитию, корпоративном управлении и воздействии на окружающую среду. Отчетность в области устойчивого развития стала важным компонентом практики ESG, поскольку она позволяет компаниям раскрывать свои нефинансовые последствия и усилия по их смягчению. На развитых рынках отчеты об устойчивом развитии все чаще становятся нормой, и компании регулярно публикуют всеобъемлющие отчеты об устойчивом развитии. Напротив, отчетность в области устойчивого развития все еще является относительно новой концепцией в России, и лишь немногие компании публикуют отдельные отчеты в области устойчивого развития. Некоторые компании добровольно отчитываются о своих действиях по обеспечению устойчивого развития, однако, большинство не раскрывает никакой информации о своей практике ESG. Также российские компании недостаточно осведомлены о преимуществах отчетности в области устойчивого развития, что снижает их готовность формировать подобную отчетность. При этом на развитых рынках компании часто свободно обмениваются информацией о своей деятельности по ESG и используют ее для улучшения своих бизнес-практик. Корпоративное управление — еще один важный аспект применения ESG. Корпоративное управление в России также остается несовершенным. На развитых рынках существуют строгие правила и стандарты корпоративного управления, которым должны следовать компании. Эти правила гарантируют, что компании работают этично и прозрачно, что необходимо для укрепления доверия с заинтересованными сторонами. В России практика корпоративного управления не столь строга, и соблюдение нормативных требований отсутствует. Это привело к таким проблемам, как низкая независимость совета директоров, конфликты интересов и отсутствие прозрачности, что может негативно повлиять на показатели ESG компании. Воздействие на окружающую среду также является проблемой для многих компаний в России. Развитые рынки предпринимают значительные шаги для снижения своего воздействия на окружающую среду, внедряя устойчивые методы, инвестируя в возобновляемые источники энергии и сокращая выбросы парниковых газов. Напротив, российские компании не торопятся внедрять устойчивые методы и неохотно инвестируют в возобновляемые источники энергии. Отчасти это связано с сильной зависимостью страны от нефти и газа, которые являются основой экономики. Кроме того, российские компании недостаточно осведомлены о преимуществах инвестирования в устойчивые методы ведения бизнеса, что снижает их готовность к переменам. Однако, в последние годы многие российские компании начали уделять большее внимание снижению своего экологического воздействия и внедрению более эффективных технологий и методов производства. Проведенное сравнение практики ESG в России с практикой на развитых рынках показывает резкие различия в отчетности по устойчивому развитию, корпоративном управлении и воздействии на окружающую среду. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 32 из 49 Страница 32 из 49 Страница 32 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling В то время как развитые рынки добились значительных успехов в улучшении своей практики ESG, Россия отстает в различных областях. Отсутствие юридических требований к отчетности в области устойчивого развития и неосведомленность российских компаний о преимуществах отчетности в области устойчивого развития препятствуют их готовности отчитываться. Несоблюдение нормативных требований и проблемы, связанные с практикой корпоративного управления в России, оказали негативное влияние на показатели ESG компании. Наконец, сильная зависимость от нефти и газа и недостаточная осведомленность о преимуществах инвестирования в устойчивые методы препятствуют снижению воздействия на окружающую среду [54]. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Чтобы улучшить практику ESG в России, правительству необходимо ввести правила и стандарты отчетности в области устойчивого развития и обеспечить соблюдение существующих правил в отношении практики корпоративного управления. Также необходимо проводить более масштабное информирование по повышению осведомленности российских компаний о преимуществах отчетности в области устойчивого развития и инвестирования в устойчивые методы. Благодаря совместным усилиям правительства, компаний и других заинтересованных сторон Россия может догнать развитые рынки с точки зрения практики ESG. Изучая практику ESG на развитых и на развивающихся рынках, мы можем сделать выводы о том, что развитые рынки, такие как США и Западная Европа, внедрили практику ESG, в то время как развивающиеся рынки, такие как Китай и Бразилия, все еще работают над разработкой и внедрением своих схем ESG. Согласно исследованию агентства ESG Ratings, рейтинг ESG в России ниже, чем у развитых рынков, но выше, чем средний балл развивающихся рынков. Это говорит о том, что хотя Россия и добивается прогресса во внедрении практики ESG, еще есть возможности для совершенствования. С точки зрения экологических факторов Россия добилась значительных успехов в сокращении выбросов парниковых газов и развитии возобновляемых источников энергии. Однако социальные факторы, такие как права человека и трудовые отношения, по-прежнему требуют внимания. Практика управления, в том числе меры по обеспечению прозрачности и борьбе с коррупцией, также являются областями, в которых Россия может проводить улучшения. Когда мы сравниваем практику ESG в России с другими развивающимися рынками, мы видим, что Россия превосходит ее в некоторых областях, таких как гендерное равенство и корпоративное управление. Тем не менее, в таких областях, как социальная интеграция и экологическая устойчивость, еще многое предстоит сделать. В целом, сравнение практики ESG в России с практикой на развитых и развивающихся рынках показывает, что Россия делает успехи во внедрении практики ESG, но предстоит еще долгий путь [55]. Для бизнеса и инвесторов в России важно продолжать уделять внимание аспектам ESG и работать над развитием более устойчивых практик. Поступая таким образом, Россия может не только улучшить свой показатель ESG, но и внести свой вклад в более устойчивое будущее для всех. Анализируя влияние ESG на корпоративную эффективность в России, мы получили подтверждение того, что инвесторы стали больше интересоваться компаниями, которые прилагают усилия для решения этой проблемы, и в результате ESG стали ключевым фактором при принятии инвестиционных решений. Одно исследование, проведенное MSCI ESG Research, показало, что компании с высоким рейтингом ESG опережают компании с более низким рейтингом в России. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling В ходе исследования была проанализирована деятельность 27 российских компаний в период с 2014 по 2018 год, и было выявлено, что компании с высокими рейтингами ESG продемонстрировали более высокую рентабельность собственного капитала. Компании с высоким рейтингом корпоративного управления продемонстрировали более высокую рентабельность активов, что свидетельствует о том, что эффективное управление является ключом к получению долгосрочной ценности. Другое исследование, проведенное Московской школой управления СКОЛКОВО, показало, что компании с более высокими показателями ESG, как правило, имеют более высокие финансовые показатели в долгосрочной перспективе. В ходе исследования была проанализирована деятельность 50 российских компаний в период с 2014 по 2017 год, и было обнаружено, что компании с высокими показателями ESG имеют более высокую Страница 34 из 49 Страница 34 из 49 Страница 33 из 49 Страница 33 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Страница 34 из 49 Страница 34 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today 2022, Том 9, № 4 2022, Vol 9, No 4 Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today рентабельность активов и более высокую рыночную стоимость. Исследование также выявило некоторую закономерность в том, что компании с более высокими социальными показателями демонстрируют более высокий уровень инноваций, что позволяет предположить, что социальная ответственность может стимулировать инновации и создавать новые возможности. рентабельность активов и более высокую рыночную стоимость. Исследование также выявило некоторую закономерность в том, что компании с более высокими социальными показателями демонстрируют более высокий уровень инноваций, что позволяет предположить, что социальная ответственность может стимулировать инновации и создавать новые возможности. Несмотря на исследования, подтверждающие связь между ESG и корпоративной эффективностью, многие российские компании по-прежнему отстают от своих мировых коллег, когда речь идет о раскрытии информации об ESG и эффективности. Опрос, проведенный KPMG в 2020 году, показал, что только 8 % российских компаний имеют комплексную ESG-стратегию по сравнению с 29 % в мире. Кроме того, исследование показало, что только 40 % российских компаний имеют специальную команду или функцию ESG по сравнению с 62 % в мире [56]. Таким образом, оценивая влияние ESG на корпоративную эффективность в России, можно сделать следующие выводы: Компании, которые отдают приоритет факторам ESG в своей бизнес-операции, оказывают положительное влияние на свои финансовые показатели. Интеграция практики ESG обеспечивает долгосрочную устойчивость, репутацию бренда и взаимодействие с заинтересованными сторонами, что в итоге способствует общему росту компании. В России акцент на ESG появился относительно недавно, и компании постепенно внедряют принципы ESG в свою деловую практику. Отсутствие осведомленности и понимания ESG среди заинтересованных сторон, включая инвесторов, правительство и потребителей, препятствует внедрению практики ESG. Необходимы правила и политика, которые стимулируют компании интегрировать методы ESG в свои бизнес-операции. Правительству необходимо ввести меры, способствующие открытости и устойчивому развитию. Инвесторы все чаще требуют от компаний раскрытия ESG, а это означает, что компаниям необходимо включать показатели ESG в свою финансовую отчетность, чтобы привлекать и удерживать инвесторов. Компании, внедрившие практику ESG, продемонстрировали устойчивость во время пандемии COVID-19, поскольку они были лучше подготовлены к преодолению сбоев в работе и снижению рисков. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com ESG и санкционное давление на Россию в 2022 году Влияние санкций 2022 года на российскую экономику, включая ВВП, инфляцию и торговые отношения, вызывает беспокойство у многих компаний, инвесторов и экономистов. Санкции, введенные США и ЕС, были направлены против различных секторов, включая финансовую и энергетическую отрасли, а также ряда конкретных компаний и частных лиц. Ожидается, что санкции 2022 года в отношении России окажут значительное влияние на практику ESG в стране. В рамках данного исследования мы уточняем потенциально возможное воздействия санкций на ESG в России. Воздействие на окружающую среду. Правительство России реализовало ряд экологических инициатив и задач, но прогресс в некоторых областях был медленным. Санкции 2022 года смогут помочь повысить осведомленность и принять меры по решению экологических проблем, поскольку многие компании вынуждены переоценивать свое воздействие на окружающую среду в условиях ограниченного доступа к мировым рынкам. Однако существует и риск того, что санкции могут привести к «гонке на выживание» по экологическим стандартам, поскольку бизнес стремится сократить расходы и сохранить рентабельность в условиях сокращения доходов. Это может привести к еще большему ухудшению состояния окружающей среды и негативным социальным последствиям, поскольку компании отдают предпочтение краткосрочным выгодам, а не долгосрочной устойчивости. Влияние на социальные вопросы. Санкции 2022 года также могут иметь серьезные социальные последствия в России. Поскольку доступ компаний к глобальным рынкам ограничен, существует риск того, что они будут вынуждены сокращать рабочие места и снижать заработную плату для сохранения прибыльности. Это может привести к усилению социального неравенства и усилению социальных волнений, поскольку граждане пытаются выживать в период экономических трудностей. Инвестируя в социальные программы и инициативы, компании могут помочь смягчить негативные последствия санкций и продемонстрировать свою приверженность устойчивым методам ведения бизнеса. Влияние на управление. Санкции 2022 года могут оказать существенное влияние на корпоративное управление в России. Поскольку компании сталкиваются с растущим давлением по поддержанию прибыльности в условиях ограниченного доступа к глобальным рынкам, существует риск того, что они будут отдавать предпочтение краткосрочным выгодам, а не долгосрочной устойчивости и методам надлежащего управления. При этом санкции могут также способствовать большей прозрачности и достоверности в частном секторе, поскольку компании вынуждены быть более прозрачными в своей деловой практике, чтобы сохранить доверие инвесторов и стейкхолдеров. Это могло бы способствовать повышению общего качества корпоративного управления в России и позиционированию страны как лидера в области устойчивого ведения бизнеса. Таким образом, санкции 2022 года против России окажут значительное влияние на практику ESG в стране. Мы приходим к выводу, что существуют потенциальные риски, связанные с санкциями, но у компаний сохраняются значительные возможности для того, чтобы сохранить свою приверженность вопросам ESG. Страница 34 из 49 Это указывает на то, что методы ESG могут помочь компаниям противостоять непредвиденным вызовам и неопределенностям. Можно сказать, что ESG оказывает положительное влияние на корпоративную деятельность в России. Компании, которые отдают приоритет практикам ESG, продемонстрировали финансовую устойчивость, долгосрочную устойчивость и взаимодействие с заинтересованными сторонами. Однако для поощрения внедрения практики ESG в России требуется больше информации, правил и политик. Данные свидетельствуют о том, что ESG является важным фактором для инвесторов, стремящихся максимизировать прибыль и снизить риски. Данные показывают, что компании с высокими рейтингами ESG, как правило, опережают компании с более низкими рейтингами в России, и что эффективное управление и социальная ответственность могут стимулировать инновации и создавать новые возможности. Однако, несмотря на исследования, подтверждающие связь между ESG и корпоративной эффективностью, многие российские компании по-прежнему отстают от своих мировых конкурентов, когда речь идет о раскрытии информации об ESG и эффективности. Это дает российским компаниям значительную возможность улучшить свои показатели ESG и обеспечить долгосрочную ценность для инвесторов. Страница 35 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2022, Том 9, № 4 2022, Vol 9, No 4 Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ESG и санкционное давление на Россию в 2022 году Работая вместе, чтобы добиться положительных изменений и продвигать устойчивые методы ведения бизнеса, инвесторы и стейкхолдеры помогут смягчить негативные последствия санкций и создать более устойчивое будущее для России. Следует отметить, что взаимосвязь между ESG и санкциями сложна и имеет как положительные, так и отрицательные последствия. Потенциальное влияние санкций 2022 года на практику ESG в России является огромным, и инвесторы должны знать о проблемах и возможностях, связанных с санкциями. Инвестирование в ESG остается важным фактором для Страница 36 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 36 из 49 Страница 36 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today инвесторов и, вероятно, станет еще более важным, поскольку влияние изменения климата и других проблем ESG становится все более очевидным. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling инвесторов и, вероятно, станет еще более важным, поскольку влияние изменения климата и других проблем ESG становится все более очевидным. Санкции 2022 года сильно повлияли на российскую экономику, особенно на корпорации, полагающиеся на международные рынки и глобальное финансирование. В ответ многие попытались уменьшить свою зависимость от внешних рынков и расширить свою деятельность внутри страны и в новых регионах, таких как Азия и Ближний Восток. Кроме того, некоторые российские корпорации отдают приоритет инициативам в области ESG, таким как сокращение выбросов углерода и улучшение социальных и управленческих методов, что становится все более важным для инвесторов. Однако при реализации этих инициатив возникают серьезные проблемы, в том числе отсутствие поддержки со стороны государства и отсутствие прозрачности в российской бизнес-среде. Несмотря на трудности, инвестиции ESG в Россию открывают возможности для долгосрочного роста, обусловленного растущим спросом на устойчивые энергетические и инновационные технологические продукты для решения экологических и социальных проблем. Кроме того, инвесторы могут поддержать компании, стремящиеся улучшить свою практику ESG, чтобы добиться положительных изменений. Одной из компаний-лидеров внедрения ESG в России является Сибур, ведущая нефтехимическая компания. СИБУР разработал комплексную стратегию ESG, которая включает в себя инициативы по сокращению выбросов парниковых газов, повышению энергоэффективности и повышению безопасности труда. Компания также поставила перед собой цель добиться более разнообразной и инклюзивной рабочей силы и повысить прозрачность своей отчетности ESG. Еще одна компания, отдающая приоритет практикам ESG в России, — ЛУКОЙЛ, крупная нефтегазовая компания. ЛУКОЙЛ разработал политику ESG и комитет по устойчивому развитию для надзора за инициативами в области ESG. Компания поставила перед собой цели по сокращению выбросов парниковых газов и повышению безопасности работников, а также по повышению прозрачности своей отчетности ESG. Московская биржа — одна из инвестиционных отраслей, которая отдает приоритет практикам ESG в России. Московская биржа представила ряд инициатив, связанных с ESG, включая запуск индекса устойчивости и принятие стандартов отчетности ESG. Биржа также установила партнерские отношения с инициативой Организации Объединенных Наций по устойчивым фондовым биржам (Инициатива SSE) для продвижения практики ESG среди зарегистрированных на бирже компаний. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 36 из 49 Еще одна инвестиционная отрасль, которая уделяет приоритетное внимание практике ESG в России, — это частная инвестиционная компания Baring Vostok. Baring Vostok разработала политику ESG и комитет по устойчивому развитию для наблюдения за инициативами в области ESG. Фирма поставила перед собой цели по сокращению выбросов парниковых газов и повышению прозрачности своей ESG-отчетности. Фирма также инвестирует в компании, которые стремятся улучшить свою практику ESG, и активно работает с портфельными компаниями, чтобы поддержать их инициативы ESG. Мы видим, что в настоящее время нормативно-правовая база для ESG в России все еще находится в стадии развития, однако это не мешает некоторым компаниям и инвестиционной отрасли отдавать приоритет практике ESG. Инвестируя в эти компании и отрасли, инвесторы могут поддержать развитие ESG-инициатив в России и способствовать позитивным изменениям. Российское правительство и неправительственные организации работают над продвижением практики ESG, но сталкиваются с трудностями. Правительство приняло принципы ESG и установило цели устойчивого развития, но эксперты считают, что Страница 37 из 49 Страница 37 из 49 50 CO Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today необходимы более жесткие правила и явные стимулы для бизнеса. Неправительственные организации продвигают устойчивое лесное хозяйство, рыболовство и развитие возобновляемой энергетики, но сталкиваются с финансовыми и нормативными ограничениями. Влияние санкций 2022 года на инициативы ESG до конца неясно и может создать проблемы для будущего финансирования и сотрудничества. Однако санкции могут также создать возможности для иностранных инвесторов взаимодействовать с российскими компаниями по вопросам ESG. Несмотря на ряд проблем, описанных выше, в России также есть значительные возможности для практики ESG. Например, поскольку страна работает над диверсификацией своей экономики за счет отказа от традиционных отраслей, таких как нефть и газ, появляется возможность уделить первоочередное внимание вопросам ESG при разработке новых отраслей и технологий. Это могло бы помочь позиционировать Россию как лидера в области устойчивых инноваций и привлечь инвестиции со стороны социально ответственных инвесторов. У российских компаний также есть возможность стать лидерами в своих отраслях и улучшить свой доступ к капиталу, продемонстрировав высокие показатели ESG. Поскольку инвесторы все чаще отдают приоритет факторам ESG в своих процессах принятия решений, компании, отдающие приоритет вопросам ESG, станут более привлекательными объектами для инвестиций. Это могло бы помочь улучшить общее качество корпоративного управления и практики устойчивого развития в России. Страница 36 из 49 Обращаясь в будущее, становится ясно, что практика ESG будет играть все более важную роль в российской экономике, однако разработка эффективных инициатив ESG потребует сотрудничества и участия частного и государственного секторов. Инвесторы и стейкхолдеры могут способствовать позитивным изменениям, поддерживая развитие устойчивого бизнеса, взаимодействуя с политиками и регулирующими органами, а также выступая за более сильные основы и стимулы ESG. Будущее внедрения ESG в России видится одновременно и сложным, и полным возможностей. Поскольку страна стремится сбалансировать экономический рост с экологической и социальной ответственностью, необходимо более тесное сотрудничество и взаимодействие во всех секторах. Уделяя приоритетное внимание факторам ESG и поддерживая развитие устойчивых предприятий и отраслей, инвесторы и другие заинтересованные стороны могут способствовать позитивным изменениям и созданию более устойчивого будущего. Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Практика ESG в российских организациях Что касается практик ESG, то российские организации традиционно отстают от своих зарубежных коллег. Отчасти это связано с исторической ориентацией на экономическое развитие в ущерб социальным и экологическим последствиям, а также с отсутствием осведомленности и регулятивным давлением по вопросам ESG. Тем не менее, глобальная тенденция к инвестированию в ESG и растущий спрос на ответственные методы ведения бизнеса начали влиять на российский рынок, что привело к некоторым улучшениям в практике ESG в последние годы. Одним из ключевых отличий подхода российских и зарубежных организаций к ESG является уровень прозрачности и отчетности. Многие иностранные компании и инвесторы приняли международно-признанные системы отчетности, такие как Глобальная инициатива по отчетности (GRI) или Совет по стандартам учета в области устойчивого развития (SASB), которые предоставляют стандартизированные показатели для оценки и сравнения эффективности ESG. В настоящее время немногие российские компании раскрывают Страница 38 из 49 Страница 38 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today информацию ESG по собственным формам, так как отсутствует стандартизированная система отчетности [57]. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Russian Journal of Resources, Conservation and Recycling 2022, Vol 9, No 4 https://resources.today информацию ESG по собственным формам, так как отсутствует стандартизированная система отчетности [57]. информацию ESG по собственным формам, так как отсутствует стандартизированная система отчетности [57]. Еще одна область, в которой иностранные и российские организации различаются, касается их подхода к управлению. Хотя корпоративное управление является ключевым компонентом ESG, российские компании исторически сталкивались с такими проблемами, как слабая независимость совета директоров, сделки со связанными сторонами и слабая защита прав акционеров. Это привело к обеспокоенности по поводу коррупции, конфликта интересов и отсутствия надзора. Иностранные инвесторы и организации часто больше внимания уделяют вопросам управления при оценке потенциальных инвестиций требуя улучшений в этой области. Что касается экологических и социальных факторов, то здесь также существуют различия между российскими и зарубежными организациями. Многие иностранные компании взяли на себя значительные обязательства по снижению своего воздействия на окружающую среду, например, поставили научно-обоснованные цели по сокращению выбросов парниковых газов, расширили использование возобновляемых источников энергии и внедрили инициативы в области экономики замкнутого цикла. Напротив, российские компании медленнее внедряют такую практику, многие из них по-прежнему полагаются на ископаемое топливо и занимаются экологически вредной деятельностью, такой как вырубка лесов и добыча и переработка нефти. Практика ESG в российских организациях С точки зрения социальных факторов, российские организации исторически боролись с такими проблемами, как права человека, трудовые отношения и участие общества в контроле бизнеса. Было несколько громких дел о нарушении трудовых прав в российских компаниях, плохих условиях труда. Хотя иностранные организации не застрахованы от этих проблем, на западных рынках, как правило, наблюдается большее регулирующее давление и общественный контроль, что привело к большему вниманию к социальным вопросам. Несмотря на эти различия, есть признаки того, что российские организации начинают более серьезно относиться к ESG. В последние годы несколько крупных российских компаний взяли на себя публичные обязательства по снижению своего воздействия на окружающую среду и совершенствованию своей социальной деятельности. Кроме того, наблюдается растущий интерес со стороны российских инвесторов к вопросам ESG, при этом количество фондов и продуктов ESG, доступных в России, неуклонно растет. Мы наблюдаем растущий интерес в России к вопросам ESG и усиление регуляторного давления приводят к некоторым улучшениям в подходе российских компаний к ESG. Поскольку ESG продолжает набирать обороты во всем мире, вполне вероятно, что мы увидим большее сближение практик ESG на разных рынках, включая Россию. В заключение можно сказать, что практика ESG приобретает все большее значение для российских организаций, особенно в условиях давления экономических санкций. Несмотря на то, что предстоит пройти еще долгий путь, многие организации предпринимают шаги по совершенствованию своих экологических, социальных и управленческих методов, что обусловлено рядом факторов, включая нормативные требования, давление инвесторов и растущее осознание преимуществ устойчивого и ответственного бизнеса. Некоторые из конкретных шагов, предпринимаемых российскими организациями для улучшения своей практики ESG, включают внедрение систем экологического менеджмента, развитие устойчивых цепочек поставок, совершенствование трудовых отношений, а также повышение разнообразия и открытости советов директоров. Эти усилия не только помогают снизить риски, связанные с санкциями и другими экономическими трудностями, но также создают возможности для организаций, чтобы построить более тесные отношения с заинтересованными сторонами, улучшить свою репутацию и ценность бренда, а также Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 39 из 49 Страница 39 из 49 50 CO Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today потенциально получить финансовую отдачу за счет более устойчивых и ответственных методов ведения бизнеса. потенциально получить финансовую отдачу за счет более устойчивых и ответственных методов ведения бизнеса. потенциально получить финансовую отдачу за счет более устойчивых и ответственных методов ведения бизнеса. Однако существуют также серьезные проблемы с внедрением практики ESG в России, в том числе отсутствие нормативно-правовой базы, культурные барьеры и ограниченная осведомленность и понимание концепций ESG среди стейкхолдеров. Кроме того, еще предстоит оценить влияние экономических санкций на российскую экономику и ее компании, в том числе те, которые отдают приоритет практикам ESG [24]. Несмотря на эти проблемы, растущий интерес к внедрению ESG в России свидетельствует о том, что устойчивые и ответственные методы ведения бизнеса становятся все более важной частью корпоративной культуры. Важно, чтобы компании уделяли приоритетное внимание практике ESG, а регулирующие органы разработали и внедрили четкие правила, которые стимулируют ответственную и устойчивую деловую практику. Кроме того, действия по повышению осведомленности и понимания концепций ESG среди заинтересованных сторон и преодолению культурных барьеров могут способствовать дальнейшему ускорению внедрения практики ESG в России. Выводы В этой работе мы исследовали концепцию ESG и ее значение для устойчивого развития. Автор проанализировал тенденции ESG, согласование ESG и ЦУР в деятельности компаний, рассмотрел примеры успешного внедрения ESG компаниями в различных странах, теоретическую основу ESG, связь между ESG и корпоративной эффективностью, а также экологические, социальные и управленческие факторы, влияющие на ESG. Также, было исследовано ESG в странах с развивающейся экономикой, в частности в России, рассмотрено влияние санкционного давления на ESG в 2022 году и практику ESG в российских организациях. ESG стала важным фактором для компаний, стремящихся привести свою деятельность в соответствие с целями устойчивого развития. Компании, применяющие методы ESG, часто имеют больше возможностей для привлечения и удержания клиентов, инвесторов и сотрудников. Практики ESG также помогают компаниям управлять рисками и повышать свою устойчивость к экономическим и экологическим деструктивным событиям. В последние годы ESG приобрела значительный импульс, поскольку инвесторы и потребители стали лучше осознавать влияние, которое компании оказывают на общество и окружающую среду. Эта тенденция, вероятно, сохранится, и компании, которые не смогут внедрить методы ESG, могут оказаться в невыгодном положении перед обществом. Мы рассмотрели примеры успешного внедрения ESG такими компаниями, как Patagonia, Unilever, Tesla и др., которые сделали устойчивое развитие основной частью своей бизнес-стратегии. Они продемонстрировали, что методы ESG могут улучшить финансовые показатели, репутацию бренда и взаимодействие с заинтересованными сторонами. В ходе нашего анализа было установлено, что ESG является мощным инструментом для компаний и стран в стремлении к устойчивому развитию. Тенденции ESG говорят о том, что инвесторы и заинтересованные стороны все чаще требуют от компаний ответственной и устойчивой деятельности, которая соответствует ЦУР. Компании, которые успешно внедряют ESG, не только выполняют свои этические и социальные обязательства, но и получают финансовые выгоды, улучшая управление рисками и укрепляя свою репутацию. Мы уверены, что ESG является мощным двигателем инноваций, эффективности и устойчивости для любого общества [58]. Страница 40 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Теоретическая основа ESG подчеркивает взаимосвязь и взаимозависимость экологических, социальных и управленческих факторов, которые требуют комплексных и системных решений. Структуры ESG обеспечивают комплексный и целостный подход к оценке и улучшению корпоративной эффективности, который выходит за рамки традиционных финансовых показателей. ESG — это не поверхностная или модная деятельность, а фундаментальная и стратегическая задача, требующая лидерства, сотрудничества и открытости. Связь между ESG и корпоративной эффективностью показывает, что компании, отдающие приоритет ESG, превосходят своих конкурентов с точки зрения прибыльности, производительности и создания ценности в долгосрочной перспективе. Факторы ESG могут быть существенными, что означает, что они оказывают значительное влияние на финансовые показатели компании, и нематериальными, что означает, что они отражают нематериальные активы компании, такие как репутация и корпоративная культура. Поэтому ESG — это не затраты, а инвестиции в будущее компании. Факторы окружающей среды, влияющие на ESG, включают изменение климата, истощение природных ресурсов, загрязнение и управление отходами. Компании, которые устраняют эти факторы с помощью методов ESG, могут уменьшить свое воздействие на окружающую среду, повысить эффективность использования ресурсов и внести свой вклад в глобальную устойчивость. Социальные факторы, влияющие на ESG, включают права человека, трудовые отношения, разнообразие и участие общества. Компании, которые учитывают эти факторы, могут повысить общественное одобрение своей деятельности, привлекать и удерживать таланты, способствовать положительному влиянию на общество. Факторы управления, влияющие на ESG, включают этику, управление рисками и корпоративное управление. Компании, которые влияют на эти факторы с помощью подходов ESG, могут повысить свою ответственность перед стейкхолдерами, открытость и эффективность, снизить юридические и репутационные риски [59]. ESG в странах с развивающейся экономикой, таких как Россия, представляет как проблемы, так и возможности. С одной стороны, внедрение ESG еще не получило широкого распространения или применения в этих странах, что может привести к экологическому и социальному ущербу, слабому корпоративному управлению. С другой стороны, практики ESG могут обеспечить конкурентное преимущество компаниям, которые их применяют, и способствовать их устойчивому развитию. Россия, в частности, сталкивается с санкционным давлением, которое ограничивает доступ к капиталу, технологиям и рынкам, увеличивает ее политические и экономические риски. Санкционное давление может стать проблемой для применения ESG в России. Компании, попавшие под санкции, могут столкнуться с трудностями в привлечении инвестиций и внедрении устойчивых методов ведения бизнеса. Несмотря на это, ESG может помочь российским организациям снизить эти риски, повысить свою устойчивость и продемонстрировать приверженность ответственным и устойчивым методам ведения бизнеса [60]. Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling В заключение можно сказать, что практика ESG становится все более важной для компаний, стремящихся привести свою деятельность в соответствие с целями устойчивого развития. Компании, применяющие методы ESG, скорее всего, будут иметь больше возможностей для привлечения и удержания клиентов, инвесторов и сотрудников, которые ценят устойчивость. Практика ESG также может помочь компаниям управлять рисками и повысить свою устойчивость к экономическим и экологическим потрясениям [61]. Применяя методы ESG, компании могут создавать долгосрочную ценность, заботясь об интересах своих заинтересованных сторон. Страница 40 из 49 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling 2022, Том 9, № 4 2022, Vol 9, No 4 ISSN 2500-0659 https://resources.today Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling Мы считаем, что ESG — это не просто тенденция, а необходимый шаг к достижению устойчивого будущего для всех. 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Страница 48 из 49 Страница 48 из 49 Kudryashov Alexander Leonidovich Financial University under the Government of the Russian Federation, Moscow, Russia E-mail: akudryashov2017@gmail.com ORCID: https://orcid.org/0000-0002-0321-1028 RSCI: https://www.elibrary.ru/author_profile.asp?id=923182 Kudryashov Alexander Leonidovich Financial University under the Government of the Russian Federation, Moscow, Russia E-mail: akudryashov2017@gmail.com ORCID: https://orcid.org/0000-0002-0321-1028 RSCI: https://www.elibrary.ru/author_profile.asp?id=923182 Keywords: sustainable development; ESG concept; ESG implementation; sanctions; ESG transformation; Russian economy; sustainable business 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2022, Том 9, № 4 2022, Vol 9, No 4 Интернет-журнал «Отходы и ресурсы» Russian Journal of Resources, Conservation and Recycling ISSN 2500-0659 https://resources.today Global ESG transformation and sustainable development trends of Russian companies under sanctions pressure Abstract. The article discusses the concept of environmental, social and governance factors (ESG) and their importance for sustainable development. In recent years, this concept has gained significant popularity as a basis for assessing the non-financial performance of companies, while investors and stakeholders are increasingly aware of the importance of ESG factors in creating long-term value, risk management and stakeholder engagement. The paper explores key trends and challenges in the current business environment, such as the proliferation of reporting forms, the need for more standardized and comparable data on ESG factors, and the importance of stakeholder participation in decision-making on the implementation of ESG policies. The author emphasizes the correspondence between the goals of sustainable development and ESG factors in the activities of companies. The study provides examples of successful implementation of ESG by companies from different sectors of the economy. The theoretical basis of the ESG concept is explored, outlining the key principles, as well as the connection with corporate performance. ESG factors are identified as key drivers of the sustainability process, and this article provides an in-depth analysis of each of these factors, highlighting the challenges and opportunities they present to implement this concept. The article also looks at the specific challenges and opportunities for ESG in emerging economies, in particular Russia, where the transition to a more resilient economy is complicated by geopolitical tensions and sanctions pressure in 2022. The article explores the issue of sanctions creating risks and opportunities for companies and investors seeking to introduce ESG into business practice. An analysis of ESG implementation practices in Russian organizations based on some case studies and empirical data is provided to illustrate the current state of ESG implementation in the country. The author identified the main problems and opportunities for companies in Russia. Overall, the article presents evidence-based conclusions and suggestions for companies, investors, policy makers and society to inform their efforts to create a more sustainable future. The author emphasizes the importance of continuous learning, the adaptation of ESG policies for business, cooperation between companies and stakeholders for the effective implementation of environmental, social and governance factors that have the potential to shape sustainable development in the 21st century. Keywords: sustainable development; ESG concept; ESG implementation; sanctions; ESG transformation; Russian economy; sustainable business ESG Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 49 из 49 Страница 49 из 49 50ECOR422 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com
https://openalex.org/W4294145292	https://www.ajnr.org/content/ajnr/43/10/1403.full.pdf	English	null	Does Gadolinium Deposition Lead to Metabolite Alteration in the Dentate Nucleus? An MRS Study in Patients with MS	American journal of neuroradiology	2,022	cc-by	6,830	ABSTRACT BACKGROUND AND PURPOSE: Repeat contrast-enhanced MR imaging exposes patients with relapsing-remitting MS to frequent administration of gadolinium-based contrast agents. We aimed to investigate the potential metabolite and neurochemical altera- tions of visible gadolinium deposition on unenhanced T1WI in the dentate nucleus using MRS. MATERIALS AND METHODS: This prospective study was conducted in a referral university hospital from January 2020 to July 2021. The inclusion criteria for case and control groups were as follows: 1) case: patients with relapsing-remitting MS, visible gadolinium deposition in the dentate nucleus (ribbon sign), .5 contrast-enhanced MR images obtained; 2) control 1: patients with relapsing- remitting MS without visible gadolinium deposition in the dentate nucleus, .5 contrast-enhanced MR images obtained; 3) control 2: patients with relapsing-remitting MS without visible gadolinium deposition in the dentate nucleus, ,5 contrast-enhanced-MR images obtained; and 4) control 3: adult healthy individuals, with no contrast-enhanced MR imaging. Dentate nucleus and pontine single-voxel 12 12 12 MRS were analyzed using short TEs. RESULTS: Forty participants (10 per group; 27 [67.5%] female; mean age, 35.6 [SD, 9.6] years) were enrolled. We did not detect any sig- niﬁcant alteration in the levels of NAA and choline between the studied groups. The mean concentrations of mIns were 2.7 (SD, 0.73) (case), 1.5 (SD, 0.8) (control 1), 2.4 (SD, 1.2) (control 2), and 1.7 (SD, 1.2) (control 3) (P ¼ .04). The mean concentration of Cr and mIns (P ¼ .04) and the relative metabolic concentration (dentate nucleus/pons) of lipid 1.3/Cr (P ¼ .04) were signiﬁcantly higher in the case-group than in healthy individuals (controls 1–3). Further analyses compared the case group with cumulative control 1 and 2 groups and showed a signiﬁcant increase in lactate (P ¼ .02), lactate/Cr (P ¼ .04), and Cr (dentate nucleus/pons) (P ¼ .03) in the case group. CONCLUSIONS: Although elevated concentrations of Cr, lactate, mIns, and lipid in the dentate nucleus of the case group indicate M C M S is a chronic demyelinating, neurodegenerative disease of the CNS that affects .2 million people worldwide.1 Contrast-enhanced (CE) MR imaging is extensively used and is vital for early diagnosis and follow-up of patients with MS because it can help fulfill the “dissemination in time” McDonald criteria. Received February 3, 2022; accepted after revision July 1. From the Departments of Radiology (M.M., S.K., M.-M.M.N., K.F., H.N., M.S., H.S., H.H.) and Neurology (M.H.H., A.A.), Tehran University of Medical Sciences, Tehran, Iran; Department of Radiology (A.M.), Iran University of Medical Sciences, Tehran, Iran; Hass School of Business (F.M.), University of California, Berkeley, Berkeley, California; Khoury College of Computer Sciences (H.K.), Northeastern University, Boston, Massachusetts; Department of Radiology and Biomedical Imaging (M.E.A.), Yale School of Medicine, New Haven, Connecticut; and Department of Radiology (D.M.Y.), Johns Hopkins University School of Medicine, Baltimore, Maryland. M. Mohammadzadeh and S. Kolahi had equal contribution as co-ﬁrst authors. Please address correspondence to David M. Yousem, MD, MBA, Johns Hopkins University School of Medicine, 600 N. Wolfe St, Phipps B100, Baltimore, MD 21287; e-mail: dyousem1@jhu.edu An MRS Study in Patients with MS Nucleus? Metabolite Alteration in the Dentate Does Gadolinium Deposition Lead to Harirchian, A. Azimi, M.E. Adin and D.M. Yousem Mohebi, H. Komaki, H. Sharifian, H. Hashemi, M.H. Firouznia, H. Naghibi, A. Mohammadzadeh, M. Shakiba, F. M. Mohammadzadeh, S. Kolahi, M.-M. Mehrabi Nejad, K. of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/43/10/1403 https://doi.org/10.3174/ajnr.A7623 doi: 2022, 43 (10) 1403-1410 AJNR Am J Neuroradiol ORIGINAL RESEARCH ADULT BRAIN ORIGINAL RESEARCH ADULT BRAIN Indicates article with online supplemental data. http://dx.doi.org/10.3174/ajnr.A7623 ABBREVIATIONS: CE ¼ contrast-enhanced; DN ¼ dentate nucleus; GBCA ¼ gadolinium-based contrast agent; Gd ¼ gadolinium; RRMS ¼ relapsing-remitting MS; SI ¼ signal intensity Indicates article with online supplemental data. ABSTRACT As a result, patients with MS are frequently exposed to the admin- istration of gadolinium-based contrast agents (GBCAs) required for CE-MR imaging.2 GBCAs have been considered very safe since the US Food and Drug Administration’s approval in 1988. The first report of gadolinium deposition as a source of the high signal in- tensity (SI) in the dentate nucleus (DN) was published in 20143 and has led to extensive investigation of this phenomenon. Initially in patients with MS, radiologists linked the suspicious increase in SI on T1WI in the deep gray matter to MS itself,4 but this etiology has since been discredited. The observed DN hyperintensity on unenhanced T1-weighted images was specifically attributed to the secondary-progressive disease MS subtype, but now there is no support for an independent association between MS and a hyper- intense DN. http://dx.doi.org/10.3174/ajnr.A7623 1403 AJNR Am J Neuroradiol 43:1403–10 Oct 2022 www.ajnr.org The GBCAs are either linear or macrocyclic based on their type of polyamino-polycarboxylic ligand. Each of these groups is composed of nonionic and ionic classes of agents based on their electric charge. Despite the rigid cage wrapping around the Gd31 in macrocyclic GBCAs, this cage is more flexible in the linear type. Because the agent’s cage contributes to its stability, linear- type GBCAs are less stable than macrocyclic agents. As a general concept, the ionic macrocyclic and nonionic linear chelates are the most and least stable GBCAs, respectively.5,6 The thermody- namic and kinetic stabilities of GBCAs are the main concerns in their safety because the stability of the gadolinium (Gd) agent determines the likelihood of its deposition.5 Although several studies have found that hyperintensity in the DN on unenhanced T1-weighted images has the highest association with previous administration of linear GBCAs,7,8 macrocyclic GBCAs can also lead to DN hyperintensity.9 The MS diagnosis was established according to an expert fel- lowship-trained neurologist based on the McDonald criteria.2 The enrolled patients were selected from a specialized MS center. This center conducts all patients’ follow-up imaging with the same pro- tocol and GBCA type (gadoterate meglumine) that is used for the imaging in this study. No patient had prior GBCA exposure before the MS diagnosis. The inclusion criteria for case and control groups were as follows: 1) Case: adult patients with relapsing-remitting MS (RRMS) with visually detectable hyperintensity in the DN (ribbon sign; DN/pons SI ratio, .1) who underwent .5 CE-MRIs. Image Acquisition and Interpretation MR Imaging Protocol. All patients were in remission at the time of the examination, and no patient had MS plaques involving the infratentorial region. Patients were scanned on a 3T MR imaging machine (Discovery MR750; GE Healthcare) using a 32-channel phased array head coil. The protocol included axial, coronal, and sagittal T2 FSE: TR ¼ 3000 ms, TE ¼ 106 ms, matrix size ¼ 352 256, FOV ¼ 230 230, section thickness ¼ 5 mm, flip angle ¼ 142°; and an axial 3D T2-weighted angiography gradient- echo sequence: TR ¼ 75ms, TE ¼ 50ms, section thickness ¼ 2mm, matrix size ¼ 320 224, and FOV ¼ 220 220. We used an axial T1 spin echo: TR ¼ 600ms, TE ¼ 10ms, section thickness ¼ 5mm, matrix size ¼ 352 256, FOV¼ 230 230 to detect high intensity in the DN. A standard dose (0.1mmol/kg) of a macrocy- clic GBCA (gadoterate meglumine) was administered to all patients during each MR imaging acquisition. To date, there is no study, to our knowledge, investigating the brain metabolite alterations following GBCA exposure in humans. A promising and relatively accessible technique is MRS because it has the unique ability to provide the chemical characteristics of MR imaging–visible lesions as well as normal-appearing brain tis- sue.18 Therefore, we aimed to investigate the potential metabolite alterations in visually detectable Gd deposition in the DN follow- ing multiple prior GBCA exposure in patients with MS using a case-control design. Preclinical and clinical studies have reported in vitro neurotoxic effects of Gd exposure due to impairment of mitochondrial function and mitochondrial metabolic alterations subsequent to gadolinium deposition.19,20 We hypothesize that Cr and lactate will show alterations due to disturbed mitochondrial metabolism. We also hoped this study would contribute to the understanding of the molecular and cellular ramifications of Gd deposition. Two fellowship-trained neuroradiologists (with 22 and 10years of experience), blinded to clinical data, independently reviewed the images. Unenhanced T1WIs were used to draw a 4-mm2 ROI in the middle of the pons and around the area with the highest inten- sity in the DN. The DN/pons SI ratio was calculated by dividing the mean SI within the DN by the mean SI within the central pons. Any disagreement was resolved by consensus. ABSTRACT 2) Control 1: adult patients with RRMS without visually detecta- ble hyperintensity in the DN who underwent .5 CE-MRIs. 3) Control 2: adult patients with RRMS without visually detecta- ble hyperintensity in the DN who underwent ,5 CE-MRIs. 4) Control 3: healthy adult individuals with no history of CE- MRI. Several studies have investigated the safety of GBCAs in pediat- ric10,11 and adult patients.12-14 The pooled evidence clearly supports the idea that GBCA administration is significantly, positively, and directly correlated with increased SI in the DN and globus pallidus on unenhanced T1WI assessments.8,9 The aforementioned correla- tion was detected regardless of the patient’s renal function.6 Studies on patients with MS have also supported this evidence.15,16 How- ever, histologic findings on brain postmortem examinations in human and animal models have not detected any damage, despite the confirmed accumulation of gadolinium in affected structures.17 Quantitative assessments of T1- and T2-weighted imaging also sup- ported an association with previous GBCA administration and gad- olinium accumulation, particularly in gray matter structures.13 Exclusion criteria were the following: 1) any MS plaques involving the infratentorial region, 2) any other previously diag- nosed neurologic diseases, 3) impaired renal function (estimated glomerular filtration rate of ,60mL/min), 4) a history of cranial irradiation, or 5) any other disease requiring CE- MR imaging. DN-to-Pons Ratio Values The relative metabolite concentrations and calculated ratios in DN/pons in all 4 studied groups are presented in Table 3. Among all variables, there were 2 statistically significant observations. First, the mean concentration of Cr on short TE (P ¼ .03) was signifi- cantly higher in case participants than in a group of control 1 and 2 participants. Second, lipid 1.3/Cr (P ¼ .04) was sig- nificantly higher in the case group than in control 3. ol 2 10) Control 3 (n = 10) D, 10.0) 30.7 (SD, 2.9) %) 3 (30%) Table 1: Demographic features of participants Variable Case (n = 10) Control 1 (n = 10) Control 2 (n = 10) Control 3 (n = 10) Age (mean) (yr) 43.4 (SD, 8.6) 36.7 (SD, 10.2) 31.7 (SD, 10.0) 30.7 (SD, 2.9) Sex (female) 8 (80%) 7 (70%) 9 (90%) 3 (30%) Table 1: Demographic features of participants Table 2: Absolute mean metabolite concentrations and calculated ratios in the DN in all 4 studied groups Studied Groups Comparis ute mean metabolite concentrations and calculated ratios in the DN in all 4 studied groups Table 2: Absolute mean metabolite concentrations and calculated ratios in the DN in all 4 studied groups Variable Studied Groups Comparisons (P Values) Case Control 1 Control 2 Control 3 Among 4 Groups Case vs Control 3 Case vs. Participant Characteristics Forty participants (10 in each group; 27 [67.5%] females; mean age , 35.6 [SD, 9.6] years; range, 20–57 years) were enrolled in this prospective study. The participants’ demographic features are presented in Table 1. Considering that all patients were scanned with the same protocol and GBCA type, any reported differences are not due to different GBCA agents or scanning techniques. DN/pons SI ratios on T1WI were 1.04, 0.98, 0.94, and 0.94 in case and control 1–3 groups, respectively (P , .001), indicating appropriate selection of case and control groups. MRS performed with short TE minimizes signal loss due to transverse relaxation, allowing higher quantification precision than long TE.18 However, on short TE, broad macromolecule resonances are presented, which could be a confounding factor for quantifica- tion.18 Because macromolecule lipid resonance and lactate resonan- ces are closely located and might overlap each other, the macromolecule confounding problem mainly occurs at the spectra of lactate.21 To confirm that the observed lactate change on short TE was clearly due to lactate, we compared the short TE and long TE spectra with TE ¼ 144, in which the lactate peak was inverted. Then, the lactate quantification was performed on short TE spectra. DN Values The absolute metabolite concentrations and calculated ratios in the DN in all 4 studied groups are presented in Table 2 and Figs 1 and 2. mIns was the only metabolite that was significantly different in all groups. The mean concentrations of mIns were 2.7 (SD, 0.73) (case), 1.5 (SD, 0.8) (control 1), 2.4 (SD, 1.2) (control 2), and 1.7 (SD, 1.2) (control 3) (P ¼ .04). We observed intergroup differences in other metabolites as well, though they were not homogeneous in all 2 2 comparisons. The mean concentrations of Cr on short TE (P ¼ .04) and mIns (P ¼ .04) were significantly higher in the case group compared with healthy individuals (control 3). Further anal- yses compared the case group with an aggregated group of control 1 and 2 participants and showed significant elevation of lactate on short TE (P ¼ .02) and lactate/Cr on short TE (P ¼ .04) in the case group and elevation of Glx/Cr (P ¼ .04) in the control groups. AJNR Am J Neuroradiol 43:1403–10 Oct 2022 www.ajnr.org 1405 MRS Evaluation This prospective case-control study was reviewed and approved by Institutional Review Board of Imam Khomeini Hospital Complex and was conducted in a quaternary referral university hospital from January 2020 to July 2021. After all patients were informed about the study goals and assured that their medical data would remain confidential, consent was obtained. Patients were also assured that unwillingness to participate in the study or leaving the study would not affect their medical care. MRS imaging with the use of pointed-resolved spectroscopy was per- formed with the following configurations: long TE ¼ 144 and short TE ¼ 35 ms, FOV ¼ 240 240, voxel size ¼ 12 12 12mm, TR1 ¼ 500 ms, and number of samples per spectrum ¼ 1024. A chemical shift selective suppression technique was used to suppress the water signal. Out-of-volume saturation bands were applied to suppress signal excited outside the FOV. No lipid suppression was applied. Manual shimming was performed. Mohammadzadeh Oct 2022 www.ajnr.org 1404 Statistical Analysis We performed the analyses in SPSS for Windows, Version 18 (IBM). All P values , .05 were considered statistically significant. Descriptive data are presented in mean (SD) for continuous vari- ables and frequency and percentage for categoric variables. To assess whether the recorded data have a normal distribution, we performed the Kolmogorov-Smirnov test. We conducted the comparisons by the following: 1) 1-way ANOVA with Tukey post hoc test and an independent 2-tailed sample t test for contin- uous variables with normal distribution and the relevant degree of freedom, 2) the Kruskal-Wallis test and the Mann-Whitney U test for the continuous variable with a significant lack of normal- ity, and 3) a x 2 test for nominal variables. RESULTS Single-voxel 12 12 12 mm MRS was performed for the evaluation of the DN and pons area. Quantification-procedure MRS imaging data were obtained in TARQUIN (Version 4.3.10; https:// sourceforge.net/projects/tarquin/) software. Quantification results for each metabolite were analyzed in the associated optimal TE with the metabolite peaks as follows: choline at 3.2ppm; NAA at 2.02ppm; Cr at 3ppm; lactate at 1.3ppm; lipid at 0.9- 1.3ppm; Glx at 2.1–2.4ppm; and mIns at 3.5ppm (Online Supplemental Data). a Statistically signiﬁcant. DISCUSSION metabolic activity in Gd-deposition regions.28 The authors found 16% and 27% lower [18F] FDG uptake in the DN and globus pal- lidus, respectively, of individuals who received GBCAs.28 Most interesting, a recent study used untargeted mass spectroscopy– based metabolomic analyses to investigate the plasma metabolite alterations after Gd administration.20 Compared with healthy con- trols, patients with Gd-deposition disease showed differences in 45 biochemicals, mostly related to mitochondrial function, similar to our findings in the brain.20 Our findings support the hypothesis that Gd deposition could be related to metabolite alterations in the DN. Despite evidence of metabolite changes among the studied groups, these findings were minor and their clinical importance should be investigated in further studies. To elaborate, we did not detect any significant alteration in the levels of NAA and Cho among the studied groups, suggesting the absence of neuronal tissue damage in the DN with visually detectable Gd deposition. Nonetheless, the lev- els of mIns, Cr, lactate, and lipid showed a significant increase in the case group, which can be interpreted as a change in cellular metabolism as discussed below. We propose that the observed increased levels of Cr in case par- ticipants might indicate a disturbance in cellular energy homeosta- sis. Cr transmission through the blood-brain barrier is minimal, and most Cr is produced in the brain using the arginine:glycine amidi- notransferase and guanidinoacetate methyltransferase enzymes.29 Cr is the essential component of high-energy phosphate metabo- lism (pCr1ADP$Cr1ATP) and plays a vital role in channeling energy into the cytosol to maintain cellular energy homeostasis.30 Therefore, because MRS measures both Cr and creatine phos- phate, compromised cellular energy production due to Gd accu- mulation could be caused by the responsive up-regulation of Cr as the substrate to compensate for an altered cellular energy system. Two studies by McDonald et al22,23 used inductively coupled plasma mass spectrometry and detected Gd deposition in the en- dothelial wall, neural tissue interstitium, and nuclei of neurons in the absence of any gross histologic changes. The concept of Gd crossing the blood-brain barrier and being deposited in neural nuclei raises concern about the potential cytotoxicity of Gd. DN-to-Pons Ratio Values Controls 1 and 2 NAA 43.0 (SD, 7.3) 44.8 (SD, 11.7) 45.3 (SD, 10.5) 36.7 (SD, 10.8) .24 .15 .59 Choline 32.8 (SD, 9.7) 25.7 (SD, 11.5) 28.8 (SD, 5.8) 23.5 (SD, 11.8) .20 .07 .27 Cr on short TE 11.6 (SD, 1.3) 9.3 (SD, 4.3) 10.1 (SD, 1.8) 8.9 (SD, 3.3) .21 .04a .06 Lactate on short TE 4.6 (SD, 2.2) 2.3 (SD, 2.1) 2.1 (SD, 1.3) 3.2 (SD, 2.8) .15 .33 .02a mIns 2.7 (SD, 0.7) 1.5 (SD, 0.8) 2.4 (SD, 1.2) 1.7 (SD, 1.2) .04a .04a .51 Glx 3.5 (SD, 1.7) 3.7 (SD, 1.7) 4.9 (SD, 1.8) 4.5 (SD, 2.7) .36 .35 .10 Lipid 0.9 4.8 (SD, 2.0) 3.9 (SD, 2.4) 4.1 (SD, 1.8) 3.5 (SD, 2.2) .65 .22 .44 Lipid 1.3 5.2 (SD, 2.7) 5.8 (SD, 4.1) 6.5 (SD, 3.6) 5.7 (SD, 3.0) .92 .73 .42 NAA/Cr 1.7 (SD, 0.5) 2.1 (SD, 0.9) 1.6 (SD, 0.6) 2.8 (SD, 2.0) .10 .11 .73 Lactate/Cr on short TE 0.4 (SD, 0.2) 0.3 (SD, 0.2) 0.2 (SD, 0.2) 0.3 (SD, 0.2) .25 .50 .04a mIns/Cr 0.2 (SD, 0.1) 0.2 (SD, 0.2) 0.2 (SD, 0.1) 0.2 (SD, 0.1) .68 .16 .99 Glx/Cr 0.3 (SD, 0.1) 0.5 (SD, 0.3) 0.5 (SD, 0.2) 0.5 (SD, 0.3) .35 .08 .04a Lipid 0.9/Cr 0.4 (SD, 0.3) 0.4 (SD, 0.2) 0.4 (SD, 0.1) 0.4 (SD, 0.1) .91 .50 .76 Lipid 1.3/Cr 0.4 (SD, 0.2) 0.9 (SD, 1.0) 0.6 (SD, 0.4) 0.8 (SD, 0.4) .43 .05 .19 a Statistically signiﬁcant. FIG 1. 1H-MR spectra from the DN in 1 patient from both case and control groups. Red lines represent the TARQUIN ﬁts. MRS at TE ¼ 35 ms shows elevated mIns peaks at 3.5 ppm, creatine peaks at 3 ppm, lactate peaks at 1.3 ppm, and Glx peaks at 2.1–2.4 ppm. FIG 1. 1H-MR spectra from the DN in 1 patient from both case and control groups. Red lines represent the TARQUIN ﬁts. MRS at TE ¼ 35 ms shows elevated mIns peaks at 3.5 ppm, creatine peaks at 3 ppm, lactate peaks at 1.3 ppm, and Glx peaks at 2.1–2.4 ppm. 6 Mohammadzadeh Oct 2022 www.ajnr.org DISCUSSION To this end, preclinical in vitro studies provided insight about the time- and dose-dependent cytotoxic and neurotoxic mechanism of Gd through disturbance of mitochondrial function and oxida- tive stress.19,24-26 The authors speculated that Gd, as a calcium an- tagonist due to their similar atomic radius, can interrupt the mitochondrial calcium metabolism leading to cellular death.27 Lactate is the product of anaerobic glycolysis and increases in stroke, encephalopathies, lactic acidosis, neonatal hypoxia, and mitochondrial myopathies.18 Neuronal metabolism appears to be mostly oxidative, and astrocytic metabolism is glycolytic according to the hypothesis of astrocyte-neuron lactate shuttle (Fig 3).31-33 Astrocytes take up glucose through the glucose transporter 1 and metabolize it to lactate. Lactate, then, is conveyed to the outside of the astrocytes and is captured by neurons via monocarboxylate transporters. Neurons oxidize intracellular lactate to pyruvate and metabolize it through the oxidative phosphorylation pathway in the mitochondria.31-33 Impaired mitochondrial function and the subsequent oxidative phosphorylation in neurons can result in accumulation of lactate. Therefore, the observed increased lactate Feng et al19 observed that Gd exposure is associated with mito- chondrial membrane depolarization, caspase-3 activation, cyto- chrome C release, lactate dehydrogenase increase, intracellular reactive oxygen species increase, adenosine triphosphate synthesis decrease, and subsequent DNA fragmentation. All the aforemen- tioned cellular mechanisms indicate mitochondrial dysfunction and oxidative stress leading to neuronal cell apoptosis. The cellular studies of GBCA exposure in humans could help to predict its potential long-term clinical consequences. However, there is only 1 study using PET/CT in human subjects that has investigated the 1406 FIG 2. Boxplots of the metabolites. Differences between case and control groups. FIG 2. Boxplots of the metabolites. Differences between case and control groups. etabolite concentrations and calculated ratios in DN/pons in all 4 studied groups Table 3: Relative mean metabolite concentrations and calculated ratios in DN/pons in all 4 studied groups Studied Groups Compari Table 3: Relative mean metabolite concentrations and calculated ratios in DN/pons in all 4 studied groups Variable Studied Groups Comparisons (P Values) Case Control 1 Control 2 Control 3 Among 4 Groups Case vs. AJNR Am J Neuroradiol 43:1403–10 Oct 2022 www.ajnr.org 140 in the case group might indicate the existence of an impaired mito- chondrial energy environment. DISCUSSION Control 3 Case vs Controls 1 and 2 NAA 0.7 (SD, 0.1) 1.1 (SD, 0.3) 0.8 (SD, 0.2) 0.9 (SD, 0.5) .13 .38 .36 Choline 0.8 (SD, 0.3) 0.8 (SD, 0.4) 0.9 (SD, 0.3) 0.8 (SD, 0.4) .98 .97 .70 Cr on short TE 1.4 (SD, 0.5) 1.1 (SD, 0.6) 1.0 (SD, 0.2) 1.8 (SD, 2.5) .54 .61 .03a Lactate on short TE 4.3 (SD, 7.4) 1.5 (SD, 2.0) 1.0 (SD, 1.6) 0.2 (SD, 0.4) .32 .25 .26 mIns 0.9 (SD, 0.5) 0.5 (SD, 0.4) 1.3 (SD, 1.5) 1.1 (SD, 1.2) .35 .69 .42 Glx 1.2 (SD, 0.8) 1.0 (SD, 0.6) 1.3 (SD, 1.0) 1.3 (SD, 0.6) .84 .72 .81 Lipid 0.9 1.3 (SD, 0.8) 1.1 (SD, 0.7) 0.8 (SD, 0.5) 1.3 (SD, 1.0) .43 .96 .13 Lipid 1.3 1.4 (SD, 1.0) 1.0 (SD, 1.3) 1.4 (SD, 1.0) 1.0 (SD, 0.5) .74 .33 .93 NAA/Cr 0.7 (SD, 0.2) 0.8 (SD, 0.4) 0.6 (SD, 0.3) 0.9 (SD, 0.5) .29 .44 .15 Lactate/Cr on short TE 0.2 (SD, 0.1) 0.2 (SD, 0.1) 0.1 (SD, 0.1) 0.2 (SD, 0.1) .95 .56 .61 mIns/Cr 0.6 (SD, 0.3) 0.4 (SD, 0.2) 1.3 (SD, 1.5) 0.7 (SD, 0.5) .11 .56 .20 Glx/Cr 1.1 (SD, 0.6) 0.9 (SD, 0.6) 1.5 (SD, 1.5) 1.2 (SD, 0.8) .67 .58 .48 Lipid 0.9/Cr 1.0 (SD, 0.6) 1.0 (SD, 0.5) 0.8 (SD, 0.4) 1.0 (SD, 0.7) .68 .83 .33 Lipid 1.3/Cr 1.5 (SD, 0.6) 1.3 (SD, 0.6) 1.8 (SD, 1.0) 0.8 (SD, 0.5) .17 .04a .45 a Statistically signiﬁcant. Comparisons (P Values) neurotransmission processes. Glutamate is released from the presyn- aptic neuron to stimulate glutamate receptors on the postsynaptic neuron. It enters the astrocytes through the synaptic gap via sodium (Na)-dependent excitatory amino acid activating transporters. Then, in the case group might indicate the existence of an impaired mito- chondrial energy environment. We also observed lower Glx levels in the case group. Glx is a mix- ture of similar amino acids that contribute to excitatory-inhibitory 1407 FIG 3. The astrocyte-neuron lactate shuttle and glutamate-glutamine cycles. After traveling through the endothelial cells, glucose (Glc) enters the astrocytes via Glc transporter 1 (GlcT1). It is metabolized to pyruvate and then lactate by lactate dehydrogenase (LDH5). The lactate is carried out- side the astrocyte and inside the neuron via the monocarboxylate transporters (MCTs). The lactate inside the neuron is metabolized in the oxygen pathway. DISCUSSION Glutamate (Glu) molecules released from presynaptic neurons are transported into the astrocytes through Na1-dependent channels. In astrocytes, the Glu is transformed into Glutamine (Gln) or a-ketoglutarate to perform oxygen metabolism in the Krebs cycle. ATP indicates adeno- sine triphosphate; ADP, adenosine di-phosphate; P, phosphate; GluR, glutamate receptor; OH/HCO3, hydroxide/bicarbonate; K+, potassium; GLT, glutamate transporter; PGK, phosphoglycerate kinase; ATPase, adenosine triphosphatase. Mohammadzadeh Oct 2022 www.ajnr.org CONCLUSIONS Apart from the proposed notions, there is another potential mechanism behind the accumulation of these metabolites. There is a common understanding that mIns and Cr are the main osmolytes in the brain, which undergo alteration under chronic osmotic changes.38 For instance, sodium-myo-inositol cotransporter-1 in the cortical astrocytes of rats was observed to up-regulate under a chronic hyperosmolar situation.39 Therefore, chronic Gd deposition in the neural tissue interstitium could result in a hyperosmolar con- dition that induces the production of these osmolytes. Such hyper- osmolarity has not been observed at clinical levels to date. We found elevated concentrations of Cr, lactate, mIns, and lipid in the DN of patients with visible Gd deposition on their unenhanced T1WIs, indicating a metabolic disturbance in the affected patients. Our findings confirm previous studies implicating a potential adverse effect of Gd deposition on mitochondrial membrane and function. However, because we observed normal levels of NAA and choline in the DN, neuronal damage might be disavowed. Nevertheless, we require further clinical correlation and support for this notion. We recommend that clinicians remain cautious in their use of Gd, and that researchers should build on these prelimi- nary findings by enrolling patients in a study with a larger sample size, with controlled and randomized design and full clinical assessment of the patients. y Our findings provide preliminary insight about a potential neurochemical alteration in patients with Gd deposition. Although we observed some degree of metabolite change in association with cellular metabolism and mitochondrial function, there were no significant changes in NAA and choline that would indicate the existence of any major neuronal damage. The authors posit that Gd deposition interrupts the mitochondrial function and results in some minor metabolite changes that are kept at the minimum level through the regulatory and compensatory cellular mecha- nisms. Nonetheless, the Gd deposition in the DN and evidence of malfunction of the mitochondrial energy pathway shown in ani- mal and in vitro studies raise the specter of subclinical neurotoxic- ity effects of Gd deposition. Disclosure forms provided by the authors are available with the full text and PDF of this article at www.ajnr.org. ACKNOWLEDGMENT The authors thank the patients and hospital staff for their collaboration. CONCLUSIONS Some animal studies have shown loss of motor coordination, including tremor, seizure, ataxia, and ster- eotyped movements and myoclonus.40,41 In a recent study, patients with Gd-deposition disease reported symptoms similar to those with known mitochondrial-related diseases.20 Although 2 other clinical studies reported lower verbal fluency in patients with MS with repetitive GBCA exposure,42,43 there has not been a compre- hensive study to indicate whether there are major adverse clinical effects attributed to multiple GBCA administrations in patients with MS.6,27 We recommend that clinicians limit CE-MR imaging to the necessary indications and conform with the latest guidelines from such reputable bodies as the Consortium of Multiple Sclerosis Centers,44 especially in those with chronic CNS diseases requiring repeat imaging. Moreover depth comparison of background infor control groups in addition to age and s to, extensive medical history, smoking sures, and other neurologic conditions ducting randomized controlled trials on the suggested background conditio potential clinical significance of gadolin CONCLUSIONS We found elevated concentrations of the DN of patients with visible Gd dep T1WIs, indicating a metabolic disturb Our findings confirm previous stud adverse effect of Gd deposition on m function. However, because we obse and choline in the DN, neuronal d Myo-inositol and its transporters may provide neuroprotection during or following brain ischemia36 and are often increased in cerebral infarction. Impairment of mitochondrial function and oxi- dative phosphorylation can simulate a relatively hypoxic condition that leads to the accumulation of mIns. The H1-mIns cotrans- porter is mainly expressed in the brain and is strongly stimulated by a decrease in pH.37 Subsequently, brain exposure to a substan- tial lactate load—as discussed before—and a favorable H1 gradient result in a significantly enhanced mIns uptake by the brain. Although mIns is known as a glial cell marker, studies have observed its elevated levels in neurodegenerative disorders without glial cell involvement, including Alzheimer disease, Huntington disease, and ataxia.18 Taken together, the aforementioned mIns observations and the associated clinical conditions raise concerns regarding the long-term sequelae of Gd deposition. agents could alter the brain metabolites, but we did not include the treatment regimens in this study because we were interested in any potential harm of Gd, even if it was caused by an interaction with medications in patients with MS. In addition, our assessment did not include clinical evaluations of the patients and was limited to a specific macrocyclic GBCA. Moreover, this study requires more in- depth comparison of background information between the case and control groups in addition to age and sex, including, but not limited to, extensive medical history, smoking history, environmental expo- sures, and other neurologic conditions. We highly recommend con- ducting randomized controlled trials that incorporate information on the suggested background conditions that could help reveal the potential clinical significance of gadolinium deposition. FIG 3. The astrocyte-neuron lactate shuttle and glutamate-glutamine cycles. After traveling through the endothelial cells, glucose (Glc) enters the astrocytes via Glc transporter 1 (GlcT1). It is metabolized to pyruvate and then lactate by lactate dehydrogenase (LDH5). The lactate is carried out- side the astrocyte and inside the neuron via the monocarboxylate transporters (MCTs). The lactate inside the neuron is metabolized in the oxygen pathway. Glutamate (Glu) molecules released from presynaptic neurons are transported into the astrocytes through Na1-dependent channels. In astrocytes, the Glu is transformed into Glutamine (Gln) or a-ketoglutarate to perform oxygen metabolism in the Krebs cycle. ATP indicates adeno- sine triphosphate; ADP, adenosine di-phosphate; P, phosphate; GluR, glutamate receptor; OH/HCO3, hydroxide/bicarbonate; K+, potassium; GLT, glutamate transporter; PGK, phosphoglycerate kinase; ATPase, adenosine triphosphatase. Lipid levels were also significantly higher in the case group than in healthy controls. A recent study also detected elevated lev- els of 7-dehydrocholesterol, the cholesterol precursor, in patients with Gd deposition.20 One of the degradation products of the leu- cine, 3-methylglutarate/2-methylglutarate, also showed increased levels after Gd deposition due to nicotinamide adenine dinucleo- tide phosphate/nicotinamide adenine dinucleotide phosphate– dependent enzyme impairment.20 Sterol synthesis and leucine deg- radation are connected via the mevalonate shunt, leading to increased sterol production due to 3-methylglutarate/2-methylglu- tarate accumulation.34,35 Hence, the authors believe that mitochon- drial metabolism impairment due to Gd deposition leads to elevated levels of sterol biosynthesis through the mevalonate shunt. it is metabolized into glutamine (by the glutamine synthase enzyme or a-ketoglutarate by glutamate dehydrogenase) to process further oxygen metabolism in the tricarboxylic acid cycle (Krebs cycle) in the mitochondria. Glutamine, then, is transported to neurons to complete further glutamate production using phosphate-activated glutaminase (Fig 3).31,33 Impairment of mitochondria functioning would lead to a compromised glutamate-glutamine cycle, which is dependent on the glutaminase enzyme in the mitochondria. In addi- tion, impairment of the tricarboxylic acid cycle results in less use of a-ketoglutarate and, accordingly, impairment of the transformation of glutamate to a-ketoglutarate. Taken together, a glutamate/gluta- mine decrease can also be interpreted as a marker for metabolic impairment of neuronal cells. Mohammadzadeh Oct 2022 www.ajnr.org 1408 agents could alter the brain metabolite treatment regimens in this study becau potential harm of Gd, even if it was c medications in patients with MS. In not include clinical evaluations of the specific macrocyclic GBCA. 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https://openalex.org/W4366215075	https://www.frontiersin.org/articles/10.3389/fnins.2023.1135995/pdf	English	null	Functional connectivity changes are correlated with sleep improvement in chronic insomnia patients after rTMS treatment	Frontiers in neuroscience	2,023	cc-by	7,397	OPEN ACCESS OPEN ACCESS EDITED BY Peng Xu, University of Electronic Science and Technology of China, China REVIEWED BY Liang Gong, Chengdu Second People’s Hospital, China Xiufeng Xu, The First Affiliated Hospital of Kunming Medical University, China CORRESPONDENCE Yi Guo xuanyi_guo@163.com SPECIALTY SECTION This article was submitted to Neuroprosthetics, a section of the journal Frontiers in Neuroscience RECEIVED 02 January 2023 ACCEPTED 28 March 2023 PUBLISHED 17 April 2023 1 Department of Neurology, Shenzhen People’s Hospital, The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology, Shenzhen, Guangdong, China, 2 Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, United States, 3 Hebrew Seniorlife, Hinda and Arthur Marcus Institute for Aging Research, Harvard Medical School, Boston, MA, United States, 4 Department of Geriatrics, Shenzhen People’s Hospital, The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology, Shenzhen, Guangdong, China, 5 Shenzhen Bay Laboratory, Shenzhen, Guangdong, China, 6 NHC Key Laboratory of Diagnosis and Treatment on Brain Functional Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Background: Repetitive transcranial magnetic stimulation (rTMS) has been increasingly used as a treatment modality for chronic insomnia disorder (CID). However, our understanding of the mechanisms underlying the efficacy of rTMS is limited. CITATION Zhu L, Dang G, Wu W, Zhou J, Shi X, Su X, Ren H, Pei Z, Lan X, Lian C, Xie P and Guo Y (2023) Functional connectivity changes are correlated with sleep improvement in chronic insomnia patients after rTMS treatment. Front. Neurosci. 17:1135995. doi: 10.3389/fnins.2023.1135995 Objective: This study aimed to investigate rTMS-induced alterations in resting- state functional connectivity and to find potential connectivity biomarkers for predicting and tracking clinical outcomes after rTMS. doi: 10.3389/fnins.2023.1135995 Methods: Thirty-seven patients with CID received a 10-session low frequency rTMS treatment applied to the right dorsolateral prefrontal cortex. Before and after treatment, the patients underwent resting-state electroencephalography recordings and a sleep quality assessment using the Pittsburgh Sleep Quality Index (PSQI). COPYRIGHT © 2023 Zhu, Dang, Wu, Zhou, Shi, Su, Ren, Pei, Lan, Lian, Xie and Guo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. chronic insomnia disorder, rTMS, EEG, functional connectivity, DLPFC TYPE Original Research PUBLISHED 17 April 2023 DOI 10.3389/fnins.2023.1135995 TYPE Original Research PUBLISHED 17 April 2023 DOI 10.3389/fnins.2023.1135995 Frontiers in Neuroscience frontiersin.org Participants Participants with CID were recruited at the Neurology Department of Shenzhen People’s Hospital. A total of 47 patients were screened for eligibility, of whom 37 gave informed consent to participate in this study. All experimental details were approved by the Ethics Committee of Shenzhen People’s Hospital (see chictr.org.cn registration: ChiCTR1900026904). Nine patients dropped out during the 1-month follow-up period; therefore, only 28 patients were included in the statistical analysis of follow-up. Repetitive transcranial magnetic stimulation (rTMS) can noninvasively modulate cortical activity by delivering a sequence of magnetic pulses. Normally, low frequency (<1 Hz) is thought to inhibit, and high frequency (≥5 Hz) to facilitate motor cortical excitability (Fitzgerald et al., 2006). The inhibitory effect of low frequency (i.e., 1 Hz) rTMS on cortical excitability has therefore led to it being increasingly considered for the treatment of insomnia disorders. All patients were required to meet the diagnostic criteria for CID according to the International Classification of Sleep Disorders, Third Edition (ICSD-3). The inclusion criteria were as follows: (1) aged 18–70 years, right-handed; (2) the sleep disturbances occur at least three times per week and present for the last 3 months; (3) PSQI ≥7 (Buysse et al., 1989); (4) scored <25 on 24-item Hamilton Depression Rating Scale (HAMD); (5) no other sleep disorders like sleep apnea, etc.; and (6) failure of at least one adequate sleep medication trial. The exclusion criteria were: (1) any contraindication to TMS (history of seizures, metallic implants, etc.); and (2) prior history of neurological or psychiatric disorders. Stimulation site is another essential factor in the field of clinical application of rTMS. Dorsolateral prefrontal cortex (DLPFC) is the most widely used rTMS target for the treatment of neuropsychiatric disorders, including depression (Eshel et al., 2020). As a node of the frontoparietal network, it plays a critical role in integrating cognition and emotion (Mars and Grol, 2007; Gong et al., 2020). According to a previous neuroimaging study, the DLPFC of insomnia patients shows hyperexcitability compared with those who were well-slept (Spiegelhalder et al., 2013). As a result, low frequency DLPFC rTMS appears to be a reasonable strategy for the treatment of insomnia (Gong et al., 2020). In addition, 40 healthy controls (HC) without sleep problems participated in the baseline assessment, serving as a reference for changes in functional connectivity. OPEN ACCESS No use, distribution or reproduction is permitted which does not comply with these terms. Results: After treatment, rTMS significantly increased the connectivity of 34 connectomes in the lower alpha frequency band (8–10 Hz). Additionally, alterations in functional connectivity between the left insula and the left inferior eye junction, as well as between the left insula and medial prefrontal cortex, were associated with a decrease in PSQI score. Further, the correlation between the functional connectivity and PSQI persisted 1 month after the completion of rTMS as evidenced by subsequent electroencephalography (EEG) recordings and the PSQI assessment. Conclusion: Based on these results, we established a link between alterations in functional connectivity and clinical outcomes of rTMS, which suggested that EEG-derived functional connectivity changes were associated with clinical improvement of rTMS in treating CID. These findings provide preliminary evidence that rTMS may improve insomnia symptoms by modifying functional connectivity, which can be used to inform prospective clinical trials and potentially for treatment optimization. 01 Frontiers in Neuroscience frontiersin.org frontiersin.org Zhu et al. 10.3389/fnins.2023.1135995 Frontiers in Neuroscience Introduction other than to demonstrate its clinical efficacy. We used the resting- state electroencephalography (rsEEG) to assess the functional connectivity characteristics in patients with chronic insomnia disorder (CID) before, after and 1-month after 10 daily rTMS sessions. rsEEG is a promising paradigm for studying abnormal functional architectures in various disorders due to its task-independent properties (Zhang et al., 2021). The overarching goal was to investigate the alterations of functional connectivity induced by rTMS in patients with CID, which is critical for linking translationally relevant discoveries that can be applied in a clinical setting. The secondary aim of this study was to examine whether the clinical improvement observed 1 month after rTMS was related to any previously identified connectivity characteristics. We hypothesized that rTMS would induce connectivity changes and that such changes would correlate significantly with symptom improvements. Insomnia is the most prevalent sleep disorder and is associated with difficulties in initiating or maintaining sleep, as well as a decline in daytime performance and cognitive impairment, leading to many health issues (Spiegelhalder et al., 2015). Hypnotics are often prescribed as a first-line treatment for acute insomnia (Glass et al., 2005); however, the long-term use of hypnotics has adverse effects and may confer a risk of dependence. Cognitive behavioral therapy (CBT) is an alternative to chronic pharmacological insomnia treatment. Although there is evidence for long-lasting improvement in sleep after CBT, the strict time commitment and insufficient number of qualified clinicians to employ it, limit its use in clinical practice (Morin and Benca, 2012). Therefore, better treatment options for chronic insomnia, a long-term pattern of difficulty sleep, are urgently needed. Insomnia is rarely an isolated psychobiological disorder and is associated with measurable aberration in functional brain mechanisms (Riemann et al., 2010). Neuroimaging studies, such as electroencephalography (EEG) or functional magnetic resonance imaging (fMRI), showed that patients with insomnia have abnormal connectivity patterns in emotional circuits (Huang et al., 2012), salience networks (Chen et al., 2014), and brain network topology in general (Li et al., 2018). Moreover, previous studies have suggested that increased cortical excitability was associated with chronic insomnia (Lanza et al., 2015; Ly et al., 2016). Insomnia is the most prevalent sleep disorder and is associated with difficulties in initiating or maintaining sleep, as well as a decline in daytime performance and cognitive impairment, leading to many health issues (Spiegelhalder et al., 2015). Participants HCs needed to meet the following criteria: (1) no history of sleep disorders; (2) PSQI <7; (3) HAMD ≤7 and Hamilton Anxiety Rating Scale (HAMA) ≤7 (Matza et al., 2010; Zimmerman et al., 2013); and (4) no neurological or psychiatric disorders. The efficacy of rTMS in treating insomnia has been investigated in several clinical studies. Most of them chose the DLPFC as the target. These studies found that 10 daily sessions of low frequency rTMS stimulation applied to the right or bilateral DLPFC resulted in a significant decrease in the Pittsburgh Sleep Quality Index (PSQI; lower index indicates better sleep) (Jiang et al., 2013; Feng et al., 2019; Shi et al., 2021). Only one study selected the right posterior parietal cortex as the stimulation site and reported similar results: 14 consecutive low frequency rTMS sessions could lower down PSQI and Insomnia Severity Index significantly (Song et al., 2019). During the study, patients were allowed to take concomitant medications, and were asked to remain constant throughout the clinical trial (see Supplementary Methods for details). Introduction Hypnotics are often prescribed as a first-line treatment for acute insomnia (Glass et al., 2005); however, the long-term use of hypnotics has adverse effects and may confer a risk of dependence. Cognitive behavioral therapy (CBT) is an alternative to chronic pharmacological insomnia treatment. Although there is evidence for long-lasting improvement in sleep after CBT, the strict time commitment and insufficient number of qualified clinicians to employ it, limit its use in clinical practice (Morin and Benca, 2012). Therefore, better treatment options for chronic insomnia, a long-term pattern of difficulty sleep, are urgently needed. fi Insomnia is rarely an isolated psychobiological disorder and is associated with measurable aberration in functional brain mechanisms (Riemann et al., 2010). Neuroimaging studies, such as electroencephalography (EEG) or functional magnetic resonance imaging (fMRI), showed that patients with insomnia have abnormal connectivity patterns in emotional circuits (Huang et al., 2012), salience networks (Chen et al., 2014), and brain network topology in general (Li et al., 2018). Moreover, previous studies have suggested that increased cortical excitability was associated with chronic insomnia (Lanza et al., 2015; Ly et al., 2016). frontiersin.org Clinical assessment All participants received a pre-treatment assessment with the PSQI, HAMA, and HAMD. We used the PSQI to measure sleep quality, as well as HAMA and HAMD to assess participant’s anxious and depressive states. Likewise, the PSQI was measured post- treatment (upon completion of the final treatment session) and at 1-month follow-up. In order to determine the magnitude of the rTMS response, the percent reduction in PSQI from pre- to post-treatment, and from pre-treatment to follow-up, were calculated (see Figure 1 for the experimental design). Estimating functional connectivity All connectivity analyses were computed at the source level using 3,003 vertices and then projected into 31 cortical regions of interest (ROI) using the Montreal Neurological Institute template (Chen et al., 2013); (see Supplementary Table 1). Here, we chose the debiased weighted phase-lag index (dwPLI) to represent the non-zero phase-lag statistical interdependencies between each pair of ROIs (Vinck et al., 2011). dwPLI is an optimized phase lag index that minimizes the influence of volume conduction and field spread, which could affect the estimation even at the source level. The connectivity between each pair of regions was calculated by averaging the dwPLI values over all possible vertex pairs. Accordingly, we identified 465 edges representing each participant’s regional pairwise connectivity. rTMS treatment However, despite the aforementioned clinical evidences, there are limited neurophysiological studies investigating the underlying mechanisms of low frequency DLPFC rTMS in patients with chronic insomnia. In this context, we conducted this single-arm, open-label, interventional study to determine the neural mechanisms of rTMS Stimulation was performed using a figure 8-shaped focal coil attached to a MagPro 100 magnetic stimulator (MagVenture, Copenhagen, Denmark). All patients received 1 Hz (10 s trains, 1 s inter-train interval, 1,360 pulses per session) rTMS treatment once 02 frontiersin.org Zhu et al. 10.3389/fnins.2023.1135995 The details of the pre-processing and source localization are described in the Supplementary Methods. Based on previous findings, the resulting EEG data were filtered into five frequency bands: theta (4–8 Hz), low-alpha (8–10 Hz), high-alpha (10–13 Hz), beta (13–30 Hz), and gamma (30–45 Hz). daily on weekdays for 2 consecutive weeks. rTMS was delivered over the right DLPFC (F4 electrode site according to the International 10–20 EEG system) at 100% of the resting motor threshold (RMT) (Mir-Moghtadaei et al., 2015). To determine the RMT, stimulus intensity was gradually increased until 5 out of 10 trials elicited motor evoked potentials with peak-to-peak amplitudes over 50 μV in the contralateral abductor pollicis brevis muscle (Rossini et al., 2015). Adverse events attributed to rTMS were documented and reported. daily on weekdays for 2 consecutive weeks. rTMS was delivered over the right DLPFC (F4 electrode site according to the International 10–20 EEG system) at 100% of the resting motor threshold (RMT) (Mir-Moghtadaei et al., 2015). To determine the RMT, stimulus intensity was gradually increased until 5 out of 10 trials elicited motor evoked potentials with peak-to-peak amplitudes over 50 μV in the contralateral abductor pollicis brevis muscle (Rossini et al., 2015). Adverse events attributed to rTMS were documented and reported. Electroencephalography To compare the difference in clinical outcomes, we analyzed the PSQI of pre-, post-treatment and follow-up using a linear mixed model with a fixed effect of time and a random intercept.hf For the HC group, EEG data were recorded once at baseline. For the CID patients, EEG data were acquired at three time points concurrently with PSQI. Equipment setting and environment were uniform across all three acquisitions. The differences between pre- and post-functional connectivity matrices were analyzed using the Network-based Statistics (NBS) (Zalesky et al., 2010), a nonparametric statistical test to control for the family-wise error rate resulting from multiple comparisons. For each comparison, 5,000 random permutations were used. EEG recordings were acquired from 64 channels under closed-eye conditions for 8 min using a BrainAmp DC amplifier (Brain Products GmbH, Germany). During the recording, the participants were asked to relax on a comfortable chair in a metal- shielded room. The data were referenced online to the FCz channel with the ground at AFz. Data were initially sampled at 5,000 Hz with impedances kept below 5KΩ throughout the data collection period. Participants were instructed to refrain from consuming any caffeinated (or energy) drinks within 24 h of the EEG recording sessions. To further investigate the association of functional connectivity changes with clinical outcomes, a multiple linear regression model was constructed between the pre- to post-network differences and the percentage PSQI change with age, sex, HAMA, and HAMD as covariates. Here, all edges that varied significantly (NBS-corrected, p < 0.01) between the pre- and post- functional connectivity matrices were treated as independent inputs. When implementing feature selection, we used the least absolute shrinkage and selection operator (LASSO) to define a low-dimensional representation of the selected connectivity features (Tibshirani, 1996). Correlation analysis between the estimated and the actual PSQI value of post- treatment and follow-up were performed using Pearson’s correlation. FIGURE 1 Experimental protocol. Healthy controls (HC) and patients with chronic insomnia disorder (CID) participated in an EEG session before rTMS treatment, as well as clinical assessments. Afterwards, patients received a course of low frequency rTMS treatment targeting the right DLPFC. Subsequently, EEG data and PSQI were collected again after treatment, and at 1-month follow-up, respectively. FIGURE 1 Frontiers in Neuroscience Discussion There were no significant differences in the functional connectivity matrices between the pre- and post-treatment measures that survived multiple corrections at four frequency bands: theta (4–8 Hz), high-alpha (10–13 Hz), beta (13–30 Hz), and gamma (30–45 Hz). Instead, a comparison of the functional connectivity of low-alpha frequency band (8–10 Hz) identified 34 edges that had been significantly changed at p < 0.01 level after NBS correction (Figure 2; also see Supplementary Table 2). Specifically, CID patients had a lower mean functional connectivity pre-treatment. On average, these rTMS-induced connectivity changes brought patients’ patterns aligned more closely with healthy controls. At follow-up, the strength of the connectivity matrix remained relatively stable (see Figure 2C). The most frequently occurring connectomes were located in the frontal lobe, preferentially involving the frontoparietal network (FPN), dorsal attention network (DAN), and ventral attention network (VAN) (see Figure 2D). Moreover, a dominant interhemispheric functional connectivity change was also notable. To better understand the regulatory role of rTMS in patients with CID, we examined longitudinal changes in functional connectivity induced by low frequency rTMS of the right DLPFC. In agreement with previous studies, our results confirmed the efficacy of this low frequency rTMS protocol for treating clinical CID. We found that rTMS to the right DLPFC was associated with widespread alterations in functional connectivity, which correlated with clinical outcomes, and this association persisted 1 month after the cessation of rTMS stimulation. These results offer promising preliminary evidence linking the altered brain function patterns observed in CID with clinical symptoms and expands on how changes in both aspects are associated. Unlike other studies that focused only on a priori brain regions, we discovered connectivity biomarkers based directly on EEG data without making any assumptions. This data-driven approach allows us to account for uncertainties in the spatial distribution of connectivity changes correlated with clinical outcomes. Our findings suggest that the significant increase in connectivity is not confined to the ones within the stimulated network, but instead spreads across networks, involving the FPN, DAN, and VAN. Remote functional connections are impacted more than local ones, indicating that rTMS ultimately modulates insomnia-related connectivity (Castrillon et al., 2020). Additionally, we observed a dominant interhemispheric functional connectivity change, which is consistent with that reported in previous studies (Watanabe et al., 2014). Longitudinal connectivity analysis All patients tolerated the rTMS well, and no adverse effects were reported. A mixed-effects model assessing PSQI differences revealed a significant main effect of rTMS treatment [F(1.84, 57.88) = 12.38, p < 0.0001]. Post hoc comparisons indicated that rTMS facilitated a significant reduction in insomnia symptoms on the PSQI between pre- and post-treatment (delta-PSQI = 3.243, p = 0.0002, Cohen’s d = 0.857), and between scores pre- and 1-month later (delta-PSQI = 3.911, p = 0.0003, Cohen’s d = 1.132) (see the Supplementary Figure 1). To gain further insight into the rTMS-induced changes in LIFJ- LINS and MPFC-LINS connectivity, we examined mean values at each time point for both HC and CID groups. Significant difference was observed between the HC and CID pre-treatment for both LIFJ-LINS (average 0.46 vs. 0.27) and MPFC-LINS (average 0.41 vs. 0.30) connections (see Figure 4). After rTMS, the connectivity of the LIFJ to the LINS increased significantly at post-treatment and follow-up. The trend of MPFC-LINS connectivity resembled a similar pattern, which increased significantly at follow-up compared to that at baseline. These active rTMS-induced changes shifted the CID patient profile closer to that of HCs (see pairwise statistics in the Figure 4 legend). Clinical results The demographic and clinical characteristics of the two groups are summarized in Table 1. There were significant differences in the PSQI, HAMA, and HAMD scores (p < 0.05) between the HC and CID groups. No significant differences were found in age or sex. Among these characteristics, the PSQI global score correlated appreciably with HAMD score (p < 0.01, r = 0.447) but was not associated with the other measures. Experimental protocol. Healthy controls (HC) and patients with chronic insomnia disorder (CID) participated in an EEG session before rTMS treatment, as well as clinical assessments. Afterwards, patients received a course of low frequency rTMS treatment targeting the right DLPFC. Subsequently, EEG data and PSQI were collected again after treatment, and at 1-month follow-up, respectively. 03 Frontiers in Neuroscience frontiersin.org Zhu et al. 10.3389/fnins.2023.1135995 TABLE 1 Demographic and clinical characteristics of the participants. Frontiers in Neuroscience Analysis of connectivity changes and clinical outcomes at follow-up Variables HC (n = 40) CID (n = 37) p-value Age, year 23–72, 46.1 ± 9.4 22–69, 48.9 ± 11.1 0.23 Sex (M/F) 18/22 15/22 0.69 PSQI 3.2 ± 1.6 15.1 ± 3.4 <0.001 HAMA 2.6 ± 1.8 13.6 ± 6.2 <0.001 HAMD 2.7 ± 1.8 13.3 ± 5.0 <0.001 RMT / 39.0 ± 12.5 Data are presented as mean ± SD. The p values were obtained by independent sample t-test and chi-square test (for sex only). HC, healthy control; CID, chronic insomnia disorder; PSQI, Pittsburgh Sleep Quality Index; HAMA, Hamilton Anxiety Rating Scale; HAMD, Hamilton Depression Rating Scale; RMT, resting motor threshold. EEG data acquired 1 month after treatment were used to validate the association between the connectomes obtained in the previous regression model and clinical measures. The connectivity changes of these two edges between the pre- and follow-up assessments were taken as independent inputs, including age, sex, HAMA, and HAMD at baseline, as covariates. The estimated PSQI score was significantly correlated with the actual PSQI score collected 1 month after rTMS completion (r = 0.41, p = 0.032) (Figure 3B). Data are presented as mean ± SD. The p values were obtained by independent sample t-test and chi-square test (for sex only). HC, healthy control; CID, chronic insomnia disorder; PSQI, Pittsburgh Sleep Quality Index; HAMA, Hamilton Anxiety Rating Scale; HAMD, Hamilton Depression Rating Scale; RMT, resting motor threshold. Discussion Of note, since all the significant edges show higher connectivity after rTMS than before it, this observation corroborates that rTMS treatment may rewire impaired intracortical connections. Regression analyses were performed to identify connections that were significantly correlated with improvement in PSQI scores at the end of rTMS treatment. That is, the functional connectivity changes between pre- and post-treatment of these 34 identified edges were taken as independent variables (see Supplementary Table 3 for coefficients of regression model). Notably, two edges appeared to be significantly correlated with PSQI change (r = 0.62, p = 0.02) (see Figure 3B): the left inferior frontal junction to left insula (LIFJ-LINS) and the medial prefrontal cortex to left insula (MPFC-LINS) (see Figure 3A). 04 frontiersin.org Zhu et al. 10.3389/fnins.2023.1135995 A B C D FIGURE 2 Functional connectivity matrices in low alpha band (8–10 Hz) and 34 identified edges. (A) Connectivity matrices of pre-treatment EEG scan. (B) Connectivity matrices of post-treatment EEG scan. (C) Connectivity matrices of the follow-up EEG scan. (D) Significant functional connectivity changes between the pre- and post- EEG scans included 34 edges. Significance was determined by network-based statistics (NBS) correction at p < 0.01. List of the brain regions are presented in the Supplementary Table 1. List of the 34 pairs showed significant differences are presented in the Supplementary Table 3. DMN, default mode network; DAN, Dorsal attention network; VAN, Ventral attention network; SOM, somatosensory network; FPN, frontoparietal network. B A B B A A C C D D FIGURE 2 Functional connectivity matrices in low alpha band (8–10 Hz) and 34 identified edges. (A) Connectivity matrices of pre-treatment EEG scan. (B) Connectivity matrices of post-treatment EEG scan. (C) Connectivity matrices of the follow-up EEG scan. (D) Significant functional connectivity changes between the pre- and post- EEG scans included 34 edges. Significance was determined by network-based statistics (NBS) correction at p < 0.01. List of the brain regions are presented in the Supplementary Table 1. List of the 34 pairs showed significant differences are presented in the Supplementary Table 3. DMN, default mode network; DAN, Dorsal attention network; VAN, Ventral attention network; SOM, somatosensory network; FPN, frontoparietal network. Neuroplasticity describes the ability of the brain to change and adapt its organization as a result of external influences. Frontiers in Neuroscience Discussion The results of the longitudinal analysis suggest that rTMS has a long-lasting plastic effect; hence, decrease in clinical symptoms and increase in functional connectivity characteristics could persist even 1-month after the completion of rTMS (Ge et al., 2020). It also proves the robustness of these two indicators, LINS-MPFC, LINS-LIFJ, over a long period of time. et al., 2014). We found that patients with CID suffered significantly higher HAMA and HAMD than those with HCs, which reflects the true situation in real life. Our study reinforces earlier findings about the insula in CID and adds to the evidence that it plays a role in the disease’s pathophysiology. The MPFC and its ample connections with other regions, play critical roles in long-range, recent, and short-term memory over a wide range of activities (Euston et al., 2012). It has also been previously discussed regarding insomnia with the conclusion that circuit dysfunction involving the MPFC was associated with poor sleep quality as measured by the PSQI (Shao et al., 2020). The IFJ is part of the cognitive control network that co-activates with the DLPFC, ventrolateral prefrontal cortex, anterior insula, and posterior parietal cortex (Sundermann and Pfleiderer, 2012). The insula is a key node in the salience network, and its abnormalities have been pointed out in many studies of insomnia (Motomura et al., 2021). An fMRI research demonstrated decreased connectivity between the amygdala and insula during resting states, indicating a possible neural mechanism responsible for the sleep- related affective disorders and dysregulation of emotional control (Li 05 frontiersin.org Zhu et al. 10.3389/fnins.2023.1135995 A B FIGURE 3 (A) Edges that were significantly correlated with percentage PSQI change before and after treatment (LIFJ-LINS, p < 0.01, MPFC-LINS, p < 0.01). (B) The estimated PSQI at follow-up were significantly correlated with the actual score (r = 0.4056, p = 0.032), with age, gender, HAMA and HAMD at baseline as the nuisance covariates. A B FIGURE 4 Significant rTMS effect of group and time. (A) Changes of the mean functional connectivity between left inferior eye junction (LIFJ) and left insula (LINS). Mixed effect model revealed significant rTMS treatment effect in the functional connection [F(1.8, 55.83) = 7.256, p = 0.0022]. Post hoc comparison showed significant differences between pre- and post-treatment (p = 0.0236), and pre-treatment to follow-up (p = 0.0076). The functional connectivity of HC was significantly higher than CID patients at baseline (t = 3.641, p = 0.0005). Discussion (B) Changes of the mean functional connectivity between medial prefrontal cortex (MPFC) and left insula (LINS). Mixed effect model revealed significant rTMS treatment effect in the functional connection [F(1.92, 59.53) = 3.862, p = 0.028]. Post hoc comparison showed a significant difference between pre-treatment and follow-up (p = 0.0497). Independent t-test showed that CID patients had significantly lower functional connectivity at baseline than HCs (t = 2.016, p = 0.0474). Error-bars represents SEM. p < 0.05, p < 0.01, p < 0.001. A A B FIGURE 3 (A) Edges that were significantly correlated with percentage PSQI change before and after treatment (LIFJ-LINS, p < 0.01, MPFC-LINS, p < 0.01). (B) The estimated PSQI at follow-up were significantly correlated with the actual score (r = 0.4056, p = 0.032), with age, gender, HAMA and HAMD at baseline as the nuisance covariates. B B FIGURE 3 (A) Edges that were significantly correlated with percentage PSQI change before and after treatment (LIFJ-LINS, p < 0.01, MPFC-LINS, p < 0.01). (B) The estimated PSQI at follow-up were significantly correlated with the actual score (r = 0.4056, p = 0.032), with age, gender, HAMA and HAMD at baseline as the nuisance covariates. A B FIGURE 4 Significant rTMS effect of group and time. (A) Changes of the mean functional connectivity between left inferior eye junction (LIFJ) and left insula (LINS). Mixed effect model revealed significant rTMS treatment effect in the functional connection [F(1.8, 55.83) = 7.256, p = 0.0022]. Post hoc comparison showed significant differences between pre- and post-treatment (p = 0.0236), and pre-treatment to follow-up (p = 0.0076). The functional connectivity of HC was significantly higher than CID patients at baseline (t = 3.641, p = 0.0005). (B) Changes of the mean functional connectivity between medial prefrontal cortex (MPFC) and left insula (LINS). Mixed effect model revealed significant rTMS treatment effect in the functional connection [F(1.92, 59.53) = 3.862, p = 0.028]. Post hoc comparison showed a significant difference between pre-treatment and follow-up (p = 0.0497). Independent t-test showed that CID patients had significantly lower functional connectivity at baseline than HCs (t = 2.016, p = 0.0474). Error-bars represents SEM. p < 0.05, p < 0.01, p < 0.001. A B A B A FIGURE 4 Significant rTMS effect of group and time. (A) Changes of the mean functional connectivity between left inferior eye junction (LIFJ) and left insula (LINS). Frontiers in Neuroscience Data availability statement CID appears to be associated with altered cortico-thalamic connectivity, which is partly responsible for cognitive regulation and circadian processes (Zou et al., 2021). Indeed, the thalamus is the pacemaker of cortical alpha rhythms. Taken together, we infer that DLPFC-rTMS regulates altered alpha connectivity by top-down control of DLPFC to the thalamus. While path analysis indicates a causal relationship, our study cannot infer causality since no connectivity with subcortical regions was observed. These results should therefore, only be considered as supportive. Further neuroimaging study is required to elucidate the potential involvement of the thalamus in the improvement of cortical alpha band connections following treatment. A previous study supports our hypothesis reporting that DLPFC-rTMS modulates the functional connectivity of the insula and thalamus in smokers (Li et al., 2017). The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Discussion Mixed effect model revealed significant rTMS treatment effect in the functional connection [F(1.8, 55.83) = 7.256, p = 0.0022]. Post hoc comparison showed significant differences between pre- and post-treatment (p = 0.0236), and pre-treatment to follow-up (p = 0.0076). The functional connectivity of HC was significantly higher than CID patients at baseline (t = 3.641, p = 0.0005). (B) Changes of the mean functional connectivity between medial prefrontal cortex (MPFC) and left insula (LINS). Mixed effect model revealed significant rTMS treatment effect in the functional connection [F(1.92, 59.53) = 3.862, p = 0.028]. Post hoc comparison showed a significant difference between pre-treatment and follow-up (p = 0.0497). Independent t-test showed that CID patients had significantly lower functional connectivity at baseline than HCs (t = 2.016, p = 0.0474). Error-bars represents SEM. p < 0.05, p < 0.01, *p < 0.001. In light of our understanding of how alpha oscillations correlate with resting wakefulness, the therapeutic benefits of rTMS typically focus on alpha rhythms. The alpha rhythms promote coordination between cortical areas and between the cortex and subcortical structures, such as the thalamus (Buzsáki and Draguhn, 2004). A highly synchronized alpha rhythms can facilitate the coordination of brain regions in an event-related, synchronized readiness manner, prior to engaging in different type of tasks (Klimesch et al., 2007). We found that DLPFC-rTMS significantly enhanced the connectivity of low-frequency alpha rhythms (8–10 Hz), but not of the entire alpha band (8–13 Hz) (see Supplementary Figure 2). In the thalamo-cortical and cortico- cortical loops, alpha rhythms carry out different functions; for example, low-frequency alpha rhythms execute inhibitory functions (Pfurtscheller and Lopes da Silva, 1999). Researchers have proposed that the lower alpha band diffusely regulates alertness and arousal in the brain (Klimesch et al., 1998). Our results show that inhibitory 1 Hz rTMS stimulation of the DLPFC increases lower alpha synchronization in the resting state. This inhibitory synchronization state could facilitates recruitment of specific regions of the cortex to transmit and retrieve task information before preparing for subsequent tasks (Klimesch, 2012). A previous study reported a similar phenomenon. rTMS targeting in the angular gyrus, core regions of the DAN, enhanced intrahemispheric alpha coherence of 8–10 Hz, suggesting the causal role of the angular gyrus in modulating of dominant low-frequency alpha rhythms under the resting conditions (Capotosto et al., 2014). 06 frontiersin.org Zhu et al. Zhu et al. 10.3389/fnins.2023.1135995 Funding This study was supported by the Natural Science Foundation of Guangdong Province (grant number 2021A1515010983), the Sanming Project of Medicine in Shenzhen (grant number SZSM202111009), the Shenzhen Science and Technology Innovation Program (grant number KCXFZ20201221173400001), and Shenzhen Key Medical Discipline Construction Fund (grant number SZXK005). Conclusion All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Despite its high prevalence, there has been little progress in the treatment of CID. We found that low frequency rTMS treatment over the right DLPFC significantly increased the EEG-derived functional connectivity in patients with CID. Furthermore, changes in connections, particularly the LIFJ-LINS and MPFC-LINS, were significantly associated with clinical measures and could predict the PSQI in the subsequent assessment at 1-month follow-up. These results could be very valuable in clinical treatment of insomnia, especially in patients that are resistant to medications or have a history of drug failure. These findings expand our understanding of neural response in patients with chronic insomnia treated with rTMS and lay the foundation for future studies. Further research using a rigorous design is required to address the aforementioned issues. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics statement The studies involving human participants were reviewed and approved by the Shenzhen People’s Hospital. The patients/ participants provided their written informed consent to participate in this study. Frontiers in Neuroscience Author contributions rTMS is a promising strategy for treating various neuropsychiatric disorders. The effects of rTMS depend on its intensity, frequency and stimulation site, in particular the stimulation site. However, there is no consensus on the optimal stimulation site for insomnia due to the prior lack of therapeutic mechanisms of rTMS. These findings offer implications for optimizing brain stimulation therapy, which should be examined in future clinical trials. Besides, as the optimal treatment parameters for insomnia have not yet been determined, the schedule implemented in this study is a commonly used protocol to ensure treatment adherence. It is essential to identify efficacious dosing parameters that are feasible for further studies. LZ contributed to the data analysis and original draft. GD, XSh, and XSu were responsible for acquisition and analysis. WW and JZ assisted with analysis and manuscript revision. XL and CL contributed to the data collections. ZP and HR contributed to the data curation. PX provided clinical study consultation. YG was responsible for the conceptualization and funding acquisition. All authors critically reviewed the final content and approved for publication. This study has some limitations. First, the lack of a sham-control group prevents us from determining whether the results can be partly explained by placebo effects. In effect, the primary goal of this study was to determine the neural mechanism of rTMS rather than to demonstrate differential clinical outcome with active versus sham conditions. Second, chronic insomnia is quite a heterogenic disorder, often comorbid with other symptoms. To ensure the homogeneity of patients enrolled in the study, we strictly enrolled patients who had difficulties with sleep for over 3 months and had failed at least one medication trial. 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https://openalex.org/W4200095646	https://bg.copernicus.org/articles/19/201/2022/bg-19-201-2022.pdf	English	null	Reply on RC1	null	2,021	cc-by	17,736	Bacteriohopanetetrol-x: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system Z ë R K d 1 L Vill 1 2 Ell C H 1 P t K l1 3 H J Witt 1 At shelf stations with [O2] > 50 µmol L−1, the BHT-x ratio was < 0.04 (in all but one case). In apparent contradiction, ladderane FAs and low abundances of BHT and BHT-x (resulting in BHT- x ratios > 0.04) were also detected in oxygenated offshore waters ([O2] up to 180 µmol L−1), whereas ladderane IPLs were undetected. The index of ladderane lipids with ﬁve cy- clobutane rings (NL5) correlates with in situ temperature. NL5-derived temperatures suggested that ladderane FAs in the offshore waters were not synthesized in situ but were transported down-slope from warmer shelf waters. Thus, in sedimentary archives of systems with known lateral organic matter transport, such as the BUS, relative BHT and BHT-x abundances should be carefully considered. In such systems, a higher BHT-x ratio may act as a safer threshold for deoxy- genation and/or Ca. Scalindua presence: our results and pre- vious studies indicate that a BHT-x ratio of ≥0.2 is a robust threshold for oxygen-depleted waters ([O2] < 50 µmol kg−1). In our data, ratios of ≥0.2 coincided with Ca. Scalindua 16S rRNA genes in all samples (n = 62), except one. Lastly, when investigating in situ anammox, we highlight the impor- tance of using ladderane IPLs over BHT-x and/or ladderane FAs; these latter compounds are more recalcitrant and may derive from transported fossil anammox bacteria remnants. Bacteriohopanetetrol-x: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system Z ë R K d 1 L Vill 1 2 Ell C H 1 P t K l1 3 H J Witt 1 biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system Zoë R. van Kemenade1, Laura Villanueva1,2, Ellen C. Hopmans1, Peter Kraal1,3, Harry J. Witte1, Jaap S. Sinninghe Damsté1,2, and Darci Rush1 1Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, the Netherlands 2Department of Earth Sciences, Geochemistry, Faculty of Geosciences, University of Utrecht, Utrecht, the Netherlands 3Department of Ocean Systems, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, the Netherlands Correspondence: Zoë R. van Kemenade (zoe.van.kemenade@nioz.nl) Received: 4 October 2021 – Discussion started: 5 October 2021 Revised: 1 December 2021 – Accepted: 5 December 2021 – Published: 13 January 2022 Received: 4 October 2021 – Discussion started: 5 October 2021 Revised: 1 December 2021 – Accepted: 5 December 2021 – Published: 13 January 2022 Abstract. Interpreting lipid biomarkers in the sediment archive requires a good understanding of their applica- tion and limitations in modern systems. Recently it was discovered that marine bacteria performing anaerobic am- monium oxidation (anammox), belonging to the genus Ca. Scalindua, uniquely synthesize a stereoisomer of bac- teriohopanetetrol (“BHT-x”). The ratio of BHT-x over to- tal bacteriohopanetetrol (BHT, ubiquitously synthesized by diverse bacteria) has been suggested as a proxy for water column anoxia. As BHT has been found in sediments over 50 Myr old, BHT-x has the potential to complement and ex- tend the sedimentary biomarker record of marine anammox, conventionally constructed using ladderane lipids. Yet, lit- tle is known about the distribution of BHT-x in relation to the distribution of ladderanes and to the genetic evidence of Ca. Scalindua in modern marine systems. Here, we in- vestigate the distribution of BHT-x and the application of the BHT-x ratio in relation to distributions of ladderane intact polar lipids (IPLs), ladderane fatty acids (FAs) and Ca. Scalindua 16S rRNA genes in suspended particulate matter (SPM) from the water column of the Benguela up- welling system (BUS), sampled across a large oxygen gradi- ent. In BUS SPM, high BHT-x abundances were restricted to the oxygen-deﬁcient zone on the continental shelf (at [O2] < 45 µmol L−1, in all but one case). High BHT-x abun- dances co-occurred with high abundances of the Ca. Scalin- dua 16S rRNA gene (relative to the total number of bacte- rial 16S rRNA genes) and ladderane IPLs. Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Research article 1 Introduction Anaerobic ammonium oxidizing (anammox) bacteria are a deep branching monophyletic group belonging to the order Planctomycetales (Strous et al., 1999). Anammox bacteria oxidize ammonium (NH+ 4 ) to dinitrogen gas (N2), using ni- trite (NO− 2 ) as an electron acceptor (Van de Graaf et al., 1995, 1997). Of the ﬁve known anammox genera, only “Candi- datus Scalindua” has been found in open marine environ- ments, of which the ﬁrst identiﬁed species was “Ca. Scalin- dua sorokinii” in the Black Sea (Kuypers et al., 2003; Schmid et al., 2003). Since then, studies have found Ca. Scalindua spp. to also be present in numerous oxygen minimum zones (OMZs) worldwide (Schmid et al., 2007; Woebken et al., 2007; Villanueva et al., 2014), where they are responsible for major losses of ﬁxed nitrogen (e.g., Thamdrup et al., 2006; Schmid et al., 2007; Jensen et al., 2008; Lam et al., 2009). Although permanent OMZs (deﬁned as O2 < 20 µmol L−1, reaching 1 µmol L−1 in the core) constitute only ∼8 % of the total oceanic area (Paulmier and Ruiz-Pino, 2009), they are responsible for 20 %–50 % of the total global nitrogen (N) loss (Gruber and Sarmiento, 1997; Codispoti et al., 2001; Gruber, 2004). Climate models predict that OMZs will ex- pand both spatially and temporally (e.g., Oschlies et al., 2018), hereby altering the biogeochemistry of the oceans. This will likely increase the potential of ﬁxed N-loss pro- cesses, such as anammox, in marine systems (Breitburg et al., 2018). Figure 1. Structures of lipid biomarkers used in this study: (a) lad- derane fatty acids (FAs) with 5 or 3 cyclobutane moieties and 18 or 20 carbon atoms. (b) Bacteriohopane-17β, 21β(H), 22R, 32R, 33R, 34S, 35-tetrol (BHT), ubiquitously synthesized by bacteria, and bacteriohopanetetrol stereoisomer (BHT-x), with an unknown stere- ochemistry of the tetrafunctionalized side chain, uniquely synthe- sized by marine anammox bacteria. (c) Ladderane intact polar lipids PC C20-[3]-monoalkylether (“PC ladderane”) and PG ether/ester linked C20[3] / C18[5] (“PG ladderane”). ) To constrain past and present N cycle variations, lipid biomarkers can be employed (see Rush and Sinninghe Damsté, 2017, for a review). Subsequently, biomarker infor- mation can be applied for predictions of future N cycling variations (e.g., Monteiro et al., 2012). 1 Introduction Anammox bacteria uniquely synthesize ladderane fatty acids (FAs) and ladder- ane glycerol monoethers, which contain three or ﬁve linearly concatenated cyclobutane rings, designated respectively as [3]- and [5]-ladderanes (Sinninghe Damsté et al., 2002, 2005; Fig. 1a). These biomarker lipids have been used to detect anammox bacteria in the natural environment (e.g., Kuypers et al., 2003; Hamersley et al., 2007; Jaeschke et al., 2010). Ladderanes containing intact polar lipids (IPLs) have also been used as biomarker lipids for the presence of anammox bacteria in marine waters and sediments (e.g., Jaeschke et al., 2010; Brandsma et al., 2011; Pitcher et al., 2011). Ladderane IPLs consist of two hydrocarbon chains, at least one being a [3]- or [5]-ladderane, esteriﬁed or ether-bound to a glyc- erol moiety, which is in turn bound to a polar head group (phosphocholine, PC; phosphoethanolamine, PE; phospho- glycerol, PG; Boumann et al., 2006; Rattray et al., 2008). Ladderane IPLs are thought to reﬂect living or recently dead anammox cells (Jaeschke et al., 2009a, 2010; Brandsma et al., 2011; Bale et al., 2014), as they rapidly degrade after cell lysis (i.e., by head group cleavage; Harvey et al., 1986). By contrast, the oldest reported ladderane FAs were found in 140 kyr old sediments underlying the Arabian Sea OMZ (Jaeschke et al., 2009b). However, partly due to the steric strain on the cyclobutane moieties, ladderane FAs degrade during diagenesis. Thus, in order to constrain the role of anammox in the N cycle during earlier time periods, a more recalcitrant biomarker is required. Recently, a stereoisomer (BHT-x) of the ubiquitous bac- teriohopanetetrol (BHT), a pentacyclic C30 triterpenoid linked to a tetrafunctionalized side chain, was reported to be uniquely synthesized by marine anammox bacteria, Ca. Scalindua (Rush et al., 2014; Schwartz-Narbonne et al., 2020; Fig. 1b). BHTs belong to the family of bacte- riohopanepolyols (BHPs), which are biological precursors of hopanoids, ubiquitously found in the geological record (Ourisson and Albrecht, 1992). Intact BHPs have been found in sediments over 50 Myr old (van Dongen et al., 2006; Tal- bot et al., 2016; Rush et al., 2019). BHT-x was found to have the same distribution as ladderane FAs in sediments of Golfo Dulce, an anoxic marine enclosure in Costa Rica (Rush et al., 2014), testifying to its potential application as an anammox marker. Moreover, since BHT is ubiquitous and the BHT-x stereoisomer was only found in low-oxygen settings, Saénz et al. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Figure 1. Structures of lipid biomarkers used in this study: (a) lad- derane fatty acids (FAs) with 5 or 3 cyclobutane moieties and 18 or 20 carbon atoms. (b) Bacteriohopane-17β, 21β(H), 22R, 32R, 33R, 34S, 35-tetrol (BHT), ubiquitously synthesized by bacteria, and bacteriohopanetetrol stereoisomer (BHT-x), with an unknown stere- ochemistry of the tetrafunctionalized side chain, uniquely synthe- sized by marine anammox bacteria. (c) Ladderane intact polar lipids PC C20-[3]-monoalkylether (“PC ladderane”) and PG ether/ester linked C20[3] / C18[5] (“PG ladderane”). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 202 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 203 Figure 2. Map of sampled stations during expeditions 64PE449 (27 January–14 February 2019) and 64PE450 (15 February– 10 March 2019), with station dots color coded according to sam- pling activities, CTD casts and nutrients (blue), and CTD casts and nutrients plus DNA and SPM sampling (red). Numbers indicate sta- tion labels of red dots. Station labels and coordinates of blue dots are given in Table S1. AC: Angolan current; BC: Benguela current; ABF: Angolan Benguela Front (∼19.8◦S). Maps were created in ODV using the ETOP01_2min global tiles map (Schlitzer, R, Ocean Data View, https://odv.awi.de, last access: 12 June 2021). potential of BHT-x to complement and extend the ladderane biomarker record. To better interpret BHT-x as a biomarker in the sedimen- tary record, either as an indicator of the presence of ma- rine anammox bacteria Ca. Scalindua spp. or as a proxy for water column deoxygenation, it is imperative to establish how BHT-x is distributed in modern marine oxygen-depleted systems. In this study, we combine measurements of BHT- x, ladderane lipids (as both IPLs and FAs) and 16S rRNA marker genes in suspended particulate matter (SPM) across a redox gradient in the water column of the Benguela up- welling system (BUS). The BUS, located along the south- west African continental margin (Fig. 2), supports one of the most productive regions in the world. The high primary productivity on the broad but shallow continental shelf re- sults in a perennial OMZ off-shelf, between ∼200–500 m below the sea surface (mbss). Additionally, annual variation in upwelling intensity leads to a seasonal, on-shelf oxygen- deﬁcient zone (ODZ; here deﬁned as [O2] < 5 µmol L−1 in bottom waters), which develops in late austral summer (Chapman and Shannon, 1987; Bailey et al., 1991; Mercier et al., 2003; Ekau and Verheye, 2005; Brüchert et al., 2006; Mohrholz et al., 2008). Previous research has indicated that in the BUS, anammox is responsible for major losses of bioavailable nitrogen (Kuypers et al., 2005; Kalvelage et al., 2011). The BUS therefore is an optimal modern marine sys- tem to assess the distribution of BHT-x in relation to that of ladderane IPLs, ladderane FAs and Ca. Scalindua 16S rRNA gene sequences, as a function of water column oxygenation. Figure 2. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Map of sampled stations during expeditions 64PE449 (27 January–14 February 2019) and 64PE450 (15 February– 10 March 2019), with station dots color coded according to sam- pling activities, CTD casts and nutrients (blue), and CTD casts and nutrients plus DNA and SPM sampling (red). Numbers indicate sta- tion labels of red dots. Station labels and coordinates of blue dots are given in Table S1. AC: Angolan current; BC: Benguela current; ABF: Angolan Benguela Front (∼19.8◦S). Maps were created in ODV using the ETOP01_2min global tiles map (Schlitzer, R, Ocean Data View, https://odv.awi.de, last access: 12 June 2021). 2.1 Hydrographic setting 50 µmol L−1) and a seasonally variable ODZ on the continen- tal shelf (∼50 mbss to seaﬂoor; [O2] < 5 µmol L−1), where the most severe oxygen depletion occurs during late austral summer (Chapman and Shannon, 1987; Bailey et al., 1991; Mercier et al., 2003; Ekau and Verheye, 2005; Brüchert et al., 2006; Mohrholz et al., 2008). In the south, the northern BUS is bordered by the Lüderitz upwelling cell around ∼26◦S (Boyer et al., 2000). The BUS is located off the southwest African coast, where the cold and nutrient-rich waters of the Benguela current (BC) are upwelled through a combination of wind-driven Ekman transport and collision with the African continental shelf. The studied area is situated in the northern part of the BUS (16–26◦S and 10–16◦E; Fig. 2). Here, changes in the offshore wind ﬁeld, which affect upwelling and hence primary production, result in seasonal variations and movements of the oxygen-depleted waters (Chapman and Shannon, 1987). The northern border of the BUS is de- lineated by the dynamic Angolan Benguela Front (ABF; ∼16–20◦S; Fig. 2), where the warm and oligotrophic wa- ters of the Angola current (AC), transporting the oxygen- poor (< 45 µmol L−1) South Atlantic Central Water (SACW) southwards, converge with the cold and nutrient-rich waters of the equatorward BC. Seasonal variations in the intensity of the AC and the BC control dissolved oxygen (DO) concen- trations in the BUS (Mohrholz et al., 1999; Brüchert et al., 2006). This results in a near permanently present OMZ lo- cated off the continental shelf (∼200–500 mbss; [O2] ∼20– 1 Introduction (2011) had earlier proposed the ratio of BHT-x over total BHT (BHT-x ratio) to be a proxy for water column de- oxygenation ([O2] < 50 µmol kg−1). These studies show the Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 2.4 Lipid extraction and analysis centrations (Figs. 3a, 4b, f). From here on, the deeper stations (> 300 mbss stations 1, 2, 8, 9, 59), sampled in the OMZ off the continental shelf, will be termed “offshore stations”, whereas the shallower stations (< 120 mbss; stations 3–6, 10, 18, 117, 140) sampled on the continental shelf will be termed “shelf stations” (Table 1). At each station, physical param- eters of the water column were recorded with a Sea-Bird SBE911+ conductivity–temperature–depth (CTD) system. The CTD system was equipped with an additional SBE 43 oxygen electrode (Sea-Bird Electronics, WA, USA) to mea- sure DO (detection limit of 1–2 µmol L−1). A NIOZ-made Rosette sampler of 24 × 12 L Niskin bottles with hydrauli- cally controlled butterﬂy lids was used to collect water for nutrient and DNA analysis. A ∼0.5 bar overpressure of N2 gas was applied to the Niskin bottles to collect water without introducing oxygen. Water samples for DNA analysis were collected into pre-cleaned acid-washed Nalgene bottles. Ap- proximately 2 L of water was ﬁltered over 0.2 µm pore diam- eter Millipore Sterivex ﬁlters from the Nalgene bottles kept on ice in a climate-controlled container set at 4 ◦C. A total of 2 mL of RNAlater (RNA protect bacteria reagent, Qia- gen) was applied to the Sterivex cartridges, which were then sealed with paraﬁlm and stored at −20 ◦C until further pro- cessing. Suspended particulate matter (SPM) for lipid anal- ysis was collected using four McLane Large Volume Water Transfer System Sampler (WTS-LV) in situ pumps, which were deployed for 4 h (∼40–900 L ﬁltered; McLane Labo- ratories Inc., Falmouth, MA, USA). Water was ﬁltered over pre-ashed GF75-grade glass ﬁber ﬁlters of 0.3 µm pore size and 142 mm diameter (Advantec MFS, Inc., USA). Filters were wrapped in aluminum foil and stored at −80 ◦C until further processing. Water column sampling depths were cho- sen based on the CTD proﬁles, focusing around and below the redoxcline (Table S1). Z. R. van Kemenade et al.: Bacteriohopanetetrol-x centrations (Figs. 3a, 4b, f). From here on, the deeper stations (> 300 mbss stations 1, 2, 8, 9, 59), sampled in the OMZ off the continental shelf, will be termed “offshore stations”, whereas the shallower stations (< 120 mbss; stations 3–6, 10, 18, 117, 140) sampled on the continental shelf will be termed “shelf stations” (Table 1). At each station, physical param- eters of the water column were recorded with a Sea-Bird SBE911+ conductivity–temperature–depth (CTD) system. The CTD system was equipped with an additional SBE 43 oxygen electrode (Sea-Bird Electronics, WA, USA) to mea- sure DO (detection limit of 1–2 µmol L−1). A NIOZ-made Rosette sampler of 24 × 12 L Niskin bottles with hydrauli- cally controlled butterﬂy lids was used to collect water for nutrient and DNA analysis. A ∼0.5 bar overpressure of N2 gas was applied to the Niskin bottles to collect water without introducing oxygen. Water samples for DNA analysis were collected into pre-cleaned acid-washed Nalgene bottles. Ap- proximately 2 L of water was ﬁltered over 0.2 µm pore diam- eter Millipore Sterivex ﬁlters from the Nalgene bottles kept on ice in a climate-controlled container set at 4 ◦C. A total of 2 mL of RNAlater (RNA protect bacteria reagent, Qia- gen) was applied to the Sterivex cartridges, which were then sealed with paraﬁlm and stored at −20 ◦C until further pro- cessing. Suspended particulate matter (SPM) for lipid anal- ysis was collected using four McLane Large Volume Water Transfer System Sampler (WTS-LV) in situ pumps, which were deployed for 4 h (∼40–900 L ﬁltered; McLane Labo- ratories Inc., Falmouth, MA, USA). Water was ﬁltered over pre-ashed GF75-grade glass ﬁber ﬁlters of 0.3 µm pore size and 142 mm diameter (Advantec MFS, Inc., USA). Filters were wrapped in aluminum foil and stored at −80 ◦C until further processing. Water column sampling depths were cho- sen based on the CTD proﬁles, focusing around and below the redoxcline (Table S1). 2.4.2 BHP and IPL analyses Deuterated diacylglyceryltrimethylhomoserine (DGTS D-9; Avanti® Polar Lipids, USA) was added as an internal stan- dard to BDE aliquots. Aliquots were then ﬁltered over 4 mm True generated cellulose syringe ﬁlters (0.45 µm, BGB, USA) and re-dissolved in a MeOH : DCM solution of 9 : 1 (v : v) before analysis. Filtered aliquots were analyzed on an Agilent 1290 Inﬁnity I ultra-high-performance liq- uid chromatographer (UHPLC) equipped with a thermostat- ted auto-injector and column oven, coupled to a Q Exac- tive Orbitrap MS with an Ion Max source and heated elec- trospray ionization probe (HESI; Thermo Fisher Scientiﬁc, Waltham, MA). Separation was accomplished with an Ac- quity BEH C18 column (2.1×150 mm, 1.7 µm, Waters) main- tained at 30 ◦C. An eluant of (A) MeOH / H2O / formic acid / 14.8 M NH3 aq with a ratio of 85 : 15 : 0.12 : 0.04 (v : v : v : v) and (B) IPA / MeOH / formic acid / 14.8 M NH3 aq with a ratio of 50 : 50 : 0.12 : 0.04 (v : v : v : v) was used, with a ﬂow rate of 0.2 mL min−1. The elution program was set at 5 % B for 5 min, followed by a linear gradient to 40 % B at 12 min and then to 100 % B at 50 min, which was maintained until 80 min. Positive ion HESI settings were as follows: capillary temperature, 300 ◦C; sheath gas (N2) pressure, 40 arbitrary units (AU); auxiliary gas (N2) pres- sure, 10 AU; spray voltage, 4.5 kV; probe heater temperature, 50 ◦C; and S-lens, 70 V. Lipids were detected using posi- 2.4.1 Modiﬁed Bligh and Dyer extraction Freeze-dried ﬁlters were extracted four times using a mod- iﬁed Bligh and Dyer extraction (BDE) method (Bligh and Dyer, 1959; Sturt et al., 2004; Bale et al., 2021). The ﬁlters were cut in ± 1 cm ×1 cm pieces and ultrasonically extracted (10 min) using a solvent mixture of 2 : 1 : 0.8 (v : v : v) methanol (MeoH), dichloromethane (DCM) and phosphate buffer; then they were sonicated for 10 min and centrifuged for 2 min at 3000 rpm. The supernatant was then transferred to another tube, while the residue was re-extracted thrice (i.e., total of four extraction rounds), where during the last two extractions the phosphate buffer was replaced with a trichloroacetic acid solution to enable optimal recovery of IPLs (Sturt et al., 2004). Phase separation between the sol- vent layer and aqueous layer was induced by adding addi- tional DCM and phosphate buffer to obtain a ratio of 1 : 1 : 0.9 (v : v : v). The bottom DCM layer, containing the lipid extract, was collected, while the aqueous layer was washed two more times with DCM. The combined DCM layers were dried under N2 gas. This extraction method was also per- formed on freeze-dried biomass from a Ca. Scalindua bro- dae enrichment culture, obtained from an anoxic sequencing batch reactor at Radboud University, Nijmegen, the Nether- lands (described in Schwartz-Narbonne et al., 2019). 2.2 Sample collection Between January and March 2019, two consecutive research expeditions in the Namibian BUS were undertaken with the R/V Pelagia (64PE449 and 64PE450). At this time, the low- oxygen waters of the SACW reached their maximum south- ward extension (Chapman and Shannon, 1987), which, com- bined with limited cross-shore bottom-water ventilation at the start of the annual upwelling cycle, led to severe oxy- gen depletion (Mohrholz et al., 2008). Sampling was per- formed at various water depths at 13 stations (11◦22′36.5′′– 14◦47′34.8′′ E and 17◦16′38.3′′–25◦12′25.0′′ S; Fig. 2; Ta- ble 1), covering a large range in water column oxygen con- https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 204 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x The BHT-x ratio was then calculated as tive ion monitoring of m/z 350–2000 (resolution 70 000 ppm at m/z 200), followed by data-dependent MS2 (resolution 17 500 ppm) of the 10 most abundant ions and dynamic ex- clusion (for 6 s) within 3 ppm mass tolerance. An inclusion list with the calculated exact masses of 165 calculated BHPs was applied. Optimal fragmentation was achieved with a stepped normalized collision energy of 22.5 and 40 (isola- tion width of 1.0 m/z) for BHP analysis (Hopmans et al., 2021) and 15, 22.5 and 30 (isolation width of 1.0 m/z) for IPL analysis (Bale et al., 2021). The Q Exactive Orbi- trap was calibrated every 48 h using the Thermo Fisher Sci- entiﬁc Pierce LTQ Velos ESI Positive Ion Calibration So- lution. The summed mass chromatograms of the ammoni- ated ([M + NH4]+; m/z 564.499) and sodiated ([M + Na]+; m/z 569.454) adducts of BHT(-x) were integrated within 3 ppm mass accuracy. The BHT-x ratio was then calculated as ple in each sequence run. Due to a lack of commercially available quantiﬁcation standards for BHT(-x) and ladder- ane IPLs, abundances are reported as the peak area response (response unit, ru) per liter of ﬁltered water. Although this does not allow for quantiﬁcation of absolute concentrations, it does allow for quantiﬁcation of the relative abundances, as the response factor should be identical across the sample set. 2.4.3 Ladderane fatty acids SPM from stations 2, 6 and 140 were additionally analyzed for ladderane FAs. BDE aliquots were saponiﬁed by adding 2 ml of KOH (1 M in 96 % MeoH) and reﬂuxing for 1 h. Then, 2 mL of bidistilled water was added, and the pH was adjusted to 3 with 1 : 1 HCL : MeOH (v : v). To collect the fatty acid fraction, 2 mL of DCM was added, after which the tube was sonicated for 5 min and centrifuged for 2 min at 3000 rpm. The fatty acid fraction (DCM layer) was col- lected, and the procedure was repeated two more times. Fatty acid fractions were then dried over a sodium sulfate (Na2SO4) column. The fractions were then methylated us- ing diazomethane to convert FAs into their corresponding fatty acid methyl esters (FAMEs). To remove polyunsatu- rated fatty acids (PUFAs), extracts were dissolved in DCM and eluted over a silica column impregnated with silver nitrate (AgNO3). Lastly, the FAME fractions were ﬁltered through a 0.45 µm PTFE ﬁlter (BGB, USA) using acetone. BHT-x ratio = BHT-x (BHT + BHT-x). (2) (2) All ladderane IPLs reported by Rattray et al. (2008) were evaluated in the BUS SPM samples and a Ca. Scalindua bro- dae enrichment culture (see Table S4 for exact masses). Only PC C20[3] monoalkylether (from here on termed “PC lad- derane”) and an ether-ester PG C20[3]–C18[5] (from here on termed “PG ladderane”) were detected in the BUS SPM sam- ples (Fig. 1c). For the PC ladderane, the exact ion mass was used for integration ([M]+; m/z 530.361). For the PG ladder- ane, the combined protonated ([M + H]+; m/z 775.491) and ammoniated ([M + NH4]+; m/z 792.517) adduct was used for integration (within 3 ppm mass accuracy). BDE of the Ca. S. brodae enrichment culture (containing both ladder- ane IPLs and BHT-x) was used as a quality control sam- Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 205 Table 1. Sampling date, cruise name, station label, coordinates and sediment depth (mbss) during expeditions 64PE449 and 64PE450. Stations are grouped according to location type and listed based on their sampling date. Location type is “shelf” when sediment depth is < 120 mbss or “offshore” when sediment depth is > 300 mbss. Table 1. Sampling date, cruise name, station label, coordinates and sediment depth (mbss) during expeditions 64PE449 and 64PE450. Stations are grouped according to location type and listed based on their sampling date. Location type is “shelf” when sediment depth is < 120 mbss or “offshore” when sediment depth is > 300 mbss. Date Cruise Station Latitude Longitude Depth Location (dd-mm-yyyy) (◦S) (◦E) (mbss) 01-02-2019 64PE449 3 23.096 14.129 120 Shelf 06-02-2019 64PE449 4 23.011 14.244 105 Shelf 07-02-2019 64PE449 5 24.020 14.303 100 Shelf 08-02-2019 64PE449 6∗ 25.072 14.596 100 Shelf 12-02-2019 64PE449 10 21.968 13.793 103 Shelf 19-02-2019 64PE450 18 17.409 11.635 100 Shelf 01-03-2019 64PE450 117 20.001 12.823 105 Shelf 07-03-2019 64PE450 140∗ 25.072 14.596 100 Shelf 03-02-2019 64PE449 1 24.056 12.843 1500 Offshore 04-02-2019 64PE449 2 24.042 13.127 720 Offshore 10-02-2019 64PE449 8 23.961 13.227 324 Offshore 11-02-2019 64PE449 9 23.962 13.226 407 Offshore 25-02-2019 64PE450 59 17.277 11.377 1000 Offshore ∗Denotes same station location, sampled 27 d apart. ∗Denotes same station location, sampled 27 d apart. tive ion monitoring of m/z 350–2000 (resolution 70 000 ppm at m/z 200), followed by data-dependent MS2 (resolution 17 500 ppm) of the 10 most abundant ions and dynamic ex- clusion (for 6 s) within 3 ppm mass tolerance. An inclusion list with the calculated exact masses of 165 calculated BHPs was applied. Optimal fragmentation was achieved with a stepped normalized collision energy of 22.5 and 40 (isola- tion width of 1.0 m/z) for BHP analysis (Hopmans et al., 2021) and 15, 22.5 and 30 (isolation width of 1.0 m/z) for IPL analysis (Bale et al., 2021). The Q Exactive Orbi- trap was calibrated every 48 h using the Thermo Fisher Sci- entiﬁc Pierce LTQ Velos ESI Positive Ion Calibration So- lution. The summed mass chromatograms of the ammoni- ated ([M + NH4]+; m/z 564.499) and sodiated ([M + Na]+; m/z 569.454) adducts of BHT(-x) were integrated within 3 ppm mass accuracy. 2.3 Nutrient analysis Samples for nutrient analysis were sub-sampled from the Niskin bottles with pre-ﬂushed 60 mL high-density polyethy- lene syringes with a three-way valve. Samples for PO3− 4 NO− 2 , NO− 3 and NH+ 4 analysis were ﬁltered over a com- bined 0.8–0.2 µm Supor Membrane Acrodisc PF syringe ﬁlter (PALL Corporation, NY, USA) into pre-rinsed 5 mL polyethylene vials and analyzed on board with an autoana- lyzer (QuAAtro39 gas segmented continuous-ﬂow analyzer, Seal Instruments). Detection limits for PO3− 4 , NO− 2 , NO− 3 and NH+ 4 were 0.005, 0.003, 0.015 and 0.019 µmol L−1, re- spectively. The ﬁxed inorganic nitrogen deﬁcit was calcu- lated as (1) N deﬁcit = 16 × [PO3− 4 ] [NO− 3 ] + [NO− 2 ] + [NH+ 4 ] , (1) N deﬁcit = 16 × [PO3− 4 ] [NO− 3 ] + [NO− 2 ] + [NH+ 4 ] , (1) in which 16 reﬂects the Redﬁeld ratio of N : P (Redﬁeld et al., 1963). Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 206 with an atmospheric pressure chemical ionization (APCI) probe (Thermo Fisher Scientiﬁc, Waltham, MA). Separation was realized with a ZORBAX Eclipse XDB C18 column (Ag- ilent, 3.0 × 250 mm, 5 µm), maintained at 30 ◦C. MeOH was used as an eluant at 0.18 mL min−1 with a total run time of 20 min. Optimal APCI source settings were determined us- ing a qualitative standard mixture of [3]- and [5]-ladderane FAMEs. Positive ion APCI source settings were as follows: corona discharge current, 2.5 µA; source CID, 10 eV; vapor- izer temperature, 475 ◦C; sheath gas ﬂow rate, 50 arbitrary units (AU); auxiliary gas ﬂow rate, 30 AU; capillary temper- ature, 300 ◦C; and S-lens, 50 V. A mass range of m/z 225– 380 was monitored (resolution 140 000 ppm), followed by a data-dependent MS2 (resolution 17 500 ppm at m/z 200), in which the 10 most abundant masses in the mass spec- trum were fragmented successively (stepped normalized col- lision energy 20, 25, 30). An inclusion list containing the exact masses of C14−24[3]- and [5]-ladderane FAMEs was used. Mass chromatograms (within 5 ppm mass accuracy) of the protonated molecules ([M + H]+) were used to inte- grate C18[3]-, C18[5]-, C20[3]- and C20[5]-ladderane FAMEs (m/z 291.232, 289.216, 319.263 and 317.248, respectively). Ladderane FAMEs were quantiﬁed by external calibration curves of isolated standards of the C20[3]- and [5]-ladderane FAMEs (Hopmans et al., 2006; Rattray et al., 2008; Rush et al., 2011). Ladderane FAME standards were isolated from an anammox enrichment culture grown in sequencing batch re- actors, containing both Ca. Scalindua wagneri and Ca. Kue- nenia stuttgartiensis (described in Kartal et al., 2006). The in- dex of ladderane lipids with ﬁve cyclobutane rings (NL5) was calculated to quantify the trends in ladderane chain lengths with respect to temperature, using Ca. Scalindua (e.g., Yang et al., 2020) and is reported to reﬂect marine community compositions well (Parada et al., 2016). Reagents were mixed with 50 times diluted DNA template (2 µL) by addition of 11.75 µL Nuclease free wa- ter, 5 µ L 5× Qiagen Phusion buffer, 2 µL dNTPs (2.5 mM), 3 µL of 515F-Y/926R primer pair (4 µmol L−1), 1 µL BSA (20 mg mL−1) and 0.25 µL Taq polymerase (5 µL−1). Neg- ative controls were included during extractions and PCR reactions. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Ampliﬁcation was performed using the follow- ing PCR program: 30 s at 98 ◦C (one cycle), 10 s at 98 ◦C followed by 20 s at 50 ◦C and 30 s at 72 ◦C (30 cycles), 7 min at 72 ◦C (one cycle), and ending with 5 min at 4 ◦C. To quantify DNA concentrations of the PCR reagents, PCR products were mixed with a Xylene Ficoll loading dye and loaded on a 2 % agarose gel, together with a homemade Es- cherichia coli quantiﬁcation standard dilution series (20, 10 and 1 ng µL−1). Gel electrophoresis was performed for 1 h at 75 V. After, the gel was stained with Ethidium bromide. Gels were imaged using GeneSys (lightning, TLUM – Mid Wave; ﬁlter, UVO32). The 400 bp bands were then quanti- ﬁed using the “QuickQuant” option. Following quantiﬁca- tion, all PCR products were pooled in equimolar amounts (40 ng DNA per sample) and loaded on a 2 % gel stained with SYBRsafe®. The 400 bp band was extracted from the gel using the QIAquick® PCR gel extraction kit. Concentra- tion of the pooled PCR product (20 ng µL−1) was quantiﬁed using Qbit (Thermo Fisher Scientiﬁc Inc.). Library prepara- tion was performed with a 16S V4–V5 library preparation kit and sequencing with an Illumina MiSeq 2×300 bp sequenc- ing platform (Illumina, San Diego, CA) at the University of Utrecht Sequencing Facility (USEQ, the Netherlands). The prokaryotic 16S rRNA gene amplicon sequences were analyzed using the Cascabel data analysis pipeline (Abdala Asbun et al., 2020). Raw forward and reverse reads were merged using PEAR (Zhang et al., 2014). Barcode reads were demultiplexed using QIIME (Caporaso et al., 2010), al- lowing a maximum of two barcode mismatches, a maximum of ﬁve consecutive low-quality base calls and a maximum un- accepted Phred quality score of 19. Reads were then ﬁltered based on length using the values of the median distribution, with an offset of 10 bp. Sequences were dereplicated with VSEARCH and subsequently clustered to operational taxo- nomic units (OTUs) using the UCLUST algorithm in QIIME, with a 97 % threshold. From each OTU, the most abundant sequence was picked as representative with QIIME (Capo- raso et al., 2010). NL5 = C20 [5] ladderane FA (C18 [5]ladderane FA + C20 [5] ladderane FA). Z. R. van Kemenade et al.: Bacteriohopanetetrol-x (3) (3) Following, the relationship between NL5 and temperature is then given by NL5 = 0.2 + 0.7 1 + e− T −16.3 1.5 , (4) (4) with temperature (T ) in degrees Celsius (Rattray et al., 2010). 2.4.4 Ladderane fatty acid analysis Puriﬁed FAME fractions were analyzed on an Agilent 1290 Inﬁnity I ultra-high-performance liquid chromatographer (UHPLC) equipped with a thermostatted auto-injector and column oven, coupled to a Q Exactive Plus Orbitrap MS, https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x A Kimura two-parameter model with gamma distributed sites was then used to calculate pairwise dis- tances between sequences and to create a maximum likeli- hood tree in MEGAX, using a bootstrap with 1000 replicates and the maximum parsimony method to create the initial tree (Kimura, 1980; Kumar et al., 2018). To estimate the relative abundance of Ca. Scalindua spp. 16S rRNA reads in rela- tion to the total amount of bacterial 16S rRNA reads, relative Ca. Scalindua spp. reads were calculated for each sample (in percent of total bacterial reads). Though this does not allow for absolute quantiﬁcation, it does allow for a comparison of relative abundances throughout the dataset of this study, as all samples were processed and analyzed in the same way. 3.2.1 BHT and BHT-x abundances and ratio A multivariate binomial regression was performed with anammox biomarker lipids and Ca. Scalindua 16S rRNA gene amplicon sequences. 16S rRNA gene amplicon se- quences were used dichotomously, deﬁned as either the presence or absence of Ca. Scalindua assigned OTUs in a given sample. Pearson’s correlations between anammox lipid biomarkers and the physiochemical parameters were also in- vestigated (Matlab, R2019a). BHT was found in all SPM samples, except one (station 117, 40 mbss). BHT abundance ranged between 1.0 × 105– 2.1 × 108 ru L−1 at the shelf stations and between 1.1 × 105– 1.8 × 107 ru L−1 at the offshore stations (Table S3). BHT-x was found exclusively in the southern part of the northern BUS. No BHT-x was observed at stations located near the ABF (station 117) nor north of the ABF (stations 18 and 59). When present, the BHT-x abundance ranged from 1.4×105– 1.6×107 ru L−1 (Figs. 4a, e, 5a, h; Table S3). At the southern stations located on the shelf (stations 3–6, 10 and 140), BHT- x was observed below ∼30 mbss. At these stations, the BHT- x abundance ranged between 2.3×105–1.6×107 ru L−1. At the southernmost shelf station, when sampled in February (station 6), BHT-x was observed between 50–85 mbss, in- creasing in abundance from 8.5 × 105 ru L−1 at 50 mbss to 8.3 × 106 ru L−1 at 85 mbss (Fig. 5a). In March (station 140), BHT-x was present at all sampled depths (35–85 mbss), rang- ing between 1.5–5.3 × 106 ru L−1 (Fig. 5a). At the southern offshore stations (stations 1, 2, 8, and 9), BHT-x was present between ∼200–400 mbss and in the bottom waters of sta- tions 1 (1500 mbss) and 2 (710 mbss). Here, the abundance ranged between 1.4 × 105–1.2 × 106 ru L−1 (Fig. 5h). The BHT-x ratio (Eq. 2) in the BUS water column ranged be- tween 0.00–0.55 (Fig. 6, Table S3). 3.1 Hydrography and seawater chemistry The location of the Angolan Benguela front (ABF) dur- ing the expedition was ∼19.8◦S, close to Walvis Ridge, where large horizontal gradients in sea surface temperature (SST, temperatures integrated between 0–30 mbss) and salin- ity (measured at the halocline) were observed, and surface isotherms and isohalines fanned out seawards (Fig. 3b and c). At the time of sampling, upwelled waters from the BC were clearly distinguishable at the stations south of the ABF (stations 1–6, 10, 117 and 140; 20◦S–26◦S), where SST and salinity are relatively low, ranging between 12.9–19.3 ◦C and 35.1–35.6 psu, respectively (Table S1). Offshore (bottom depth ∼300–1500 mbss), a weak OMZ was present between ∼100–500 mbss, with [O2] levels down to ∼40 µmol L−1. In the shallow waters on the continental shelf (bottom depth < 120 mbss) an ODZ was present, with [O2] decreasing rapidly with depth from 30 mbss onwards, with nearly anoxic waters ([O2] ∼1.5–5.5 µmol L−1) below ∼50–80 mbss. The strongest oxygen depletion was found at the shallowest sta- tions (< 100 mbss; stations 4, 5, 6, 10, 117 and 140), while at the slightly deeper shelf station 3 (∼120 mbss), [O2] in bottom waters was not lower than ∼20 µmol L−1 (Figs. 4b, 5c). The inﬂuence of the AC was apparent at stations north 2.5 DNA extraction and phylogenetic analysis Millipore® Sterivex™ﬁlters were extracted for DNA us- ing the Qiagen DNeasy Powersoil kit®. PCRs of the DNA templates were conducted with the Qiagen® PCR reagents (Taq PCR Master Mix Kit). The universal prokaryotic primer pair, forward 515F-Y and reverse 926R (Parada et al., 2016) was used to target the V4–V5 small subunit ribosomal RNA region and was modiﬁed with 12 different nucleotide bar- codes at both the forward and reverse primers. The 515F- Y/926R primer pair has been found to successfully target Taxonomy was assigned based on the SILVA database (SSU 138 Ref NR 99), using the VSEARCH tool. OTUs rep- resentative of the order Brocadiales (to which Ca. Scalin- dua spp. belong) were extracted with QIIME using ﬁl- ter_taxa_from_otu_table.py. The ﬁltered sequences were im- ported in the SILVA NR99 SSU 138 Ref database using the ARB parsimony tool in the ARB software package (ARB SILVA, Germany) to assess the phylogenetic afﬁliation of the partial 16S rRNA gene sequences. Afﬁliated sequences Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 207 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x of the ABF (stations 18 and 59), where SST and salinity are relatively high (Fig. 3b and c), ranging between 16.9–22.9 ◦C and 35.7–35.9 psu, respectively (Table S1). In these waters, [O2] declined to ∼20 µmol L−1 between ∼50–500 mbss (Figs. 4f, 5j). During the expeditions, water column nutrient concentrations in the BUS ranged between 0.0–4.9 µmol L−1 for NO− 2 , 0–40 µmol L−1 for NO− 3 and 0.1–9.8 µmol L−1 for NH+ 4 (Fig. 5d–f, k–m; Table S2). Nearly all sampled stations exhibited an N deﬁcit (Eq. 1) throughout the water column, with the strongest deﬁcit being observed at the shelf stations (Fig. 4d, h; Table S2). The N deﬁcit was highest at station 6, reaching a deﬁcit of ∼49 µmol L−1 at 85 mbss. When sta- tion 6 was resampled in March (station 140), the deﬁcit had decreased to a maximum of ∼23 µmol L−1 at 10 mbss. The only station in the BUS where no N deﬁcit was observed was station 117. were checked for homology and imported in MEGAX us- ing the BLAST search query (Kumar et al., 2018). The 12 OTUs with the largest number of reads and 27 reference se- quences were aligned, based on 422 bp, with the Clustal W alignment tool. A Kimura two-parameter model with gamma distributed sites was then used to calculate pairwise dis- tances between sequences and to create a maximum likeli- hood tree in MEGAX, using a bootstrap with 1000 replicates and the maximum parsimony method to create the initial tree (Kimura, 1980; Kumar et al., 2018). To estimate the relative abundance of Ca. Scalindua spp. 16S rRNA reads in rela- tion to the total amount of bacterial 16S rRNA reads, relative Ca. Scalindua spp. reads were calculated for each sample (in percent of total bacterial reads). Though this does not allow for absolute quantiﬁcation, it does allow for a comparison of relative abundances throughout the dataset of this study, as all samples were processed and analyzed in the same way. were checked for homology and imported in MEGAX us- ing the BLAST search query (Kumar et al., 2018). The 12 OTUs with the largest number of reads and 27 reference se- quences were aligned, based on 422 bp, with the Clustal W alignment tool. 3.2.3 Ladderane FAs Ladderane FAs (i.e., C18[3]-, C18[5]-, C20[3]- and C20[5]- ladderane FAs) were analyzed as FAMEs in the SPM from an offshore station (station 2) and of the southernmost shelf station, sampled in February (station 6) and in March (sta- tion 140). Summed ladderane FA concentrations ranged be- tween 15–320 pg L−1 (Fig. 7; Table S5). At offshore sta- tion 2 (Fig. 7a), of the six sampled depths, ladderane FAs were observed at 125, 250 and 710 mbss, with peak con- centrations at 250 mbss (320 pg L−1). At the southernmost shelf station sampled in February (station 6; Fig. 7b), the highest ladderane FA concentrations were observed at the deepest sampling depth (85 mbss, 140 pg L−1). Concentra- tions an order of magnitude lower were found at the oxycline (30 mbss; 20 pg L−1) and at 60 mbss (30 pg L−1), while no ladderane FAs were observed at 35 or 40 mbss. At this lo- cation in March (station 140; Fig. 7c), ladderane FAs were present throughout the oxygen-depleted interval of the wa- ter column (35–85 mbss), with total concentrations ranging 3.3 16S rRNA gene analysis The 16S rRNA gene amplicon sequencing (422 bp fragment) analysis was conducted on SPM collected at 13 stations from various water column depths, including the same or similar depths as analyzed for lipids. A total of 10 283 136 bacterial reads were recovered from the BUS stations (excluding sin- gletons), of which 0.14 ‰ could be taxonomically assigned to the genus Ca. Scalindua (Arb SILVA 132R database). These sequences were further analyzed to assess the distri- bution and phylogeny of Ca. Scalindua in the BUS. Nega- tive controls did not contain reads taxonomically assigned to Ca. Scalindua spp. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 208 Figure 3. Isosurface plots of (a) minimum observed oxygen concentration in water column (µmol L−1), (b) sea surface temperature (SST, ◦C) and (c) maximum observed salinity in water column (psu). ABF: Angolan Benguela Front (∼19.8◦S). Maps were created in ODV using the ETOP01_2min global tiles map 150×75 weighted average gridding (Schlitzer, R, Ocean Data View, https://odv.awi.de, last access: 12 June 2021). Figure 3. Isosurface plots of (a) minimum observed oxygen concentration in water column (µmol L−1), (b) sea surface temperature (SST, ◦C) and (c) maximum observed salinity in water column (psu). ABF: Angolan Benguela Front (∼19.8◦S). Maps were created in ODV using the ETOP01_2min global tiles map 150×75 weighted average gridding (Schlitzer, R, Ocean Data View, https://odv.awi.de, last access: 12 June 2021). between 20–60 pg L−1. The NL5 index was calculated for all SPM samples that contained both C20[5] and C18[5] FAs (Table S5). For station 2, the NL5 was 0.9 at 250 mbss and 0.5 at 710 mbss. For stations 6 and 140, the NL5 ranged be- tween 0.2–0.4. No evidence was found for the presence of short-chain biodegradation products (i.e., C14[3]-, C14[5]- , C16[3]-, C16[5]-ladderane FAs; Rush et al., 2011) nor C22−24-ladderane FAs. ited number of shelf stations located south of the ABF (sta- tions 5, 10, 117 and 140), below ∼30 mbss waters. Concen- trations ranged between 1.1 × 104–6.4 × 105 ru L−1 for the PG ladderane and between 2.1 × 104–4.2 × 105 ru L−1 for the PC ladderane (Table S3). At station 5, PC and PG ladder- anes were both present. At stations 10, 117 and 140 the PG ladderane was less abundant or absent at the water column depths where the PC ladderane was found. From here on, their summed abundances are reported as “ladderane IPLs” (Figs. 4c, g, 5b, i). 3.2.2 Ladderane IPLs The presence of all ladderane IPLs reported for the Ca. Scalindua brodae enrichment culture (Table S4) and those previously reported for Ca. Scalindua spp. (Rattray et al., 2008) was evaluated in the BUS SPM samples. How- ever, at the time of sampling, only the PC and PG ladderanes (Fig. 1c) were detected in the BUS water column. Further- more, these ladderane IPLs were found in SPM from a lim- https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 209 gure 4. Longitudinal (a–d) and latitudinal (e–h) section plots showing interpolated concentrations of (a, e) BHT-x (ru L−1), (b, f) dissolve ygen (µmol L−1), (c, g) ladderane IPLs (ru L−1) and (d, h) nitrogen deﬁcit (µmol L−1). Contour lines for O2 and N deﬁcit concentration e shown. Figure 4. Longitudinal (a–d) and latitudinal (e–h) section plots showing interpolated concentrations of (a, e) BHT-x (ru L−1), (b, f) dissolved oxygen (µmol L−1), (c, g) ladderane IPLs (ru L−1) and (d, h) nitrogen deﬁcit (µmol L−1). Contour lines for O2 and N deﬁcit concentrations are shown. detected in 11 out of 13 stations, in SPM collected at the shelf (< 120 mbss; stations 3–6, 10 and 140) and at offshore stations (> 300 mbss; stations 1, 2, 8 and 9), but were not detected at stations located north of the ABF (stations 18 and 59). At shelf stations (Fig. 5g), the relative Ca. Scalin- dua spp. gene read abundance ranged between 0 ‰–2.7 ‰, with the highest abundances found below 50 mbss. In sur- face waters (< 30 mbss), no Ca. Scalindua spp. 16S rRNA gene sequences were detected, except at station 140, where Ca. Scalindua spp. were present throughout the water column (0.03 ‰–0.53 ‰). At offshore stations, the relative abun- dance of Ca. Scalindua spp. 16S rRNA gene copies ranged between 0 ‰–1.5 ‰, with the highest relative abundances found in bottom waters at station 2 at 700 and 710 mbss (Fig. 5n). At offshore stations 1, 8 and 9, Ca. Scalindua spp. were detected between 50–390 mbss. 3.3.1 Distribution of Ca. Scalindua spp. 16S rRNA sequences in the BUS The relative abundances of Ca. Scalindua spp. 16S rRNA gene sequences with respect to the total number of bacte- rial 16S rRNA gene sequences was calculated to estimate the distribution of Ca. Scalindua in the BUS (Fig. 5g, n). Se- quences taxonomically assigned to Ca. Scalindua spp. were Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 210 Figure 5. Concentration and relative abundance proﬁles in the water column of the BUS at shelf (top panels) and offshore (bottom panels) stations. (a, h) BHT-x abundance (ru L−1); (b, i) ladderane IPL abundance (ru L−1); concentrations in µmol L−1 of (c, j) oxygen [O2], (d, k) nitrate [NO− 3 ], (e, l) nitrite [NO− 2 ] and (f, m) ammonium [NH+ 4 ]; and relative abundance of Ca (g, n). Scalindua spp. reads in per mill of total bacterial reads (NB circles are plotted on bottom x axis with a different scale). Figure 5. Concentration and relative abundance proﬁles in the water column of the BUS at shelf (top panels) and offshore (bottom panels) stations. (a, h) BHT-x abundance (ru L−1); (b, i) ladderane IPL abundance (ru L−1); concentrations in µmol L−1 of (c, j) oxygen [O2], (d, k) nitrate [NO− 3 ], (e, l) nitrite [NO− 2 ] and (f, m) ammonium [NH+ 4 ]; and relative abundance of Ca (g, n). Scalindua spp. reads in per mill of total bacterial reads (NB circles are plotted on bottom x axis with a different scale). Figure 5. Concentration and relative abundance proﬁles in the water column of the BUS at shelf (top panels) and offshore (bottom panels) stations. (a, h) BHT-x abundance (ru L−1); (b, i) ladderane IPL abundance (ru L−1); concentrations in µmol L−1 of (c, j) oxygen [O2], (d, k) nitrate [NO− 3 ], (e, l) nitrite [NO− 2 ] and (f, m) ammonium [NH+ 4 ]; and relative abundance of Ca (g, n). Scalindua spp. reads in per mill of total bacterial reads (NB circles are plotted on bottom x axis with a different scale). Figure 5. Concentration and relative abundance proﬁles in the water column of the BUS at shelf (top panels) and offshore (bottom panels) stations. (a, h) BHT-x abundance (ru L−1); (b, i) ladderane IPL abundance (ru L−1); concentrations in µmol L−1 of (c, j) oxygen [O2], (d, k) nitrate [NO− 3 ], (e, l) nitrite [NO− 2 ] and (f, m) ammonium [NH+ 4 ]; and relative abundance of Ca (g, n). Scalindua spp. reads in per mill of total bacterial reads (NB circles are plotted on bottom x axis with a different scale). Figure 6. 3.3.2 Ca. Scalindua phylogeny Ca. Scalindua 16S rRNA reads (422 bp) were assigned to 66 operational taxonomic units (OTU 1–66) based on 97 % se- quence similarity (Table S6). Most of the Ca. Scalindua spp. https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Scatter plots showing relationship between BHT-x ratio (BHT-x / (BHT + BHT-x) and oxygen concentration (µmol L−1) for stations located on the shelf (a) and stations located offshore (b). The BHT-x ratio observed in the Ca. S. brodae enrichment culture is indicated with the black dotted line at 0.38. The grey area corresponds to the standard deviation of the ratio (± 0.02) between different HPLC/MS runs. Figure 6. Scatter plots showing relationship between BHT-x ratio (BHT-x / (BHT + BHT-x) and oxygen concentration (µmol L−1) for stations located on the shelf (a) and stations located offshore (b). The BHT-x ratio observed in the Ca. S. brodae enrichment culture is indicated with the black dotted line at 0.38. The grey area corresponds to the standard deviation of the ratio (± 0.02) between different HPLC/MS runs. Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 211 Figure 7. Water column depth proﬁles of stations (a) 2, (b) 6 and (c) 140, showing distributions of (from left to right) ladderane FAs (pg L−1), ladderane IPLs and BHT-x (ru L−1), nitrite and ammonium concentrations (µmol L−1), oxygen and nitrate concentrations (µmol L−1), and temperature (◦C) and salinity (psu). Star symbols indicate water column depths where SPM was collected for DNA analysis. Filled stars represent depths where Ca. Scalindua spp. 16S rRNA gene sequences were detected. Stations 6 and 140 were sampled at the same location 27 d apart (station 6 in February and station 140 in March). https://doi org/10 5194/bg 19 201 2022 Biogeosciences 19 201 221 2022 Figure 7. Water column depth proﬁles of stations (a) 2, (b) 6 and (c) 140, showing distributions of (from left to right) ladderane FAs (pg L−1), ladderane IPLs and BHT-x (ru L−1), nitrite and ammonium concentrations (µmol L−1), oxygen and nitrate concentrations (µmol L−1), and temperature (◦C) and salinity (psu). Star symbols indicate water column depths where SPM was collected for DNA analysis. Filled stars represent depths where Ca. Scalindua spp. 16S rRNA gene sequences were detected. Stations 6 and 140 were sampled at the same location 27 d apart (station 6 in February and station 140 in March). Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 https://doi.org/10.5194/bg-19-201-2022 4.1.1 Anammox markers are constrained to the ODZ in the southern BUS shelf waters During expeditions 64PE449 and 64PE450, the seasonal ODZ had developed in the southern BUS shelf waters (20– 26◦S) between ∼50 mbss and the seaﬂoor, with [O2] down to ∼1.5–5.5 µmol L−1. Nutrient analyses revealed a large N deﬁcit in the ODZ (N deﬁcit > 10 µmol L−1; Fig. 4d), sug- gesting major losses of bioavailable N by anammox and/or denitriﬁcation. Relatively high BHT-x abundances are de- Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 212 reads (88 %) could be assigned to 12 OTUs (OTU-1 to OTU- 12) ranging from the relative most abundant to least abundant OTU (Fig. S1). To estimate the phylogenetic relationship of these 12 OTUs to Ca. Scalindua spp. sequences from other OMZs, a maximum likelihood tree was constructed with ref- erence sequences from various other OMZs and anammox enrichment cultures (Fig. 8). In addition, the pairwise dis- tances between these sequences (based on 422 bp) were cal- culated (Table S7). The phylogeny of the BUS OTUs re- vealed a cluster of 10 OTUs (OTUs 1–4, 6–9, 11 and 12; Fig. 8), with an overall sequence identity of 96 %. OTU-10 displays a relatively large evolutionary divergence (> 12 %; Fig. 8) and limited sequence identity (88 %) compared to the other BUS OTUs. The highest sequence identity of the BUS OTU cluster is observed with environmental sequences isolated from the Guaymas deep sea hydrothermal vent sed- iment (98 %) and the Black Sea suboxic waters (53 mbss; also 98 %), which in turn both exhibited the highest se- quence identity with Ca. Scalindua sorokiini (again 98 % in both cases). Sequence identity with Ca. Scalindua brodae and Ca. Scalindua spp. sequences detected previously in the Namibian OMZ (Kuypers et al., 2005) was 96 %. The lowest sequence identity is seen with Ca. Scalindua wagneri (93 %) and the Arabian Sea (94 %). OTU-5 is placed outside of the BUS cluster (Fig. 8) and shows the highest sequence identity with Gulf of Mexico and Indian Ocean sediments (98 % and 97 % respectively). Sequence identity of OTU-5 in relation to the BUS OTU cluster is 94 %. tected at the southern shelf stations (stations 3–6, 10, 140) below 30 mbss (Fig. 5a). The 16S rRNA gene analysis in- deed indicated on-shelf presence of Ca. Scalindua spp. in the water masses below 30 mbss, with the highest abun- dance of Ca. Scalindua spp. 16S rRNA gene sequences rel- ative to the total bacterial 16S rRNA gene sequence pool below ∼50 mbss (Fig. 5g). In addition to the Ca. Scalin- dua spp. 16S rRNA gene, ladderane IPLs are thought to re- ﬂect living or recently dead anammox cells (Harvey et al., 1986; Jaeschke et al., 2009a; Brandsma et al., 2011; Bale et al., 2014). 4 Discussion In the BUS, seasonal shifts in upwelling intensity create large spatiotemporal variability in oxygen concentrations (Bailey, 1991). Anammox has been reported previously in the low- oxygen BUS water column (Kuypers et al., 2005), which therefore presents an ideal location to investigate anammox biomarkers. We assessed the distribution of BHT-x across the redox gradient in the BUS to provide further insights into the application of BHT-x as a biomarker for Ca. Scalindua spp. and its ratio over total BHT (BHT-x ratio) as a proxy for de- oxygenation in dynamic upwelling systems. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Ladderane IPLs are found on-shelf (stations 4, 5, 10, 117 and 140) between 30 mbss and the seaﬂoor. The N deﬁcit was signiﬁcantly correlated with both BHT- x and ladderane IPLs (r(60) = 0.53, ρ = < 0.001; Table 2), and on-shelf N deﬁciencies were accompanied by relatively high BHT-x and ladderane IPL abundances (Fig. 4). This suggests anammox was at least in part responsible for loss of bioavailable N. In summary, the co-occurrence of BHT-x with Ca. Scalindua spp. 16S rRNA reads, ladderane IPLs and on-shelf N deﬁciencies indicates the presence of living (or re- cently dead) anammox cells in the BUS shelf waters (below ∼30 to 50 mbss), consistent with earlier reports of anammox activity on the Namibian continental shelf waters (Kuypers et al., 2005). Additionally, Ca. Scalindua spp. 16S rRNA gene se- quences and anammox biomarkers were detected in the more oxygenated surface shelf waters (above 50 mbss), at ambi- ent [O2] up to ∼45 µmol L−1 (and up to ∼90 µmol L−1 in one case), surpassing earlier established oxygen limits for anammox. Culturing studies have indicated that anammox bacteria are already inhibited at [O2] as low as 1 µmol L−1 (Strous et al., 1997). In the environment, Ca. Scalindua spp. has been shown to remain active at [O2] up to 9– 20 µmol L−1 in the Namibian and Peruvian OMZs (Kuypers et al., 2005; Hamersley et al., 2007; Kalvelage et al., 2011) and up to ∼9 µmol L−1 in the Black Sea (Jensen et al., 2008). A possible explanation has been provided by Woe- bken et al. (2007), who showed that Ca. Scalindua spp. colo- nize microscopic particles in the BUS, which provide suit- able anaerobic micro-niches. Nevertheless, this was found to be restricted to ambient [O2] levels below 25 µmol L−1. Likely, our evidence for the presence of anammox bacteria in the more oxygenated BUS shelf waters reﬂects material transported upwards from the deeper ODZ. Upwelled waters from the BC were clearly distinguishable at stations south of the ABF (stations 1–6, 10, 117 and 140; 20–26◦S), as indi- cated by the relatively low SST and salinity at the halocline (Fig. 3b, c; Table S1). 4.1.2 Absence of anammox biomarkers near and north of the Angolan Benguela Front At the end of austral summer (i.e., the timing of expeditions 64PE449 and 64PE450), the ABF reaches its most south- ern point and is generally found around 20◦S. At this time, Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 214 Table 2. Pearson’s correlation matrix between BHT-x, oxygen (O2), ammonium (NH+ 4 ), nitrite (NO− 2 ), ladderane IPLs (IPLs), nitrogen deﬁciency (N def.), temperature (Temp), salinity (Sal) and BHT-x. r values indicate Pearson’s correlation coefﬁcient. p values indicate the signiﬁcance level (two-tailed), with bold numbers indicating that correlation is signiﬁcant at the 0.05 signiﬁcance level. BHT-x IPLs BHT-x ratio NO− 2 NH+ 4 N def. Temp. Sal. O2 BHT-x r value – 0.65 0.59 0.12 0.17 0.53 0.02 −0.01 −0.33 ρ value < 0.001 < 0.001 0.36 0.19 < 0.001 0.88 0.92 0.01 IPLs r value 0.65 – 0.52 −0.02 −0.03 0.53 0.09 0.08 −0.29 ρ value < 0.001 < 0.001 0.88 0.83 < 0.001 0.51 0.57 0.02 BHT-x ratio r value 0.59 0.52 – −0.04 0.13 0.63 −0.04 −0.06 −0.35 ρ value < 0.001 < 0.001 0.74 0.31 < 0.001 0.76 0.66 0.01 NO− 2 r value 0.12 −0.02 −0.04 – 0.62 0.07 0.23 0.3 −0.3 ρ value 0.36 0.88 0.74 < 0.001 0.61 0.08 0.02 0.02 NH+ 4 r value 0.17 −0.03 0.13 0.62 – 0.49 0.17 0.17 −0.32 ρ value 0.19 0.83 0.31 < 0.001 < 0.001 0.20 0.19 0.01 N def. r value 0.53 0.53 0.63 0.07 0.49 – 0.04 −0.01 −0.37 ρ value < 0.001 < 0.001 < 0.001 0.61 < 0.001 0.76 0.95 < 0.005 Temp. r value 0.02 0.09 −0.04 0.23 0.17 0.04 – 0.9 −0.18 ρ value 0.88 0.512 0.759 0.076 0.197 0.757 < 0.001 0.159 Sal. r value −0.01 0.08 −0.06 0.3 0.17 -0.01 0.9 – −0.29 ρ value 0.92 0.57 0.66 0.03 0.19 0.95 < 0.001 0.02 O2 r value −0.33 −0.29 −0.35 −0.3 −0.32 −0.37 −0.18 −0.29 – ρ value 0.01 0.02 0.01 0.02 0.01 < 0.005 0.17 0.02 of the ABF (stations 18 and 59). The absence of anammox biomarkers here is thus concurrent with the latitude of the ABF (∼19.8◦S) and the most severe oxygen-depleted waters (∼26◦S), known to occur at this time. During austral winter, the ABF is located furthest north (∼14–16◦S), and the most severe oxygen depletion occurs between 16–20◦S (Chapman and Shannon, 1987; Boyer et al., 2000). Considering the sea- sonal northward shift of the ABF and oxygen-depleted wa- ters during austral winter, the occurrence of anammox bacte- ria and associated biomarkers would likely shift northwards too, at this time of year. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 213 Figure 8. Maximum likelihood phylogenetic tree based on partial (422 bp) 16S rRNA gene sequences displaying the relationship o Ca. Scalindua spp. sequences of the 12 most abundant OTUs detected in the BUS (in red/bold; see Fig. S1 for relative abundance of eac OTU) with other sequences from marine OMZs and sediments (in black) and anammox bioreactors (in black/bold). Bootstrap values highe than 50 % are indicated in the nodes. The scale bar represents 10 % estimated sequence divergence. The outgroup is formed by 16S rRNA gene sequences of Planctomyces maris and Isosphaera pallida. NBCI accession numbers are indicated in between parentheses. Figure 8. Maximum likelihood phylogenetic tree based on partial (422 bp) 16S rRNA gene sequences displaying the relationship of Ca. Scalindua spp. sequences of the 12 most abundant OTUs detected in the BUS (in red/bold; see Fig. S1 for relative abundance of each OTU) with other sequences from marine OMZs and sediments (in black) and anammox bioreactors (in black/bold). Bootstrap values higher than 50 % are indicated in the nodes. The scale bar represents 10 % estimated sequence divergence. The outgroup is formed by 16S rRNA gene sequences of Planctomyces maris and Isosphaera pallida. NBCI accession numbers are indicated in between parentheses. and north of the ABF, oxygen depletion occurred only down to ∼20 µmol L−1 (between 150–500 mbss; Fig. 4f), which would likely inhibit anammox (Strous et al., 1997; Woebken et al., 2007; Kalvelage et al., 2011). Coincidently, evidence for the presence of Ca. Scalindua spp., as indicated by 16S rRNA gene sequences and BHT-x and IPL ladderane concen- trations, was completely lacking at stations sampled north the strongest oxygen depletion is known to occur around ∼24–26◦S, while less severe oxygen depletion is observed near the ABF (Chapman and Shannon, 1987; Boyer et al., 2000). At the time of sampling, large horizontal gradients in SST and salinity existed around ∼19.8◦S, fanning out sea- ward (Fig. 3b, c), indicating that the ABF had developed at this latitude. The ODZ did not extend past the frontal zone, https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 4.1.4 Seasonality in anammox biomarker distributions The Lüderitz upwelling cell has been identiﬁed as one of the most intense upwelling regions in the BUS. In austral win- ter, the water column near the cell is relatively oxygenated, due to the upwelling of oxygen-rich South Atlantic Central Water (Bailey et al., 1991). However, low-oxygen conditions and even anoxia prevail during austral summer due to the res- piration of sinking organic matter supplied by phytoplankton blooms (Bailey et al., 1991; Brüchert et al., 2006). Conse- quently, continental shelf waters between 24–26◦S display large temporal variations in DO concentrations under the in- ﬂuence of the Lüderitz upwelling cell. At the time of sam- pling, the Lüderitz upwelling cell was apparent at ∼26◦S, appearing as a water mass with a low SST, low salinities (Fig. 3b, c) and high chlorophyll α concentrations (Table S1). Here, the water column was sampled once in February (sta- tion 6) and once in March (station 140) to explore the occur- rence and distribution of anammox lipid biomarkers and 16S rRNA gene sequences, as the ODZ developed on the conti- nental shelf (sediment depth 100 mbss). Yet, in apparent contradiction, high concentrations of lad- derane FAs were detected at offshore station 2 at 125, 250 and 710 mbss, with peak concentrations at 250 mbss. In ad- dition, BHT-x was observed at 250, 310 and 710 mbss, with the highest abundance found at the lowest depth (Fig. 7a). To determine the provenance of ladderane FAs observed at sta- tion 2, the NL5 index was used (Table S5). The NL5 index is correlated to the in situ growth temperature of anammox bac- teria (Rattray et al., 2010). At station 2, NL5-derived temper- atures (21.0 ◦C at 250 mbss and 15.6 ◦C at 710 mbss) were substantially higher (i.e., ca. 10 ◦C) than CTD-measured temperatures (11.2 and 5.5 ◦C, respectively), indicating lad- derane FAs were not synthesized in situ. In contrast, at shelf stations 6 and 140, NL5-derived temperatures (7.4–15.0 ◦C; between 30–85 mbss) were close to CTD temperature mea- surements (12.5–13.4 ◦C; between 30–85 mbss), indicating in situ synthesis of ladderane FAs. This suggests that ladder- ane FAs observed offshore likely originated in the warmer shelf waters and were transported down-shelf. ( p ) In February (Fig. 7b), the nutrient, oxygen and tempera- ture proﬁles show a highly stratiﬁed water column. 4.1.3 Lateral transport of anammox biomarkers to oxygenated offshore waters are thought to reﬂect living or recently dead anammox cells; e.g., Jaeschke et al., 2009a). Subsequently, lateral offshore transport of organic matter in the nepheloid layer of the wa- ter column taking place in the BUS (Mollenhauer et al., 2007; Blumenberg et al., 2010) may have transported the more re- calcitrant ladderane FAs and BHT-x from the ODZ on the shelf to offshore waters, whereas ladderane IPLs would not withstand this transport. Afﬁrmatively, there is an absence of evidence for living anammox bacteria (e.g., 16S rRNA gene sequences and IPL ladderanes; Fig. 7a) at 125 and 250 mbss at station 2, further strongly suggesting an allochthonous ori- gin of ladderane FAs and BHT-x. In the more oxygenated offshore waters (up to ∼180 µmol L−1), BHT-x was observed at stations 1, 2, 8 and 9, whereas ladderane IPLs were not detected, and the relative abundance of the Ca. Scalindua spp. 16S rRNA gene was extremely low (Fig. 5). Potentially, ladderane IPLs (and hence fresh anammox bacterial cells) were present, but simply below the detection limit of our method. However, various studies have reported a high sensitivity of IPLs when analyzed using HPLC-ESI-MS. Especially IPLs with a PC head group were found to have a high response factor, likely due to the charged quaternary amine moiety on the PC head group (Sturt et al., 2003; van Mooy and Fredricks, 2010; Wörmer et al., 2015). In light of these results, it is unlikely that BHT-x was detected while the PC ladderane remained below the detection limit. Rather, it seems that a living anammox community was absent in offshore waters. Indeed, the offshore N deﬁcit was limited (< 4 µmol L−1; Fig. 4d) and earlier reports (Kuypers et al., 2005) did not ﬁnd anammox bacteria to be active in the BUS at an offshore station where bottom waters exceeded 20 µmol L−1. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 4.1.3 Lateral transport of anammox biomarkers to o genated offshore aters 215 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x mox bacteria (e.g., Strous et al., 1999; Jetten et al., 2009) could explain why biomarker and 16S rRNA gene evidence for the presence of anammox bacteria was sparse at the time of sampling. + Salinity, temperature or nutrient (NO− 2 and NH+ 4 ) concen- trations were not seen to inﬂuence biomarker distributions in the BUS: i.e., no correlation was observed between these physiochemical parameters and BHT-x or ladderane IPLs (Table 2). This agrees with earlier ﬁndings. Ca. Scalindua spp. have an optimal temperature range of 10–30 ◦C (Awata et al., 2012, 2013), well within the temperature range found in the BUS. Furthermore, changes in salinity have not been found to affect abundance of Ca. Scalindua spp. (Awata et al., 2012, 2013), and Ca. Scalindua spp. are known to have an extremely low afﬁnity for NO− 2 and NH+ 4 (Awata et al., 2013). In our study, only [O2] returned a weak but signiﬁcant negative correlation with BHT-x (r(60) = −0.33, ρ = 0.01) and ladderane IPLs (r(60) = −0.29, ρ = 0.02). At station 117, located just south of ABF, [O2] was be- low ∼20 µmol L−1 at ∼50 mbss (down to ∼3 µmol L−1 at 85 mbss; Fig. 5c), yet evidence of anammox was sparse. Sta- tion 117 was the only BUS station where N was not lim- ited (N deﬁcit < 0 µmol L−1; Fig. 4h; Table S2), revealing that loss of bioavailable nitrogen by anammox and/or den- itriﬁcation was absent or limited here. No BHT-x was de- tected, and only very low abundances of ladderane IPLs (8.6 × 104 ru L−1; Fig. 5b) and Ca. Scalindua spp. 16S rRNA gene sequences (0.001 ‰; Fig. 5g) were detected at 85 mbss. Possibly, the water column at station 117 had only recently become oxygen depleted. If so, the slow growth rate of anam- https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x consistent with previously reported seasonality: lower tem- peratures and increased upwelling commence in austral au- tumn, resulting in decreased SSTs (Monteiro et al., 2008; Louw et al., 2016). Indeed, the strong redoxcline observed in February was absent in March. SST in March was also ∼1.5 ◦C lower than observed in February, indicating water column mixing and weakened stratiﬁcation. Likewise, the nutrient-rich sub-thermocline waters mixed with the surface waters, resulting in similar NO− 2 , NO− 3 and NH+ 4 concentra- tions throughout the water column. Additionally, salinity was relatively high throughout the water column (35.2–36.2 psu), indicating the late summer (February–April) salinity maxi- mum (S > 35.1 psu) had set in, which is known to co-occur with the oxygen minimum (Monteiro et al., 2008). Con- formingly, in March, surface waters (< 10 mbss) were more oxygen-depleted than observed in February. Ca. Scalindua spp. 16S rRNA genes were detected at all sampled depths, in- cluding 10 mbss. Likewise, ladderane IPLs, BHT-x and lad- derane FAs were also present throughout the water column at all sampled depths (35–85 mbss). The ladderane FA and BHT-x concentrations were slightly lower then observed in February at 85 mbss, which may indicate that particulate ma- terial sank to the seaﬂoor, was degraded or was transported elsewhere prior to the occurrence of an established anammox community in March. Ca. Scalindua spp. 16S rRNA gene sequences were also detected in offshore waters. Yet, co-occurrence with BHT-x was limited (only in four of the 19 offshore SPM samples), and the extremely low relative abundance of Ca. Scalindua spp. 16S rRNA gene sequences here (0 ‰–0.4 ‰; Fig. 5n) and BHT-x concentrations (factor of 10 to 100 lower than at shelf stations) make it unlikely that anammox bacteria formed an active community. Rather, lateral organic mat- ter transport, discussed in Sect. 4.1.3, seems to contribute to the BHT-x concentrations observed offshore. Considera- tions must thus be taken when interpreting low abundances of BHT-x, as these may inaccurately suggest the presence of living Ca. Scalindua. Our ﬁndings suggest a strong temporal variability in the presence of anammox bacteria and their synthesized lipids at 26◦S, corresponding to a large shift in hydrographic char- acteristics of the water column. In all likelihood, anammox bacteria only became an established community at the end of austral summer, once the oxygen minimum had set in. 4.1.4 Seasonality in anammox biomarker distributions A strong oxycline is present around ∼20 mbss, with near-anoxic con- ditions in the bottom waters (down to ∼3 µmol L−1). The 16S rRNA amplicon sequences of Ca. Scalindua spp. and BHT-x were detected below 40 and 50 mbss, respectively, with (relative) abundances increasing with depth. Ladderane FAs followed a similar distribution, increasing in concentra- tion with water column depth. However, ladderane IPLs were not detected throughout the water column, which may indi- cate that anammox bacteria were not yet a dominant feature in the water column community. Possibly, BHT-x and ladder- ane FAs at this station were laterally transported from more southern shelf sites (Mollenhauer et al., 2007; Blumenberg et al., 2010). The accumulation of ammonium in the bottom waters (Fig. 7b), corresponding to a very high N deﬁcit of 38 µmol L−1 (Fig. 4h; Table S2), would suggest that deni- triﬁcation was more active than anammox (Richards et al., 1965). p Mollenhauer et al. (2007) showed that radiocarbon ages of lipid biomarkers in the BUS increased with distance from shore and water depth, as a consequence of lateral organic matter transport over the Namibian margin. In fact, most of the organic matter deposited offshore was found to derive from the shelf (Mollenhauer et al., 2007). In addition, Blu- menberg et al. (2010) observed a decoupling of bio- and geohopanoids in BUS sediments, likely reﬂecting laterally transported fossil organic matter. The degradation rate of ladderane FAs and BHT-x is slower than that of ladderane IPLs (i.e., ladderane FAs have been observed in sediments of 140 kyr BP; Jaeschke et al., 2009b, and BHT in sediments over 50 myr BP; Talbot et al., 2016, whereas ladderane IPLs In March (Fig. 7c), the same sampling location showed distinct differences in physiochemical properties. This is https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 216 consistent with previously reported seasonality: lower tem- peratures and increased upwelling commence in austral au- tumn, resulting in decreased SSTs (Monteiro et al., 2008; Louw et al., 2016). Indeed, the strong redoxcline observed in February was absent in March. SST in March was also ∼1.5 ◦C lower than observed in February, indicating water column mixing and weakened stratiﬁcation. Likewise, the nutrient-rich sub-thermocline waters mixed with the surface waters, resulting in similar NO− 2 , NO− 3 and NH+ 4 concentra- tions throughout the water column. Additionally, salinity was relatively high throughout the water column (35.2–36.2 psu), indicating the late summer (February–April) salinity maxi- mum (S > 35.1 psu) had set in, which is known to co-occur with the oxygen minimum (Monteiro et al., 2008). Con- formingly, in March, surface waters (< 10 mbss) were more oxygen-depleted than observed in February. Ca. Scalindua spp. 16S rRNA genes were detected at all sampled depths, in- cluding 10 mbss. Likewise, ladderane IPLs, BHT-x and lad- derane FAs were also present throughout the water column at all sampled depths (35–85 mbss). The ladderane FA and BHT-x concentrations were slightly lower then observed in February at 85 mbss, which may indicate that particulate ma- terial sank to the seaﬂoor, was degraded or was transported elsewhere prior to the occurrence of an established anammox community in March. dua spp. sequence presence in the BUS. This test showed that the presence of BHT-x signiﬁcantly predicts the pres- ence of Ca. Scalindua spp. in 78.8 % of all cases (ρ < 0.001), showing BHT-x is a suitable biomarker for Ca. Scalindua spp. even in complex upwelling regions such as the BUS. However, in the BUS, Ca. Scalindua spp. 16S rRNA gene se- quences constituted only a small portion of the total bacterial pool (max. 2.7 ‰; Fig. 5g). Low abundance of marine anam- mox bacteria in comparison to other phylogenetic groups in marine ecosystems has been reported previously (Woebken et al., 2007) and is likely caused by slow cell division rates (Strous et al., 1999; Jetten et al., 2009). Even so, it cannot be excluded that well-known PCR biases might also have led to a low coverage of Ca. Scalindua spp. reads. Unequal ampli- ﬁcation efﬁciency of PCR products could result in the prefer- ential ampliﬁcation of certain 16S rRNA genes, whilst others might be inhibited for ampliﬁcation (e.g., Pinto and Raskin, 2012). 4.2 Application and constraints on the use of BHT-x as a biomarker for Ca. Scalindua In the BUS, sequences taxonomically assigned to Ca. Scalin- dua spp. were detected at 11 out of 13 stations (Fig. 5g, n). A phylogenetically closely related cluster of Ca. Scalin- dua OTUs could be identiﬁed (i.e., the BUS OTU clus- ter indicated in Fig. 8). The BUS OTU cluster displayed a large sequence identity to Ca. Scalindua sorokinii isolated from the Guaymas deep sea hydrothermal vents and the Black Sea suboxic waters (98 %; Table S7) and Ca. Scalin- dua brodae (97 %; Table S7). BHT-x was originally re- ported to be uniquely synthesized by marine anammox, us- ing Ca. S. brodae enrichment cultures (Rush et al., 2014; Schwartz-Narbonne et al., 2020). In accordance with these reports, BHT-x was observed at the same 11 stations where Ca. Scalindua 16S rRNA gene sequences were detected. In addition to being a useful biomarker for Ca. Scalindua, BHT-x has been applied as a proxy for low oxygen concen- tration in marine systems. Saénz et al. (2011) proposed the ratio of BHT-x over total BHT as a proxy for suboxic–anoxic conditions (deﬁned as [O2] < 5 µmol kg−1), since BHT-x was only found in low-oxygen settings, whereas BHT is ubiquitously synthesized by mostly aerobic bacteria. The discovery that BHT-x is, to our knowledge, uniquely syn- thesized by marine anammox (Rush et al., 2014; Schwarz- Narbonne et al., 2019), provided further evidence for this ap- plication of the BHT-x ratio, as it reﬂects the contribution of BHT-x synthesized by the anaerobic “Ca. Scalindua spp.” to the total BHT pool. At BUS shelf stations, when [O2] was > 50 µmol L−1, the BHT-x ratio remained below 0.04, in all but one case (sta- tion 5 at 30 mbss). However, at ﬁve offshore sites where [O2] was > 50 µmol L−1 (up to ∼180 µmol L−1), BHT-x ra- tios > 0.04 were observed. Likely, transported BHT-x de- rived from the ODZ on the shelf (see discussion Sect. 4.1.3) and the markedly low BHT concentrations (Table S3) con- At shelf stations (stations 3–6, 10, 117, 140), the presence of BHT-x co-occurred with the detection of Ca. Scalindua spp. 16S rRNA gene sequences at all depths except station 5 at 30 mbss. A multivariate binomial regression was per- formed to determine if the relative abundance of BHT-x can be used to predict the likelihood of 16S rRNA Ca. Scalin- Biogeosciences, 19, 201–221, 2022 https://doi.org/10.5194/bg-19-201-2022 Z. R. 4.2 Application and constraints on the use of BHT-x as a biomarker for Ca. Scalindua van Kemenade et al.: Bacteriohopanetetrol-x Figure 9. Relationship between dissolved oxygen concentration and the BHT-x ratio in suspended particulate matter (SPM) col- lected from the water columns of northern Chile (Matys et al., 2017); the Cariaco Basin, Peru Margin and Arabian Sea (Sáenz et al., 2011); and the Benguela upwelling system (BUS; this study). Figure adapted from Sáenz et al. (2011) and Matys et al. (2017). Z. R. van Kemenade et al.: Bacteriohopanetetrol-x Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 217 Z. R. van Kemenade et al.: Bacteriohopanetetrol-x higher fractional abundance than observed in the enrichment. Even so, our results (using modiﬁed Bligh and Dyer extrac- tions and UHPLC-HESI-MS) are found to align well with those from other marine systems, as investigated by Sáenz et al. (2011; Arabian Sea, Peru Margin and Cariaco Basin; us- ing Soxhlet extractions and UHPLC-APCI-MS analysis) and Matys et al. (2017; Humboldt current system; using modiﬁed Bligh and Dyer extractions and UHPLC-APCI-MS analysis). Nonetheless, in order to apply this threshold (BHT-x ratio ≥0.2) to infer low-oxygen conditions (< 50 µmol kg−1) in sedimentary records, this signal must be retained in the sedi- ment (i.e., not become diluted by BHT settling from the oxic zone of the water column). Matys et al. (2017) found that BHT II isomer ratios (i.e., BHT-x ratios) observed in sur- face sediments of the Humboldt current system were com- parable to those observed in the OMZ core of the overly- ing water. In accordance, Berndmeyer et al. (2013) showed that BHPs recorded in the sediment of the Gotland Deep mir- rored those of the suboxic zone of the water column. Hence, it is likely that BHT-x ratios observed in the low-oxygen zone of the water column are retained in the underlying sed- iments. Moreover, considering the large variety in marine settings (four different upwelling regions and one restricted anoxic basin) and in methodologies, a BHT-x ratio of ≥0.2 is thought to provide a robust threshold in sedimentary records to estimate past low-oxygen conditions (< 50 µmol kg−1) of the overlying water column, hereby accounting for potential allochthonous BHT-x material. Figure 9. Relationship between dissolved oxygen concentration and the BHT-x ratio in suspended particulate matter (SPM) col- lected from the water columns of northern Chile (Matys et al., 2017); the Cariaco Basin, Peru Margin and Arabian Sea (Sáenz et al., 2011); and the Benguela upwelling system (BUS; this study). 4.2 Application and constraints on the use of BHT-x as a biomarker for Ca. Scalindua Figure adapted from Sáenz et al. (2011) and Matys et al. (2017). tributed to the relatively high BHT-x ratio signal observed here. When considering both offshore and on-shelf sites, when [O2] was > 50 µmol L−1, the BHT-x ratio remained below 0.2, in all but one case (station 5 at 30 mbss). In addition, a ratio of ≥0.2 corresponded in all cases (ex- cept one) with the presence of the Ca. Scalindua spp. 16S rRNA gene, which was not the case for ratios > 0.04. In the water columns of the Arabian Sea, Peru Margin and Cariaco Basin, Saénz et al. (2011) found that when [O2] was > 50 µmol kg−1 (∼50 µmol L−1), the BHT-x ratio (i.e., BHT-II ratio) remained below 0.03 (with one exception). In Matys et al. (2017), the highest BHT-x ratios (> 0.2; i.e., BHT-II ratios) were detected in waters of the Humboldt cur- rent system with [O2] < 3 µmol kg−1. Yet, elevated ratios, thought to originate from transported organic matter, were also observed in oxygenated waters (> 200 µmol kg−1) be- low the OMZ. Combining our results (BUS oxygen concen- trations converted to µmol kg−1; Fig. 9) with the ﬁndings of Sáenz et al. (2011) and Matys et al. (2017) shows that when [O2] is < 50 µmol kg−1, the BHT-x ratio (i.e., BHT-II ratio) is ≥0.2 (except in one sample from the Cariaco Basin). References Data availability. Unassembled sequences are submitted to NCBI under BioProject number PRJNA761075. Individual sequences of OTUs 1–12 are published in GenBank under accession numbers OK086296–OK086307. All the data used in this study are provided in the Supplement. Abdala Asbun, A., Besseling, M. A., Balzano, S., van Blei- jswijk, J. D. L., Witte, H. J., Villanueva, L., and En- gelman, J. C.: Cascabel: A Scalable and Versatile Am- plicon Sequence Data Analysis Pipeline Delivering Repro- ducible and Documented Results, Front. Genet., 11, 489357, https://doi.org/10.3389/fgene.2020.489357, 2020. Awata, T., Tanabe, K., Kindaichi, T., Ozaki, N., and Ohashi, A.: In- ﬂuence of temperature and salinity on microbial structure of ma- rine anammox bacteria, Water Sci. Technol., 66, 958–964, 2012. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-19-201-2022-supplement. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-19-201-2022-supplement. Awata, T., Oshiki, M., Kindaichi, T., Ozaki, N., Ohashi, A., and Okabe, S.: Physiological characterization of an anaerobic ammonium-oxidizing bacterium belonging to the “Candidatus Ca. Scalindua” group, Appl. Environ. Microbiol., 79, 4145– 4148, https://doi.org/10.1128/AEM.00056-13, 2013. Author contributions. ZRvK wrote the manuscript. PK and DR were in charge of the research expeditions. ZRvK, DR and PK per- formed the sample collection. ZRvK performed the laboratory work and data analysis. LV and HJW contributed to the data analysis of the 16S rRNA gene sequences. ECH optimized UHPLC mea- surements. ECH and DR contributed to the lipid data analysis. DR, JSSD, LV and ZRvK designed and conceptualized the project. All co-authors provided critical feedback and helped shape the research, analysis and manuscript. Bailey, G. W.: Organic carbon ﬂux and development of oxygen deﬁ- ciency on the modern Benguela continental shelf south of 22◦S: Spatial and temporal variability, Geol. Soc. Spec. Publ., 58, 171– 183, https://doi.org/10.1144/GSL.SP.1991.058.01.12, 1991. Bale, N. J., Villanueva, L., Fan, H., Stal, L. J., Hopmans, E. C., Schouten, S., and Sinninghe Damsté, J. S.: Occurrence and activity of anammox bacteria in surface sediments of the southern North Sea, FEMS Microbiol. Ecol., 89, 99–110, https://doi.org/10.1111/1574-6941.12338, 2014. Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests. Bale, N. J., Ding, S., Hopmans, E. C., Arts, M. G. I., Villanueva, L., Boschman, C., Haas, A. F., Schouten, S., and Sinninghe Damsté, J. S.: Lipidomics of Environmen- tal Microbial Communities. I: Visualization of Component Distributions Using Untargeted Analysis of High-Resolution Mass Spectrometry Data, Front. Z. R. van Kemenade et al.: Bacteriohopanetetrol-x 218 ane IPLs were constrained to the shelf stations. Calculating the temperature-sensitive NL5 index for ladderane FAs in- dicated that offshore ladderane FAs were not synthesized in situ and likely originated from the shelf. This must be taken into consideration when using BHT-x and ladderane FAs as lipid biomarkers for in situ water column anammox. Lastly, at shelf stations, when [O2] was > 50 µmol L−1, the BHT-x ratio remained below 0.04, in all but one case. Yet, laterally transported BHT-x resulted in high offshore BHT-x ratio val- ues (> 0.04) in oxygenated waters. We therefore suggest us- ing a BHT-x ratio threshold of ≥0.2 to infer low-oxygen con- ditions (< 50 µmol kg−1) in sedimentary records of dynamic upwelling systems: when comparing BUS BHT-x (i.e., BHT- II) ratios with those from other marine settings (four different upwelling regions and one restricted anoxic basin; Saénz et al., 2011; Matys et al., 2017), it was observed that when [O2] was > 50 µmol kg−1, the BHT-x ratio remained below 0.2 (in all but one case). and Dyer extractions. Marianne Baas is thanked for deploy- ing the in situ pumps during the second expedition. In addi- tion, we thank Karel Bakker and Jan van Ooijen for the on- board NUTS analyses. We are also grateful for the support Denise Dorhout and Monique Verweij have delivered in the lipid lab and Maartje Brouwer and Sanne Vreugdenhil in the molecular labs. Lastly, Tom Vaessen is thanked for taking the time to discuss statistics. and Dyer extractions. Marianne Baas is thanked for deploy- ing the in situ pumps during the second expedition. In addi- tion, we thank Karel Bakker and Jan van Ooijen for the on- board NUTS analyses. We are also grateful for the support Denise Dorhout and Monique Verweij have delivered in the lipid lab and Maartje Brouwer and Sanne Vreugdenhil in the molecular labs. Lastly, Tom Vaessen is thanked for taking the time to discuss statistics. Financial support. This research has been supported by the Soehn- gen Institute of Anaerobic Microbiology (grant no. 024.002.002). Review statement. This paper was edited by Sebastian Naeher and reviewed by two anonymous referees. 5 Conclusions This study reveals a strong spatiotemporal variability in the presence of anammox bacteria (as reﬂected by their 16S rRNA gene sequences) and their membrane lipids in the Benguela Upwelling System (BUS), which corresponds to differences in hydrographic characteristics of the water col- umn. By elucidating the distribution of BHT-x across a large oxygen gradient, and comparing it to distributions of ladder- ane IPLs, ladderane FAs and Ca. Scalindua spp. 16S rRNA gene sequences, we assessed the suitability of BHT-x as a lipid biomarker for Ca. Scalindua spp., as well as its ratio over total BHT as a proxy for low-oxygen water column con- ditions. On the continental shelf, BHT-x co-occurred with the detection of Ca. Scalindua spp. 16S rRNA genes in all but one case, further highlighting its suitability as a lipid biomarker for marine anammox in the sedimentary record of upwelling regions. Shifts in the anammox lipid biomarker distribution at the southernmost shelf station (∼25◦S), sam- pled 27 d apart, implied that anammox bacteria only became an established community in the shelf waters at the end of austral summer, when oxygen depletion was most severe. At the offshore stations, ladderane FAs and low concentrations of BHT-x were also observed to accumulate in relatively oxy- genated waters ([O2] up to ∼180 µmol L−1), while ladder- However, different extraction and/or analytical techniques may impact the BHT-x ratio. The BHT-to-isomer ratio de- rived from an acetylated culture analyzed by Peiseler and Rohmer (1992) using HPLC (0.1) was different than that measured by Schwartz-Narbonne et al. (2019) in an aliquot of the same non-acetylated culture using UHPLC (0.2). Fur- thermore, to date, it is unknown if and how the relative ratio of BHT and BHT-x synthesis by Ca. Scalindua spp. is inﬂu- enced by environmental conditions and phylogeny. As BHT- x ratios observed in the BUS surpassed the ratio observed in the Ca. Scalindua brodae enrichment (Fig. 6), Ca. Scalindua spp. detected in the BUS might have synthesized BHT-x in a https://doi.org/10.5194/bg-19-201-2022 Biogeosciences, 19, 201–221, 2022 Z. 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https://openalex.org/W2797104178	https://repository.lboro.ac.uk/articles/journal_contribution/The_coherent_shear_wave_in_suspensions/9243446/1/files/16827281.pdf	English	null	The coherent shear wave in suspensions	Journal of physics. Conference series	2,018	cc-by	3,987	This content was downloaded from IP address 158.125.80.192 on 29/05/2018 at 11:39 PAPER • OPEN ACCESS Related content Ultrasonic Generation by Ferromagnetic Resonance in Evaporated Ni–Fe Alloy Films. 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I Minoru Takahashi, Yoshiki Sunakawa and Yukio Shibata - Measurement System for Longitudinal and Shear Waves in Solids under Shock Compression in Several Tens of GPa Region Combined with the Keyed-Powder Gun Tsutomu Mashimo and Kunihito Nagayama - Observation of Induced Shear Acoustic Phonons by Brillouin Scattering Shigeo Murata, Takayuki Kawamoto, Mami Matsukawa et al. - The coherent shear wave in suspensions View the article online for updates and enhancements. Observation of Induced Shear Acoustic Phonons by Brillouin Scattering Shigeo Murata, Takayuki Kawamoto, Mami Matsukawa et al. - 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 The coherent shear wave in suspensions M Alam1, F Luppé1, V J Pinfield2 and P Maréchal1 1 Laboratoire Ondes et Milieux complexes, Normandie Univ, UNILEHAVRE, CNRS, LOMC, 76600 Le Havre, France 2 Chemical Engineering Department, Loughborough University, Loughborough, UK, LE11 3TU M Alam1, F Luppé1, V J Pinfield2 and P Maréchal1 1 Laboratoire Ondes et Milieux complexes, Normandie Univ, UNILEHAVRE, CNRS, LOMC, 76600 Le Havre, France 2 Chemical Engineering Department, Loughborough University, Loughborough, UK, LE11 3TU M Alam1, F Luppé1, V J Pinfield2 and P Maréchal1 1 Laboratoire Ondes et Milieux complexes, Normandie Univ, UNILEHAVRE, CNRS, LOMC, 76600 Le Havre, France 2 Chemical Engineering Department, Loughborough University, Loughborough, UK, LE11 3TU 1 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd francine.luppe@univ-lehavre.fr Abstract. We consider a disordered suspension of spherical silica particles in water. For a particle size of a few hundred nanometres and concentration (volume fraction) around 0.15 to 0.2, experiments conducted in the MHz range have shown that the non ideal nature of water must be taken into account for the "longitudinal" coherent wave attenuation to be understood, because of wave conversions, from longitudinal to shear and then back to longitudinal, occurring at each pair of scattering events. We are interested here in the properties of the "shear" coherent wave that are given by the expansion of its squared wavenumber, around that in the absence of particles, in powers of the concentration. At 1 MHz and a particle radius of 0.05 m, we show that convergence of the modal series involved in that expansion may be reached after three terms: we use ten terms subsequently. We study the evolution of both the effective shear velocity and attenuation with concentration, as well as that of the effective shear viscosity deduced therefrom. 1. Introduction We study the propagation of the "shear" coherent wave in a disordered suspension of silica spheres in water. Forrester [1] et al. have shown that for particle radii smaller than a few micrometres the attenuation of the "longitudinal" coherent wave in such a suspension could be well predicted by a multiple scattering model, as long as the shear-mode effects, due to water viscosity, were taken into account, as in Ref. [2]. Including those effects leads to the introduction of another coherent wave, called the "shear" coherent wave (with quotes omitted in the following), because its wavenumber S reduces to that, kS, of the shear wave in (viscous) water in the absence of particles. It is in the properties of this shear coherent wave that we are interested here. The physical properties of both water and silica are the same as in Ref. [1], and the numerical study is conducted at one frequency only, f = 1 MHz. All particles have the same radius a = 0.05 m, so that letting kC denote the compressional wavenumber in water in the absence of particles, while low frequency approximations may be used when dealing with compression waves, one must be careful with shear waves, as 4 2 10 C k a , and 0.1 1 Sk a i . The properties of the shear coherent wave are deduced from the calculation of its wavenumber S, as a function of the concentration (volume fraction) c of solid, using the multiple scattering model of Ref. [2], and section 2 is dedicated to numerical issues that need to be taken care of in doing this. The 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 16th Anglo-French Physical Acoustics Conference (AFPAC) 1234567890 ‘’“” g y g IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 effective shear velocity and attenuation are studied as a function of the concentration in section 3, and preliminary work on the definition of a prospective effective shear viscosity is conducted in section 4. effective shear velocity and attenuation are studied as a function of the concentration in section 3, and preliminary work on the definition of a prospective effective shear viscosity is conducted in section 4. 2. The effective shear wavenumber 2. The effective shear wavenumber ective shear wavenumber expansion 2.1. Effective shear wavenumber expansion After Eqs. (16,29-32) of Ref. [2], the effective "shear" wavenumber S may be expanded around kS in powers of the concentration (c = 4n0 a3/3), ff p After Eqs. (16,29-32) of Ref. [2], the effective "shear" wavenumber S may be expanded around kS in powers of the concentration (c = 4n0 a3/3), 2 SS 1S 1 3 2 S S 1 c k k a (1) 2 2c 2 SS 1S 1 3 2 S S 1 c k k a (1) 2 2 2 SS SC 3 S 1S 2 2 6 2 2 S S S c O c k k k a (2) (1) 2 2 2 SS SC 3 S 1S 2 2 6 2 2 S S S c O c k k k a (2) (2) The first order term (in concentration), SS 1 , involves a modal series of the form 0 SS n n n f T , while The first order term (in concentration), SS 1 , involves a modal series of the form 0 SS n n n f T , while m 0 0 SS SS nm n m n m g T T and 0 0 SC CS nm n m n m h T T , SS 2 and SC 2 involve respectively series of the form 0 0 SS SS nm n m n m g T T and 0 0 SC CS nm n m n m h T T , SS 2 and SC 2 involve respectively series of the form 0 0 SS SS nm n m n m g T T and 0 0 SC CS nm n m n m h T T , SS 2 and SC 2 involve respectively series of the form 0 0 SS SS nm n m n m g T T and 0 0 nm n n m h T with fn, gnm, hnm more or less complicated functions of n and m, and the pq nT (p,q=S,C) the modal scattering coefficients of a single particle for an incident compressional (p=C) or shear (p=S) wave into either a compressional (q=C) or a shear (q=S) one. They are defined in Ref. 2. The effective shear wavenumber [2] as associated with the Debye potentials of either compressional or shear waves, with no dependence with the azimuthal angle (see Eqs. 5,6 in Ref. [2]) . Their decay with the increase of mode number n is shown in Figure 1. with fn, gnm, hnm more or less complicated functions of n and m, and the pq nT (p,q=S,C) the modal scattering coefficients of a single particle for an incident compressional (p=C) or shear (p=S) wave into either a compressional (q=C) or a shear (q=S) one. They are defined in Ref. [2] as associated with the Debye potentials of either compressional or shear waves, with no dependence with the azimuthal angle (see Eqs. 5,6 in Ref. [2]) . Their decay with the increase of mode number n is shown in Figure 1. 1 2 3 4 5 6 7 8 9 10 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 n log( \|Tn (CC)\| ) log( \|Tn (CS)\| ) log( \|Tn (SS)\| ) log( \|Tn (SC)\| ) 1 2 3 4 5 6 7 8 9 10 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 n log( \|Tn (CC)\| ) log( \|Tn (CS)\| ) log( \|Tn (SS)\| ) log( \|Tn (SC)\| ) 2 2 16th Anglo-French Physical Acoustics Conference (AFPAC) 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 igure 1. The modal scattering coefficients (amplitudes) as functions of mode number. The scattering coefficient of a compressional wave into a compressional one is the fastest to decrease, that of a shear wave into a shear one the slowest. The first one is also the lowest in magnitude, while the second one the largest. However, all coefficients are smaller than 10-10 as soon as n is larger than 4, and we can suppose that all the series involved in Eqs. (1,2) should have achieved convergence after summing up 5 terms at most. 2. The effective shear wavenumber Convergence of the 2 0 0 N N SS SS SS N nm n m n m g T T series may be studied through the plot of its real vergence of the 2 0 0 N N SS SS SS N nm n m n m g T T series may be studied through the plot of its real and imaginary parts versus N, as done in Figure 2, where all the  terms are plotted against N, showing that N=2 should be sufficient for all series to reach convergence. While this information could be useful to carry out analytic approximations of Eqs. (1,2), a value of N=10 has been used nevertheless to obtain all the subsequent results. 1 2 3 4 5 6 7 8 9 10 -5 -4 -3 -2 -1 0 1 2 3 4 5 x 10 -3 N e(  1N (SS) ) a) 2 4 6 8 10-5 -4 -3 -2 -1 0 1 2 3 4 5x 10 -3 m(  1N (SS) ) 1 2 3 4 5 6 7 8 9 10 -5 -4 -3 -2 -1 0 1 2 3 4 5 x 10 -3 N e(  1N (SS) ) a) 2 4 6 8 10-5 -4 -3 -2 -1 0 1 2 3 4 5x 10 -3 m(  1N (SS) ) 1 2 3 4 5 6 7 8 9 10 -5 -4 -3 -2 -1 0 1 2 3 4 5 x 10 -3 N e(  1N (SS) ) a) 2 4 6 8 10-5 -4 -3 -2 -1 0 1 2 3 4 5x 10 -3 m(  1N (SS) ) 1 2 3 4 5 6 7 8 9 10 -3 -2 -1 0x 10 -6 N e(  2N (SS) ) b) 2 4 6 8 10-5 -4 -3 -2 x 10 -5 m(  2N (SS) ) 1 2 3 4 5 6 7 8 9 10 -4 -3 -2 -1 0 x 10 -7 N e(  2N (SC) ) c) 2 4 6 8 10-2 -1 0x 10 -5 m(  2N (SC) ) Figure 2. Convergence of the first and second order terms of the effective wavenumber expansion Eqs. (1,2). N is the order at which all modal series are truncated. 2. The effective shear wavenumber 1 2 3 4 5 6 7 8 9 10 -3 -2 -1 0x 10 -6 N e(  2N (SS) ) b) 2 4 6 8 10-5 -4 -3 -2 x 10 -5 m(  2N (SS) ) e( (SC) ) 1 2 3 4 5 6 7 8 9 10 -4 -3 -2 -1 0 x 10 -7 N 2N c) 2 4 6 8 10-2 -1 0x 10 -5 m(  2N (SC) ) c) b) Figure 2. Convergence of the first and second order terms of the effective wavenumber expansion Eqs. (1,2). N is the order at which all modal series are truncated. Eq. (2) corresponds to the expansion of 2 S 2 Sk in powers of the concentration c, up to order 2. This expansion was carried out [2] under the assumption of a small enough concentration for all successive orders to be decreasing in magnitude. However, each term of a given order r in Eq. (2) is multiplied, 3 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 not only by the small quantity cr, as one would have expected, but by a larger one, 3 S r r c k a , so that, not only by the small quantity cr, as one would have expected, but by a larger one, 3 S r r c k a , so that, while the delta terms relating to the terms of order 2 in concentration (Figure 2) are two orders of magnitude smaller than the delta term relating to the order 1 term in concentration, Figure 3 shows that the whole term of order 2 in concentration in Eq. (2) is no longer smaller than the whole term of order 1 in concentration as soon as the concentration gets higher than about 0.25. This shows clearly the limit of the model, and the subsequent analysis considers only concentration up to 0.2. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 c c \|1 (SS) / (kSa)3\| c2 \|2 (SS) / (kSa)6\| c2 \|2 (SC) / (kSa)6\| c2 \|(2 (SS)+2 (SC))\| / (kSa)6\| Figure 3. Magnitude of the terms in the effective shear wavenumber expansion in concentration. Figure 3. 2. The effective shear wavenumber Magnitude of the terms in the effective shear wavenumber expansion in concentration 2.2. Properties of the effective shear wave 2.2. Properties of the effective shear wave The effect of the concentration of scatterers on the velocity and the attenuation of the effective shear wave is shown in Figure 4. Not surprisingly, the introduction of solid scatterers in water increases the shear velocity and decreases the shear wave attenuation, compared to the situation in the absence of scatterers. The deviation from the corresponding values in the absence of scatterers, however, is less than 10 %. As the effective wavenumber expansion in concentration is limited to order 2 in concentration, both the velocity and attenuation curves exhibit a quadratic behavior, but the concentration ccrit at which the velocity reaches a maximum is slightly lower than that at which the attenuation is lowest. As the quadratic behavior of the curves is the most obvious for concentration values around ccrit, it might be an indication that, for this particular frequency under study, order 3 in concentration can no longer be neglected for c  ccrit. Figure 5 shows that the real and imaginary parts of the effective shear wavenumber are practically equal, just like those in the absence of scatterers, and the next section discusses the possibility of defining an effective shear viscosity by analogy with a pure viscous fluid. 4 16th Anglo-French Physical Acoustics Conference (AFPAC) 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 0 0.05 0.1 0.15 0.2 1 1.02 1.04 1.06 1.08 1.1 c e ( kS ) / e ( S ) 0 0.05 0.1 0.15 0.2 0.9 0.92 0.94 0.96 0.98 1 m ( S ) / m ( kS ) Figure 4. Ratios of the effective shear wave velocity (solid line, left vertical axis) and attenuation (dotted line, right vertical axis) to those of the shear wave in the absence of scatterers, versus concentration. 0 0.05 0.1 0.15 0.2 1 1.02 1.04 1.06 1.08 1.1 c e ( kS ) / e ( S ) 0 0.05 0.1 0.15 0.2 0.9 0.92 0.94 0.96 0.98 1 m ( S ) / m ( kS ) Figure 4. 3. Towards the definition of an effective shear viscosity 3. Towards the definition of an effective shear viscosity In a viscous fluid such as water, and supposing the exp(-it) time dependence, ous fluid such as water, and supposing the exp(-it) time dependence, (3) 2 2 S , , k i       (3) so that the shear wavenumber has equal real and imaginary parts. The fact that the effective shear wavenumber calculated in the preceding section almost satisfies this condition suggests that one could define an effective medium, at least from the shear wave point of view, with a real effective mass density eff and a (practically) real effective shear viscosity eff obeying 2 S 2 eff S eff . k       (4) (4) There have been many published works regarding the effective density of a random configuration of scatterers in a host matrix, with nearly as many formulas for that density. All agree on the complex nature of the effective mass density, as well as on its real static limit, which is given [4,5] by a simple volume average in the case of an elastic matrix,   eff water Silica 1 c c       , (5) (5) and by a different one [4] in the case of an inviscid fluid matrix. and by a different one [4] in the case of an inviscid fluid matrix. Taking into account the shear viscosity of water through Eq. (3) is equivalent to considering water as a solid with a purely imaginary second (shear) Lamé coefficient , and thus adopting Eq. (5) with Eqs. (2, 4) allows an effective viscosity, eff, to be obtained. This effective viscosity eff, which is almost real, is plotted in Figure 6 (black curve), and has an imaginary part of less than 2 % of the viscosity of pure water . The other curves in Figure 6 correspond to a few hydrodynamic models of viscosity for hard sphere suspensions in water that are described in Ref. [5]. While all hydrodynamic models provide quite different results, they all show a monotonic increase of the effective viscosity with concentration, which is not the case in our calculation (black curve). This dramatically different behavior may be explained from the difference in the assumptions of the hydrodynamic models and the one used here. 2.2. Properties of the effective shear wave Ratios of the effective shear wave velocity (solid line, left vertical axis) and attenuation (dotted line, right vertical axis) to those of the shear wave in the absence of scatterers, versus concentration. 0 0.05 0.1 0.15 0.2 0.986 0.988 0.99 0.992 0.994 0.996 0.998 1 1.002 1.004 c m( S ) / e( S ) Figure 5. The effective shear wavenumber: ratio of its imaginary part to its real part versus concentration. 0 0.05 0.1 0.15 0.2 0.986 0.988 0.99 0.992 0.994 0.996 0.998 1 1.002 1.004 c m( S ) / e( S ) Figure 5. The effective shear wavenumber: ratio of its imaginary part to its real part versus concentration. 5 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP P 16th Anglo-French Physical Acoustics Conference (AFPAC) 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 1234567890 ‘’“” g y ( ) g IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 1234567890 ‘’“” OP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/0 3. Towards the definition of an effective shear viscosity All of them correspond to static expressions of the effective mass density and viscosity, whereas our calculation, while using a static approximation for the effective density (Eq. (5)), has been obtained from a multiple scattering model at intermediate frequency 4 2 10 C k a , and 0.1 1 Sk a i . It is quite clear that one should use here a frequency dependent approximation of the effective mass density, but this is a topic of further investigation. A comparison of our model calculations of the effective compressional and shear wavenumbers with those obtained from various self consistent models such as [6,7] is also an area for future work. 6 6 16th Anglo-French Physical Acoustics Conference (AFPAC) 1234567890 ‘’“” 16th Anglo-French Physical Acoustics Conference (AFPAC) IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 1017 (2018) 012003 doi :10.1088/1742-6596/1017/1/012003 0 0.05 0.1 0.15 0.2 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 c eff /  e(eff / ) Einstein Batchelor Ruiz-Reina Figure 6. The effective shear viscosity as a function of concentration. Hydrodynamic models follow Ref. [5]. Figure 6. The effective shear viscosity as a function of concentration. Hydrodynamic models follow Ref. [5]. 4. Conclusion This work is a preliminary study of the properties of the shear coherent wave in suspensions of spherical particles. It highlights the truncation of the series in partial wave orders as well as the problem with convergence of the concentration series. Although an effective viscosity has been determined, its validity requires use of a frequency dependent effective mass density. Further work will include the introduction of self consistent models such as in Refs. [6, 7] and their relation with the model used here. The relation between hydrodynamic models and multiple scattering effective theories should be also investigated further, as begun by the authors of Ref. [8]. [1] Forrester D M, Huang J, Pinfield V J and Luppé F 2016 Nanoscale 8 5497-5506 [2] Luppé F, Conoir J M and Norris A N 2012 J. Acoust. Soc. Am. 131 1113-20 [3] Pinfield V J 2007 J. Acoust. Soc. Am. 122 205-21 [4] Berryman J G 1980 J. Acoust. Soc. Am. 68 1809-19 [5] Challis R E and Pinfield V J 2014 Ultrasonics 54 1737-44 [6] Mei J, Liu Z, Wen W and Sheng P 2007 Phys. Rev. B 76 134205 1-20 [7] Jin C 2012 J. Acoust. Soc. Am. 132 615-20 [8] Valier-Brasier T, Conoir JM and Coulouvrat F 2015 J. Acoust. Soc. Am. 138, 2598-2612 References [1] Forrester D M, Huang J, Pinfield V J and Luppé F 2016 Nanoscale 8 5497-5506 [2] Luppé F, Conoir J M and Norris A N 2012 J. Acoust. Soc. Am. 131 1113-20 [3] Pinfield V J 2007 J. Acoust. Soc. Am. 122 205-21 [4] Berryman J G 1980 J. Acoust. Soc. Am. 68 1809-19 [5] Challis R E and Pinfield V J 2014 Ultrasonics 54 1737-44 [6] Mei J, Liu Z, Wen W and Sheng P 2007 Phys. Rev. B 76 134205 1-20 [7] Jin C 2012 J. Acoust. Soc. Am. 132 615-20 [8] Valier-Brasier T, Conoir JM and Coulouvrat F 2015 J. Acoust. Soc. Am. 138, 2598-2612 7 7

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